Proceedings of the
Symposium on Analytical
Methodology for
Determining Immunotoxicity of
Chemicals, Including Pesticides
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SYMPOSIUM OF ANALYTICAL METHODOLOGY FOR DETERMINING
IMMUNOTOXICITY OF CHEMICALS, INCLUDING PESTICIDES
Proceedings of a Conference
Association of Official Analytical Chemists
93rd Annual Meeting, Washington, D.C.
OCTOBER 16, 1979
Co-Sponsored by:
U.S. Environmental Protection Agency
and
Association of Official Analytical Chemists
Edited by:
Thomas S. S. Mao, Ph.D.1
Senior Chemist and Toxicologist
Hazard Evaluation Division
Office of Pesticide Programs
U.S. Environmental Protection Agency
Washington, D.C. 20460
Jack H. Dean, Ph.D.2
Chief of Immunology Laboratory
Environmental Biology Branch
National Institute of Environmental
Health Sciences, NIH
Research Triangle Park, NC 27709
Organizing Committee
Thomas S. S. Mao, Ph.D.
Office of Pesticide Programs
U.S. Environmental Protection Agency
Washington, D.C. 20460
Jack H. Dean, Ph.D.
National Institute of Environmental
Health Sciences, NIH
Research Triangle Park, NC 27709
Howard T. Holden, Ph.D.
Senior Scientist, Laboratory of
Immunodiagnosis
Office of Immunology Programs
National Cancer Institute, NIH
Bethesda, MD 20205
^Visiting Professor
Graduate Programs, Department of Environmental Sciences,
Rutgers - The State University of New Jersey, New Brunswick
Campus, New Jersey 08903
2Director of Toxicology, Sterling-Winthrop Research Institute
Division of Sterling Drug, Inc.
Rensselaer, N.Y. 12144
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FOREWARD
The papers in this compilation were given at the Symposium
on Analytical Methodology for Determining Immunotoxicity of
Chemicals, Including Pesticides - held during the 93rd Annual
Meeting of the Association of Official Analytical Chemists in
October 1979. Publication of these Proceedings was underway in
1980, but was not completed for reasons that are unclear at
this time. Since several inquiries have been received about
this document, and since EPA was a co-sponsor of this symposium,
we are supporting the production of a limited number of copies
of this Proceedings to complete the record.
The opinions, findings, conclusions, and recommendations
expressed herein are those of the authors and speakers, and do
not necessarily reflect the views of the Environmental Protection
Agency.
Hazard Evaluation Division
Office of Pestice Programs
U.S. Environmental Protection Agency
Preparation of this document was completed
prior to the January 22, 1982 effective date
of the EPA Administrator's Order 2200 and
consequently did not necessarily undergo the
peer review procedures described therein. The
document received peer review according to
procedures in place prior to that date and
received complete administrative review.
April 1989
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'TUESDAY
MORNING, OCTOCEfl 16
SYMPOSIUM ON ANALYTICAL METHODOLOGY FOR
DETERMINING IMMUNOTOXICITY OF CHEMICALS. IN-
CLUDING PESTICIDES
1:30.12:00 Persian / Room
THOMAS S. S. MAO.
8:30 OFFICIAL WELCOME Peter E. McGrath, Ph.D.
Director - Hazard Evaluation Division
Pesticide Programs - Office of Toxic Subtances
U. S. Environmental Protection Agency
8:35 INTRODUCTORY REMARKS Kenneth W. Sell, M.D., Ph.D..
Scientific Director
National Institute of Allergy & infectious Diseases - NIK
8:50 (40) Essentials of the Immune Response. H. T. Holden, Laboratory of Immuno-
diagnosls, National Cancer Institute, NIH, Bethesda, MD 20205
9:20 (41) Approaches 'for Assessing Immunobioloplcal Effects Induced bv Chemicals
ef Environmental Concern. J. H. Dean, M. I. Luster, & G. A. Doorman,
Mstional Institute of Environmental Health Sciences, NIH, RTP, NC 27709
9:50 (42) Organochlorlne Induced Immune Suppression. L. D. Loose, J. B. Silkvorth,
S. P. Mudzinski, & T.T. Charbonneau, Albany Medical College, Albany, NY 12208
10:20 Coffee Break
10:30 (43) The Effects of DES on Immune Response of Adult Female Mice. M. I. Luster,
G. A. Boorman, R. Leubke, R. Wilson, & J. H. Dean, National Institute of
Environmental Health Sciences, Research Triangle Park, N.C. 27709
11:00 (44) The Use of Quantitative Micro-Cytotoxlclty Assay to Detect the
Inhibitory Effects of Chemicals on the Immune System. W. A. Stylos,
T. S. S. Mao, & M. A. Chirigos, National Cancer Institute, NIH, Bethesda,
Md, 20014 and U.S. Environmental Protection Agency, Washington, D.C. 20460
11:30 (45) Approaches to Assess Altered Host Resistance. P. C. Hu, R. J.
Smialowicz, & D. E. Gardner, Health Effects Research Laboratory, U.S.
Environmental Protection Agency. Research Triangle Park, N.C. 27711
TUESDAY
AFTERNOON, OCTOBER 16
SYMPOSIUM ON ANALYTICAL METHODOLOGY FOR
DETERMINING IMMUNOTOXICITY OF CHEMICALS,
INCLUDING PESTICIDES
1:30-5:00 Persian I Room
HOWARD T. HOLDEN. Pr.iidmg
1:30 (73) Immunotoxicity Studies of Food Chemicals. D. L. Archer, B. G. Smith,
& J. A. Wess, Food and Drug Administration, Department of Health, Education,
and Welfare, Cincinnati, OH 45226
2:00 (74) Modification of Lymphocyte Transformation (LT) by Pb . Cd . and Cr .
N. J. Baiter, J. A. Bellanti, & I. Gray, Department of Biology and Inter-
national Center for Interdisciplinary Studies of Immunology, Georgetown
University, Washington, D.C. 20007
2:30 (75) Approaches to Investigate Effects of Heavy Metals on Immune Responses.
L. D. Koller, School of Veterinary Medicine, University of Idaho, Moscow,
ID 83843
3:00 (75A) The Potential of Immunologlc Methods in Environmental Testing Programs.
B. S. Zwilling, F. W. Chorpenning, M. S. Rheins, A. Koestner, E. S. Panke,
S. P. Somers, & L. B. Campolito, Dept. of Microbiology and Comprehensive
Cancer Center; The Ohio State University, Columbus, OH 43210
3:30 Coffee Break
Jack H. Dean, Presiding
3:40 PANEL ON THE PRESENT STATUS OF GOALS OF IMMUNOTOXICITY METHODOLOGY
AND OVERVIEW OF IMMUNOTOXICOLOGY
D. L. Archer ( FDA ) M. Luster (NIEHS - NIH)
J. A. Bellanti (Georgetown U.) 0. J. Plescia (Rutgers U.)
P. C. Hu (U.S. EPA, NC) J. Seifter (U.S. EPA, Wash. DC)
L. D. Koller (Univ. of Idaho) W. A. Stylos (NCI - NIH)
L. D. Loose (Albany Med. College) B. S. Zwilling (Ohio State U.)
4:40 CLOSING REMARKS: The Present Status of Coals of Immunotoxicity Methodology
Otto J. Plcscla, Ph.D.
Professor of Dept. of Immunology and Immunochemistry
Uaksmnn Institute of Microbiology
Rutgers - Th*« Sr.ite t'nlvprnlty of Now Jersey
New Brunswick, New Jersey 08903
4:50 CLOSING REMARKS: Overview of Tmniunotoxlcology
Joseph J. Bellanti, M.D.
Professor & DlrccLor of Intcrnntlon.il Center for
Interdisciplinary Studies of Immunology & Profcacor of
Georgetown University School of Me ill cine
K.mliiniTOii , ll.r.. ?r'l"ir.7
ii
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TABLE OF CONTENTS
Page
Foreward .
Acknowledgement ,
Introductory Remarks - Kenneth W. Sell, M.D.
Approaches for Assessing Immune Alterations Induced
by Chemicals of Environmental Concern - Jack H. Dean,
Michael I. Luster, Gary A. Boorman, and Jack A. Moore ... 4
Modification of Cell-Mediated Immunity by Poly-
chlorinated Biphenyl (AroclorR 1016) and Hexachloro-
benzene - Jay B. Silkworth and Leland D. Loose 24
Modification of Lymphocyte Transformation by Trace
Heavy Metals - Nancy J. Baiter, Joseph A. Bellanti
and Irving Gray 72
The Use of Quantitative Micro-Cytotoxicity Assay to
Detect the Inhibitory Effects of Chemicals on the
Immune System - W. A. Stylos, T. S. S. Mao, and
M. A. Chirigos 89
Development of Radioimmunoassays for Chlorinated
Biphenyls, Dibenzofurans and Dibenzo-p-Dioxins -
M. I. Luster, P. W. Albro and K. Chae, and
James D. McKinney 109
Approaches to Assess Altered Host Resistance -
Ping C. Hu, Ralph J. Smialowicz and Donald E. Gardner . . . 125
Approaches to Investigate Effects of Heavy Metals on
Immune Responses - Loren D. Koller 155
Immunotoxicity Testing of Food Chemicals: Different
Results May Be Obtained with In Vitro and In Vivo
Exposure to Gallic Acid - DougTas L. Archer an3
Bennett G. Smith 169
Chemical Carcinogenesis and Immunity (Effects of
Methylnitrosourea on the Normal Iramunologic Function
of Rats) - Bruce S. Zwilling, Frank W. Chorpenning,
Adalbert Koestner and Melvin S. Rheins 179
Mitogenic Studies of the Effects of 1,3-Bis(2-Chloroethyl)-
1-Nitrosoure.a (BCNU) and Indomethacin on the Lymphocytes
from Normal and Tumor-Bearing Mice - William A. Stylos
and Thomas S. S. Mao 196
111
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TABLE OF CONTENTS
(Continued)
Page
Panel Discussion on the Current Status of the Developing
Discipline of Immunotoxicology - Jack H. Dean 207
Iramunotoxicology in Perspective - Otto J. Plescia 212
Closing Remarks: Immunotoxicity Symposium -
Joseph A. Bellanti 214
Appendix 215
IV
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ACKNOWLEDGEMENT
In memory of the late Joseph Seifter, M.D. - An outstanding
scientist and scientific decisionmaker- for his staunch support
for health sciences and understanding of the public impact of
toxicological science. Dr. Seifter endorsed this Symposium from
its beginning.
We, the Organizing Committee, would like to take this
opportunity to thank Dr. William A. Coniglio of the EPA science
staff for his earlier advice and negotiation in November, 1985.
The manuscripts for this proceedings were partially edited in
the Information Services Branch, Program Management and Support
Division, Office of Pesticide Programs and were then left there
in January, 1986 in order to make further plans with the Hazard
Evaluation Division, OPP. We would also like to thank Ms. Barbara
McDowell of EPA, Office of Visual Aids (Graphics) for designing
the art work for the cover.
The Association of Official Analytical Chemists (AOAC) co-
sponsored this Symposium by including it in their 93rd Annual
Meeting in Washington, D.C. The Organizing Committee for this
Symposium appreciated AOAC's support which partially financed this
Immunotoxicity Symposium. Dr. David B. McLean, Executive
Director of AOAC, was very helpful to the Symposium's Organizing
Committee for consultation during the early stage of planning
and designing of the Symposium. We also would like to thank
Dr. McLean at this time for his generosity and kindness in
giving this committee the free choice and decision as to where
we would like to publish all the papers which were presented
in the 93rd AOAC Annual Meeting. Ms. Kathleen M. Fominaya,
Program Assistant of AOAC, assisted us in various ways during
the carrying-out stage of this Symposium, so we thank her very
much for her kindness in this matter.
Naturally, the Organizing Committee and Editors of this
Proceedings are very grateful to all authors of papers,
written remarks and comments, who took the time to present
their hard-working research data in the meeting and made their
great efforts in writing it into the final manuscripts for
publication in this Proceedings. Therefore, we sincerely
express our appreciation to the authors not only for their
excellent research work but also for their support, understanding
and patience exhibited while waiting for the final publication
of this Proceedings.
The Organizing Committee and Editors
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INTRODUCTORY REMARKS
Kenneth W. Sell, M.D., Ph.D.1
Scientific Director, National Institute of Allergy
and Infectious Diseases
National Institutes of Health
Bethesda, Maryland 20205
Good Morning Ladies and Gentlemen. I an encouraged to see that so
many people have gathered to express their interest in the effects of
chemicals on the immune system jnd host immunity ID nan.
Today, this conference will address topics that suggest that
chemicals, Insecticides and other manufactured agents may either
Influence, augment or suppress the Immune system. These environmental
factors provide an Important source of toxicity in man as measured by
host immunity. These Immune processes are Important In the control of
Infectious diseases, autoimmunity and probably the ontogeny of cancer.
For those of you who are not Immunologists, I would like to remind you
that immunology is a relatively young science. Of course, the immune
nature of man was known as long ago as ancient Greece. During the
plagues, only those individuals who had already suffered from the
plague and survived were assigned to be nurses in the wards In the
hospitals. Even then It was known that once recovered, the plague
victim could not get the disease a second time. In their pragmatic
fashion, they had discovered immunity. It wasn't until about the
early 1800's that the process was placed on a firm scientific basis by
Jenner. Each of you have already heard of his experiments which
utilized the cow pox virus to provide a mild Infection which would
subsequently prevent the devastating and virulent disease produced by
the smallpox virus in man. Using vaccination, smallpox has now been
irradicated from all populations in all countries in the world. A
more firm basis and understanding of microbiology and immunology,
however, awaited the discoveries of Pastuer. At about the time of our
Civil War, he began to identify microorganisms and suggested the "germ
theory". While his discoveries provided the sound fundamental basis
for our understanding of microbial processes, we must remember that
the support for his work came from the wine industry interested in
microorganisms which helped in the fermentation process. Pastuer,
therefore, was an applied scientist who made long-reaching and
Important basic discoveries which are fundamental to our understanding
of biomedical processes of Infectious diseases. Those Interested in
studying the environmental effects of chemicals and drugs as they
interact with the Immune system should take this lesson very
seriously. Applied science and basis science walk hand In hand to
unravel the underlying mechanisms of biological defects and disease.
At the turn of this century, fhrllch and Metchnikoff took the next
step to help us understand the operation of the Immune system. They
suggested mechanisms by which cells and antibodies could Interact and
noted that some cells could phagocytose or engulf the Invading
organisms thereby protecting the body from infectious damage. Paul
Ehrlich not only suggested that serum proteins circulated which co.uld
react with Invading foreign antigens, he also was among the first 'to
Professor and Chairman, Department of Pathology & Laboratory Medicine, Biory
University School of Medicine, Itocn 703, Woodruff Mem. Bldg., Atlanta, GA 30322
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point out that we did not react with the constituents of our own
bodies and to suggest that we have self-tolerance to the antigens of
our own tissues.
Later In the 1920*s Landsteiner discovered blood group antigens. This
allowed us to develop an understanding of the Immunogenlc or antigenlc
nature of our own cells and tissues, suggesting that while we did not
react against our own tissues that these cells did In fact contain
antigens which could stimulate others to produce an Immune response
similar to or Identical to that response seen when an Individual Is
exposed to Invading mlcroblal agents.
In the past few decades, Immunology as a science has developed at an
amazing pace. In my view, much of this development has been
stimulated by the development of tissue and organ transplantation.
The transplant surgeon saw a need for reconstructive and repalratlve
surgery which required the transplantation of human tissues. When the
tissue grafts failed, they turned to their basic scientist colleagues
to provide assistance In the understanding of graft rejection. Gowans
soon pinpointed the heart of the Immune response to be located in the
lymphocyte. The processes of tolerance, graft versus host disease,
histocompatiblHty antigens and their role 1n graft rejection and
specific immunosuppresslon all developed because of the requirements
for understanding of transplantation. Serend1p1tously, the study of
tissue antigens led us to an Intense Investigation of the major
hlstocompatlblHty complex. This genetic system, one of the most
complex, Interactive, genetic loci In man, soon was shown to be the
genetic heart of Immune cell Interaction and function. The
Immunoregulatory genes which were located within the MHC (chromosome 6
of man) provide the basis for understanding genetic susceptibility to
autoimmune and infectious disease processes.
In order to provide prolonged graft survival, we began to develop
drugs and chemicals which could suppress the Immune system so that
graft rejection would not occur. Chemical Immunosuppresslon has
proved to be very successful. Certain medications such as
azathioprine and prednisone have proved sufficiently immunosuppressive
so that kidney transplants, even when not matched and not related, can
be accepted by a patient with over 50 percent graft survival. Many
other drugs, of course, have been studied for their effect on the
immune system. Cytoxan acts much as radiation does. It provides
direct cytotoxidty or killing of lymphocytes which are responsible
for graft rejection. Unfortunately, it also affects all rapidly
dividing mitotic cells so that 1t causes great damage to tissues such
as the bone marrow.
The study of Immunosuppressive drugs leads us directly to the
understanding of problems with other chemicals, medications or
environmental factors which may also affect the Immune system and In
some cases cause Immunosuppresslon. Clearly, the iatrogenic process
of chemotherapy In the field of cancer often results in the
significant and serious side effect related to the destruction of
lymphocytes and the consequent Immune suppression. We know that when
the Immune system is suppressed, as 1s necessary in transplantation,
there Is a significant problem In response to Invading organisms,
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- 3
particularly viruses, fungi and protozoa. Perhaps even more
importantly we now understand the immune system must be significant in
our protection against cancer. When chronic, long-term
immunosuppression is used to prevent graft rejection, as many as four
to six percent of the patients subsequently develop a denovo tumor or
cancer. Fortunately, most of these cancers are easily treated. Their
occurrence, however, suggests to us that Interference with the immune
process makes us susceptible to neoplastic changes. We, therefore,
can see that there may be significant problems from exposure to
chemicals, pharmaceutical agents, pesticides or other drugs that may
affect the immune system resulting in increased susceptibility to
infection and cancer.
We must remember, however, that environmental agents and
pharmaceutical products may, in fact, enhance the immune response.
Some years ago in our laboratory, for Instance, we showed that
microwaves (an environmental pollutant) actually stimulated certain
subsets of B lymphocytes causing the expression of increased numbers
of complement receptors on their surface. Other chemical agents and
pharmaceutical agents have also been discovered which will stimulate
the immune system. This stimulation, however, itself may not be
beneficial. Excess stimulation could lead to autoimmune disease and a
pathologically overreact!ve immune system.
The problem, therefore, is a complex one. Many drugs, chemicals,
insecticides and pharmaceutical agents can affect the immune system,
either stimulating or suppressing the various cells involved in the
immunity reaction. The problems you face, therefore, which involve an
understanding of the interaction of chemicals with the immune system
will provide a challenging area of research. Fortunately, the modern
tools of science allow us to inspect the various aspects of the immune
system with greater precision than ever before. The challenge to the
research scientist is now accompanied by an array of investigational
tools that allow the inquiring mind to at least begin to answer these
perplexing problems.
Thank you for your kind attention. I, as you, look forward to the
very exciting information which will be presented here today as an
introduction to the discussion of the effect of drugs and chemicals on
the immune system.
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APPROACHES FOR ASSESSING IMMUNE ALTERATIONS INDUCED
BY CHEMICALS OF ENVIRONMENTAL CONCERN
Jack H. Dean1, Michael I. Luster? Gary A. Boorman? and Jack A. Moore8
Environmental Biology Branch and Environmental Chemistry Laboratory, National
Institute of Environmental Health Sciences, National Institutes of Health, Research
Triangle Park, North Carolina 27709
At Present;
Department of Toxicology
Sterling-Winthrop Research Institute
Division of Sterling Drug, Inc.
Rensselaer, N. Y. 12144
o
Iraraunotoxicology Group
Systemic Toxicology
National Institute of Environmental Health Sciences
P.O. Box 12233
Research Triangle Park, N. C. 27703
^Chemical Pathology Branch
National Institute of Environmental Health Sciences
P.O. Box 12233
Research Triangle Park, N. C. 27703
Office of Peaticides and Toxic Subtances
U.S. Environmental Protection Agency
Washington, D. C. 20460
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ABSTRACT
* —.
The observations of altered host resistance and immunologic dysfunction following
low level exposure of rodents and man to various chemical pollutants have prompted
the evaluation of Immunologic approaches for application to routine assessment of
alterations in immunocompetence following chemical exposure. Most routine toxicology
procedures nay lack the sensitivity achievable through functional assays provided by
modern immunology. The panel of assays selected for immunotoxicologic evaluation
should include procedures to study impaired immune responsiveness, hypersensitization,
and altered host resistance. The assay panel selected should also lend itself to
certain practical considerations such as simplicity, reproducibility, cost and
application to routine toxicology studies without sacrificing biological relevance.
With these considerations, an assay panel is described to screen for imrnunologic
effects. Methodologies are also described which can be used to further define the
mechanisms responsible for the immunobiological effects observed. Approaches for
evaluating host susceptibility following challenge with small numbers of bacteria
or transplar.table tumor cells (LD1Q or TD|Q) are described. Information provided
by this test panel should provide a reasonable and sensitive data base from which
judgments can be made regarding the safety of the test drug or chemical. These
immunologic assays may represent more sensitive endpoints than currently employed
in general toxicity assessment since functional and cell-cooperation responses are
examined using bone marrow and lymphoid cells.
INTRODUCTION
There is increasing evidence in the literature suggesting a relationship
between neoplasia or altered host resistance to infectious agents and immunologic j
5
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dysfunction in experimental rodent models. Likewise individuals born with severe
immunologic dysfunction or receiving chemotherapy or radiation therapy to sustain
organ or marrow transplants have been found to have altered host resistance and a
higher frequency of neoplasia (1-4). It is believed that general immunologic
competence of the thymus-dependent lymphocyte component and mononuclear phagocyte
system provide host resistance to foreign organ transplants and to neoplastic
transformed cells. This complex component of host resistance was termed immune
surveillance by Burnet (5).
Immunologic dysfunction as indicated by depressed humoral (antibody) or cell-
mediated immunity has been reported in rodents exposed to low concentrations of
certain chemicals and drugs (Table 1) (see reviews 6-9). Chemicals which have
reportedly induced immunologic dysfunction in rodent studies include among others
2,3,7,8-tetrachlorodibenzo-p_-dioxin (10-14), polychlorinated biphenyls (15-22),
polybrominated biphenyls (23), gallic acid (24), hexachlorobenzene (21,22,25),
certain organo- and heavy metals (26-30). Immunologic dysfunction also has been
reported in rodents exposed to chemotherapeutic drugs such as cyclophosphamide,
adriamycin, actinomycin, 5-fluorouracil methothioprine (31-35). Immune impairment,
similar to that observed in rodents, was recently observed in humens inadvertently
exposed to polybrominated biphenyls (36). Several studies have shown that chemical
exposure resulting in immune dysfunction can alter host resistance to certain
bacteria (37), viruses (37,38), parasites (22) and transplantable tumor cells (35).
There are a variety of mechanisms which could be invoked to describe how
chemicals or drugs might alter immure function. The mechanisms responsible for
chemical-induced immune dysfunction are listed in Table 2. The chemical may directly
affect the developing cells, be selectively toxic and cause lysis, merely impair
the functional response of the target cell or operate through an effect on non-
lymphoid target organ (i.e., adrenal glands).
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Also of major concern is the observation that some chemicals can induce
immediate or delayed-type hypersensitivity (e.g., penicillin, DNCB, DNFB) and
thus new chemicals should be evaluated for this potential.
Panel for Evaluating the Immunobiologic Effects
A test for evaluating immunologic effects should meet several criteria if it
1s to be considered useful and definitive. The test should be relevant to the
human experience and adaptable to practical considerations such as expense,
simplicity, time required for completion, reproducibility, uniformity and applica-
tion to routine toxicology studies. Certain compromises are associated with the
development of routine screening techniques. Ideally, sensitive inrounologic assays
that will identify and assess the risk potential of a chemical or drug are desirable,
however, in general, no single assay can accomplish this task and as such, a panel
of selected assays that have been validated in experimental rodent models and human
clinical studies are recommended.
The assays that are utilized or under development in our laboratories at the
National Institute of Environmental Health Sciences assess immunologic dysfunction
following chemical exposure are listed in Table 3. These procedures are reproducible
and easily standardized. A major emphasis was placed on assays that could be
automated, routinized and require only microquantitates of cells or body fluids.
All of these assays can be performed on six groups of animals containing 20-40
animals per group and includes a control and 3 dosage levels of the test chemical.
If the in vivo and in vitro data obtained from such carefully planned studies
using these screening tests are negative, there can be reasonable confidence in
the safety of the drug or chemical under the conditions defined.
A major limitation in risk assessment involves extrapolation of dose response
curves from effect to no-effect levels or from rodent model systems to humans.
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However, if extrapolations are made on^ the conservative side using data from ap-
propriate assays, they will offer the best and roost relevant estimate possible.
If warranted, additional tests to examine the mechanisms by which a chemical
or drug impairs or potentiates immune function can be performed following evaluation
of the results in the screening tests (Table 4). These assays can provide addi-
tional Information regarding mechanisms of immunotoxidty and means to circumvent
the undesired effects. If the pathophysiology responsible for the effect or the
target cell 1s defined, possibly new analogs of the chemical can be synthesized
which provide the desirable effects without the undesirable ones (e.g., synthetic
penicillin). ,
Mice
Female, BgC3F1 (C57BL/6NxC3H) mice weighing 18-22 grams were obtained through
the National Cancer Institute production contracts (Charles River, Wilmington, MA)
and were used throughout the immunotoxicity studies. It has been established that
hybrids are less variable among individual mice than either outbred or inbred
strains (39).
Tumor Susceptibility
Sarcoma PYB6 was kindly provided by Dr. Lloyd Law of the National Cancer
Institute and was induced by Polyoma virus transformation in C57BL/6 mice. This
tumor is carried in the parental strain by passage at 2 week intervals via sub-
cutaneous injection. Single cell suspensions are prepared by careful dissection
and injected subcutaneously at 5 x 10* into chemically exposed BCF, mice. This
tumor cell inoculum produces 10-20% tumor takes (T^0_20) *n control non-exposed
B6C3F1 m
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Details of Selected Methods
Several methods will be described in detail since these are recently developed
or modified procedures which appear to offer sensitive endpoints for detecting an
immunologic effect following chemical exposure.
Isotopic Delayed Hypersensitivity Response (DHR) Assay
BgC^Fj mice were immunized with keyhole limpet hemocyanin (Pacific Bio-Marine
Co., Venice, CA) by subcutaneous (sc) Injection of 100 pg of protein in incomplete
Freund's adjuvant, followed by a subsequent subcutaneous injection in adjuvant
9 days later. One to 6 weeks following the final immunization, mice can be admin-
istered to the test chemical followed by challenge with recall antigen using the
radiometric assay described originally by Lefford (42) and in detail as employed in
this laboratory by Luster et al. (40). Alterations of the DHR response to T-cell
dependent antigens following chemical exposure promises to be a sensitive in vivo
parameter of immune dysfunction (35). Depressed DHR responses have been observed
•i in mice following administration of cyclophosphamide (35), TCDD (50), DES (49), and
PCB (23). Recent clinical studies demonstrated a strong correlation between
depressed DHR to recall antigens and an increased susceptibility to bacterial
sepsis and wound infections in surgical patients (51).
Cunningham's Antibody Plaque Forming Cell (PFC) Assay
The IgM antibody PFC response to the T-dependent antigens on sheep erythrocytes
was performed as described by Cunningham (52) and more recently in detail by Dean
et al. (46). Following chemical exposure mice were immunized by intravenous
injection of 0.2 ml of 5% SRBC. Four days later the mice were sacrificed and their
spleen removed. Single cell suspensions of spleen cells were prepared and aliquoted
(2 x 106) along with SRBC (0.3 of 10% suspension) complement (0.1 ml undiluted),
and RPMII640 to give a final volume of 1 ml. Thirty yl of this suspension was
added to Cunningham chambers. The chambers were sealed and incubated at 37eC for
1 hour. Antibody plaques in the SRBC lawn were counted using an inverted microscope
9
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Katz et al. (53) described an automated method for counting plaques on Cunningham
» **
slides using a modified Artek colony counter. This automated counting procedure
1s currently being evaluated in our laboratory.
Numerous studies have described depression of the T-dependent antibody PFC
response to SRBC following exposure to cyclophosphamide (35), heavy metals (8), and
DES (49.50). The PFC assays are more sensitive and quantitative than measurements
of serum antibody ttters.
Bone Marrow Progenitor Cell Assays
Within the past decade in vitro and In vivo culture techniques have been
developed to examine the proliferative capacity of bone marrow cells including
hematopoietic stem cells, macrophage-granulocyte progenitors, megakaryocytes
precursors, erythroid precursors and T and B lymphocytes (55). As a result, a
wealth of information concerning hematopoiesis has been obtained (56). For
example, chronic myelogenous leukemia, polycythemia rubra vera and myelofibrosis-
myeloid metaplasia have been shown to be neoplastic disorders of hematopoietic stem
cells (57). Bone marrow culture techniques are also used to assess potential
hematopoietic toxicity of various chemotherapeutic agents (58,59). The antileukemia
agent, Myleran, reduces both marrow cellularity, splenic stem cells (CFU-S) prolif-
eration, and granulocytic progenitor cells (CFU-C) (60). Short term exposure of
mice to Busulfan decreases bone marrow stem cells, a defect that persists for at
least 95 weeks, while bone marrow cellularity and peripheral blood counts remain
normal (61). Antimicrobial agents such as trimethoprim and sulphamethoxazole also
inhibit human erythroid and granulocytic progenitors at therapeutic levels (62). In
toxicology, however, little attention has been devoted to the use of these hemo-
poietic culture techniques, even though hemopoietic cells are sensitive to some
chemicals in vitro at picomolar concentration (63). Recent studies have shown
that environmental chemicals such as TCDD (48), and diethylstilbestrol (49,50) all
10
-------
cause alterations in the numbers of bone marrow hemopoietic stem cells and macrophage-
granulocyte progenitors in mice. These recent findings suggests that bone marrow
culture assays may be important parameters for immunological assessment.
SUMMARY
The proceeding pages describe approaches and an assay panel for identifying
and defining immunological dysfunction in mice following chemical exposure. These
immunologic procedures have the potential of increasing the sensitivity with which
one can measure toxicity to chemicals, since they measure functional responses at
t
the cellular level. Some chemicals may indeed be selectively toxic to bone marrow
cells or lymphocytes (e.g., TCDD, DES), although it is unlikely that most toxic
chemicals will possess this potential. It can be assumed, however, that certain
cells of the immune system are more sensitive to chemical assault then less rapidly
proliferating cells of other organ systems. It is also possible that the demands
made on bone marrow and lymphoid cells by evaluating functional assays may serve to
enhance our resolution for assessing toxicity at a cellular level. Enough conjecture
on how assays of immunologic function might improve toxicity assessment, the facts
are that if the tests described above are carefully performed they should provide
valuable information regarding the potential of the suspect chemical to produce an
Immunological effect. This information should help expand the data base from which
a decision can be made regarding the safety of the test chemical.
11
8
-------
TABLE.!.
PARTIAL LISTING OF CHEMICALS AND DRUGS REPORTED TO ALTER IMMUNE FUNCTIONS
Chemicals
Halogenated Aromatic Hydrocarbons -
Pesticides -
Arsenicals -
Organometals -
Heavy Metals -
2,3,7,8-tetrachlorodibenzo-p-dioxin,
dibenzofuran, polychlorinated biphenyl,
polybrominated biphenyl, hexachlorobenzer
DDT, dleldrln, carbaryl, carbofuran,
methy!parathion
Sodium arsenlte, arsenate, arsenic triox
Methylmercury chloride, di-N-octyltindi-
chlorlde, di-N-butyltindichloride
Lead, nickel, cadmium, mercury,
chromium, cobalt
Drugs
Alkylating Agents -
Antibiotics -
Folic Acid Antagonists -
Pyrimidine Nucleoside Analogs
Thiopurines -
Cyclophosphamide, nitrogen mustards
Actinomycin, adriamycin
Methotrexate
5-Fluorouracil
6-Mercaptopurine, ezathioprine
12
-------
-10-
TABLE 2
MECHANISMS OF CHEMICAL INDUCED IMMUNE DYSFUNCTION
I. Causes depletion of responding cell type(s)
A. Blocks maturatlonal development of cell
B. Chemical interference at cell surface
C. Directly toxic or cytolytic for cell
II. Induces functional defect(s)
A. Blocks recognition or activation of cell
B. Blocks essential metabolism of cell
C. Activates supressor cell
III. Hormonal effect
A. Effects adrenal or other endocrine gland
13
-------
TABLE 3
PARAMETERS TO EXAMINE AND PROCEDURES TO PERFORM TO MEASURE IMMUNOLOGIC DYSFUNCTION FOLLOWING CHEMICAL EXPOSURE IN RODENTS
1
Parameter
Pathotoxlcology
Host Resistance
Procedure Performed
Detailed Reference of
Immunology Procedure
Delayed Hypersensltlvlty
Lymphocyte Proliferation
Humoral Immunity
Hematology Profile-hemoglobin, red blood cell
count, white blood cell count, differential
Liver Chem1str1es-SGPT, trlglycerldes, cholesterol
Serum Proteins-albumin, globulin, A/G, total proteins
Lymphold Organ Weights-spleen and thymus
Histology-liver, thymus, lung, kidney, spleen
Tumor Challenge- TD^gn
Llsterla monocytogenes challenge-LDjQ on
Endotoxin hypersons1t1v1ty
Radlometrlc DHR to T-cell dependent antigen
One-way mixed leukocyte culture using pool of
dllogenelc stimulator cells
Wtogens-PHA, Con A, LPS
Immunoglobulln levels
Tlter of serum antibody to T-dependent antigen
Plaque forming eel! response-T-dependent antigen
(35)
(40)
(41)
(42)
(43)
(44)
(45)
(46)
-------
Macrophage Function
Bone Marrow Colony Forming Units
Phagocytlc Index
L>sosomal enzymes-lysozyme, add phosphatase,
superoxide dismutase
Cytostasls of tumor target cells
Cytolysls of tumor target cells
CFU-S-muHipotent, hematopoietlc stem cells
CFU-GM-granulocyte/macrophage progenltor
CFU-M-megakarocytes progenltor
CFU-E-erythrocytes progenitor
Histology and myelold/erythrold ratio
(47)
(47)
K>
I
1
The assays described In this panel can all be performed on six groups of animals.
-------
TABLE 4
PROCEDURES TO FURTHER STUDY THE MECHANISM OF CHEMICALLY INDITED IMMUNE DYSFUNCTION
Altered Parameter
Further Procedures to Perform
Pathotoxicology
Host Resistance
Cell Mediated Immunity
Antibody Mediated
Immunity
Bone Marrow Toxicity
Bioaccumulation study
Hormone levels
Virus challenge
Staphylococcus or Streptococcus challenge (B-cell dependent)
DHR using T-cell independent antigen
Supressor cell studies with mitogens
Helper cell studies
T-cell mediated cytotoxicity
Mishell-Dutton assay
Local production of antibody
Titer of serum antibody-T-cell independent antigen
Serum levels of colony stimulating factor (CSF)
CSF and prostag!andin synthesis by macrophages and
bone merrow stronial ceT>s
16
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-14-
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23
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Modification of Cell-Mediated Immunity by
Polychlorinated Biphenyl (AroclorR 1016) and Hexachlorobenzene^
Jay B. Silkworth2
Leland D. Loose3
24
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This work was supported by a fellowship to J. B. Silkworth from the
Monsanto Fund and, in part, by a joint program between the Gesellschaft
F. Strahlen-und Umweltforschung mbH, Munich, Germany and the Institute
of Comparative and Human Toxicology, Albany Medical College, Albany,
New York (U.S.A.).
f\
*• Present address: Toxicology Section, Division of Laboratories and
Research, New York State Health Department, Empire State Plaza, Albany,
New York 12201.
Pfizer Company, Inc.
Central Research
Eastern Point Poad
Groton, Connecticut 06340
25
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Abbreviations used in this paper: CML, cell-mediated lymphocytotoxicity;
MLR, mixed lymphocyte response; GVHR, graft-versus-host response; HCB,
hexachlorobenzene; PCB, polychlorinated biphenyl; PHA, phytohemag-
glutinin; IPS, lipopolysaccharide; PMN, polymorphonuclear neutrophil;
HBSS, Hanks balanced salt solution; FBS, fetal bovine serum.
-------
ABSTRACT
This investigation was designed to determine whether chronic
exposure to two polyhalogenated aromatic hydrocarbons, AroclorR 1016,
(a polychlorinated biphenyl, PCB), or hexachlorobenzene, (HCB), alters
the cell-mediated immune responsiveness of mice. Furthermore, the
experimental design assessed the influence of the compounds on each of
the three required developmental phases of a cell-mediated immune
response. It was determined that PCB, at the concentration used in
this study, had minimal effect on the cell-mediated immune responsive-
ness of mice. In contrast, however, dietary administration of HCB to
mice resulted in a decreased graft-versus-host reactivity and lympho-
cytotoxicity of isolated splenic lymphocytes and it is suggested that
the site of the HCB-induced lesion is located in the effector phase of
the immune response.
27
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INTRODUCTION
Evidence has indicated that host resistance and antibody-mediated
immunity may be altered by short-term exposure to environmental poly-
halogenated aromatic hydrocarbons (1-8). There is also some evidence
that short-term exposure to these compounds may influence cell-mediated
immunity (9-12).
Since the involvement of the cell-mediated immune system in the
expression of environmental chemical toxicity is evident following short-
term exposure, it is appropriate to determine the sensitivity of the
cell-mediated immune system to modification following chronic exposure
to xenobiotics, to delineate the mechanism of functional alteration of
the immune system, and simultaneously develop a test system for the
assessment of the potential toxicity of new chemicals.
The development of an immune response depends upon the proper
function of three basic phases: (a.)» initial recognition of the anti-
gen, (b.) , activation, which includes proliferation and differentiation
of reactive clones, and (c.)> the expression of immunity (Figure 1).
This concept can be developed further with what is already known of the
mechanism of cell-mediated immune responses and applied to the assessment
of environmental chemical immunotoxicity.
Available jji vivo and in vitro techniques allow the examination of
the developing cell-mediated immune response and perhaps clarification
of the mechanisms of polyhalogenated aromatic hydrocarbon immunotoxicity
(Figure 1) and shall serve as the protocol for this paper.
Injection of Immunocompetent cells into an immunoincompetent animal
expressing histoincompatible antigens results in a GVHR. It has been
28
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FIGURE 1
DEVELOPMENTAL PHASES OF A CELL-MEDIATED IMMUNE RESPONSE AND ASSAYS
WHICH CAN BE USED FOR THEIR FUNCTIONAL ASSESSMENT
RECOGNITION
( T-cell, B-cell,
macrophage )
ACTIVATION
( proliferation,
differentiation,
clonal expansion )
EXPRESSION
( cytotoxlclty,
lynphoklnes,
soluble factors )
GRAFT-VERSUS-HOST RESPONSE
MITOGEN RESPONSE
MIXED LYMPHOCYTE RESPONSE
LYMPHOCYTOTOXICITY
ro
NO
-------
established that the GVHR is an expression of cell-mediated immunity (13) and
that the response is mediated by the T cell (14-16). The GVHR has three
prerequisites. First, the recipient must be unable to react against the
donor cell. Experimentally this may be accomplished by the injection of
immunocompetent parental strain cells injected into young Fl hybrids.
Second, the donor and recipient cells must be histoincompatible. The
injection of parental strain cells into Fl hybrid recipients satisfies
this requirement. Third, the donor cells must be immunocompetent.
The severity of the graft-versus-host response induced by immuno-
competent cells from control and treated animals will serve as an assess-
ment of the functional status of the recognition, activation, and expres-
sion phases of the immune response as indicated by the development of a
completed response, i.e. splenomegaly (17), and will reflect the functional
ability of the splenic lymphocytes which are involved (18).
The mixed lymphocyte response (MLR) is generally accepted to
represent the recognitive and proliferative phases of the cell-mediated
immune reaction (19-21). The presentation of alloantigen of irradiated
stimulator cells to responder cells from control and chemical-exposed
mice results in a proliferative response by the responder cells which can
o
be quantitated by (JH)-thymidine incorporation. Since the intensity of
the response is primarily dependent on the disparity of H-2 and Mis
antigens expressed by the stimulator and responder cell populations,
DBA/2 (H-2d, Mis1) cells can be used as stimulator cells to provide a
stimulus to C57BL/6 (H-2b, Mis2) mice.
Folyclonal mitogens such as PHA and LPS are thought to activate
lymphocytes by binding to glycoprotein receptors on the cell surface (22).
-2-
30
-------
Although these mitogens seem to bind only certain glycoproteins which may
be present on either T lymphocyte subpopulations, B lymphocytes or only
on accessory cells such as macrophages, their effects are independent of
antigen binding specificity since polyclonal activation by mitogens
results in B lymphocyte differentiation to cells producing immunoglobulins
of many idiotypes (16) or results in T lymphocyte differentiation into
cells which express non-specific cytotoxicity. The B lymphocyte mitogen,
LPS, activates the B lymphocyte non-specifically at sites other than Ig
receptors and the activation is direct (23).
The measurement of mitogen-induced ( H)-thymidine incorporation in
lymphocytes is, therefore, an assessment of the activation phase of the
immune response with the advantage of by-passing specific antigen initial
recognition (16). The interpretation of such an experimental design must
include the consideration of the role of the macrophage in mitogen-
induced T lymphocyte activation. The use of the B lymphocyte mitogen,
LPS, in this study, in addition to the T lymphocyte mitogen, PHA, may aid
in clarifying the nature of the chemical-induced lesion, if any, by
exposing a tendency to alter lymphocyte activation in general, or, T or
B lymphocyte activation in particular.
Cell-mediated lymphocytoxicity (CML) can be assayed by several in
vitro techniques including short-term Chromium release from Chromium-
labelled target cells and is an assessment of the effector phase of cell-
mediated immunity (16, 24). Although the recognition and activation
phases of the response to alloantigens may be intact as indicated by
comparable values in MLR ( H)-thymidine incorporation between cells from
control and chemical-treated animals, the cytotoxic mechanisms may,
-3-
31
-------
nevertheless, be functionally impaired. Thus, cytotoxic lymphocytes from
C57BL/6 mice immunized with the DBA/2 tumor, P815, will be tested for
their ability to recognize and kill cells which express alloantigen (H-2d).
Another DBA/2 tumor, F388, will be used as a target cell to obviate anti-
tumor antigen activity directed against P815 tumor antigen. The PHA-
induced AKR blast splenocyte (H-2 ) will be used as a target cell to
detect chemical-induced non-specific killing and the C57BL/6 tumor, EL-A,
(H-2 ) will be used to detect chemical-induced alteration of recognition
of self.
In summary, an experimental protocol has been designed to determine
whether chronic exposure to environmental polyhalogenated aromatic hydro-
carbons alters the cell-mediated immune responsiveness of mice. Further-
more, the experimental design will assess each of the three required
developmental phases of a cell-mediated immune response and, perhaps,
clarify the mechanism by which environmental chemicals cause modulation
of the immune system.
MATERIALS AND METHODS
Animals
Male C57BL/6Tex mice with a histocompatibility gene complex denoted
as H-2b, male B6D2F1 (H-2b'd) mice (BDFl), pregnant C57BL/6Tex mice which
had been mated with D6B2A/2 (H-2d1 mice (DBA) and male DBA mice were
supplied by Timco, Texas. Male AKR mice (H-2k) were supplied by Jackson
Laboratories, Bar Harbor, Maine. Mice were purchased at an initial
weight of 18-20 grams.
All mice were housed in the Animal Facility at Albany Medical
College. Control animals were fed Wayne Mash . Experimental animals
-4-
-------
were fed a diet containing either 167 ppm AroclorR 1016 (PCB) or 167
ppm hexachlorobenzene (HCB) in WayneR MashR. Food and water were
provided ad libitum. Body and organ weights were determined for each
experimental period and the relative organ weights were calculated.
An analysis of variance and the Student's t-test were used to determine
statistical significance of differences between control groups and
experimental groups at p<0.05.
Chemicals
The test chemicals used were the polychlorinated biphenyls
AroclorR 1016 (Monsanto), a distillation product of AroclorR 1242 which
contains approximately 42% chlorine by weight but from which has been
removed most isomers containing five or more chlorine atoms per molecule;
hexachlorobenzene (CgC^) (HCB) (Eastman practical grade) was purified
by passage through activated charcoal and then recrystallized 2 times
from boiling benzene. Both compounds were dissolved in acetone for
incorporation into Wayne MashR and mixed for at least 2 hours. Liquid-
gas chromatographic analyses were conducted to assure the proper concen-
tration of the test chemicals in the diets.
Tumor Cells
The methyIcholanthrene-induced DBA/2 lymphoma, P388, was a gift
from Dr. R. Megirian, Albany Medical College, Albany, NY. The
benzo(oOpyrene-induced C57BL/6 lymphoma, EL-4 was a gift from Dr. L.
Flaherty, New York State Health Department, Albany, NY. The spontaneous
DBA/2-derived mastocytoma, P815, was a gift from the Cell Distribution
Center, Salk Institute, San Diego, CA. All tumor lines were maintained
-5-
33
-------
in ascites form by weekly intraperitoneal (ip) inoculation and serial
passage.
Spleen Cell Isolation Procedure
Mice were killed by cervical dislocation and weighed. A midline
ventral incision into the peritoneal and thoracic cavities exposed the
major visceral organs for gross examination jin situ and allowed a blood
sample to be taken from the abdominal vein. Spleens were removed
aseptically and weighed in a sterile tissue culture dish (Falcon 3001,
35 x 10 mm, Oxnard, CA). The thymus, lung and liver were then removed
and weighed. Approximately 10-60 mg samples of each tissue, in addition
to 100-200 pi of the final spleen cell suspension (0.5-1 x 106 cells)
and 100-200 pi serum, were frozen in glass vials for later liquid-gas
chromatographic analysis for PCB or HCB content. Only those groups of
mice used in mixed lymphocyte cultures were used for body and organ
weight data (n=5-6 per group). The spleen was then teased across
sterile #60 mesh stainless steel into cold HBSS and transferred into
polypropylene tubes (Falcon 2059, 17 x 100 mm, Oxnard, CA). Cells which
remained in suspension after a 5 minute settling period were used,
thereby discarding unwanted debris and cell aggregates. Cell yield and
viability were assessed immediately using trypan blue (0.4% Trypan Blue
in saline (GIBCO)), mixed 1:10 with the cell suspension and appropriately
diluted with 3% acetic acid and counted using a hemacytometer. The cells
were then washed 3 times in cold HBSS and spun at 180 x G and suspended
to a concentration of 5 x 106/ml with RPMI 1640 media (GIBCO) and 10%
heat inactivated (57°C, 30 min) fetal bovine serum (FBS) (GIBCO). Cell
-6-
-------
suspensions were kept on ice and recounted just before they were dis-
pensed into cultures to assure proper concentrations. In addition, a
portion of the isolated spleen cells was diluted to a concentration of
5 x 10 /ml with media and 200 jil of this suspension was pelleted onto
a glass slide using a Cytospin (1000 rpm x 5 min) (Shandon Elliott,
Camberley, Surrey, England). The cells were stained with Wright's
stain and counted by differential cell count.
Graft-Versus-Host Response (GVHR)
A GVHR was induced in neonatal (<24 hr) BDF1 mice by the ip
injection of 1 x 10 spleen cells isolated from either control or
chemical-treated C57BL/6 mice following 3, 6, 13 or 37 weeks of
dietary administration of the test chemical. Spleen cells from 4-10
donor C57BL/6 mice from each diet group were used for the GVHR assay
after each diet treatment interval. The degree of splenomegaly was
determined for each of the 4-18 neonatal recipient BDFl mice which were
used for each diet group after each diet treatment interval. The
inoculum was administered in a volume of 0.5 ml RPMI 1640. Injection
of spleen cells isolated from control BDFl mice (isogeneic) served as a
negative control for the GVHR. The split-litter procedure (25) was
used to obviate experimental error due to variation between litters and
to allow comparison of test chemicals within litters. Spleen and body
weights of the neonates were determined on the ninth day of maternal
rearing following inoculation with spleen cells. Results are expressed
as the spleen index which was calculated by dividing the relative spleen
weight of neonates inoculated with cells from control or chemical-
-7-
35
-------
treated donors by the mean relative spleen weight of non-injected
littermates. A spleen index of greater than 1.3 was considered to be a
positive GVHR (26). Analysis of variance and the Student's t-test were
used to determine statistical significance between positive GVHR
responses.
Mixed Lymphocyte Response (MLR)
One-way MLR assays were conducted using a modification of the
procedure described by Rich and Rich (27). Responder splenocytes from
either control or chemical-treated adult male C57BL/6 mice (4-6 mice
per group) were either cultured alone, for the determination of the
rate of background DNA synthesis, or co-cultured with equal numbers
(5 x 10 cells) of allogeneic stimulator splenocytes from control DBA
mice. Cultures were contained in a final volume of 0.2 ml RPMI 1640
(GIBC01 supplemented with 2 raM L-glutamine (GIBCO), 10% FBS and 50-100
units penicilin (GIBCO"> and 50-100 pg streptomycin (GIBCO) per ml (P-S)
in 96-well flat bottom microtiter plates (Falcon 3040, Microtest II
with lids) in a humidified atmosphere of 57. C02 at 37°C (National
Appliance, model 3341, Portland, OR) for 1-6 days. Viability and cell
number of microcultures was determined daily. Splenocytes used as the
stimulator cells were irradiated (2000 rads, Gammator Cs-137 Irradiator,
Model M-38-1, Isomedix Inc., Parsippany, NJ), washed 3 times in cold
HBSS and suspended to the proper concentration in media.
DNA synthesis was assayed by the addition to each culture of 1.0
;iCi tritiated thymidine (NET-027, methyl-(3H)-thymidine, Spec. Act.
6.7 Ci/mM, New England Nuclear Corp., Boston, MA (NEN)) for the last 18
-8-
36
-------
hours of culture. Cells were lysed with a water wash and the DNA was
collected by aspiration onto glass fiber filter strips using an auto-
mated sample harvester (Skatron, Flow Laboratories, Rockville, MD).
The filter discs were dried and the radioactivity of triplicate cul-
tures was measured in 3 ml universal LSC cocktail (AquasolR-2, MEN)
using an antomatic liquid scintillation spectrometer (Packard Tri Carb
Model 3390, Downers Grove, IL) (counting period, 2-5 min, (3H) channel,
50% gain, 50-1000 window). Data are presented as the arithmetic mean +
standard error of the counts per min (cpm) of triplicate cultures. An
analysis of variance and the Student's t-test were performed to deter-
mine the statistical significance of the differences between the means
of control groups and experimental groups at p^O.05.
Mitogen-Induced Blast Transformation
Mitogen responsiveness assays were conducted using the same method
as employed in mixed-lymphocyte cultures, however, either 40 pg/ml
phytohemagglutinin (PHA-M, B grade, Calbiochem, La Jolla, CA) or 10 ug/ml
gram negative bacterial lipopolysaccharide (LPS, Salmonella typhosa,
Westphal, Difco, Detroit, MI) were added to the cultures as a stimulus
in place of allogeneic cells when the cultures were first established.
Cell-Mediated Lymphocytotoxicity
Immunizations
Control and chemical-treated male C57BL/6 mice (4-6 mice per
group) used in cytotoxicity assays were inoculated ip with 3 x 107 live
P815 mastocytoma cells in 0.5 ml BBSS 10 days prior to spleen cell
isolation. Tumor cells used for the immunization were harvested from
-9-
37
-------
DBA mice inoculated ip 7 days earlier with 1 x 10 P815 mastocytoma cells.
Labelling of target cells
P388 and EL-4 tumor cells were obtained by peritoneal lavage with a
22 gauge needle using 5 ml HBSS from isogeneic mice inoculated with live
tumor cells 5-7 days earlier. PHA-induced blast lymphocytes were pre-
pared by culturing spleen cells from adult male AKR mice for 2 days
(1 x 106/ml) with 40 pg PHA-M/ml. Prior to labelling, target cells were
washed 2 times in HBSS and suspended to 2-20 x 106/ml in media without
serum. The cells were labelled by incubating 0.5 ml cell suspension
with 100-200 pCi 51Cr (0.1-0.2 ml, NEZ-030S, sodium chromate in saline
solution, Spec. Act. 200-500 Ci/g, NEN) in a 50 ml conical test tube on
an aliquot shaker in a humidified atmosphere of 5% C02 at 37°C for 45-60
min. The labelled cells were then washed 5 times with 20 ml HBSS
supplemented with 10% FBS, counted and suspended to a concentration of
2 x 105/ml.
Cytotoxicity assay
Spleen cells from control and chemical-treated non-immunized
C57BL/6 mice and from mice immunized with P815 mastocytoma cells 10 days
earlier were suspended in media RPMI 1640 supplemented with 2 mM-glutamine
and 10% FBS and dispensed, in duplicate, into 96-well, flat bottom micro-
titer trays in 100 pi volumes. Spleen cells were not pooled. Chromium-
labelled target cells (P388, AKR blast, and EL-4) were then added in 100
pi volumes of the same media to the appropriate wells. The effector
cell:target cell ratio was 100:1 in all cultures, i.e., 2 x 106
effector cells:2 x 10 target cells per well. In addition, C57BL/6
-10-
38
-------
effector cells were mixed with P388 target cells at ratios of 30:1 and
10:1 for later construction of titration curves for the determination of
the ED50 for specific lysis (28). Spontaneous release of the label was
determined by incubating target cells in media only. The microtiter
plates were spun at 25 x G for 2 min to increase cell-cell contact and
minimize reaction time and then incubated without rocking in a humidified
atmosphere of 57. CC>2 at 37°C. To examine the kinetics of the cytotoxic re-
sponse, the specific lysis was determined after 3 and 5 hours of incuba-
tion at which times the plates were spun at 500 x G for 10 min at 7°C.
One hundred microliters of the supernatant of each well was transferred
into a polystyrene gamma counting tube (15.6 x 125 mm, Amersham,
Arlington Heights, IL) and counted for 1 min in an automatic gamma
counting system (Searle, Model 1185 Series, Searle Analytical Inc.,
Waltham, MA) with the window centered at 3221 KeV and with a width of
100 KeV.
The experimental release (ER) of label was determined by calculating
the mean counts per min (cpm) of the supernatant samples of duplicate
cultures, less background counts, of each culture containing effector
cells and labelled target cells and multiplying by 2 to account for the
volume of supernatant actually counted. The spontaneous release (SR) for
each target cell type was determined by calculating the mean cpm of
quadruplicate cultures of labelled target cells, less background, and
multiplying by 2 to account for the fraction of the total volume of
supernatant actually counted. The maximal release (MR) of label was
determined by mixing 100 pi labelled target cells with 300 jil distilled
water in polypropylene tubes (Falcon 2058) which were passed through 4
-11-
39
-------
freeze-thaw cycles. The mean cpm, less background counts, was then
calculated and multiplied by 4 to account for the volume actually
counted. The total incorporated label was determined by calculating
the mean cpm, less background counts, of eight 100 pi samples of
labelled target cells. The percent specific Cr release was then cal-
culated as follows (29): 7. specific 51Cr release - (ER-SR)/(MR-SR) x
100. Data is presented as the arithmetic mean + standard error of the
percent specific Cr release calculated for individual mice (n*3-6)
of the control and experimental groups. An analysis of variance and
the Student's t-test were used to determine statistical significance
of the difference between the means of control groups and experimental
groups at pOO.05.
Histopathology
The mice were killed with ether, the peritoneal and thoracic
cavities were opened and their contents examined jln situ. The thymus,
lung, spleen and liver were removed in the stated order. The thymus,
lung and representative liver and spleen sections were fixed in 107.
neutral buffered formalin. The tissues were embedded in parafin, cut
at 6 microns and stained with Hematoxylin-eosin. In addition, a
measured piece of spleen tissue was frozen for liquid-gas chromatography.
Organochlorine Residue Analysis
B
Liquid-gas chromatographic analysis of Aroclor 1016 and HCB was
conducted using a modification of the procedure described by Loose e_t al..
(8) and Holden and Marsden (30).
-12-
40
-------
RESULTS
There were no significant differences from control values in body
weight, relative spleen, thymus and lung weights (Figure 2) or in the
number of spleen cells isolated (data not presented1} from mice which
were fed a diet containing 167 ppm Aroclor* 1016 (PCB), hereafter
referred to as PCB-treated mice, for up to 40 weeks. However, after 3
weeks of dietary exposure to PCB, there was an 11% increase of the
relative liver weight above control values which returned to the control
value after 6 weeks exposure. Although histology was not conducted on
the 3 and 6 week groups, the increase in relative liver weight may be
histologically associated with parenchyma! cell centrilobular hyper-
trophy which was observed in mice which were fed PCB for 13 and 40 weeks.
Dietary administration of 167 ppm HCB to male C57BL/6 mice (HCB-
treated mice) resulted in a 147. decrease in weight gain by 24 weeks
which became even more pronounced (21%) by 40 weeks. The decrease in
weight gain was not related to food consumption which was comparable for
all groups throughout the study. The relative liver weights of HCB-
treated mice were significantly greater (23% and 22%) after 3 and 6 weeks,
respectively, rose to a peak of 178% greater than controls by 13 weeks
and decreased to 114% and 92% greater than controls at 24 and 40 weeks,
respectively, of dietary exposure to HCB. The increase in relative liver
weight was due primarily to an increase in absolute liver weight and was
histologically associated with marked centrilobular and midzonal paren-
chyma 1 cell hypertrophy. The relative spleen weight of HCB-treated mice
was 34% greater than control values at 6 weeks, and became significantly
-13-
41
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BODY WEIGHT
(gromt)
RELATIVE SPLEEN
WEIGHT (%)
RELATIVE THYMUS
WEIGHT CM
RELATIVE LUNG
WEIGHT (%)
RELATIVE LIVER
WEIGHT (%)
40
35
30
25
20
15
1.0
3 6
13
24
40
WEEKS ON DIET
Figure 2. Body weights. relative spleen Mights, relative tbvams weights,
relative lung weights and relative liver weight* of C57BL/6 Bice fed
either a control diet, or a diet containing Aroclor 1016 (KB) or
hexachlorobenzene (HCB) for 3, 6, 13, 24 and 40 wavks; •-control;
A BKB; | -HCB. See Materials and Methods section for experimental
details, n-5-6 for all points. P (KB) or H (HCB) Indicates ststlstlcal
significance froa control values at p£0.05.
42
-------
greater (777. and 557.) at 24 and 40 weeks, respectively. Total spleen
cell yield was significantly greater than controls (977.) only after 6
weeks exposure (data not presented). Relative thymus weights were not
significantly altered from control values, however, they were consis-
tently lower (197., 297. and 247.) than control values following 6, 13 and
24 weeks exposure to HCB but had returned to control values after 40
weeks. There were no histological alterations of thymic cortex. The
relative lung weights of HCB-treated mice were significantly greater
(227., 307. and 327.) than control values at 13, 24 and 40 weeks dietary
exposure and was histologically associated with an increase in the
thickness of alveolar septa.
Graft-Versus-Host Response
The inoculation of neonatal BDF1 mice with 10 spleen cells from
C57BL/6 mice which were fed either a control diet or a diet containing
167 ppm PCS for 3, 6, 13 or 37 weeks or a diet containing 167 ppm HCB for
3, 6 or 13 weeks resulted in a positive graft-versus-host response in
all groups. No significant effect of chemical exposure of the donors
on the GVH response was demonstrated (Table I). However, exposure to
HCB for 37 weeks resulted in a significant reduction of 207. in the
graft-versus-host activity of HCB-treated cells. Spleen cells from
normal BDF1 mice did not produce a GVHR in neonatal BDF1 mice.
Spleen Cell Jn Vitro Background DNA Synthesis,
Viability and Differential Counts
Spleen cells isolated from mice which were fed PCB for 3, 6, 13,
24 and 40 weeks and cultured in media for 1-6 days did not show any
-14-
43
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TABLE I
GRAFT-VERSUS-HOST ACTIVITY OF SPLEEN CELLS PROM C57BL/6 MICE FED EITHER A CONTROL
DIET, PCB 1016 OR HCB FOR 3-37 WEEKS AND INJECTED INTO NEONATAL BDFl MICE.8
DONOR CELL DIET WEEKS ON DIET
STRAIN
C57BL/6 CON
C57BL/6 PCB
C57BL/6 HCB
BDFl CON
2
2. 78+. 21
2. 77+. 25
2. 38+. 28
1.10+. 10
6_
2. 07+. 28
2. 57+. 34
2. 26+. 2 4
0.91+.02
i! 11
2. 21+. 12 2. 24+. 12
2. 13+. 11 2. 63+. 20
*
2.25+. 12 1. 80+. 09
0.93+.04
Data presented as mean spleen index +_ standard error. See Material and
Methods section for experimental details. There were 4-10 donor mice and 4-18
recipient mice for each diet group and each diet Interval.
* Asterisk indicates statistical significance from control value at p^O.Ol.
-------
alterations in DNA synthesis during the culture periods as compared to
control cultures (Figure 3). In addition, the number of viable cells
per culture for each day of culture, which was determined only in the
3 and 40 week groups, was comparable to control values at both diet
duration intervals tested (data not presented). These results indicate
that PCB did not alter culture viability. Also, there were no shifts
from control values in the population densities of small or large
lymphocytes, polymorphonuclear leukocytes or macrophages in either the
3 or 40 week groups (data not presented).
Spleen cells from mice which were fed 167 ppm HCB for 3 weeks
(Figure 3") and cultured in media for 1-6 days, demonstrated no altera-
tions in DNA synthesis as compared to control values, for the culture
period. However, spleen cells from mice which were fed HCB for 6, 13,
24 and 40 weeks demonstrated a pattern of increased DNA synthesis during
the first day of culture. It was first observed in the 6 week group
(205% increase). In the 13 week group, DNA synthesis was elevated 279%
during the first day of culture and fell to below the control value by the
third day. The increase in DNA synthesis during the first day of culture
in the 24 week group was significantly above (14317.) control values
and remained above control values through the second day of culture.
In the 40 week group, DNA synthesis was 6287. above the control value
on the first day of culture but was comparable to the control values
thereafter. There were no differences from control values in the
number of viable cells throughout the culture period in the 3 or 40
week groups (data not presented). In addition, in the 3 and 40 week
group there were no alterations from control values in the cell popula-
tion densities of small or large lymphocytes, polymorphonuclear leuko-
-is- 45
-------
BACKGROUND
COUNTS
MXEO
LYMPHOCYTE
RESPONSE
LPS
RESPONSE
SO .
>o
10 .
WEEKS ON DIET
is
»*-*-*-•
40
•0
n
O 40 ,
• >0 .
* CO .
" 10 ,
0
NO DATA
PHA
RESPONSE
« 120
o
10 .
»
S CO .
0 90 .
0 ,
A.
r
•o ,
S 4 S « I t > 4 ft • I t 3 4 8 « I t 3 4 S «
DAY OF CULTURE
I t 3 4 S €
Figure 3. (3H)-thymidine incorporation in CFM in cultures of spleen
cells from C57BL/6 mice fed either • control diet, or • diet cootlining
Aroclor 1016 (PCB) or hexachlorobenzene for 3-40 weeks end cultured
•lone (background counts'*, with alloantigen (mixed lymphocyte response^
or with mitogens (PHA and LPS responses). See Materials and Methods
section for experimental details, n-4-6 for all points. P (PCBt or
H (HCB) indicates statistical significance from control values at
p£0.05.
46
-------
cytes and macrophages (data not presented).
Mixed Lymphocyte Response (MLR)
Spleen cells isolated from C57BL/6 mice which were fed either a
control diet or a diet containing 167 ppm PCB 1016 or HCB for 3, 6, 13,
24 or 40 weeks responded with a transient increase in DNA synthesis
when stimulated in a one-way mixed lymphocyte culture with equal numbers
of irradiated spleen cells from DBA mice (Figure 3). Peak DNA synthesis
by C57BL/6 spleen cells from control, PCB- and HCB-treated groups sti-
mulated by DBA cells occurred on the sixth day of culture in the 3 week
group, on the fifth day of culture for spleen cells from PCB- and HCB-
treated mice but on the fourth day for spleen cells from control mice
in the 6 week group, on the fourth day of culture in the 24 week group,
and on the third day of culture in the 40 week group.
Spleen cells from C57BL/6 mice which were fed PCB for 24 weeks
responded to irradiated DBA cells significantly greater than controls
on the first day of culture only. This was the only significant
difference from the control response in mixed lymphocyte response
observed in mice which were fed PCB for up to 40 weeks (Figure 3").
Alloantigen-induced DNA synthesis in spleen cells from C57BL/6
mice which were fed HCB for 3 weeks was significantly greater than
control values for days 1, 2, 3 and 5 of culture when stimulated with
DBA alloantigen. Although DNA synthesis in mixed lymphocyte cultures
of C57BL/6 spleen cells with DBA cells is significantly greater than
the control values on the first two days of culture after exposure to
HCB 24 weeks and only on the first day of culture after exposure to HCB
-16-
47
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for 40 weeks, the increase is due to the elevated background synthesis
observed at the initiation of cultures as previously noted, and does not
reflect an alteration of alloantigen reactivity.
Mitogen Responsiveness
Figure 3 also shows the mitogen-induced DNA synthesis of cultured
spleen cells from C57BL/6 mice which were fed a diet containing 167 ppm
PCB or HCB for 3-40 weeks. A transient increase in tritiated thymidine
(t3H}-TdR) incorporation which followed the addition of 40 ug/ml PHA-M
(a T cell mitogen) at the initiation of culture and which generally
peaked on day 2, but peaked on day 3 in control and PCB-treated cultures
in the 13 week group, was observed in all control and experimental
cultures. There were no significant changes from control values in the
( H)-TdR incorporation in cultures of splenocytes from mice which were
fed PCB for 3, 6, 13 or 40 weeks. However, following 24 weeks of dietary
exposure to PCB, a significant increase in DNA synthesis was observed in
PHA-stimulated splenocytes on days 1, 3 and 6 of cultures and was above
control values, but not statistically significant, on days 2, 4 and 5 of
culture.
PHA-induced DNA synthesis in splenocytes from mice which were fed
HCB was significantly greater than control values on day 1 of culture
after 3 weeks exposure to HCB, significantly above control values on days
1 and 3 of culture in the 24 week group and above control values on the
first day of culture in the 40 week group. There were no alterations in
the 6 and 13 week groups. The increase in DNA synthesis on day 1 of
culture in the 24 week group and on day 1 in the 40 week group are
-17-
48
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associated with the high background ( H)-TdR incorporation rate previously
mentioned and, therefore, probably do not represent chemical-induced
alterations of the response to PHA.
The addition of 10 fig/ml LPS (a B cell mitogen) at the initiation
of cultures of spleen cells from control mice or mice fed either PCB
or HCB for 3-40 weeks resulted in an increase in the (3H)-TdR incorpora-
tion rate, which generally peaked on days 2 or 3, in all control and
experimental cultures. LPS-induced DNA synthesis was significantly
greater than control values on the sixth day of culture of splenocytes
from mice which were fed PCB for 3 weeks but there were no chemical-
induced alterations observed in mice which were fed PCB for 6, 13, or
24 weeks. However, exposure to PCB for 40 weeks resulted in a profound
decrease in LPS-induced DNA synthesis on day 4 of culture.
LPS-induced DNA synthesis by spleen cells from mice which were fed
HCB for 3 weeks was above control values on days 1 and 5 of culture but
there were no alterations observed in mice which were fed HCB for 6 or
13 weeks. An increase in DNA synthesis was observed on the first day of
culture of cells from mice which were fed HCB for 24 weeks, however, the
increase is associated with the elevated background (3H)-TdR incorpora-
tion rate previously mentioned and, therefore, does not represent chemical-
induced alteration of the response to LPS. However, exposure to HCB for
40 weeks resulted in a significant decrease in LPS-induced DNA synthesis
on days 4 and 5 of culture.
Cell-Mediated Lymphocytotoxicity (CML)
Sensitized spleen cells from C57BL/6 (H-2b) mice fed a control diet
-18-
49
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for 3-40 weeks and immunized with 2 x 107 live P815 (H-2d) tumor cells
10 days prior to CML assays exhibited specific lysis of ^1Cr-
labelled cells which expressed the H-2d alloantigen (Figure 4), i.e.,
P388 tumor (H-2d). There was no lysis of allogeneic AKR (H-2k) PHA-
blast cells against which they were not immunized, which indicated that
lysis was specific, and there was no lysis of syngeneic EL-4 (H-2b)
tumor cells which indicated the absence of any alteration of cytotoxicity
directed against self (data not presented). Also, specific lysis of
labelled target cells by non-sensitized effector cells was less than 5%
in all trials.
The mean spontaneous release of label over the 5 hour incubation
period for all intervals was 13% for the P388 tumor, 10% for the EL-4
tumor, and 447. for the AKR PHA-blast cell. Maximum freeze-thaw release
of label was 83%-88% for all labelled cells.
When the effector cells were from immunized mice which were fed a
control diet for 3, 6, and 13 weeks (Figure 4) an effector cell:target
cell ratio of 100:1 resulted in the progressive specific release of
^1Cr from labelled P388 tumor cells of approximately 43% and 69% after
3 and 5 hours incubation, respectively. However, effector cells from
immunized mice which were fed a control diet for 24 and 40 weeks caused
the progressive specific Cr release from P388 tumor cells of approxi-
mately 26% and 57% after 3 and 5 hours incubation, respectively, in the
24 week group and 167. and 36% after 3 and 5 hours incubation, respectively,
in the 40 week group. These results may indicate an age-related decrease
in specific cytotoxicity in the control population which began after 13
weeks of experimental housing and approximately 18 weeks of age. In
-19-
50
-------
II
t4
4O
100
•0
•0
40
tO
0
100
•0
•0
40
to
0
5 too
-• 00
u eo
•k
u 40
M
i to
i •
100
• 0
•0
40
to
0
too
•0
• 0
40
to
0
CONTMOL
»c§
NCR
100-1
INCUIATION fCHIOD ( NOU*S 1
rigor* 4. Specific lyti* of M88 time* e«lli by cplM* Mils ftc»
C57BL/6 aic* fed «lth«r • control dice, PCI 1016 or 1C! (or 3, 6. 13.
24 md 40 w«k« nd either iiHal**d (aelid liaea) with P815 tumor
cell* or not liiMiniMd (duhed line*); »-cootrol; A*Kl; | -HCB.
Sec Hiterlel* md Method* eectioo for experimental deteile. n-3-6
for eolid line* end n-1 for daehed line*. B Indlcetee etatietlcal
algaificence froej control value* et p
-------
addition, an effector cell dose-response was observed when effector
cell:P388 target cell ratios of 30:1 and 10:1 were used.
There were no significant alterations from control values of the
specific lysis of labelled target cells by spleen cells from immunized
C57BL/6 mice which were fed 167 ppm PCB 1016 for 3 to 40 weeks (Figure 41 .
There were no significant alterations from control values of the specific
lysis of labelled target cells by spleen cells of immunized C57BL/6 mice
which were fed 167 ppm HCB for 3, 13, 24, or 40 weeks (Figure 4). However,
the incubation of sensitized spleen cells from mice exposed to HCB for 6
weeks with labelled P388 target cells at an effector:target cell ratio
of 100:1 resultsd in the significantly decreased specific lysis of only
9% and 17% (20% and 17% of the control value) after 3 and 5 hours incuba-
tion, respectively. Similarly, effector:target cell ratios of 30:1
resulted in the significantly decreased specific lysis of 4% and 7%
(18% and 15% of the control) after 3 and 5 hours incubation, respectively,
and at ratios of 10:1, a significantly decreased specific lysis of 1% and
2% (11% and 10% of the control values) after 3 and 5 hours, resepctively.
The ED50, that is, the number of cells required to cause 50% lysis as
determined by linear regression analysis of the dose-response values for
each treatment group of the 6 week exposure period, was calculated for 3
and 5 hours incubation. The control EDSOs were 2.25 x 106 and 1.4 x 106
for 3 and 5 hours, respectively. The EDSOs for the PCB-treated group
were 2.13 x 106 and 0.92 x 106 for 3 and 5 hours, respectively. The EDSOs
for the HCB-treated group were 11.39 x 106 and 6.01 x 106 for 3 and 5 hours,
respectively, and represents a 5 fold and 6 fold decrease from control values
after 3 and 5 hours incubation in effector cell ability to cause the
specific lysis of the target cells. In addition, there was no alteration
-20-
52
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from the control response when labelled EL-4 tumor cells were the target
cells (data not presented).
Residue Analysis
Electron capture liquid-gas chromatography of serum, tissue and
isolated spleen cells revealed that PCB concentrations did not follow
the same pattern of disposition in all samples (Table II). The concentra-
tion of PCB in the liver decreased from the 3 week value during the 3-13
week period after which it increased to a steady state level. The con-
centration of PCB in the spleen consistently decreased from the 6 week
value. The concentration of PCB in the thymus remained within 16% of
the median value during 40 weeks of exposure. The concentration of PCB
in the serum, although varying significantly from each previous measure-
ment, remained within 26% of the median value throughout the observation
period. And the concentration of PCB in spleen cells remained within
22% of the median value throughout the observation period.
HCB concentrations reached maximal levels in liver during the 13 to
24 weeks exposure period. The concentration of HCB in the spleen reached
its highest level by 6 weeks after which it decreased. The HCB concentra-
tion in serum and thymus reached its highest value by 24 weeks of exposure.
The concentration of HCB in the spleen had reached its highest level by
6 weeks, after which it decreased. The concentration of HCB ir spleen
cells increased from 3-24 weeks, after which it decreased, and the kinetics
did not correlate with those of whole spleen concentrations.
The ranges of the ratios of the concentrations of HCB to PCB within
the same exposure periods were: liver, 4-11:1; spleen, 4-9:1; thymus,
-21-
53
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TABLE 11
PCB AND UCB RESIDUE ANALYSIS'
Weeks On Experimental Diets
PCB
IICB
TISSUE
Liver (pptn)
Spleen(ppm)
Thymus(ppm)
Serum(ppm)
Spleen, cells
(ng/10 cells)
Liver (ppm)
Spleen(ppm)
Thyuus (ppm)
Serun(ppm)
Spleen, Cells
(ng/10 cells)
3
13
+ 1 .1
— — — —
20
+ 2.9
.18
+ .01
80
+23
56
+ 5.6
— — — —
18
+ 1.7
3.5
+0.2
5.4
+ 1 .5
6
9.1
+2.3
12
+0.2
19
+ 2.0
. 14
+ .01*
95
+ 18
49
+ 1 1
59
+ 17
28
+6.0
4.1
+0.4
6.3
+ 1.4
13
4.6
+0.5
6.2
+0.9*
19
.23
+ .02*
92
+ 10
187
+ 12*
40
+ 1.3
47
+ 9.6
7.9
+ 1.0*
15
+2.4*
24
19
+2.9*
4.0
+0.8
19
.14
+ .01*
203
+42
18
+ 3.3*
70
+ 17
8.0
+0.8
27
+2.6*
40
21
+6.3
3.4
25
+ 5.2
.22
+ .01*
61
+8.0
115
+ 17
31
+ 7. 1
58
+ 11
4.5
+0.6*
12
+ 3.3*
Data presented as mean concentration of PCB or HCB wet weight + standard
error; See Materials and Methods section for experimental details; n-1-5
* Asterisk Indicates statistical significance from previous sampling Interval
at pjCO.05 by analysis of variance and Student's t-test.
Ul
-------
1-4:1; serum, 20-56:1; and spleen cells, 1:5-15. Neither PCS nor HCB was
found in random samples of tissue from mice which were fed a control diet
for up to 40 weeks.
DISCUSSION
The influence of the chronic dietary administration of two common
environmental contaminants (PCB and HCB) on certain cell-mediated immune
responses has been investigated. PCB, at the concentration used in this
study, did not alter, in any consistent manner, the graft-versus-host
response, the mixed lymphocyte response, or cytotoxic activity of spleen
cells isolated from experimental animals.
The time and the magnitude of the peak response of spleen cells from
PCB-treated mice in all mixed lymphocyte cultures were comparable to the
response of spleen cells from control animals. These results indicate
that PCB did not alter the initial recognition of alloantigen, as
measured by DNA synthesis, during the activation phase of the response
to alloantigen.
A 37% decrease in LPS-induced spleen cell DNA synthesis was observed
after 40 weeks dietary exposure in the absence of simultaneous impairment
of PHA-induced DNA synthesis. Loose ejt al. (8> reported a 69% decrease
in the number of plaque forming cells (PFCs) per million spleen cells
during the primary antibody response to sheep erythrocytes (SRBC) in mice
fed PCB 1242, a form of polychlorinated biphenyl which has a greater
diversity in the isomer content of chlorine by weight than Aroclor 1016
used in the present study, for 6 weeks. The antibody response to SRBC
-22-
55
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requires B lymphocytes, T lymphocytes and macrophages (31) and, there-
fore, the cell type which was the target cell for PCB toxicity, if indeed
there was only one, could not be defined. Since LPS is known to
directly stimulate DNA synthesis in cultures of B lymphocytes depleted
of T lymphocytes and macrophages (32) PCB-induced impairment of B
lymphocyte activation is probably not directly due to either impaired
T lymphocyte helper activity or macrophage function. These results
implicate the B cell as a possible target cell in PCB toxicity.
In the present study, the impairment of B lymphocyte responsiveness
to mitogen, however, was not observed until after 40 weeks of PCB
exposure, whereas Loose et al. (8) demonstrated a decrease in the number
of plaque forming cells after 6 weeks of PCB exposure. While the temporal
differences in these observations may be due to differences in the
experimental animal strains used and/or the differences in the isomer
contents of the PCBs used, it is also possible that the plaque assay,
which detects the presence of plasma cells derived from B lymphocytes
responsive to sheep erythrocyte antigens, is more sensitive and, there-
fore, better able to detect alterations from control responses than the
LPS mitogen assay, which detects polyclonal B lymphocyte activation, due
to the inherent individual variation of the response of the larger
proportion of responding cells in the culture.
Although PCB-induced alterations of B lymphocyte mitogen responsive-
ness were observed in the present study, there were no alterations of
cytotoxic activity against cell surface alloantigen by sensitized lympho-
cytes from PCB-treated mice and there was no increase in non-specific
killing. These results suggest that PCB did not interfere with the
-23-
56
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effector phase of the cell-mediated immune response.
There were no alterations from control values of body weight or
relative spleen, thymus or lung weights or in the number of spleen cells
isolated from PCB-treated mice throughout the entire experiment. The
relative liver weight was significantly greater than control values
during the first 6 weeks of PCB exposure and is consistent with the
findings of other investigators (7) and is due to marked proliferation
of smooth endoplasmic reticulum (SER).
The findings of the present study extend the previous evidence
that PCBs alter humoral immunity reported by Vos and Van Genderen (9),
Roller and Thigpen (5), and Loose e_t al. (8) and suggest that PCBs can
express selective toxicity on different portions of the immune system.
They also indicate that the target site for PCBs causing the depression
of antibody-mediated immunity is a mechanism and/or a cell type which is
not shared by the components of cell-mediated immunity. Since Vos and
Van Driel-Grootenhuis (7' reported that exposure of guinea pigs to PCB
resulted in a decrease in the relative thymus weight and a decreased
delayed type hypersensitivity skin reaction, the present findings may
also indicate species differences in the susceptibility to PCB-induced
immunotoxicity. It is possible that a regulatory cell which normally
acts to balance both the cell-mediated and the humoral-mediated arms of
the immune system is functionally impaired by PCBs and allows an imbal-
anced response to occur.
Unlike PCB exposure, HCB exposure resulted in the alteration of all
the parameters of immune responsiveness measured in this study. A 207.
reduction of GVH activity was observed after 37 weeks dietary exposure to
-24-
57
-------
HCB. This result may indicate that splenomegaly is not sensitive enough
to detect a functional alteration of GVH activity even though other
aspects of immune responsiveness are modified. For example, Vos and
Moore (10) reported that 4 weekly doses of 25 /Jg/kg TCDD reduced the
GVH activity of spleen cells of young mice by 257, whereas the responsive-
ness to PHA was reduced 67%. However, it may also indicate that the
cells involved in the graft rejection response are resistant to acute
chemical-induced functional alteration and that only chronic chemical
exposure results in detectable immunotoxicity.
Chemical-induced alteration of immune function may not always
result in an impaired lymphoid activity. Instead, enhanced activity of
certain aspects of immune reactivity may occur and result in an improper
or unbalanced overall response, as seen, for example, in hypersensitivity
reactions.
A 32-76% increase in alloantigen-induced DNA synthesis was observed
in cultures of spleen cells from mice fed HCB for 3 weeks. These
results may suggest that a population of non-specifically primed lympho-
cytes was present in the cultures of HCB-treated spleen cells and were
able to support a greater response to alloantigen than control cultures.
Since the kinetics of the response were the same as the control response,
it is apparent that the increase in the rate of DNA synthesis during the
culture period was due to a stimulus present in the culture and not due
to a pre-existing stimulus in vivo- This does not seem to be the case
with the increase in the background rate of DNA synthesis observed
following longer periods of exposure to HCB and which will be discussed
later. There were no HCB-related alterations in alloantigen-induced DNA
-25-
58
-------
synthesis, however, during the 40 week exposure period which followed.
Vos e_t al_. (11) reported that HCB did not alter skin rejection times
of rats exposed to HCB pre- and post-natally until 5 weeks of age.
However, allograft rejection also involves effector cell function and,
therefore, evaluates the effector phase, in addition to the recognition
and activation phases of the immune response.
A 517. increase in PHA-induced DNA synthesis was observed following 24
weeks exposure to HCB on day 3 of culture. These results compare with the
findings of Vos e_t_ al. (11) that HCB did not profoundly alter PHA res-
ponsiveness of spleen cells from rats following pre- and post-natal
dietary exposure to 100 ppm HCB through 5 weeks of age although a slight
enhancement of the response was noted. In addition, chemical-induced
enhancement of lymphocyte response to a T cell mitogen may represent an
alteration in splenic T/B ratios and lead to an inappropriate response to
an antigen. For example, if suppressor T cell function is increased, due
to the presence of an excessive number of T lymphocyte precursors, B
lymphocyte differentiation into a sufficient number of antibody producing
plasma cells may also be impaired. Furthermore, the present study extends
the findings of Vos et al. (11) by indicating that chronic exposure of
experimental animals to toxic compounds may be necessary to detect certain
immunological dysfunctions.
» A decrease of 50-537. in LPS-induced DNA synthesis observed after 40
weeks exposure to HCB, although not a measure of T lymphocyte function,
extends the findings of Loose e£ al. (8) that the number of PFCs per 106
spleen cells was decreased 537. below control values after 6 weeks expo-
sure to HCB and since plaque forming cells are of the B cell lineage,
-26-
59
-------
chemical-induced B lymphocyte dysfunction may be indicated. In the same
study, Loose also reported a 24% to 427. decrease in IgGj, IgA, and IgM
in mice fed HCB. These results provide more evidence that there is a
disparity in the sensitivity between T and B lymphocytes to HCB and that
humoral immune functions may be more sensitive than cell-mediated immune
parameters to the toxicity of HCB.
A 75-797. reduction in the specific cytotoxicity directed against
cell surface alloantigen by sensitized spleen cells from mice exposed to
HCB for 6 weeks is associated with the time when the highest concentration
of HCB was detected in the spleen. Impairment of lymphocytotoxicity in
the absence of alteration of the recognition and activation phases of the
immune response indicates HCB-induced functional alteration of the
effector phase. This hypothesis is supported by the decreased GVH
activity demonstrated in HCB-treated mice since there is evidence which
indicates that the effector cells involved in GVH activity are the same
subpopulation of cells as those which are responsible for CTL (16). It
is possible, however, and difficult to exclude with certainty, that HCB-
induced alteration of host resistance (3, 11) to infectious agents, such
as bacteria, viruses and protozoa, may result in a delayed response or
even dysfunction of the mechanisms of host defense and, in turn, may have
interfered with the cytotoxicity assay. In addition, a 977. increase
above control values in the number of spleen cells isolated was also
observed. The high concentration of HCB in the spleen may have caused
impaired differentiation of precursor cytolytic cells into effector cells
or the compound may have interfered with the cytolytic mechanism itself.
The 14317. increase in background DNA synthesis observed during the
-27-
60
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first 24 hours of culture of spleen cells from mice treated with HCB
for 24 weeks was consistent with a pattern which began to develop after
6 weeks of HCB exposure. The increased rate of DNA synthesis was no
longer present after 2 days of culture and suggests that the population
of cells which were synthesizing DNA may have completed synthesis and
entered the mitotic and differentiation phases (although the synchrony
seems too great), may no longer be viable in the culture, or, most
probably, have lost the stimulant for the initiation of DNA synthesis.
There are two possible explanations of enhanced DNA synthesis. First,
pathogenic organisms present in the HCB-treated animals, due to a
decreased host resistance, but not present in control or PCB-treated
animals, would result in a chronic condition of lymphoid activation, but
would have, perhaps, been eliminated from the cell preparation during the
isolation procedure. The absence of bacteria in the blood and spleens
of the HCB-treated animals suggests that DNA synthesis is not due to
bacterial infection. Furthermore, there was no increase in the number of
splenic PMNs, which would have indicated bacterial infection. Viral
infection of the HCB-treated animals is a strong possibility and is
indicated by the increase in the relative lung weight and altered
pulmonary histology. Second, the enhanced rate of DNA synthesis may be
due to a compound with mitogenic properties present in vivo which is the
result of HCB exposure. The compound could be HCB itself, a metabolite
of HCB not present in mitogenic concentrations in vitro or an HCB-
induced cellular product which is mitogenic.
The body weights of mice fed HCB for 13 to 40 weeks were consis-
tently less than control mice. A decrease in the amount of adipose
-28-
61
-------
tissue was apparent on gross examination and suggested an alteration in
lipid metabolism or food consumption, but could not be attributed to a
decrease in food consumption due to the technical imprecision of the
feed weigh-back method available. A decrease in the body weight and food
consumption by rats fed 1000 mg/kg HCB for 3 weeks was reported by Vos
£t al. (11). The increase in relative spleen weights of mice fed HCB for
24 to 40 weeks could suggest either immune reactivity since the percentage
of large lymphocytes present in the spleen cell suspensions from mice
treated with HCB for 40 weeks was increased (although the increase was not
great enough to be statistically significant) and was associated with an
increase in the frequency of (3H)-TdR-labelled spleen cells in the HCB-
treated mice and/or in increase in hemopoiesis, since erythrocytes were
considerably more abundant in the spleen cell preparations from HCB-treated
mice than from control mice. The decrease in the relative thymus weights
observed after 6, 13, and 24 weeks of exposure to HCB, although not
statistically significant, may be associated with the thymo-toxic
properties seen with other chlorinated hydrocarbons such as TCDD (10). In
addition, slight cortical atrophy of the thymus was reported by Vos (11)
in rats fed 2000 mg/kg HCB. However, no histological alterations of the
thymus were observed in this study.
The increase in the relative liver weight observed throughout the
study is typical of many chlorinated hydrocarbons such as PCB and
reflects marked proliferation of SER. It is difficult to delineate the
physiological response from hepatotoxicity (33), however, and it is
possible that alterations of hepatic function may be directly or
indirectly associated with altered lymphoid function.
-29-
62
-------
The greatest tissue concentrations of PCB were in thymus and liver
and indicates that the thymus, a primary lymphoid organ, may concentrate
PCB to levels above serum levels and may act as a storage depot for PCB.
Since Vos and Moore (10) found that TCDD had a greater influence on
CMI following pre- and post-natal exposure than following adult exposure,
in addition to the lack of CMI alteration found in the present study in
which adult animals were used, these data may indicate that certain
compounds are toxic to CMI function only if exposure occurs during the
development of the immune system.
The highest tissue level of HCB was localized in the liver and did
not increase significantly after 13 weeks of exposure. The highest con-
centration of HCB in the spleen was observed following 6 weeks of expo-
sure and was coincident with the observed decrease in lymphocytotoxicity
of spleen cells sensitized against alloantigen and with the time when
the relative spleen weight was the highest value recorded. After 6 weeks,
the spleen concentration had declined to, and remained at, less than one-
half its highest value and indicates an alteration in the biohandling of
the compound. Both spleen and thymus concentrated HCB to values above
serum levels and it is of interest to note that as the spleen concentra-
tion of HCB decreased with prolonged exposure, the concentration of thymic
HCB increased. Since the relative thymus weight decreased during the
3-24 week period of exposure, while the concentration of HCB in the thymus
increased, it is possible that thymic affinity for HCB increased with
continued exposure or that the compound has great avidity for a certain
component of thymic tissue and as other components are mobilized during
thymic weight loss, the HCB is retained. The concept of thymic receptor
-30-
63
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sites for chlorinated hydrocarbons, such as TCDD, which seems to be
under genetic control, has been suggested by Poland e_£ al. (34) and
may be related to the altered CMI responses observed in this study.
The influence of the dietary administration of HCB on cell-mediated
immune functions, in contrast to those observed with PCB, could reflect
the different patterns of absorption of these chemicals. In rats,
latropolous e_t al. (35) demonstrated that 48 hours after oral adminis-
tration of a single dose of either C-labelled dichlorobiphenyl (DCB5*,
a chlorinated biphenyl, or HCB, that DCB is transported to the liver by
the venous portal system. In contrast, HCB is primarily absorbed by the
lymphatic system. This pattern of absorption of HCB results in the
direct exposure of thoracic duct lymphocytes to high concentrations of
chemical before any detoxification by the liver or dilution in the blood
is possible. Thoracic duct lymphocyte adsorption or absorption of HCB
may help to explain the high concentration of HCB found in lymphoid
tissue and its toxicity.
In the present study, the dysfunction of cell-mediated immunological
parameters, associated with thymus weight reduction and spleen weight
increase, may reflect a thymus-dependent toxic expression. That is, the
thymo-tropic properties of HCB result in high concentrations of HCB in
mature thymus tissue where it exerts thymo-toxic effects. And there is
evidence which suggests that the immature thymus may be more susceptible
to permanent damage than the mature organ (10). Reduction in thymus
weight may be due to a reduction of thymocyte development and since non-
primed T lymphocytes are short-lived cells (36), splenic T lymphocytes
may not be replaced and, therefore, T lymphocyte-mediated splenocyte
-31-
64
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function may be impaired.
In the assessment of the mechanism of action by which HCB caused the
suppression of effector phase function in the development of a cell-
mediated response, the data of the present study suggest that the lesion
is not due to an impaired ability to recognize specific cell surface
antigens. Also, the lesion is probably not due to impairment of the
mechanism of lymphocyte activation since T lymphocyte mitogen responsive-
ness, which is thought to by-pass initial specific recognition, is not
impaired. Therefore, the HCB-induced lesion probably exists beyond
antigen recognition and activation and exists within the effector phase
since lymphocytotoxicity and GVH reactivity, which are both measures of
effector cell function, were impaired following exposure to HCB in this
study.
In summary, the influence of two environmental polyhalogenated
aromatic hydrocarbons, PCB and HCB, on the development of cell-mediated
immune response has been investigated and it is concluded that:
P HCB is a more potent immunomodulator than PCB,
21 neither PCB nor HCB interact in a detectable and deleterious
manner with the mechanisms of the initial antigen recognition or activa-
tion phases of a cell-mediated immune response, and
3) immune dysfunction is related to exposure time to the chemical.
Furthermore, it is suggested that:
1) the mechanism of action of the immunotoxicity of HCB is within
the effector phase of the immune response,
2) PCB has a more profound influence on parameters of antibody-
mediated immunity than on cell-mediated immunity,
-32-
65
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3s! environmental chemicals can have specific mechanisms of
toxicity and, therefore, can influence antibody-mediated immunity while
it has no detectable effect on cell-mediated immunity, and
4) a single assay of immune function may not be appropriate to
detect chemical-induced immune dysfunction.
66
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Rozman, F. Coulston, and F. Korte. 1975. Absorption, transport
and organotropism of dichlorobiphenyl (DOS'*, dieldrin, and
hexachlorobenzene (HCB) in rats. Envir. Res. 10:384.
36. Sprent, J. 1975. In: The Lymphocyte: Structure and Function.
New York, Marcel Dekker, Inc., P. 105.
-38-
7 1-
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Modification of Lymphocyte Transformation by Trace Heavy Metals
Nancy J. Baiter, Joseph A. Bellanti and Irving Gray
Department of Biology and International Center for
Interdisciplinary Studies of Immunology, Georgetown
University, Washington, D. C. 20007
72
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INTRODUCTION
A number of recent studies have demonstrated that a variety of chemicals of
environmental concern, including heavy metals (1,2,3), have effects on host
immunocompetence. This becomes a public health concern in as much as changes
in host immunocompetence may be reflected in such things as altered host
resistance to infectious agents or altered immunosurveillance against malignancy.
Various systems have been used to demonstrate that a chemical has an effect
on host immunocompetence. For the most part, these involve exposure of the host
to the chemical via a route which approximates the route of environmental exposure,
followed by challenge of the host with an infectious agent, tumor cell load, or other
test antigen. Immunocompetence may then be assessed by measuring such parameters
as time to death, antibody titers, or a secondary humoral or cell-mediated response
following in vitro challenge of cultured lymphocytes with the sensitizing antigen.
However, most of these methods are cumbersome, requiring the long-term
maintenance of large numbers of animals on the experimental regimens, and therefore,
do not readily lend themselves for use as a screening test.
We report here on the use of the lymphocyte transformation test (LTT) to assess
the effects of lead and cadmium on lymphocyte responses. The LTT was chosen
because antigen-induced blast transformation is central to both humoral and cell-
mediated immune responses. Therefore, an agent which is demonstrated to alter
lymphocyte metabolism at this level has the potential for profoundly affecting
specific immune responses, suggesting that it would be worthy of further immuno-
toxicologic investigation.
73
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- 2 -
The LTT is a simple, rapid and inexpensive test to perform and may be
readily modified to assess the effects of drugs or chemicals of environmental
concern. Lymphocytes are cultured in vitro with an antigen to which the host has
been sensitized and the blast transformation response assessed by measuring any
of a number of parameters including DNA, RNA or protein synthesis, various
enzymatic activities or morphology. By stimulating in vitro blast transformation
with a non-specific mitogen such as PHA or LPS, rather than specific antigen,
the preimmunization of animals with antigen can be bypassed.
The interpretation of the results of experiments measuring the effect of
addition of exogenous agents on lymphocyte transformation requires a consideration
of the variables which are operating in this test system. In this report, we
demonstrate the factors involved in the interpretation of the results of studies on
the effect of Pb2+ and Cd2+ on unstimulated and LPS-stimulated lymphocyte
transformation. These findings are discussed in relation to the existing data on
the effects on host immunocompetence of in vivo exposure to these metals.
MATERIALS - METHODS
The methods used in these studies have been described in detail previously (4,5).
Briefly, spleens from male Balb/c mice, 8-12 weeks old, were used as a source of
lymphocytes. Mononuclear cells were separated by Ficoll-Hypaque flotation.
Cultures, established in 12 x 75 mm polystyrene tubes, contained 1 x 106 viable
cells in 1 ml of RPMI supplemented with antibiotics and 10% heat inactivated fetal
calf serum. Cadmium and lead, as chloride salts, were added to give final
concentrations of 10"7, 10~6, 10~5, KT4 andl(T3M; LPS (Escherichia coli.
055:BS, Difco) was used at a final concentration of 10 /jg/ml. Cultures, in
triplicate, with each concentration of metal (or no metal), with or without LPS, ~J A
-------
- 3 -
were incubated at 37°C in a humidified atmosphere of 5% CCfc in air. Cell
viability, DNA, RNA and protein synthesis, and blast cell morphology were
quantitated at the end of a 72-hour culture. Cell viability was determined by
trypan blue exclusion. DNA, RNA and protein synthesis were determined by
measuring the incorporation of an appropriate radiolabelled precursor
(3H-thymidine, 3H-uridine, or 3H-alanine, respectively), added 18 hours before
the end of the incubation, into TCA-insoluble material. Blastogenesis was
assessed histologically in monolayers of washed cells fixed with methyl alcohol
and stained by the Hemal Stain technique.
The effect of metal on unstimulated and mitogen-stimulated lymphocyte
response was quantitated by calculation of the £1 and SIR respectively according
to the following definitions:
_ Response in Presence of Metal
£1 ~
SIR =
Response in Absence of Metal
Response in Presence of LPS and Metal
Response in Presence of LPS alone
Response for DNA, RNA and protein synthesis is cpm; for blastogenesis,
percentage blast cells.
RESULTS
Cadmium and lead, present in lymphocyte cultures, were associated with
similar dose-dependent cytotoxic effects (Figure 1). With neither metal nor
mitogen present, 80% of the cells recovered after the 72-hour incubation period
were viable. Both Cd2+ and Pb2*, at all concentrations tested, except for
75
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-4-
10~7M Cd2+, significantly reduced cell viability. At 1(T3M, Cd2+ was
associated with a recovery of 27% viable cells, and Pb2* with 23%. In cultures
with LPS (data not shown) LPS alone was associated with a reduction in viability
to 64%; both Cd2+ and Pb2+ caused a dose-dependent reduction In viability to
levels slightly below those where no mltogen was present.
The effect of Pb2+ on lymphocyte DNA, RNA and protein synthesis are
represented in Figure 2. Lead appears to have no effect on the parameters
measured except perhaps at 10~3M where it is inhibitory. The £1 values are
calculated from the cpm of precursor incorporation with no correction for the
decreased viability associated with the Pb2+ treatment. However, If each of
the precursor Incorporation values is corrected to 100% viable cells, and the
£1 values recalculated and plotted against Pb2+ concentration (Figure 3),
Pb2+, at all concentrations above KP^M, appears to stimulate lymphocyte
transformation by each of the parameters measured. DNA, RNA and protein
synthesis in the presence of 10~5 to 10~^M Pb2+ Is approximately two times that
measured in the absence of Pb2+. The effect of Pb2+ on lymphocyte transformation
was also assessed by a direct morphologic enumeration of blast cells (Figure 4).
In agreement with the pattern observed when the macromolecular synthesis values
were corrected for cytotoxicity, Pb2+ at 10~4M and 10"3M, was associated with
an Increase In blast transformation.
The effect of Pb2+ on LPS-induced lymphocyte transformation is shown in
Figures 5 and 6. When the ratios are uncorrected for metal-induced cytotoxicity
(Figure 5), Pb2+ has no apparent effect on lymphocyte transformation parameters
76
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-5-
except at 10~3M where macromolecular synthesis is slightly Inhibited, and at
10-4 j^ IQ-SM Pb2+ where there is an apparent enhancement of DMA synthesis
but no effect on RNA or protein synthesis. When the SIR values are corrected
for the metal-induced cytotoxicity (Figure 6), Pb2+ is associated with a marked
enhancement of lymphocyte transformation. The enhancement was particularly
dramatic for DNA and protein synthesis, but much less so for RNA synthesis.
When blast transformation was assessed histologically, Pb2+ had no measurable
effect on LPS-induced transformation.
The effect of Cd2+ on lymphocyte transformation was assessed in similar
experiments and the results shown in Figures 7-10. Cadmium added to cultures
of unstimulated lymphocytes appears to stimulate DNA and protein synthesis at
10~7 and 10-6M, but totally inhibits all macromolecular synthesis at 10"4 and
10~3M (Figure 7). This pattern is unchanged when the precursor incorporation
values are corrected for Cd2+-induced cytotoxicity (Figure 8). However, when
blast transformation is assessed morphologically (Figure 9), Cd2+ appears to
stimulate blastogenesis at 10~4 and 10~3M. The effect of Cd2+ on LPS-induced
lymphocyte transformation again shows a similar pattern (Figure 10). At low
concentrations of Cd2+, there is a slight enhancement of DNA and protein synthesis;
at high concentrations, there is a total inhibition of all macromolecular synthesis.
Correcting the SIR values for Cd2+-induced cytotoxicity does not alter this pattern
(data not shown). As with Pb2+, in the presence of LPS, Cd2+ had no measurable
effect on the percentage of cells with blast cell morphology.
77
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- 6-
PISCUSSION
Both lead and cadmium, added to cultures of mouse splenocytes, significantly
affect both unstimulated and LPS-stimulated lymphocyte transformation as
estimated by DNA, RNA and protein synthesis. For each metal, the effect on
each of these three parameters is similar, suggesting that the metals are having a
generalized effect on lymphocyte transformation rather than a specific effect on a
particular macromolecular synthetic pathway.
The interpretation of experiments testing the effects of Pb2+ on lymphocyte
transformation is dependent on an analysis of the role of Pb2+-induced cytotoxicity
in the observed results. When the uncorrected values for radiolabelled
precursor incorporation are used to calculate the El and SIR values, Pb2+ appears
to have no effect on either unstimulated or LPS-stimulated lymphocyte transformation
except at the highest concentrations of Pb2+. However, Pb2+ was associated with a
significant, dose-dependent reduction in cell viability. Ibis cytotoxic effect
means that fewer cells per culture are capable of macromolecular synthesis
which, in turn, is reflected in a lowered incorporation of radiolabelled precursors.
Therefore, in order to assess the effect of Pb2+ on transformation, independent
of the cytotoxic effect, the El and SIR values were recalculated using label
incorporation values which were corrected to 100% viable cells. Using this
analysis, by each of the parameters measured, Pb2+ at concentrations above
10~6M, is associated with an enhancement of both unstimulated and LPS-
stimulated lymphocyte transformation. In the LPS-stimulated cultures, there
is an apparent discrepancy between the effect of Pb2+ on DNA, RNA and protein
synthesis when the SIR values are corrected for cytotoxicity. A re-examination
78
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- 7 -
of the original data revealed that the viabilities recorded in the experiments
where DNA and protein synthesis were measured were unusually low while the
viabilities in the experiments measuring RNA synthesis were unusually high.
Since different investigators were involved in these studies, this discrepancy
most likely represents individual variation in the performance of the trypan blue
exclusion test and demonstrates the subjective nature of this assay. If, however,
each of the values of radiolabelled precursor incorporation is corrected by the
appropriate viability measure obtained by averaging the viabilities obtained by
all experimenters over the course of numerous experiments, the SIR plots for
DNA, RNA and protein synthesis are similar with a peak enhancement at
10 M Pb2+ of approximately 2.5 times that observed in cultures containing
no metal.
m an attempt to determine whether the Pb2+-induced Increases In macro molecular
synthesis were associated with an actual Increase In blast transformation, the
effect of Pb2+ on the percentage of cells exhibiting blast cell morphology was
also determined. Although such an analysis lacks sensitivity and is subjective
in that blast cells are morphologically similar to other cells, especially macrophages,
such an analysis does have certain advantages. In particular, if It Is assumed that
the cytotoxic effect of Pb2+ is independent of the state of differentiation of the
lymphocyte, then the percentage of the total cells having blast cell morphology
will be the same regardless of viability (I.e., the distribution of blast cells in
the non-viable fraction of cells is the same as that in the viable fraction). In non-
mitogen stimulated cultures, Pb2+ was associated with an increase In blast
transformed cells consistent with the viability-corrected results of experiments
measuring macromolecular synthesis. In LPS-stimulated cultures, however,
79
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-8-
Pb2+ had no measurable effect on the percentage of cells exhibiting blast cell
morphology inspite of the enhanced macromolecular synthesis which was observed.
Since LPS is far more mltogenlc than Pb2*, it is possible that the relatively
small effect of Pb2+ would be overshadowed in an insensitive assay such as that
involved in the evaluation of cells with blast morphology.
The interpretation of experiments measuring the effect of Cd2+ on lymphocyte
transformation is the same whether or not the precursor incorporation values
are corrected for the Cd2+-induced cytotoxiclty. Thus, although the dose-
dependent cytotoxic effects of Pb2+ and Cd2+ are similar, the correction to 100%
viable cells does not alter the interpretation of the results of experiments measuring
the effect of Cd2+ on transformation as it does with Pb2+. In experiments assessing
the effect of Cd2+ on the percentage of cells exhibiting blast cell morphology,
Cd2+ at 10 "^ and 10'^M appeared to be blastogenic. ft is difficult to reconcile
this finding with the results of the macromolecular synthesis studies. A possible
explanation is that Cd2+ may have a direct effect on the assay systems used to
measure macromolecular synthesis, for example, by inhibiting the transport of the
radiolabelled precursors across the cell membrane. The mechanisms of the
cadmium-induced inhibition of macromolecular synthesis are currently under
investigation in our laboratory.
Thus, the in vitro exposure of lymphocytes to cadmium clearly results in a
functional modification; whether or not the in vitro exposure to lead affects
lymphocyte function depends on the interpretation of the experimental results in
light of the cytotoxicity associated with Pb2+ exposure. However, in terms of
the development of methodology useful in immunotoxicologic assessment, it is
essential to know the relationship between these data and the effects of in vivo
exposure to Pb2+ and Cd2+ on lymphocyte responses. In studies in our laboratory (6) nr\
-------
- 9 -
as well as those of others (7), in_ vivo exposure of mice to Pb2+ of Cd2+ had
no effect on the viability of cells recovered from the spleen of metal-treated
mice although we have observed that the total number of cells recovered per
spleen is slightly lower following metal exposure.
We have studied the effect of in vivo metal exposure on unstimulated and
and antigen-stimulated lymphocyte transformation (as assessed by measurement of DNA
synthesis) in Balb/c mice exposed to Pb2"1" or Cd2+, 0.1-5 ppm, in drinking water for
six weeks (6). The results of these studies have paralleled those of the in vitro
exposure studies reported here: Pb2+ was associated with a dose-dependent
increase in both unstimulated and antigen-stimulated DNA synthesis; Cd2+ exposure
was associated with a profound reduction in stimulated and unstimulated transformation.
Roller and his associates (7,8) have performed similar experiments in CBA mice
exposed to 13-1300 ppm Pb2+ or 3-300 ppm Cd2+ in drinking water for 10 weeks.
These investigators report that Pb2*1* exposure generally inhibits mitogen- or
antigen-induced lymphocyte DNA synthesis while Cd2+ exposure inhibits transformation
at 3 ppm but enhances DNA synthesis at 30 and 300 ppm. These investigators did not
report any data concerning the effect of metal exposure on unstimulated DNA
synthesis. In comparing these two experimental systems, it must be noted that
the level of metal exposure, strain of mouse, and mitogen dose were different.
In fact, since it does appear that the effect of metal exposure may vary depending
on the strain of mouse, it may be valid only to compare in vitro and in vivo
exposure studies within the same strain.
81
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- 10 -
Thus, it appears that in vitro exposure studies may be useful in predicting the
effect of in vivo exposure on lymphoproliferative responses within the same
species. However, the relationship between these effects and the effect of
metals on functional lymphocyte responses, e.g. antibody synthesis or lymphokine
production remains to be determined. The fact that Pb2+ and Cd2+ modulate
lymphocyte metabolism, as described in this report, suggests that further
investigation into their effects on humoral and cell-mediated immune responses
is appropriate.
82
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lOOf-
10
to
40
70
I
I
W6 lo'5
Mctil Conctntrilion (Ml
Figure 1
• DNA
»RNA
o Protein
I
10"7 «rUT
Concentration Pb2*(Ml
Figure 2
10
83
-------
• DNA
• RIM
o PnKein
10
10'
10-
10 •
Concentration "b2" (Mi
Figure 3
o Uncorrected (composnei
• Corrected (composite!
• Blast Cells
uf7 W* io'5
Concentntion Pb*'(M>
Figure 4
10'
84
-------
• DMA
* RNA
10
,-7
10 10
Concentration
10
10
,-3
Figure 5
ll.ti
W.5I
10'
10"* 10
Conctnlrttien »
W"3
Figure 6
85
-------
UNA
10V 10- VT
Conctntritlon CI**(M»
Figure 7
• DM
• UNA
e Prottin
^ 2
Cencinlritien
Figure 8
86
-------
- t
UP
• IlKtCflll
Conctntntion Cd**(M)
Figure 9
• DM
* RNA
o Protein
10
Conccntrilien Cfl2->(Ml
10-
Figure 10
87
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BIBLIOGRAPHY
1. Keller, L.D. Some immunological effects of lead, cadmium and
methylmercury. Drug Chem. Toxlcol. 2: 99, 1979.
2. Cook, J.A., E.O. Hoffman and N.R. DiLuzio. Influence of lead and
cadmium on the susceptibility of rats to bacterial challenge.
Pro. Soc. Exp. Biol. Med. 150; 741, 1975.
3. Treagan, L. Metals and the immune response; a review. Chem. Pathol.
Pharmacol. 12.: 189, 1975.
4. Shenker, B.J., W.J. Matarazzo, R.L.Hirsch and I. Gray. Trace metal
modification of immunocompetence. I. Effect of trace metals in cul-
tures on in vitro transformation of B lymphocytes. Cell. Immunol.
34: 19, 1977.
5. Gallagher, K., W.J. Matarazzo, and I. Gray. .Trace metal modification
of immunocompetence. II. Effect of Pb2*, Cdz , or Cr* on RNA
turnover, hexokinase activity and blastogenesis during B lymphocyte
transformation in vitro. Clin. Immunol. Immunopathol. 13: 369, 1979.
6. Lavoie, D., N.J. Baiter and I. Gray. Manuscript in preparation.
7. Keller, L.D., J.G. Roan and N.I. Kerkvliet. Mitogen stimulation of
lymphocytes in CBA mice exposed to lead and cadmium. Environ. Res.
I£? 177, 1979.
8. Keller, L.D., J.G. Roan and N.I. Kerkvliet. Evaluation of data from
mitogen studies in CBA mice: comparison of counts per minute, stim-
ulation index and relative proliferation index. Am. J. Vet. Res.
40: 863, 1979.
88
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THE USE OF QUANTITATIVE MICRO-CYTOTOXICITY ASSAY TO DETECT
THE INHIBITORY EFFECTS OF CHEMICALS ON THE IMMUNE SYSTEM
W. A. Stylos1, T. S. S. Mao2, and M. A. Chirigos3
123
National Cancer Institute ' ' , National Institutes of Health,
U.S. Public Health Services, Bethesda, Maryland 20205 and
The Waksman Institute , Rutgers - The State University of New
Jersey, New Brunswick Campus, New Jersey 08903
At present;
Immunobiology Study Section, Division of Research Grants,
National Institutes of Health, U.S. Public Health Services,
Bethesda, MD 20205
2
Graduate Programs, Cook College, Rutgers - The State University
of New Jersey, New Brunswick Campus, New Jersey 08903
3
U.S. Army Medical Research Institute of Infectious Diseases,
Fort Detrick, Frederick, Maryland 21701
89
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Abstract
The technique of quantitative determination of cellular
cytotoxicity both in the presence (antibody-dependent cellular
cytotoxicity, ADCC) or absence (cell-mediated cytotoxicity,
CMC) of antibody generally utilizes lymphocytes as effector
cells. By these means cytotoxicity, primarily of T lymphocytes
can be used to quantitatively assess cell killing. It has been
shown that peritoneal macrophages, stimulated with thiogly-
collate and reacted with target cells in the presence of
antibody to these target cells that one can quantitatively
assess target cell killing by the thioglycollate-activated
macrophages. Using this assay in some preliminary experiments,
we have been able to detect significant levels of cytotoxicity
by thioglycollate-activated peritoneal macrophages.
Procedure
I. Effector cells - Macrophages are harvested from the
peritoneum. For use in the cytotoxicity studies, 2-3 days
prior to the setting up of the assay, the mice are injected
I.P. with 2 ml of glycollate. The peritoneal exudate cells
(PEC) are treated with ammonium chloride to lyse RBCs. The PEC
are then plated on Petri dishes and incubated @37°C for 90* in
a 5% CC>2 incubator, the nonadherent cells are decanted and
the adherent macrophages are isolated by either; (1) incubating
on ice for 60-90', or (2) by scraping with a rubber policeman.
The viability of the macrophages is determined by Trypan blue
dye inclusion. In our tests approximately 1x10^ macrophages
are used per microtiter plate well in an effector to target
ratio of 10:1 (1,2).
II. Target cells - in our preliminary studies we have used
various iri vitro passaged target cells. Including; LSTRA - a
viral-induced mouse leukemia syngeneic in BALBlc mice and P815
a mouse plasmacytoma syngeneic in DBA mice. Both of these cell
lines are cultivated in RPMI-1640 median containing 10% PCS and
SxlO'^M 2-mercaptoethanol. Prior to use in the assay, cell
viability is determined by Trypan blue dye exclusion (3,4).
III. Antibody - Antibodies to either LSTRA or P815 target cell
lines were produced in BALBc or DBA mice, respectively. For
antibody production mice received approximately 1x10®
irradiated (12000R) cells emulsified in complete Freunds
adjuvant and 3 or 4 booster injections with 1x10** irradiated
cells emulsified in incomplete Freund's adjuvant. One week
after the last booster injection the mice were exsanguinated,
and antibody titers determined. The negative antibody activity
was determined by (1) titratron of cell killing of a fixed
amount of target cells with various dilutions of the antiserum
in the presence of guinea pig complement, and by indirect
immunofluorescence using the anti-P815 or anti-LSTRA
90
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-2-
as 1st antibody/ followed by FITC-labeled goat anti-mouse
inununoglobin as a second antibody.
IV. Labeling of target cells - approximately IxlO7 viable
target cells are incubated with 100-105 uCi {specific activity
400-600 mC/mg) for 1 hour at 37°C, 5% C02 with constant
agitation. The labeled target cells are tbrn washed 3x with
RPM1 1640 media to remove excess 51Cr(5).
V. Conduct of the test - IxlO4 51Cr-label«d target cells
(in 50 ul) are added to each microtiter plate well, 50 ul of
antibody (~1:100 dilution) is then added to IxlO5 thiogly-
collate-activated macrophages (100 ul) which are then added and
the microliter plates are incubated from 16-24 hours at 37°C-5%
C02. Prior to the 16-24 hour incubation period, the
microliter plates are centrifuged at 300 x g for 5'. After the
16-24 hour incubation the microliter plates are again
centrifuged at 300x g for 5'and 100 ml of the supernatant fluid
is assayed for radioactivity in a gamma scintillation counter (5)
VI. Theory and Application - using the assay described above
only two parameters are needed in order to demonstrate the
immunoinhibition of cytotoxicity by drugs or other agents.
Tnese are, optimal dose of drug (nontoxic levels to tissue -
but adversely effective on the immune system) and the optimal
time of reactivity. Once these have been determined the dosage
of drug and time for administration relative to a 2-3 day
activation time for the macrophages, the cytotoxicity of a drug
or agent can be quantitatively determined.
TL
Total Count
Theoretical Max
Count
71160
71657
70663
71160+287
Pen hour
BCNU-Treatea
Animals
FT
Preeze-Thaw-
Actual Max
Count
50352 60-90%
48392
48028
48924+722
How it was treated 6
In vivo-i.p. Injection
Normal
SR
Spontaneous Release
of 5/ci
19415
20207
18711
19444+432
% Specific release
- Exptl-SR X
FT-SR
Pyran-Treated
In vivo by i.p.
insect
91
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-3-
26162 28564 30691
26233 29039 32254
27034 29298 30931
26476+280 28967+215 31292+496
t—4.71 t—4.38
df-4 df-4
P-<0.01 P»<0.02
% Specific Release
1. Normal 28967-19444 « 9523 x 100 - 32% Spec. Rel.
48924 - 19444 - 29480
2. BCNU-Treated 26476-19444* 7032 x 100 - 24% Spec. Rel.
29480 29480
3. Pyran-Treated 31292-19444 11848 x 100 « 40% Spec. Rel.
29480 29480
Conditions of Cytptoxicity Assay ADCC
MICE-DBA/2N
Pyran-treated - Pyran 25 mg/kg - i.p., 6 days prior to onset of
cytotoxicity test (6) .
BCNU treated - BCNU 30 mg/kg - J»fc ,6 days prior to onset of
cytotoxicity test (7) .
PEC-source animals 2 ml thioglycollate medium I.P. - 2 days prior
to onset of cytotoxicity test.
Target cells - P815 in vitro cultivated syngeneic to DBA/2N mice.
Effector cells PEC (primarily macrophagesr 1 x
cells/well, effector : target ratio 10:1.
Labeling of cells - 100 ul of 51 Ci- (specific activity 350
mc/mg) + 1 x 107 p815 cells incubate .1 hr - 37°C - 5% CC>2
with gentle agitation - then wash 3x with RPMl 1640 media - 10%
to get rid of unbound 51Cr.
Antibody anti-P815 - raised in DBA mice in response to initial
+ 3 booster injections - initial injection IxlO8 irradiated
(12,000 R) + complete Freund's adjuvant/mouse S.C. booster
injection 1 x 1U° irradiated cells + incomplete Freund's
adjuvant S.C.
-------
-4-
Conduct of assay
TC
13199
13061
12922
X-13061+80
NS
1234
1245
1253
SR
11298
11345
11322
11322+14
NS+I
1697
1694
1699
IS
3225
3326
3426
864
981
923
923+34
IS+I
2391
2538
2684
1244+6
1697+1
t—79.8
df-4
P» 0.001
3326+58
t—7.68
df-4
P- 0.002
2538+85
% SPECIFIC RELEASE « Expt. Value - SR
F7- SR
DNS 1244-923 - 321 x 100 * 3%
11322-923 10399
2. NS +I«1697-923 « 774 x 100 «
11322-923 10399
7% / Increase in % Sp. Rel,
3. IS = 3326-923
11322-923
« 2403 x 100
10399
23%
3. IS+I
2538-923
1615 x 100
10399
16% / Decrease in % Sp. Rl.
11322-923
CONDITIONS OF CYTOTOXICITY ASSAY
Normal Mice: DBA - o - 10 weeks old target cells
Immune Mice DBA - Inoculated c 5x10' EL-4 cells or days
previous to conduct of the test
Effector Cells - I or N spleen cells -
Macrophage depleted- (by adherence for 60-90' at 37°C 5%
C02) use non-aoherent cells. RBC depleted - by incubation at
room temp for 5' with 0.11M ammonium chloride. Ficoell-Hypaque
densely gradient centrifugation. Lymphocytes band at
interface-300 x g - 30.' Density of gradient « 1.09 g/ml.
Lymphocytes washed x 3 c RPM11640 medium + 10% FCS. Used at a
ratio of 100:1 (effector to target ratio) (8).
93
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-5-
Labelling of cells
Ix 107 target cells - EL-4 cells in a volume of 20.2 ml
incubated with ~ 200 ml of 51Cr (specific activity ~
300-400 mc/mg) for 1 hr at 37°C - 5% CO?. Then washed 3 x c
RPN1 1640 medium.
Conduct of assay -
In each microliter well - 0.1 ml of targets IxlO5
51Cr-labelled EL-4 cells + 0.1 ml of 1 x 107 effector cells
(normal or immune spleen cells) incubate at 37°C-50% CC>2 - 4
hrs. Before starting the incubation, centrifuge plate x 300
RPM for 10* to allow effector and target cells to come into
close contact. Then incubate for 4 hrs. Centrifuge plate
again at the same speed (300 RPM-10') and collect 0.1 ml of
supernatant. Count in a & scintillation counter.
The I on page (1) represent the actual data for the
variously by treated samples. The various controls mean the
following:
Total count (TC) - This is the theoretical maximum count
obtainable under the conditions of a particular experiment.
Freeze-thaw (FT) tnis is the actual maximum releasable
count for a particular test.
Spontaneous Release (SR) this is the amount of radioactive
label that escapes from ^Cr-labeled cells when target-
labeled cells are incubated witn medium alone.
Interpretation of Data.
In this particular experiment - tnis is a lymphocyte
cytotoxicity assay in an allogeneic system (as opposed to the
other experiment which is ADCC using peritoneal exudate cells
[primarily macrophage,] and antibody to the target cells).
This particular experiment is important in that it shows the
activity of the inhibitor, in this case indomethacin actually
stimulates under normal conditions, and inhibits under immune
conditions, i.e., increases the specific release of 51Cr with
normal spleen cells, while inhibiting the specific release of
51Cr with immune [injected with tumor cells] spleen cells. -
Can generalize a lot from this data!
-------
DESCRIPTION OF P815 ANTIBODY USED IN ADCC
I. PRODUCTION:
ANTI-P815 WAS DEVELOPED IN SYNGENEIC DBA MICE BY REPEATED I.P. INJECTIONS
OF IRRADIATED <10,OOOR) P815 CELLS. THE INITIAL INOCULATION CONTAINED 1 x 10*
IRRADIATED P-815 CELLS, EMULSIFIED WITH COMPLETE FREUND'S ADJUVANT AND
INJECTED I.P. IN DBA MICE. THREE SUBSEQUENT BOOSTER INOCULATIONS WITH THE
IRRADIATED P-815 CELLS EMULSIFIED WITH INCOMPLETE FREUND'S ADJUVANT WERE
ADMINISTERED I.P. AT BIWEEKLY INTERVALS. TEN DAYS AFTER THE FINAL BOOSTER
INJECTION THE MICE WERE EXSANGUINATED, AND THE ANTISERUM USED IN THE ADCC
ASSAYS.
II. TITER AND SPECIFICITY:
DETERMINED BY CYTOTOXICITY STUDIES IN THE PRESENCE OF G.P.C
- % CELLS DEAD
1 x 106 P815 CELLS - 0.2*1 + 10 jut P815 Ab (UNDID +10 -ul G.P.C 24.7 + 2.3
1-10 " 24.3 + 0.9
1-50 " 30.3 + 0.9
1-100 " 72.0 + 3.6
1X10* NORMAL SPLENIC CELLS 1-10 " 1.1+0.9
vO
LP
-------
Table 1
ADCC
PERITONEAL EXUDATE
CELLS FROM NORMAL 5tCr LABELED PB15 TARGET CELLS MOUSE ANTI - P815 Ab
OR IMMUNE DBA MICE SYNGENEIC IN DBA MICE +
•f
5.0 x 10s VIABLE CELLS - 0.05 ML 5.0 x 104 VIABLE CELLS - 0.05 ML 1-50 DILUTION OF Ab-0.05ML
REAGENTS ADDED TO WELLS OF MICROTITER PLATES, CENT. BO x 0-10 , INCUBATE AT
37°C - 5% CO2 - 95% HUMIDIFIED AIR FOR 4 OR 24 HOURS.
AT THE END OF 4 OR 24 HOURS MICROTITER PLATES CENT. AT 80 x g - 10 .100 MI OF
SUPERNATANT FLUID REMOVED FROM WELLS, ADDED TO TUBES AND PLACED IN
GAMMA SCINTILLATION COUNTER TO DETERMINE AMOUNT OF 51Cr RELEASED.
CONTROLS: SPONTANEOUS RELEASE (SR), THE AMOUNT OF 61Cr RELEASED BY
LABELED TARGET CELLS INCUBATE WITH MEDIUM ALONE.
TOTAL COUNT, THE THEORETICAL MAXIMUM AMOUNT OF 51Cr RELEASE BY
A GIVEN ALIQUOT OF LABELED TARGET CELLS.
MAXIMUM RELEASE (MR), THE ACTUAL MAXIMUM AMOUNT OF 51Cr RELEASED
BY A GIVEN ALIQUOT OF LABELED TARGET CELLS THAT ARE FROZEN AND THAWED
THREE TIMES.
R4 _ JEW. CPM — 5RCPM x Km
CALCULATIONS: % SPECIFIC 51Cr RELEASED " MR PPM _ sn rPM
VO
0\
-------
Table 2
TYPICAL DATA FROM ADCC ASSAYS
CPM CrlN
SUPERNATANT FLUID
0)
NORMAL
MICE
X - 28967 ±216
CONTROL : (CPM IN SUPERNATANT)
TOTAL COUNT: 5? • 71160 ±287
MAXIMUM RELEASE: 5? • 48924.+722
SPONTANEOUS RELEASE: X • 19441432
(2)
BCNU-TREATED
MICE
(3)
PYRANT
TREATED MICE
X- 264761280 X- 312921498
vO
-------
Table 3
CALCULATIONS OF % SPECIFIC RELEASE
(1) NORMAL 289617 - 19444 _ 9523 ,inn,-»o*
48924 - 19444 ~ "29480~ W 3Z%
(2) BCNU-TREATED 26476-19444 _ 7032 100-24%
48924-19444" 29480 * "
(3) PYRAN-TREATED 31292.-19444 - 11845 x100 = 40%
48924-19444 ~~ "29480
-------
Table 4
STATISTICAL TREATMENT OF DATA
(2) (1) (3)
BCNU- NORMAL PYRANT
TREATED MICE TREATED
CPM IN SUPERNATANT 26162 28564 30691
26233 29039 32254
27034 29298 30931
5T+SEM 264764280 289674215
P-<0.02
VO
-------
Table 5
CMC
I. EFFECTOR CELLS
SPLEEN CELLS FROM NORMAL OR IMMUNE MICE ARE INCUBATED IN A
HUMIDIFED ATMOSPHERE OF 5% CO2 AND 95% AIR AT 37°c FOR 1 HR TO
REMOVE ADHERENT CELLS. THE NONADHERENT CELLS ARE COLLECTED
AND INCUBATED AT RT FOR 5 MIN. WITH 0.11M AMMONIUM CHLORIDE TO
DEPLETE RBCs. THE NONADHERENT, RBC-DEPLETED CELLS WERE LAYERED
OVER FICOLL-HYPAQUE MEDIUM (SPECIFIC GRAVITY — 1.09 g/ml), AND
CENTRIFUGED AT 300 x g FOR 30' AT RT. THE INTERFACE CONTAINING
THE SEPARATED LYMPHOCYTES WERE COLLECTED, WASHED 3x. CHECKED
FOR VIABILITY AND BROUGHT TO THE DESIRED CONCENTRATION FOR
USE IN THE CMC ASSAY.
II. TARGET CELLS
1 x 107 IN VITRO CULTURED P-815 CELLS IN A 0.2411 VOLUME ARE!
INCUBATED WITH 100-150/iCi 51Cr {SPECIFIC ACTIVITY 300-400mC/mg)
FOR 1 HOUR WITH OCCASIONAL SHAKING. THE CELLS ARE THEN WASHED
3x, CHECKED FOR VIABILITY AND BROUGHT TO THE DESIRED CONCENTRATION.
III. EFFECTOR CELLS AND LABELED TARGET CELLS (AT EFFECTOR: TARGET
RATIOS OF 5:1 TO 200:1) ARE INCUBATED AT 37°c - 5% CO2 - 95% HUMIDIFIED
AIR FOR 4 OR 24 HOURS.
IV. CONTROLS AND CALCULATIONS ARE THE SAME AS SHOWN IN THE
PREVIOUS SLIDE FOR ADCC.
-------
Table 6
TYPICAL DATA FROM CMC ASSAYS
CPM 51Cr IN
SUPERNATANT FLUID
NS
0)
X = 1244±6
CONTROL: (CPM IN SUPERNATANT)
TOTAL COUNT: 7 - 13061 ±.80
MAXIMUM RELEASE: JT - 11322±14
SPONTANEOUS RELEASE: R - 923 ± 34
NS + I
(2)
7-1697*1
IS
(3)
3326158
IS+I
(4)
2538185
-------
O
Table 7
CALCULATIONS OF % SPECIFIC RELEASE
(1) NORMAL SPLEEN - = * 10° " 3%
(2) NORMAL SPLEEN 1697-923 fe 774 x 100 » 7%
+ INDOMETHALIN 10399 Io399
(3) IMMUNE SPLEEN j *j$Jr X 10° " »«
(4) IMMUNE SPLEEN 2338-923 , 1615 x 100 - 16%
+ INDOMETHALIN 10399 ' 10399
-------
Table 8
STATISTICAL TREATMENT OF DATA
(1) (2)
NS NS + I
CPM IN SUPERNATANT 1234 1698
1245 1694
1253 1699
X + SEM 1244 ±6 1697 i
\ /
P = < 0.001
-------
-6-
This article describes several variations of techniques
which are suitable for the quantitative determination of
cell-mediated immune reactions. Using these techniques it is
possible to accurately determine minute traces of small
molecular weight substances or drugs. In addition to the
determination of extremely small amounts of the materials to be
detected, the described techniques have been shown to be
reproducible by statistical methods. Finally, the use of these
techniques for determining trace amounts of potentially harmful
compounds or drugs is apparent.
104
-------
REFERENCES
1. Schultz, R. M., Papamatheakis, J. D., Luetzeler, J. and Chirigos, M. A. (1977)
Macrophage involvement in the protective effect of pyran copolymer
against the Madison lung carcinoma (M 109). Cancer Research ^J7: 350-364.
2. Schultz, R. M., Chrigos, M. A., PavUdes, N. A. and Younger, J. S. (1978)
Macrophage activition and ant -tumor activity of Brucella abortus ether-
extract, Bru pel. Cancer Treatment Reports, 62_:1937 - 1941.
3. Dean, J. H., Padarathsingh,M. L. and Keys, L. (1978) Response of raurine
leukemia to combined BCNU-tnaleic anhydride-vinyl ether MVE) adjuvant therapy
and orrelation with macrophage activation by MVE in the ^n Vitro growth
inhibition assay. Ibid . ,62_: 1807-1816.
4. Stylos, W. A., Chirigos, M. A., Lengel, C. R. and Weiss, J. F. (1978)
T and B lymphocytes of irridiated,tumor-bearing mice treated with an
immunostimulator, pyran. Cell Immunol. 41:168.
5. Kiessling,R., Hochman, P. S., Haller, 0., Shearer, G. M. , Wigzell, H.,
and Cudkowicz, G. (1977) Evidence for a similar or common mechanism for
natural killer cell activity and resistance to hemopoietic grafts. Eur. J.
Immunol., 7^:655-663. /
6. Papamatheakis, R. M., Schultz, R. M. , Chirigos, M. A. and Massicot, J. G.
(1978) Cell and tissue distribution of ^C-labeled pyran copolymer. (1973)
Cancer Treatment Reports, 62:1845-1851.
7. Mao, T. S. S. and Chirigos, M. A. (1978, March) Mitogenic effects of pyran
copolyraers on lymphocytes. Federation Procedings, 37(3):829.
-------
Pages 106 - 108 are intentionally blank.
-------
Running Head: RIAs for Polychlorinated Aromatic Hydrocarbon
&
Title: DEVELOPMENT OF RADIOIMMUNOASSAYS FOR CHLORINATED BIPHENYLS,
DIBENZOFURANS AND DIBENZO-p-DIOXINS
Authors: M. I. Luster,1 P. W. Albro and K. Chae
James D. McKlnney
Laboratory of Environmental Chemistry
National Institute of Environmental Health Sciences
P.O. Box 12233
Research Triangle Park. N.C. 27709
At Present: IiTiTunotodaology Group, Systemic Toxicology, National
Institute of Environmental Health Sciences, P.O. Box 12233, Research
Triangle Park, NC 27703
109
-------
ABSTRACT
Radioimmunoassays are described for quantHating a number of toxic
polychlorlnated aromatic hydrocarbons considered chemical pollutants
from environmental samples. The assays have been developed to minimize
the need for mass spectral analysis and can be easily performed in most
clinical laboratories. Assays are presently developed for 2,3,7,8-
•
tetrachlorodibenzo-p_-diox1n, 2,3,7,8-tetrachlorodibenzofuran; 4-monoch-
lorobiphenyl, 3,4,3',4', and 2,6,2l,6'-tetrachlorobiphenyl. Extensive
cross reactivity studies for the various antisera are described as well
as comparative analysis of tissue samples with gas chromatography and/oi
mass spectrometry.
110
-------
-1-
INTRODUCTION
Several of the chlorinated aromatic hydrocarbons Including dibenzo-
£-dioxins, dibenzofurans, and bipehnyls are environmental contaminants,
presently of considerable concern (1). 2,3,7t8-Tetrachlorod1benzo-£-
dloxln (TCDD) and 2,3,7,8-tetrachlorodlbenzofuran (TCDF) are probably
the most toxic members of these classes with an acute oral LD50 1n the
guinea pig of 1 (2) and 5-10 vg/kg body weight (3), respectively. The
polychlorlnated bipehnyls (PCBs) are much less toxic although their
toxldty will vary due to their 1somer1c composition (4). Concern over
PCBs has resulted from their widespread appearance in tissues of humans
and wildlife (5) and their apparent carcinogenecity in laboratory animals
(6). Furthermore, various PCBs are contaminated with the more toxic
dibenzofurans (7).
Until recently the only analytical technique with sufficient sensitivlt
and specificity for determination of dibenzo-£-dioxins and dibenzofurans
in environmental samples has been high resolution mass spectrometry (MS).
Recently gas chromatography (GC) combined with low or medium resolution
MS in either electron impact or chemical ionization modes have been used
to estimate levels of various d1benzo-p_-diox1ns and dibenzofurans (8).
Due to the cost and complexity of GC-MS instrumentation; the high degree
of technical skill and experience needed in assays of this type;and the
lack of a confirmatory technique not based on MS, we have developed
assays based on the highly sensitive, relatively specific and simple
technique of radiommunoassay (RIA).
Ill
-------
-2-
DESIGN AND METHODS
The chemicals specifically adapted for RIAs Included TCDD, TCDF, 4-
monochlorobiphenyl (4-MCBP). 2,6,2',6'-tetrachlorob1phenyl (TCBP) and
3»4,3',4'-TCBP. Since these chemicals (haptens) have no chemically
reactive functional groups, derivatives were synthesized that .retained
most of the structural feature of the native chemical but with the
addition of a reactive site. While a variety of compounds have been
derivatized, those compounds used as haptens in the RIAs to be described
include l-amino-3,7»8-triCDD, 4-amino-2,7,8-tr1CDF, 4-amino-4-chlorobi-
phenyl. 2-amino-4,5,3',41-TCBP, and 3-amino-2,6,2'.6'-TCBP. The synthesis
of the amino-triCDD (9) and amino-TCBPs (10) have been described. The
4-amino-4-monochlorobiphenyl was purchased from K&K Laboratories, Inc.
while the synthesis of the amino-triCDF will be described in a subsequent
publication (Luster, et. al., in preparation). These derivatives were
coupled to carrier proteins, either thyroblogulln or albumins, prior to
immunization in rabbits to afford them immunogenecity. The coupling
procedure was accomplished by reacting the amono-derivative of these
polychlorinated aromatic hydrocarbons with the acid chloride of mono
methyl adipate followed by converting the adipamide to a mixed anhydride.
Detailed characteristics of this coupling procedure has been described
elsewhere (11).
Radioactive compounds were also synthesized from the amino derivativ
by initially converting the derivatives to amides with 5-bromovaleryl
125
chloride followed by substitution with iodine (Na'"I; New England
Nuclear; 17Ci/mg). Following purification over silica gel, the final
112
-------
-3-
products contained >95% of the radioactivity in a single spot during
thin layer chromatography on silica gel in benzene and had estimated
specific activities of, approximately, BOCi/mmole.
In the assay, a dilution of antiserum capable of binding about
125
20pg of I-labeled compound (*40JJ of the tracer dose) was preincubated
»
with the detergent emulsion of sample extract (standards or unknowns).
125 "
The mixture was then incubated with the I-labeled derivative long
•
enough for equilibrium binding to occur (*72 hrs.). Goat antibodies
against rabbit y-Qlobulin were then added to the mixture to precipitate
the antibody-hapten complex (double antibody method) and the amount of
125
I in the precipitate was determined in a gamma counter.
The extent to which preincubation with the test material inhibited
125
or decreased the amount of I precipitated relative to the amount
precipated in the absence of test material was a measure of the amount
of chlorinated aromatic hydrocarbon in the test sample. Details of
these procedures including materials, characterization of labeled and
unlabeled antigens, Immunizations, tissue clean-up as well as assay
conditions have been described in our other publications (11-14).
RESULTS AND DISCUSSION
A feature of the assay method is the use of nonionic detergents to
solubilize the extremely hydrophobia aromatic hydrocarbons in a manner
permitting their binding to antibodies. Numerous detergents were compared
for their ability to solubilize 14C-TCDD or 14C-3.4,3'.4I-TCBP. Only
113
-------
-4-
two Triton X-305 and Cutscum provided sufficient micelle formation to
negate insolubility and had minimal effects on inhibiting precipitation
in the double antibody RIA. In general, lower levels of hapten could be
measured using Triton X-305 than with Cutscum; however, a wider range of
concentrations could be assayed using Cutscure than Triton X-305. Cutscum
was employed-in the TCOD and TCDF assays while Triton X-305 was employed
in the PC6 assays.
Cross reactivity was evaluated in the various assays to give an
indication of possible interferences to be expected from compounds other
than those being analyzed (Tables I-II). The potential for interference
in these assays is self-limiting, since only those amounts of hydrophobic
compounds capable of being solubilized are actually presented effectively
to the antibodies. Antisera against the various PCBs were relatively
specific to the immunizing antigen although enough cross reactivity was
obtained to their corresponding non-aminated TCBPs to indicate that the
RIA possessed sufficient sensitivity to analyze these biphenyls in
unknown samples (Table I). Antisera to 4-MCBP cross reacted extensively
not only with 4-MCBP, but several other low chlorinated biphenyls indicating
a lack of specificity.
Table II summarizes cross reactivity studies utilizing antisera
produced against l-amino-3,7,8-triCDD and 4-amino-2,7,8-triCDF- With
these particular antisera in the presence of cutscum, the sensitivity of
these assays were in the order of 100 pg and 20 pg for TCDO and TCDF,
respectively, when no interfering contaiminants were present. These
values are based on non-overlapping ranges of blank and test replicates.
114
-------
Antisera from various rabbits immunized with l-amino-3,7,8-tr1CDD revealed
distinct cross reactivity pattens with some antisera capable of discriminate
TCDD from other chlorinated d1benzo-£-d1oxin isomers while other antisera
were capable of distinguishing TCOD from TCDF. Antisera unable to
distinguish various dibenzo-p_-dioxin Isomers, but discriminating TCDD
from TCDF can be used in a screening assay to reduce the number of
samples that must receive the much more elaborate mass spectrometrie
•
assay. When a single dibenzo-£-diox1n isomer is found present or
greatly predominate, an antiserum relatively specific for TCDD can be
used to confirm the quantitative data from mass spectral analysis or if
used alone to monitor exposure to TCDD in an environment known to contain
this Isomer. In the latter case monitoring results would only be presumpti\
in nature.
While only several antisera from rabbits Immunized against 4-amino-
2,7,8-trlCDF have been extensively examined at this time, it appears
that equal, if not greater, specificity and sensitivity can be obtained
with the TCDF assay than the TCDD assay. From Table II it appears that
while this particular TCDF antiserum is fairly specific for TCDF, some
cross reactivity occurs with structurally similar compounds such as
3,4,3',4'-tetrachlorobiphenylene. The inability of S.A.S'.A'-TCBP to
cross react with the antiserum in conjunction with the cross reactivity
to TCDD and blphenylene would indicate that not only are the chlorine
positions important for antibody recognition but also the rigidity of
the molecule. The cross reactivity noted with 2,3,7,8-TBrDF would
suggest that the antisera cannot readily discern one halogen from another.
115
-------
-6-
Comparative analysis of environmental samples between RIA and GC,
with either negative chemical 1on1zat1on MS or electron capture detector
as measuring devices are currently underway. At present, the PCB RIAs
would not readily lend themselves to tissue analysis since only the more
hydrophobic or higher chlorinated PCB homologs are likely to be retained
(15). Prior to this use, antlsera will have to be generated against
several of the higher chlorinated homologs (e.g. 2,4,5,2',4',5'-hexaCBP).
None the less, the*PCB assays have been used to quantltate the percent
of several PCB congeners present 1n Aroclors 1242 and 1248 (Table III)
and In the case of the TCBPs, are 1n fairly good agreement with GC
analysis, these assays have also been adapted to "Fingerprint analysis"
to determine the probable Aroclor number in a non-biological sample
(immersion oil) as well as the percentage of Aroclor in this sample
(14).
The applicability of the TCDD and TCDF RIAs for tissue analysis was
examined with liver and adipose samples from Rhesus monkeys that had
been used 1n toxlcity studies involving TCDD (16) and TCDF (17).
Sample clean-up size ranged fron 3 to 50 mg of tissue although only a
fraction of the sample was used in the RIA. The results are summarized
in Table IV. Of the three techniques, GC with an electron capture
detector has the greatest potential for Interference, and thus can only
suggest an upper limit for the amount of TCDD or TCDF in a sample. In
37
the absence of a suitable internal standard (e.g. [C1]-TCDD), the
mass spectral technique gives results whose accuracy depends upon how
soon the unknown sample was run after preparing the standard curve.
1 16
-------
-7-
In contrast, the unknown and standard curve are run simultaneously in
the immunoassay procedure. Considering the many differences in the
principles involved in the three assay methods, the agreement of results
in Table IV was acceptable.
These RIAs have by no means been brought to their maximum -limits of
specificity or sensitivity. Further improvements may be realized by
increasing the specific activity of the radioiodinated compounds, improv-
ing sample cleanup'procedures with respect to recovery as well as removal
of interfering contaminants and increasing the avidity of the various
antisera. At present, the assays should be applicable to screening
samples to minimize the demand for mass spectrometric analysis by elimina-
ting "negative" samples, and routine monitoring of exposure in environ-
ments where specific polychlorinated aromatic hydrocarbons are suspect
or known to be present.
117
-------
-8-
TABLE I
Percent Cross Reactivity In PCB Assays*
Compound Tested
Cross Reactivity with Antlsera To;a
2.6.2\6'-TCBP 3,4,3',4'-TCBP 4-MCBP
Biphenyls
Biphenyl
4-C1
4-F
3,3,'-C12
3.4-Cl2
2,3,2',3'-Cl4
2,4,2',4'-Cl4
2,6,2',6I-C14
3,4,3',4'-Cl4
3.5.3',5'-Cl4
3,4,3',4'-Me4
3,4,2',4',5'-Cl5
2,3,6,2',3I,6'-C16
3,4,5,3',4I,5'-C12
Decachlorobi phenyl
S-.A.S'.A'-C^BIphenyl ether
Various PCDFs and PCDDs
65
65
32
5.0
2.4
27
10
12.5
43
ND
1.9
3
2.4
ND
aCross reactivity = (ng compound causing 20% displacement of label
(values not shown) per ng immunizing ligand causing 20% displacement of label)
x 100.
b(-) = Compound with less than 0.14% cross reactivity.
c(+/_) » Compounds with 0.15-1.49% cross reactivity.
ND = not done.
*Adapted from Reference 14.
118
-------
-9-
TABLE II
Percent Cross Reactivity 1n TCDD and TCDF RIA
Compound Tested
Cross Reactivity With Antisera To:'
TCDD TCDF
DBD's
Unchlorinated
2,3,7-Cl3
1,2,3,4-C14
2,3,7,8-Cl4
1,2.3.6,7,8-C16
DBF's
Unchlorinated
2,3,8-Cl3
2,3.7,8-Cl4
2,3,4,6,7.8-Clg
2,3,7,8-Br4
Others
.3,4,3',4'-TCBP
3,4,3',4'-Cl4-Biphenyler,e
3,4,3I,4'-Cl4-Biphenyl ether
3,4,3'.4'-(CH3)4
RIA Working Proteins
72
20
100
9-991
2
2
25-921
ND
40
6
ND
100
2
39
2
24
aCross reactivity = 100 x(Inhibition of I-binding by test compound/inhibition
by equal amounts of TCDD or TCDF
Cross-reactivity will vary with different antisera
CND « not done
119
-------
-10-
TABLE III
Comparison of PCB RIAs for Determining the Concentration of Their
Specific Homolog in Aroclors 1242 and 1248 with Gas Chromatography.
Biphenyls Analyzed Antisera Used in Aroclor 1242 Aroclor 1248
by GC RIA GC(%) RIA(X) GC(%J RIA(X)
4-MCBP 4-Am1no-4'-MCBP 0.23 4.0 trace 0.6
3,4,3'4'-TCBP plus 2-Amino-4,5.3',4l-TCBP 0.34 0.24 *2.2 %3.5
2,6,2',6'-TCBP S-Amlno^.e^'.S'-TCBP 0.17 0.24 trace 0.7
120
-------
-11-
TABLE IV
Comparison of RIA with 6C-EC or GS-M5 for Determination of
TCDD and TCDF In Monkey Tissue
Assay Animal No.a
TCDD
373 .
373
380
380
391
•391
TCDF
378
489
396
Tissue
Adipose
Liver
Adipose
Liver
Adipose
Liver
Adipose
Adipose
Adipose
ppb
RIA ' GC-MS
NDb ND
NA -c
41 28
20
1800 1740
94
8
30
52
GC-EC
ND
ND
87
17
1810
30
ND
46
42
aMonkeys 373 and 378 were controls and dosed orally with corn oil alone.
Monkeys 380 and 391 received a single oral dose of 70 yg/kg and 350 ug/kg
TCDD in corn oil,respectively,while monkeys 489 and 396 received a single
oral dose of 0.5 mg/kg and 1.5 mg/kg TCDF in corn oil, respectively
bND *= not detected
c- * not done
121
-------
-12-
REFERENCES
1. W. J. Nicholson, and J. A. Moore, Eds. Health Effects of Haloqenated
Aromatic Hydrocarbon. Anna! N.Y. Acad. Sci.. Vol. 320 (1979), pp
1-730.
2. 6. A. Schwetz, J. H. Norn's, G. L. Sparchu, V. K. Rowe, P. J.
Gehring, J. L. Emerson and C. G. Gerblg. "Toxicology of Chlorinated
Dibenzo-£-d1oxins". Environ. Health Perspec. Vol. 15 (1973) pp
87-99.
3. J. A. Moore, B. N. Gupta and J. G. Vos, "Toxicity of 2,3,7,8-
Tetrachlorodibenzofuran- Preliminary Results", EPA-560-6-75-004
(1976), pp 77-80.
4. J. D. McKlnney, K. Chae, B. N. Gupta, J. A. Moore and J. A. Goldstein.
"lexicological Assessment of Hexachlorobiphenyl Isomers and 2,3,7,8-
Tetrachlorodibenzofuran in Chicks. 1. Relationship of Chemical
Parameters", Toxicol. Appl. Pharmacol. (1976), Vol. 36, pp 65-80.
5. R. W. Risebrough, P. Rieche, D. B. Peakall, S. G. Herman, and M. W.
Kirven. "Polychlorinated Biphenyls in the Global Ecosystem", Nature
Vol. 222 (1968), pp 1098-1099.
6. R. D. Kimbrough, and R. F. Under. "Induction of Adenofibrosis and
Hepatomas of the Liver of BALB/cJ Mice by Polychlorinated Biphenyls
(Aroclor 1254)". J.. Natl. Cancer Inst. Vol. 53 (1974), pp 547-552.
7. J. G. Vos, J. H. Koeman, H. L. van der Maas, M. C. ter Noever de
Brauw and R. H. de Vos". Identification and toxicological evaluation
of chlorinated dibenzofuran and chlorinated napthalene in two com-
mercial polychlorinated biphenyls". Cosmet. Toxicol. Vol. 8 (1970),
pp 625-673.
122
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-13-
8. J. R. Hass, M. D. Friesen, D. J. Harvan and C. E. Parker, "Determinatioi
of Polychlorinated dibenzo-p_-dioxins in biological samples by
negative chemical ionization mass spectrotnetry, Anal. Chem. Vol. 50
(1978), pp 1474-1479.
9. K. Che, L. K. Cho, and J. D. McKinney. "Synthesis of l-Amino-3.7,8-
trichloro-dibenzo-£-dioxin as Haptenic Compounds", J.. Agric. Food
Chem. Vol. 25 (1977), pp 1207-1209.
10. S. K. Chaudhafy and P- W. Albro. "A convenient method for the prepara-
tion of 2-amino-4,5,3l,4'-tetrachlorobiphenyl". Org. Prep. Proc. Intl.
Vol. 10 (1978), pp 46-55.
11. P. W. Albro, M. I. Luster, K. Chae, S. K. Chaudhary, G. Clark,
L. D. Lawson, J. T. Corbett, and J. D. McKinney. "A Radioimmunoassay
for Chlorinated Dibenzo-p_-Dioxins". Toxicol. Appl. Pharmacol., Vol.
50 (1979), pp 137-146.
12. P- W. Albro, and B. J. Corbett. "Extraction and Clean-up of Animal
Tissues for Subsequent Determination of Mixtures of Chlorinated
Dibenzo-£-Dioxins and Dibenzofurans," Chemosphere. Vol. 7 (1977),
381-385.
13. M. I. Luster, P. W. Albro, K. Chae, G. Clark, and J. D. McKinney.
"Radioimmunoassay for Mono-(2-ethylhexyl)Phthalate in Unextracted
Plasma", Clin. Chem.. Vol. 24 (1978), pp 429-432.
14. M. I. Luster, P. W. Albro, G. Clark, K. Chae, S. K. Chaudhary,
L. D. Lawson, J. T. Corbett, and J. D. McKinney. "Production and
Characterization of Antisera Specific for Chlorinated Biphenyl Species:
Initiation of a Radioimmunoassay for Aroclors". Toxicol. Appl.
Pharmacol., Vol. 50 (1979), pp 147-155.
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-14-
15. J. D. McKinney. "Toxicology of Selected Symmetrical Hexachloro-
biphenyl Isomers: Correlating Biological Effects with Chemical
Structure". In Proceedings of the Natl. Conf. on PCBs. EPA (1975)
pp 73-76.
16. E. E. McConnell, J. A. Moore, and D. V. Dalgard, "Toxicity of
2,3,7,8-tetra-chlorodibenzo-£-dioxin in Rhesus monkeys (Macaca
mulatta) following a single oral dose," Toxicol. Appl. Phartnacol.,
•
Vol. 43 (1978), pp 175-187.
17. E. E. McConnell, and J. A. Moore, "Toxicopathology Characteristics
of the Halogenated Aromatics". Ann. N.Y. Acad. Aci., Vol. 320 (1979),
pp 138-150.
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1-3
Approaches to Assess Altered Host Resistance
Ping C. Hu,4 Ralph J. Smialowlcz and
Donald E. Gardner5
Health Effects Research Laboratory
United States Environmental Protection Agency
Research Triangle Park, N.C. 27711
2
This paper has been reviewed by the Environmental
Protection Agency and approved for publication.
Approval does not signify that the contents neces-
sarily reflect the views and policies of the Agency,
nor does mention of trade names, commercial pro-
ducts or commercial processes constitute encorse-
ment or recommendation for use.
Requests for reprints should be addressed to:
Dr. Ping C. Hu
Inhalation Toxicolgoy Branch
HERL, EPA, MD-82,
Research Triangle Park, N.C. 27711
. At Present:
Department of Microbiology, The University
of North Carolina at Chapel Hill, 535 Clinical
Sciences Bldg 229H, Chapel Hill, NC 27514 i 25
Environmental Sciences, Northrop Services, Inc.,
P.O. Box 12313, Research Triangle Park, NC 27709
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Approaches to Assess Altered Host Resistance
Introduction
There is abundant evidence in the literature of a relationship
between an increased susceptibility to viral, fungal or bacterial
infections and neoplasia in Inmunosuppressed experimental animals and
human patients. Individuals born with primary immunologic deficiencies,
such as athymic hunans and nude mice, have been found to have increased
susceptibility to bacterial, viral or fungal infections and neoplasia
•
(14,23). Immune deficiency can also be resulted from malnutrition,
X-ray irradiation, chemotherapeutic agents, and exposure to certain
chemicals (32). In the past few years, imnunosuppression by environ-
mental chemicals has become an increasing concern of environmental
toxicologists. Unfortunately, this aspect of toxicology has until
recently received limited attention. Furtheremore, methodologies and
approaches for routine assessment of immunobiological effects have been
questioned. In this paper, we described two immunological approaches
which may be useful in the assessment of effects of toxic substances on
immune system: 1) A solid-phase radioimmunoassay for the detection of
specific antibody in the lung of hamsters infected with Mycoplasma
pneumoniae; and 2) A tumor susceptibility assay for measuring the inci-
dence of progressing tumors in mice.
I. Solid-Phase Radioimmunoassay for the Detection of Specific Antibody
in the Lung Lavage.
The lung occupies a unique position with respect to the relation-
ship between the man and his environment. An adequate oxygen supply for
126
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an average adult requires the Inhalation of approximately 15 Kg of air
daily. Unfortunately, even so called "fresh air" carries a variety of
injurious materials including infectious agents, toxic chemicals,
noxious gases, and solid particulates. However, the lower respiratory
tract is normally sterile, emphasizing the remarkable efficiency of the
defense mechanisms of this organ system. Respiratory defense mechanisms
are directed against the pathogenic potential of inhaled materials.
Alterations of these defense mechanisms may result in lung diseases
(17,26). One of the prime respiratory defense mechanisms is the immuno-
logic response in the lung to foreign antigens. This protective mechan-
ism produces specific antibodies and have several important immuno-
logical functions. For instance, IgM which appears in the early stage
of an immune response may serve to aggutinate the Invading microorganisms,
thus to reduce its invasiveness; IgA prevents the attachment of infecti-
ous agents to the respiratory tract and hence reduces their opportunity
to establish themselves in the respiratory tract; and IgG functions to
provide a competent mechanism for opsonization and lysis of microorganisms
in concert with complement. Thus, it is essential that the evaluation
of the toxicity of inhaled toxics or pollutants on pulmonary defense
mechanisms should include experiments to determine if lung immunity is
altered. Currently the measurement of the antibody-mediated immune
responses in the lung, is most commonly approached by measuring the
number of specific antibody-forming cells in bronchus-associated lymphoid
tissue either by the direct immunofluorescent technique or by the hemo-
lytic plaque assay (2,10). A solid-phase radioimmunoassay which can be
used to measure the level of specific antibody in the lungs of small
experimental animals will be very useful in the assessment of the immuno-
logical effects of environmental toxicants.
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II. Tumor Susceptibility Assay
Another important area of immunotoxicology of potential environ-
mental concern is the effect of chemicals which may result in the enhance-
ment of tumorigenicity in the host. Neoplastic cells arise frequently
either spontaneously or due to chemical and viral induction, but in most
cases such cells are eliminated by Immune mechanisms. Evidences exist
that exposure to certain chemicals can result in immune alterations in
experimental animals (32) which often leads to altered host resistance
to bacterial (12,13,31), viral (8,11), or transplantable tumor cell
challenge (6) as well as spontaneous tumor development. Studies have
also shown that low-level chronic exposure of humans to certain chemicals
may induce immunologic alterations similar to which occurs in experimen-
tal animals (1). Burnet (5) first proposed the concept of immune
surveillance that the immune system, principally the thymus-dependent
lymphocytes and accessory macrophages, provided primary resistance to
neoplastic or transformed cells. In recent years, exposure to chemicals
has been shown to increase tumor frequency (30) and to reduce host
resistance to transplantable syngeneic tumor cells in experimental
animals (6). Humans treated by chemotherapy to sustain organ transplants
have also been shown to have a higher frequency of spontaneous tumors
(27). Consequently, neoplastic cells may arise in normal animals or
humans undergoing such exposure or treatments which may abrogate the
immune system.
A number of ^n vitro and |n. vivo tests have been developed which
allow us to assess the functional integrity of the immune response.
Ultimately, however, the most relevant tests are those in vivo tests
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which assess an animal's ability to handle neoplastic diseases following
exposure to toxic substances. These tests are most desirable because
they measure the cumulative effects on all aspects of the immune response
rather than focusing on a single aspect as do the in vitro tests. The
Moloney sarcoma model described in this paper may serve this purpose
very well. This tumor susceptibility test involves the use of Moloney
sarcoma virus-transformed cells (MSC) (17). Injection of BALB/c mice
with MSC cells produces either regressing or progressing tumors depend-
ing upon the number of tumor cells administered. The host's immunologic
mechanisms are thought to be important in the mediation of regression
(15). Various forms of immunosuppression, such as treatment with corti-
sone, X-irradiation, or thymectomy, can predispose animals, given regres-
sing doses of tumor cells, to develop progressing tumors. In addition,
it has recently been shown (15) that T-lymphocytes from lymph nodes
draining Moloney sarcomas of animals with regressing tumors possess
cytolytic activity against tumor cells. The cytolytic activity of these
lymphocytes disappear when the tumor grows progressively. This informa-
tion substantiates the concept that the change of a regressing state to
a progressing state of Moloney sarcoma is due to a failure of the immune
system. Hence, this model system may be a very sensitive system for the
assessment of immunotoxicity of chemicals of environmental concern.
Materials and Methods
Animals: Young adult male Syrian golden hamsters (80-90g body
weight) and BALB/c mice (18-22g body weight) were obtained from Charles
River Laboratory Animals, Inc. and used throughout the studies.
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Organisms: The Mycoplasma pneumom'ae strain (M-129) used in this
study was originally isolated from a patient with mycoplasmal pneumonia,
and was provided by Dr. Wallace A. Clyde (University of North Carolina).
The organisms were maintained in Hayflick's medium containing 20% of
agamma horse serum (19). Active cultures used to infect animals were
grown in the same medium on the inner surface of glass prescription
bottles at 37°C for 36 to 48 hours. The attached monolayer of organisms
were washed three times with phosphate-buffered saline (PBS), pH 7.2,
and scraped into M199 medium (GIBCO, Grand Island, N.Y.) containing 1%
agamma horse serum and 10 mM HEPES. This suspension was used to gener-
ate aerosol to infect the hamsters.
Infection of Hamsters: Hamsters were infected by inhalation of
aerosolized M. pneumoniae. The design of the nose-only inhalation
chamber and the procedures of generating aerosol of M. pneumoniae organ-
isms have been described elsewhere in detail (20). Briefly, 4 ml of
mycoplasma suspension containing approximatley 10 CFU/ml was used to
generate an aerosol to infect 8 or 16 animals in a single experiment.
Infected animals were sacrificed periodically for pathological examina-
tion, viable mycoplasma counting, and for preparation of lung lavage
fluid for radioimmunoassay.
Lung Lavage; To obtain lung lavage, animals were anesthetized by
intraperitoneal injection of nembutal (150 mg/kg body weight) and
exsanguinated by intracardiac puncture. The trachea was exposed and
cannulated with a 18-gauge Angiocath Teflon catheter (Deseret Pharmaceuti-
cal Co., Sandy, Utah), and the lungs were lavaged with 3 ml of pre-
warmed PBS. The fluid was injected and retrieved three times. Approxi-
mately 2.5 ml of fluid was recovered. The lavages were centrifuged at
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1,000 rpm for 10 minutes to remove cells, and the supernatants were
centrifuged again at 15,000 rpm for 30 minutes in a Sorvall RC-2B centri-
fuge to remove debris. The supernatant was removed and stored at -80 C
until time of assay.
Preparation of Solid-phase Antigens; Cultures of Mycoplasma
pneumonias were scraped into 25 ml of fresh Hayflick's medium, and
clumps of organisms were dispersed by passing the suspension three times
through a 26-gauge needle. The suspension was then filtered through a
0.45 u Millipore filter, and 0.2 ml of the filtered suspension was added
to individual wells of microtiter plates (Falcon, microtest II, Flat-
bottom). The microtiter plates were incubated at 37°C for 36 to 48
hours in 5% C02 - 95% air until a monolayer of uniformed colonies
appeared in each well. The supernatant was removed by suction, and
the attached organisms were fixed with 0.15% glutaldehyde in 0.2 M
Sorensen's phosphate buffer, pH 7.2, for 5 minutes at room temperature.
After fixation, the antigen monolayer was rinsed with 0.15 M glycine in
0.02 M Sorensen's phosphate buffer containing 1% agamma horse serum.
The rinsing fluid was again removed and 0.2 ml of PBS, pH 7.2, contain-
ing 2% agamma horse serum was added to each well and the plates were
stored at 4°C.
Immune Serum Against M. pneumoniae; Rabbits were immunized with M_.
pneumoniae as described by Powell and Clyde (28). Log phase cultures of
M. pneumoniae were centrifuged at 14,000 x g for 30 minutes, washed with
PBS, pH 7.2, and resuspended in fresh PBS to a concentration of approxi-
o
mately 10 CFU/ml. Rabbits were inoculated subcutaneously twice at 14-
day intervals with 1 ml of the organism suspension emulsified in equal
amounts of complete adjuvant (Difco, Detroit, Michigan). Three weeks
131
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later, 1 ml of organism suspension was given intravenously. Rabbits
were bled 2 weeks after the intravenous injection. Serum was prepared
and stored at -20°C. Before use, the serum was heat-inactivated at 56°C
for 30 minutes.
125
Preparation of I-labeled Antisera: IgG fractions of goat anti-
rabbit IgG and rabbit anti-hamster immunoglobulin were purchased from
Microbiological Associates, Inc. (Rockville, MD). lodination of antisera
125
with I-iodine was carried out as described by Bolton and Hunter (3).
Immediately following iodination, samples were eluted through a Sephadex
C_-50 column to separate the unbound iodine. The protein fractions were
pooled, and stored at -4°C until used. Greater than 95% of the radio-
activity is trichloroacetic acid (TCA) precipitable.
Radioimmunoassay: Microtiters with fixed M. pneumoniae served as
the solid-phase antigen. The stored plates were incubated at 37°C for
30 minutes and rinsed once with PBS containing 2% agamma horse serum
(PBS-AHS). To individual wells, 100 ul of anti-mycoplasma antiserum
diluted with PBS-AHS was added, and incubated at 37°C for 2 hours.
Non-bound material was removed as before by washing with PBS-AHS.
125I-labeled anti-immunoglobulin (50 ul), diluted with PBS-AHS, was
added to each individual well and allowed to react for 30 minutes at
37°C. The wells were then washed 3 times with PBS, and then swabbed with
cotton tipped applicators.to remove the adherent mycoplasmas. Appli-
cator tips were then placed in glass scintillation vials and counted for
radioactivity in a Packard Gamma Counter (Packard Instruments Co.,
Downeas Grove, 111.)
Tumor Cell Line: Moloney sarcoma cell (MSC) line was kindly
supplied by Dr. Steven Russell (University of North Carolina). This
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8
cell line was originally derived from a male BALB/c mouse infected with
Moloney sarcoma virus (25). MSC cells were maintained in minimal essen-
tial medium supplemented with 10% fetal calf serum. Active cultures
used to challenge BALB/c mice were grown in the same medium, harvested
by trypsinization (0.25%) and resuspended in serum free medium. The
concentration was adjusted to 2.5 x 10 cells/ml. It was shown experi-
mentally, that a challenge dose of 5 x 10 cells (20 yl) produces regres-
sing tumors in untreated mice (29). Higher dose, 10 cells, will produce
progressing tumors and eventually death.
Tumor Susceptibility Assay: Regressing sarcomas were induced in
male BALB/c mice by injecting 5 x 10 MSC cells in 20 vl intramuscularly
between the gastrocnemius and the tibia of the right hind leg. Animals
were palpated twice weekly for tumor development.
Exposure to Testing Chemicals; Cyclophosphamide (80 mg/kg body
weight) was dissolved in sterile saline and was given intraperitoneally
at times indicated in the result section. Animals treated with NiCK
were administrated via various routes as indicated in Table 4. Untreated
control mice were injected with equal volume (0.2 ml) of buffered saline.
Lymphocytes Proliferation Assay: Twenty-four hours after the infec-
tion with the test substance or saline, the mice were killed by cervical
dislocation and spleen cell suspensions were prepared in RPMI medium
with 25 mM HEPES (GIBCO, Grand Island, N.Y.) containing 100 units/ml
penicillin, 100 pg/ml streptomycin and 5 percent heat Inactivated fetal
calf serum. The cells were adjusted to a concentration of 5 x 10
viable (i.e. trypan blue excluding) cells/ml in RPMI. Cells were cul-
tured in steriVe flat bottom microtitic plates (Falcon Plastics, Oxnard,
CA). Triplicate cultures containing 5 x 10 cells in a 0.2 ml volume of
medium with or without mitogen were prepared for spleen cells from
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Individual control and treated mice. The T-lymphocyte mitogens
used were purified phytohemagglutinin (PHA, Burroughs Wellcome, Co.,
Research Triangle Park, N.C.) and concanavalin A (Con A, Miles Lab,
Kankakee, 111.); the B-lymphocyte mitogen was bacterial lipopolysacchar-
ide of Escheridia coli 0.28:B12 (IPS, Difco Lab, Detroit, Michigan).
Cultures were incubated for 72 hrs in a humidified atmosphere of 95% air
and 5% C02 at 37°C. Eighteen hrs before harvesting, 20 yl of 0.5 yCi of
methyl-3H-thymidine (3H-TDR, specific activity 6.7 Ci/mM, New England
Nuclear, Boston, Mass.) were added to cultures. The cells were collec-
ted onto glass fiber paper strips with a MASH II automated harvester
(Microbiological Associates, Bethesda, MD) and DMA was precipitated with
ice cold 5% TCA. Samples were counted in a Model 2650 Tri-Crab liquid
scintillation counter (Packard Instrument Co., Downers Grove, 111).
Data collected as counts per minute (CPM) were log,Q transformed and
analyzed statistically using analysis of variance. Results presented in
the text are given as the arithmetic mean CPM +_ one standard error.
Primary Antibody Response
BALB/c mice were injected intramuscularly in the thigh 24 hrs prior
to an intraperitoneal immunization with sheep red blood cells (SRBC).
Four days after immunization the animals were killed and spleen were
removed. The spleen cells producing IgM anti-SRBC antibody were assayed
using a modification of the direct-plaque-forming-assay (PFC) described
by Jerne and Nordin (25).
Results
Sensitivity of RIA Assay: To test the sensitivity of the solid
phase RIA, dilutions of 1:200 through 1:25,600 of rabbit anti-mycoplasma
134
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10
serum were incubated with the fixed antigen in microtiter wells. A
125
constant amount of I-iodine-labeled goat anti-rabbit IgG was added to
each well to detect the absorbed anti-mycoplasma antibodies. As shown
in Figure 1, even for the highest dilution (1:25,600), a higher count
was obtained for the immunized serum than for normal serum. This result
indicates that the solid-phase RIA technique is highly sensitive and can
be used to detect a very small amount of specific antibody. In addition,
the linear relationship of concentration versus radioactivity counts
indicates that the radioactivity count is directly related to the amount
of specific antibody present in the serum, since the concentration of
the second antibody was kept constant in all wells.
Reproducibility of RIA Assay: Figure 2 shows the reproducibility
of this RIA technique. The linear relationship between the dilutions of
the first antibody and radioactivity counts can be demonstrated over a
wide range of labeled second antibody dilutions. Also, the results show
that storage of the fixed mycoplasma antigen at 4°C for up to 2 months
did not appear to affect the titration. Microscopic examination of the
stored plates indicated there was little, if any, detachment of the
fixed mycoplasma antigens in the wells. It is possible that the shelf
life of the antigen plates can be extended for an even longer time
without jeopardizing its effectiveness.
Effects of Antigen Concentration : It was essential to determine
if the concentration of the initial mycoplasma inoculum affects the
results of antibody titration. Dilutions of an Initial mycoplasma
Q
suspension (^7.5 x 10 CFU/ml) were used to innoculate microtiter wells.
The plates were incubated for 48 hours and the mycoplasma were fixed.
135
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11
The fixed organisms serve as solid phase antigen. As shown in Figure 3
the slopes of the lines from experiments using 1:2 and 1:4 dilutions are
essentially the same as the slope of the non-diluted suspension. This
result at least suggests that within a 4-fold difference, the size of
the initial innoculum will not cause any significant effect on the
titration of the antibody. M. pneumoniae is a slow growing organism and
the total yield of mass is very low. This can be a disadvantage when
using lysate-coated tubes as the solid-phase antigen (18) because of the
large amounts of organisms required. Therefore, the use of mycoplasma
organisms fixed in microtiter wells as the antigen has the advantages of
being economical as well as time-saving.
Titration of Anti-Mycoplasma Antibody in Lung Lavage and Serum from
Hamsters Infected with M. Pneumoniae: Hamsters were infected with
aerosolized M. pneumoniae organisms, and the lung was lavaged with 3 ml
of PBS at pH 7.2. The cells were removed by low speed centrifugation
(150 x g, 10 minutes) and debris by high speed centrifugation (20,000 x
g, 30 minutes). The clear supernatants were stored at -70°C until
125
tested by RIA. The second antibody used in these assays was I-
lodine-labeled rabbit anti-hamster immunoglobulins. Blood samples were
also collected from these animals to prepare sera when they were sacri-
ficed, and stored in the same manner.
Figure 4 shows that the level of specific antibody against M._
pneumoniae in the lung became evident by the second week following
infection and rose dramatically by the third week. The specific anti-
body titer remained at that level for 6-7 weeks and then started to
decline by the 8th week when this experiment was terminated.
136
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12
A significant advantage of this RIA technique when it is applied to
detect the antibodies in lung lavage fluids is that the lavage fluid
need not be concentrated. This allows one to assay a greater number of
samples with minimal efforts. However, when the same procedure was
applied to titrate the antibodies in serum, a problem was encountered.
The background counts were extremely high. This problem was solved by
centrifugation of the serum samples at 15,000 rpm for 30 minutes to
remove the lipids which tended to "stick" to the microtiter wells increas-
ing the non-specific binding of the second antibody. Also, the serum
samples were diluted 1:100 with PBS to reduce the viscosity which also
appeared to affect the absorption of the labeled second antibody.
Figure 5 illustrates the results for serum samples which appear to
follow a similar pattern as that of the lung lavage fluid. However, the
highest antibody response in the serum occurs one week later.
Effects of Cyclophosphamide on the Production of Progressing Tumors:
To validate the concept that chemicals with immunosuppressive potential
may enhance the production of progressing tumors in animals which received
a regressing dose of syngenetic tumor cells, BALB/c mice were given a
therapeutic dose of Cyclophosphamide (80 mg/kg) 1 day prior to or 4 and
o
8 days following the administration of 5 x 10 MSC cells. This lower
level of tumor cell challenge normally induces regressing tumors and
would be a more sensitive measure of the chemical modulation of normal
host immune surveillance and resistance. The results are summarized in
Table 1. It is obvious that the incidence of progressing tumors was
increased in all Cyclophosphamide-treated groups. Animals treated with
Cyclophosphamide 1 day prior to the tumor cell challenge have the highest
incidence of progressing tumors, while those receiving the drug treatment
at day 4 or day 8 produced a fewer number of progressing tumors.
137
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13
Figure 6 illustrates the histopathology of the primary tumor (A)
and progressive tumor (B). A clear difference is the absence of infil-
trating mononuclear iinflammatory cells in the progressing tumor. Neo-
plastic cells are uniform in size, spindle-shape and are closely opposed
to form interlacing, parallel arrays. Tumor cells in animals bearing
progressing tumors are capable of metastasizing to other organs. Figures
6C and 6D show metastasized tumors in the spleen and in the lung.
Immunologic Effects of Nickel Chloride: Nickel (Ni), along with
other heavy metals, has been shown to have immunologic effects (16,22,24).
Although it is generally agreed that inorganic metals primarily affect
the humoral immune response, the effects of Ni and other heavy metals on
the cell-mediated immune component have not been thoroughly examined.
For example, rats exposed pre- and postnatally to lead (Pb) have been
shown to have decreased thymic weights, suppressed responsiveness of
lymphocytes to mitogen stimulation by PHA and Con A and impaired delayed
hypersensitivity reactions (9). These results indicate that low level
transplacental exposure to Pb causes suppression of the cell-mediated
immune function. We have recently found that NiCl2 inhibits the mitogen-
stimulated response of both T- and B-lymphocytes (Table 2). The primary
antibody response to the T-lymphocyte dependent antigen, SRBC (Table 3)
was also found to be inhibited by NiClg. An attempt was made to deter-
mine whether NiCl2 may enhance the susceptibility to tumor cell challenge.
Mice were exposed to NiCl» by either intraperitoneal Injection or stomach
intubation. As shown in Table 4, a single dose of NiCl2 by either route
at 10 mg/kg body weight did not affect the resistance to tumor cell
challenge.
138
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14
Discussion
The solid phase radioimmunoassay for the measurement of anti-
pneumonia antibody was designed to provide a means of measuring the
immunoglobulin-specific antibody response. M. pneumonia is a common
disease producing agent of the human respiratory tract (7). Because the
organism is difficult to grow, the identification of a recently acquired
infection must frequently depend on detection of a serological manifes-
tation. Numerous serologic assays have been developed to detect anti M..
pneumom'ae antibody. Few, however, have possessed the capability of
measuring immunoglobulin class-specific activity. The currently avail-
able techniques of immunof1uorescence (10) or radioimmuno-precipitation
(4) suffer the drawbacks of requiring subjective technician assessment
or difficulties in reproducibility. The ability to quantitate immuno-
globul in-specific antibody responses may be important in assessing
primary versus recurrent infection and in measuring the immune response
in body fluids other than serum. Although the primary objective of the
present studies is to develop a technique for rapid assessment of
specific antibody in the lung of small laboratory animals following
experimental infection, it appears to be feasible that that same technique
can be extended for clinical use. The results of studies with this RIA
assay have demonstrated that it is a simple and sensitive indicator of
anti-mycoplasma antibody activity. This technique takes advantage of
the glass or plastic-adhering properties of M. pneumoniae which allows
one to visibly inspect microtiter wells for the presence of antigen thus
bypassing the problem of inconsistent adherence of soluble antigens to
the plastic surface. In addition, the use of the whole organism as the
antigen is economical and time-saving.
139
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ib
For this mycoplasma-hamster model, determination of class-specific
antibody requires highly purified anti-hamster class-specific immuno-
globulin anti-sera which are not currently available. Attempts are
being made in this laboratory to prepare anti-sera against class-specific
hamster immunoglobulins in order to examine the time course of the
occurrence of class-specific Immunoglobulins in the lung of animals
following the experimental Infection.
The preliminary data obtained 1n the tumor susceptibility assay
employing Moloney sarcoma cells (MSC) described in the present studies
indicates that this assay may be a promising method for the detection of
immune dysfunction.
Treatment with an immunosuppressive agent such as cyclophosphamide,
produced progressing tumors in animals which otherwise would produce
only regressing tumors (Table 1). While this study was in progress,
Dean et al (6) described a similar tumor susceptibility assay using sMKA
cells. They reported that the tumor susceptibility was a very sensitive
assay for the immunosuppressive effects of cyclophosphamide. The
commonly employed mitogen-induced lymphoproliferative responses (LP) and
the antibody plaque-forming cell response (RFC), were also found to be
good indicators for the detection of immunosuppression by this agent. In our
hands, the LP and PFC responses (Tables 2 and 3) were also found to be
sensitive indicators of NiClg immunosuppression.
The MSC cell model described in the present studies is based on the
same concept as the sMKA model described by Dean et al. (6). These
tumor systems have the advantage of providing a measure of the cumulative
effects of agents on several components of the immune system. This is
contrary to most in vitro tests, which have a serious defect in that
they are limited to evaluate only a single component of the host's
1 40
immune system. '
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16
The MSC assay provides an additional advantage to the evaluation of
toxic effects on the immune system because this model can produce either
regressing or progressing tumors depending upon the dose administered.
Consequently, it appears that in this system at least, a critical number
of cells can be dealt with effectively by an Inmunologically competent
host. Only when this postulated upper limit is exceeded does progres-
sion ensue in spite of host responses against the neoplasm. Consequently,
this tumor model system lends itself to delineation of the effects of
toxics on the Immune system. By examining the various segments of the
immune system, particularly those associated with cell-mediated immunity
in animals bearing regressing tumors, one may come to a better under-
standing of the host defense mechanisms against neoplastic diseases. By
employing a tumor model system such as the MSC assay along with other
immunologic tests insights into the inter-relationship of the various
components of the immune system as they relate to the effects of toxic
substances on health can be realized. This information will help us to
assess the immunotoxicity of environmental chemicals.
141
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Legends
Figure 1. Specific antibody titration. Rabbit anti-mycoplasma anti-
serum (first antibody) (•) or normal rabbit serum (o) was diluted and
added to wells followed by addition of 1£ I-goat anti-rabbit IgG
(second antibody). The pooled second antibody (see text) was diluted
1:10.
Figure 2. Reproducibility of RIA Technique. Fresh microtiter plates
were prepared 2 days prior to the assay. Aged plates were prepared and
stored at 4 C for 2 months prior to use. Concentration of the first
antibody used was same as described in Figure 1. The dilution factors
indicated were for the second antibody.
'Figure 3. Effect of Antigen Concentration on RIA. Various dilutions of
the initial mycoplasma suspension were used to inoculate the microtiter
plates, incubated for 48 hours and fixed. Inserted numbers of the bar
graphs indicate the dilution factors of the first antibody. Concentra-
tion of the second antibody was a constant for all assays.
Figure 4. Detection of Antibody in Lung of Infected Hamsters. Hamsters
were infected with M. pneumoniae aerosols, and sacrificed at indicated
times. The antibody in the lung lavage fluids was assayed by RIA.
I-labeled rabbit anti-hamster immunoglobulins were used as the second
antibody.
Figure 5. Detection of Antibody in Serum of Infected Hamsters. Assay
procedure was same as described in Figure 4 except that the sera were
diluted 100 x with PBS.
Figure 6. Photomicrographs of Moloney Sarcoma. Heavy inflammatory
infilteration is evident in primary tumor (A), while absent in progres-
sing tumor (B). The neoplastic cells in progressing tumor are uniform in
size, spindle-shaped, and are closely opposed to form interlacing,
parallel arrays. C and D show the metastaisis of Moloney sarcoma in the
spleen and the lung from an animal bearing progressing tumor. (H + E
stain, A,B 450x, C.D. 125x)
142
-------
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DILUTION OF IST ANTIBODY
Figure 1
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DILUTION OF IST ANTIBODY
-2
Figure 2
-------
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Figure 3
LD
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LUNG LAVAGES
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WE.EKS, POST-.INFECTION
Fioure 4
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146
Figure 5
-------
,>** \ •• \1r w/yrvy,:*.. j;. y>. (-y
-------
TABLE 1
EFFECT OF CYCLOPHO^HAMIDE ON THE PRODUCTION OF PROGRESSING TUMORS IN
BALB/C MICE WHICH RECEIVED REGRESSING DOSES OF MSC CELLS
TREATTCNT NUMBER OF ANIMALS WITH
PROGRESSING TUTORS
EXP, 1
5 x ID5 CELLS J/1Q
5 X ILr CELLS AND CY*
ON DAY -1 8/9
EXP, 2
CYCLOPHOSPHAMIDE ON DAY 4 Q/20
5 X ID5 CELLS 1/20
5 X IQr CELLS AND CY ON DAY 4 6/20
5 X ILr CELLS AND CY ON DAY 8 5/20
* DOSE OF CYCLOPHOSPHAMIDE (CY> WAS 80MG/KG BODY WEIGHT IN ALL
148
-------
TABLE 2
EFFECT OF NIO_2 ON THE MITOGEN STIMULATED RESPONSE OF MURINE
SPLEEN CEULSA
MITOGEN (UG/CULTURE) F'bvN UPTAKE OF VTHYMIDINE (CPM x 10? ± SE)
CONTROL TREATED
0 1,97 ±0,3 2,57 ±0,6
PW (0,1) 73,5 ±2,0 56,1 ± 3,5B
CbNA (0,1) 89,8 ± 5,7 66,9 ± 8,3B
LPS (5,0) 36,9 ±1.9 28,3 ± 2.5B
A 10 VG/GRAM NlQ_2 WAS ADMINISTERED INTRAPERITONEALLY 21 HOURS PRIOR TO
EUTHANIZATION OF MICE, EIGHT FEMALE BALB/C MICE PER GROUP,
B P < 0,05, ANALYSIS OF VARIANCE,
NO
-------
TABl£3
EFFECT OF NICL2 ON THE PRIWRY ANTIBODY RESPONSE OF MICEA
NiCL2 DOSE PFC/1# PFC x N?/SPLEEN
(vG/G BODY WT.) SPLEEN CELLS (x ± SE)
(x±SE)
SALINE 725 ± 133 1,66 ± 0,40
5 505 ± 71 1,18 ± 0,18
10 266 ± 158 0,51 ± 0,
A NlQ.2 ADMINISTERED INTRAMUSCULARLY 21 HOURS PRIOR TO
IMMUNIZATION (I,P.) WITH SRBC, Six FEMALE BALB/C MICE
PER GROUP,
B P < 0,05, ANALYSIS OF VARIANCE,
-------
TABLE 4
EFFECT OF NICLo ON TT€ ENHANOTIT OF PROGRESSING TUMORS IN BALB/C MICE
TREATMENT
NONE
NiCLo*
L INTRAPERITONEAL
STOMACH PERTUBATION
NiCL2*
L INTRAPERITONEAL
STOMWCH PERTIBATION
TUMOR CELLS
ADMINISTERED
SxlO3
5x10*
5X103
NONE
NONE
f*JMBER OF ANIMALS WITH
PROGRESSING TUMORS
2/20
Q/20
2/20
0/10
0/10
*
10 MG/KG BODY WEIGHT
-------
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1. Bekesi, J. G., J. Roboz, H. A. Anderson, J. P. Roboz, A. S.
Fishbein, I. J. Selikoff, and J. F. Holland. 1979. Impaired immune
function and identification of polybrominated biphenyls (PBB) in blood
compartments of exposed Michigan dairy farmers and chemical workers.
Drug Chem. Toxicol. 2.:179-191.
2. Bice, D. A., D. L. Harris, C. T. Schnizlein, and J. L. Mauderly.
1979. Methods to evaluate the effects of toxic materials deposited in
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3. Bolton, A. E. and W. M. Hunter. 1972. A new method for the radio
iodination of proteins to high specific activities. Biochem. J. 133:
529-
4. Brunner, H. and R. M. Chanock. 1973. A radioimmunoprecipitation
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5. Burnet, F. M. 1970. The concept of immunological surveillance.
Prog. Exp. Tumor Res. 13_:l-27-
6. Dean, J. H., M. L. Padarathsingh, T. R. Jerrello, L. Keys, and
J. W. Northing. 1979. Assessment of immunobiological effects induced
by chemicals, drugs or food additives. II. Studies with cyclophospha-
mide. Drug Chem. Toxicol. 2_:133-153.
7. Denny, F. W., W. A. Clyde and W. P. Glezen. 1971. Mycoplasma
pneumoniae disease: Clinical spectrum, pathophysiology, epidemiology,
and control. J. Inf. Dis. 123:74-92.
8. Fairchild, G. A. 1974. Ozone effect on respiratory deposition of
vesicular stomatitis virus aerosols. Amer. Rev. Resp. Dis. 109:446-451.
9. Faith, R. E., M. I. Luster, and C. A. Kimmel . 1979. Effects of
combined pre- and postnatal lead exposure on cell mediated immune function.
Clin. Exp. Immunol. 35:413-420.
10. Fernald, G. W., W. A. Clyde, and J. Binenstock. 1972. Immunoglobul in-
containing cells in lungs of hamsters Infected with Mycoplasma pneumoniae.
J. Immunol. 108:1400-1408.
11. Gainer, J. J., and T. W. Pry. 1972. Effects of arsenicals on
viral infections in mice. Amer. J. Vet. Res. 33:2299-2307.
12. Gardner, D. E. and J. A. Graham. 1977. Increased pulmonary disease
mediated through altered bacterial defenses. In Pulmonary Macrophage and
Epithelial Cells. Ed. C. L. Sanders and R. P. Schneider. ERDA Symposium
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13. Gardner, D. E., F. J. Miller, J. W. IlUng, and J. M. Kirtz.
1977. Increased infectivity with exposure to ozone and sulfuric acid.
Toxicol. Letters 1:59-64.
14. Gatti, R. A., and R. A. Good. 1971. Occurrence of malignancy in
immunodeficiency diseases: A literature review. Cancer 28:89-98.
15. Gillespie, G. Y., C. B. Hansen, and S. W. Russell. 1978.
Resurgence of killing and 1n vivo protection mediated by lymphocytes
cultured from lymph nodes (Training Moloney sarcomas. Br. J. Cancer
38:366-374.
16. Graham, J. A., D. E. Gardner, F. J. Miller, M. J. Daniels, and
D. L. Coffin. 1975. Effect of nickel chloride on primary antibody
production in the spleen. Environ. Health Persp. 12:109-113.
17. Green, G. M., G. J. Jekeb, R. B. Low and G. S. Davis. 1977.
Defense mechanisms of the respiratory membrane. Amer. Rev. Resp. Dis.
115:479-514.
18. Horowitz, S. A. and G. H. Cassell. 1978. Detection of antibodies
to Mycoplasma pulmonis by an enzyme-linked immunosorbent assay. Infect.
Immuno. 22:161-T7UT
19. Hu, P. C., A. M. Collier, and J. B. Baseman. 1977. Surface
parasitism by
Med. 145:1328
parasitism by Mycoplasma pneumoniae of respiratory epithelium. J. Exp.
-1343.
20. Hu, P. C., J. M. Kirtz, D. E. Gardner, and D. A. Powell. 1980.
Experimental infection of the respiratory tract with Mycoplasma pneumoniae.
Env. Health. Persp. In press.
21. Jerne, N. E. and A. A. Nordin. 1963. Plaque formation in agar by
single antibody-producing cells. Science 140:405.
22. Koller, L. D. 1979. Some immunological effects of lead, cadmium
and methyl mercury. Drug. Chem. Toxicol. 2_:99-110.
23. Lischer, H. W., H. W. Pinnett, and A. M. DiGeorge. 1967.
Lymphocytes in congenital absence of the thymus. Nature (London).
214:580-582.
24. Luster, M. I., R. E. Faith and C. A. Kimmel. 1978. Depression of
humoral immunity in rats following chronic developmental lead exposure.
J. Environ. Path. Toxicol. 1:397-402.
25. Massicot, J. G., W. A. Moods, and M. A. Chirigos. 1971. Cell
line derived from a murine sarcoma virus (Moloney pseudotype)-induced
tumor: cultural, antigenic and virological properties. Appl. Microbiol.
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26. Miller, S. D. and A. Zarkower. 1974. Alterations of murine
immunological responses after silica dust inhalation. J. Immunol.
113:1533-1543.
27. Penn, I. 1978. Development of cancer in transplant patients.
Adv. Surgery 12:155-191.
28. Powell, D. A. and W. A. Clyde. 1975. Opsonin-reversible resistance
of Mycoplasma pneumonias to in vitro phagocytosis by alveolar macro-
phages. Infect. Immun. 11:5411-550.
29. Russell, S. W. and C. G. Cochrane. 1974. The cellular events
associated with regression and progression of murine (Moloney) sarcomas.
Int. J. Cancer 13_: 54-63.
30. Theiss, J. C., M. B. Shimkin, and L. A. Poirier. 1979. Interaction
of pulmonary adenomas in strain of mice by substituted organohalides.
Cancer Res. 39^:391-395.
31. Thigpen, J. E., R. E. Faith, E. E. McConnell and J. A. Moore. 1975.
Increased susceptibility to bacterial infection as a sequela of exposure
to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Infect. Inmun. 12_: 1319-1324.
32. Vos, J. G. 1977. Immune suppression as related to toxicology.
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154
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Approaches to Investigate Effects of
Heavy Metals on Immune Responses
Loren D. Roller 1
School of Veterinary Medicine
University of Idaho
Moscow, Idaho 83843
At Present:
College of ^
Sciences Center, Oregon State University, Corvallis, OR 97331
College of Veterinary Medicine and Environmental Health
155
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ABSTRACT
Immunotoxicology is a recently developed but rapidly growing
discipline. Recent studies have demonstrated that many metals
such as lead, cadmium, mercury, methylmercury, arsenic, cobalt,
and nickel are immunosuppressive. This articles describes immuno-
logical methods applicable for investigating the effect heavy
metals produce on the immune system of experimental animals.
Factors which should be considered for these investigations
include choice of experimental animals, method of exposure, gross
and microscopic examination of lymphoid tissue, clinical pathology,
challenge with infectious agents and toxins, antibody titers,
antibody synthesis, B and T lymphocyte functions, macrophage
activity, tumor growth, and tumor immunity. Problems associated
with certain techniques are discussed.
156
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-2-
The field of immunotoxicology is in its infancy but in the
past few years considerable data has been accumulated on many
chemicals in various species of animals utilizing a variety of
immunoassays. These investigations have only scratched the
surface of what could be a serious problem for human health.
However, the data which has been collected often does not consider
the entire immune response nor reaction by other body systems
which may influence those responses. Further, baseline data
concerning mechanisms by which these compounds react on the
immune system of a host is either inconclusive or lacking for
most groups of compounds. Lead, for example, appears to manifest
the most immunosuppressive properties of the many environmental
contaminants examined to date except for perhaps tetrachlorodibenzo-
p-dioxin (TCDD) (1). Many immune function assays have a relatively
low sensitivity index and, therefore, require considerable suppres-
sion or enhancement to detect significant differences. For
example, mitogen and mixed lymphocyte cultures usually display
considerable variation between similar samples. The chromium
release assay generally has a natural cytotoxicity of 30% leaving
a small range to demonstrate impaired cytotoxicity. Therefore, a
known immunosuppressant should be included in the assessment of
susceptibility of animals to infectious agents, immune parameters,
carcinogenicity and contribute information for developing tech-
niques and methods for future investigations.
Many factors must be considered for immunotoxicological
investigations. Some of these include choice of animal, method
157
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-3-
of exposure to the toxicant, gross and microscopic examination of
lymphoid tissue, clinical pathology, challenge with infectious
agents and toxins, antibody titers, antibody synthesis, B and T
lymphocyte functions, properties of macrophages, tumor growth and
tumor immunity. Each of these features will be discussed for
their application in immunotoxicology.
The animal selected for study depends on several different
factors. First, -extensive immunology data have been compiled in
the mouse while toxicology data has been established primarily in
the rat. Second, it must be realized that all species of animals
may not respond to a particular pollutant in the same manner.
Third, the response in males and females is frequently dissimilar.
Fourth, the availability of infectious agents and tumor models
should be considered. Fifth, susceptibility to spontaneous
diseases and neoplasms is particularly important for long term
studies. Finally, when space is limited, the number of animals
housed per cage could influence selection of an animal species.
Another point which must be considered is the method of
exposure. It has been established that the immune response can
vary according to route of exposure. Therefore, when considering
the response in an intact animal, administration of the pollutant
should simulate natural exposure in an attempt to extrapolate
data from experimental animals to man. The concentration of the
compound selected preferably should include a level estimated to
produce an effect decreasing to a definite non-toxic amount and
finally establishing a no effect level, even if the dosage is.
158
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-4-
below maximum permissible levels for humans. Exposure to the
compound should extend for a prolonged period of time (chronic)
to insure the response is not delayed or develops after accumula-
tion of the pollutant in the body. Further, the type of compound
or chemical with which the contaminant is combined may regulate
its action. Also, the particular isomer responsible for deterring
the immune response needs to be identified. Finally, the fetus
and neonates often are more susceptible and may react differently
than do weanlings and adults.
Alteration of the immune system by environmental pollutants
may be suspected by gross and histopathological examination of
lymphoid tissue but usually is not conclusive without conducting
specific immunoassays. On gross examination, all lymphoid organs
should be observed and the thymus, spleen, bursa of Fabricus
(chicken), and perhaps larger lymph nodes weighed. Cells from
these organs could also be counted (electronic) to determine
concentration or examined by a dye exlcusion test (trypan blue)
for viability. Cell distribution within organs can be enumerated
by histologic examination. Two rather obvious alterations which
may occur are atrophy of the thymus and depletion of lymphoid
follicles in the spleen. More subtle changes are often difficult
to detect. Measurements of serum globulins by electrophoretic
separation may also indicate an altered immune response.
It must be kept in mind that the above procedures are super-
ficial observations of the immune system and do not measure
responses to specific antigens. A pollutant may reduce serum
159
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-5-
globulins but unless there is an inhibition of the lymphocyte or
macrophage response to antigen challenge (infectious agent,
carcinogen, etc.). then the compound more than likely would not
be detrimental to health. Therefore, it becomes necessary to
determine the effect of the contaminant on specific aspects of
the immune response by immunological methods.
One method of initially ascertaining if an environmental
contaminant may.affect the immune response of an animal is to
challenge an exposed animal with a LD50 dose of an infectious
agent. This procedure is simple, reliable, and establishes if
the compound actually interferes with the course of disease.
Infectious agents most commonly used are bacteria and viruses.
If a virus is used, it must be remembered that the ensuing response
may be affected by interferon. Some of the common bacteria
utilized are Salmonella typhimurium, Salmonella bern, Streptococcus
pneumoniae and Listeria monocytogenes. Encephalomyocarditis
virus has also been used. Other methods of infectivity include
challenge by the endotoxins of S_. typhosa, S_. enteriditis and
Escherichia coli. Parasitic diseases such as the Plasmodium
species also appear to be appropriate. Metals reported to affect
the susceptibility of animals to infectious agents include arseni-
cals (2), methylmercury (3), lead (4,5,6,7,8), cadmium (4,5,6,8)
and cobalt (9).
When a compound is suspected of altering the immune response
of a host, the humoral, cell-mediated and macrophage systems must
be examined either collectively or individually. Antibody responses
160
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-6-
to many antigens require cooperation between at least two types
of lymphocytes for optimal expression. One cell type is thymus-
derived (T cell). T cells may amplify., help or suppress B cells
as well as function as cytotoxic cells. T cells do not produce
antibody. The other cell type is bone-marrow-derived (B cell)
which matures independently of thymus influence. B cells differ-
entiate into antibody-producing cells and are often influenced by
T cells. A third cell type is the macrophage which has an impor-
tant role as an accessory cell by cooperating with T cells in
aiding the response of B cells to antigens.
Immunoassays used to examine humoral immunity include those
which measure antibody titers (immunodiffusion, complement fixation,
serum neutralization, hemagglutination, passive hemagglutination,
radioimmunoassay, enzyme linked immunosorbent assay, etc.), anti-
body synthesis (Jerne plaque) and B lymphocyte receptors (EA = FC
and EAC = C3). Humoral immunity may be determined after initial
exposure to the antigen (primary response) or after re-challenge
with antigen (secondary or memory response). Staining of surface
immunoglobulin will identify B lymphocytes and enumerate percent
B cells. Many antigens, both T dependent and T independent, have
been utilized in these investigations. Some of these antigens
are tetanus toxoid, sheep red blood cells, bovine serum albumin,
bovine gammaglobulin, Salmonella typhi, pseudorabies virus,
influenza virus, keyhole limpet hemocyanin and lipopolysaccharide.
Metals responsible for reducing circulating antibody titers to
infectious agents are lead, cadmium, mercury (10), and methylmercury
161
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-7-
(11). Lead (12,13), cadmium (14,15) and methylmercury (16,17)
also inhibit antibody synthesis. More recently it has been shown
that both lead and cadmium inhibited the activity of B lymphocyte
receptors (18). Lead, cadmium and methylmercury have also been
demonstrated to affect immunological memory (19).
Cell-mediated immunity is generally measured by delayed
hypersensitivity and graft vs. host reactions ill vivo as well as
by mixed lymphocyte culture, mitogen stimulation, and assessment
of helper, suppressor and cytotoxic activity of T cells in vitro.
Another technique occasionally used is antibody-dependent cell-
mediated cytotoxicity which requires K cells (surface Fc recep-
tors) for cytotoxic expression. Soluble products of T lympho-
cytes (lymphokines), of which there are many, also convey cell-
mediated activity. Lymphocytes exposed to cadmium (20,21) and
lead (22) exhibited altered responses to mitogenic stimulation.
Lead (22) also impaired delayed hypersensitivity.
Macrophages are characterized by several properties. Many
procedures are available to explore phagocytic properties in vivo
and in vitro. Some antigens phagocytized in vivo are Listeria
monocytogenes and collodial carbon while L. monocytogenes and
sheep red blood cells have been used in in vitro methods. Other
methods determine the ability of macrophages to digest phagocytized
materials by measuring metabolic and enzyme activity in these
cells. Membrane, EA (Fc receptor), and EAC (C1 receptors) activity
also measures macrophage function. Further, some macrophages are
cytotoxic while others secrete a variety of soluble factors which
162
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-8-
regulate humoral and cell-mediated immunity. Lead (23), cadmium
(24) and nickel (25) have been reported to alter phagocytic
properties of macrophages.
Immune surveillance postulates that tumor cells arise in the
normal organism at an enormous frequency and they are regularly
eliminated by immune mechanisms (26). This theory challenges
research to demonstrate and characterize immunological failure as
the primary responsibility for tumor development. However, the
immunological surveillance theory has been occasionally disputed.
Nevertheless, resistance against development of virus-induced
tumors is mainly due to immune responses against viral antigens
and is often mediated through T cell dependent mechanisms.
Therefore, the nonrejectability of spontaneous tumors may be
overcome by modifying the target cell. This could perhaps be
accomplished by chemical coupling, somatic cell hybridization,
viral xenogenization, or even manipulation of the specific immune
response to Ir genes that can influence the recognition of tumor-
associated membranes. Not only do T cells modulate tumor immunity,
but K cells and macrophages are also involved as well as antibodies
secreted from B cells. Furthermore, antitumor antibodies may
actually enhance tumor growth by blocking mechanisms. Therefore,
it is imperative to determine the effect environmental contaminants
may have on the process of tumor development and the ensuing
immune response as well as the entire complex immune system.
The influence of environmental contaminants on neoplasia can
be assessed by using various tumor systems. Neoplasms can be
163
-------
-9-
induced by oncogenic viruses, transplacentally, transplanted or
occur spontaneously. Certain strains of mice and rats manifest a
high incidence of spontaneous tumors while others have a low
incidence. Cytotoxic T lymphocytes are the immune cells respon-
sible for killing tumor cells. This can be detected in vivo by
the Winn test (27) and in vitro by the chromium release assay
(28). Other forms of defense against tumor invasion are natural
cytotoxicity, antibody-dependent cell-mediated cytotoxicity,
serum blocking factors as well as helper and suppressor T lympho-
cytes. Lead has been reported to influence viral induced (29)
and transplanted tumors (30) while cadmium produced a significant
effect on transplanted tumors (31). Methylmercury, indirectly,
by transplacental exposure also affected the incidence, latency
and distribution of transplacental induced carcinogens (32).
In summary, metals may alter one specific or several segments
of the immune response. It is often necessary to examine each
parameter (humoral, cell-mediated and macrophage) to assess the
extent that each is involved. Finally, species differences
definitely should not be overlooked. The effect of compounds are
often expressed differently in various species of animals. A
close relationship should exist for each disease between man and
the experimental animal tested when developing a model for extra-
polation of data. Therefore, the immune response should be
examined in several species of animals after exposure to a metal
in order to provide realistic and comparable information.
164
-------
-10-
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25. Graham, J.A., Miller, F.J., Daniels, M.J. et al.
(1978). Influence of Cadmium, Nickel and Chromium on Primary
Immunity in Mice. Environ Res 16: 77.
26. Klein, G., and E. Klein (1977). Immune Surveillance
Against Virus-Induced Tumors and Nonrejectability of Spontaneous
167
-------
-13-
Turaors. Contrasting Consequences of Host Versus Tumor Evolution.
Proc Natl Acad Sci 74: 2121.
27. Winn, H.J. (1961). Immune Mechanisms in Homotransplan-
tation. II. Quantitative Assay of the Immunologic Activity of
Lymphoid Cells Stimulated by Tumor Homografts. J Immunol 86:
228.
28. Tamerius, J.D., Garrigues, H.J., Hellstrom, I. et al.
(1978). An Isotope Release Assay and a Terminal-Labelling Assay
for Measuring Cell-Mediated Allograft and Tumor Immunity to Small
Numbers of Adherent Target Cells. J Immunol Methods 22: 1.
29. Gainer, J.H. (1973). Activation of Rauscher Leukemia
Virus by Metals. J Natl Cancer Inst 51: 609.
30. Kerkvliet, N.I. (1979). Personal Communication.
31. Kerkvliet, N.I., Roller, L.D., Beacher, L.G., and
Brauner, J.A. (1979). Effect of Cadmium Exposure on Primary
Tumor Growth and Cell-Mediated Cytotoxicity in Mice Bearing MSB-6
Sarcomas. J Nat Cancer Inst. In press.
32. Nixon, J.E., Koller, L.D., and Exon, J.H. (1979).
Effect of Methylmercury on Transplacental Tumors Induced by
Sodium Nitrite and Ethylurea in Rats. Fd Cosmet Toxicol. In
press.
168
-------
DMLJNOTCKICnY TESTING OF POOD CHEMICALS: DIFFERENT RESULTS MKX BE
OBTAINED WITH IN VITRO AND IN VIVO EXPOSURE TO GALLIC ACID
Douglas L. Archer and Bennett G. Smith
Department of Health, Education and Welfare
Food and Drug Administration
Division of Microbiology
1090 Tusculum Avenue
Cincinnati, Ohio 45226
At Present:
^•Division of Microbiology, Center for Food Safety and Applied
Nutrition, Food and Drug Administration, Washington, DC 20204
169
-------
ABSTRACT
Previous work demonstrated that Iri vitro treatment of lymphoid
cells with gallic acid (GA), 3,4,5-trihydroxybenzoic acid, suppressed
the following in vitro tests: 1) the thymus-dependent antibody re-
sponse (PFC) to sheep erythrocytes (SRBC) and 2) T-cell mitogen-
induced DNA synthesis and immune interferon (IIP) production. Admini-
stration of GA to animals in vivo followed by in vitro testing as de-
scribed previously yielded some contrasting results in that; 1) GA
failed to suppress the primary antibody response to SRBC and 2) GA
failed to suppress mitogen-induced DNA synthesis significantly. GA did,
however, suppress the induction of IIP by one T-cell mitogen, staphy-
lococcal enterotoxin A (SEA). Possible reasons for such discrepancies
in data due to different methods of chemical exposure are discussed.
170
-------
INTRODUCTION
In vitro immunologic tests for immunotoxicity have been used ex-
tensively for screening purposes. Few studies have attempted to compare
results obtained by In vitro exposure of cells to toxicant and subsequent
1n vitro functional testing with 1n vivo exposure to toxicant and sub-
sequent jm vitro functional testing.
Previous studies determined that gallic acid (6A), a food consti-
tuent, suppressed the following macrophage-dependent T-lymphocyte
functions 1n vitro: a) the direct anti-sheep erythrocyte (SRBC) plaque
forming cell (RFC) response (1, 2), b) T-cell mitogen-induced DNA
synthesis (1) and c) T-cell mitogen-induced lymphokine immune interferon
(IIF) production (3). Macrophage involvement in 6A-1nduced suppression
of the PFC response to SRBC (1) and IIF production in response to
staphylccoccal enterotoxin A (SEA) (3) were directly demonstrated. More
recently, it was shown that in vivo administration of GA affected sub-
sequent in vitro IIF production in a manner similar to cortisone acetate
(CA), but that GA had no effect on DNA synthesis induced by T-cell mito-
gens, whereas CA had a suppressive effect (4). The different results
obtained by in vitro and in vivo exposure led to an investigation of
the effects of GA exposure in vivo on the subsequent ^n vitro anti-SRBC
PFC response. The results suggest that while in vitro exposure tests
may be useful for screening purposes, they lack the ability to predict
functional loss when toxicants are administered in vivo.
171
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IN. VITRO AND IN. VIVO IMMUNOTOXICITY TESTING Page 2
MATERIALS AND METHODS
Animals. Adult female (6-8 weeks old) BDF! (C57B1/6 x DBAZ^ mice
were supplied by the Laboratory Supply Co., Indianapolis, Ind.
Chemical treatment of animals. GA, CA, or antithymocyte serum (ATS)
were administered subcutaneously at 10 mg, 5 mg, and 0.1 ml re-
spectively, in both flanks of test animals as detailed elsewhere (4),
48 hr prior to splenectomy.
Cell cultures. Thymus, lymph node, and spleen cell dispersions were
made in RPMI 1640 (Microbiological Associates, Walkersville, Md.).
Cells were enumerated in a hemacytometer and culture viability was de-
termined by trypan blue dye exclusion. Spleen cells from specifically
treated animals were split into three groups; one portion was used for
DNA synthesis determinations in response to mitogens, another portion
was used for IIP assays and the third portion was used for determining
the primary antibody response to SRBC.
Mitogens. The source and purity of the mitogens used, SEA, concanavalin
A (ConA), and phytohaemagglutinin-P (PHA), have been detailed elsewhere
( 4 ).
In vitro antibody production- Cultures for the direct in vitro primary
IMS—JSSI^C^S^™.^—^—^fc^^B^B—^—l™»"^—.—i^^^^««^—>^—^^— ^^"™~ H^^^^B^^^
antibody responses to SRBC were performed exactly as described by Mi shell
and Dutton ( 5 ), and direct PFC determinations were performed according
to the method of Cunningham and Szenberg ( 6 ).
DNA synthesis determinations. Mitogen-induced rates of DNA synthesis
of thymus, lymph node, and spleen cells were determined using a micro-
culture method as described ( 4 ).
172
-------
U[ VITRO AND _IN VIVO IMMUNOTOXICITY TESTING Page 3
RESULTS AND DISCUSSION
The data presented in Table I summarize the effects of in vitro
exposure of splenocytes to 6A on various in vitro Immunologic responses
as praviously described ( 3 ). It is apparent from the data that GA
abrogates the following in vitro systems: the primary, thymus-
dependent antibody response, and mitogen-induced T-cell DNA synthesis
and lymphokine production. The data also show that 2ME is capable of
reversing the effect of GA on all three systems; this has been discussed
elsewhere (1,2,3). The data further suggest that GA's ability to suppress
the RFC response supersedes that of SEA; SEA exerts its suppressive
effect by activation of suppressor T-cells ( 7 ). The fact that 2ME
can reverse the suppressive effect of gallic acid, but not the sup-
pression induced by SEA, suggests that GA- and SEA-induced suppression
of the PFC response are mechanistically dissimilar.
The data presented in Table II summarize the effects of in vivo
exposure of splenocytes to gallic acid (and cortisone acetate) on
subsequent in vitro immunologic responses. A comparison of the data in
Table II with that presented in Table I reveals that in contrast to
cells exposed in vitro, cells exposed to gallic acid in vivo are
capable of mounting both a PFC response to SRBC, and normal rates of
mitogen-induced DNA synthesis. The only immunologic parameter affected
by in vivo gallic acid was SEA-induced IIF production. In this regard
alone, GA was similar to CA, which effectively abrogated IIF production.
173
-------
IN_ VITRO AND IN. VIVO IMMUNOTOXICITY TESTING Page 4
CA has long been established as an agent lymphocytotoxic to T-cells of
recent thymic origin ( 8 ). In contrast to GA however, splenocytes
from animals exposed to CA in vivo were unable to mount an anti-SRBC
PFC response and exhibited a depressed DNA synthesis rate when activated
by SEA. The partial restoration of the CA-abrogated PFC response was
consistently obtained by adding 2ME to splenocytes at the start of
culture; whether 2ME is "replacing" a macrophage function, or perhaps
eliciting a mitogenic signal required for an SRBC PFC response is un-
clear.
It should also be noted that not only did in vivo exposure to GA
fail to abrogate the PFC response, it significantly enhanced it (Table
II). This is consistent with the recent finding that GA abrogates Tl
suppressor cell function in vivo ( 4 ). The Tl suppressor cell (pre-
sumably an Ly Tf2+3+cell) has been shown to be induced by helper T
cells to exert feedback suppression on the PFC response (9). Loss of
Tl cell function in vivo would predictably lead to increased numbers of
anti-SRBC PFC in vitro since no feedback would occur.
The data further demonstrate the dangers of extrapolating .in
vitro test data concerning chemical effects on the immune response
to the whole animal. GA obviously has direct effects on lymphoid
cells in vitro which are not entirely manifest In vivo. This may be due
to factors such as sequestering of lymphoid cells from the chemical,
metabolic alteration of the chemical, or compensatory mechanisms in the
lymphoid organs. This does not detract from the utility of in vitro
174
-------
IN. VITRO AND _IN. VIVO IMMUNOTOXICITY TESTING Page 5
testing, however. Compounds which adversely affect the in vitro immune
response should probably receive priority for 1n vivo testing. Also
with regard to ingested compounds, the immune response of the gastro-
intestinal tract, not the spleen, should be examined, as this will be
where the unaltered toxicant will likely impinge at its highest concen-
tration.
175
-------
IN. VITRO AND IN. VIVO IMMUNOTOXICITY TESTING Page 6
REFERENCES
1. Archer, D. L., J. A. Bukovic-Wess, and B. 6. Smith. 1977. Sup-
pression of macrophage-dependent T-lymphocyte function(s) by gallic
acid, a food additive metabolite. Proc. Soc. Exp. Biol. Med. 156:
465.
2. Archer, D. L., and J. A. Wess. 1979. Chemical dissection of the
primary and secondary in vitro antibody response with gallic acid
and butylated hydroxyairiTsole. Drug Chem. Toxicol. 2:155.
3. Archer, D. L., and H. M. Johnson. 1978. Blockade of mitogen in-
duction of the interferon lymphokine by a phenolic food additive
metabolite. Proc. Soc. Exp. Biol. Med. 157:684.
4. Archer, D. L., B. 6. Smith, J. T. Ulrich, and H. M. Johnson. 1979.
Immune interferon induction by T-cell mitogens involves different
T-cell subpopulations. Cell. Immunol. 48:420.
5. Mishell, R. I., and R. W. Dutton. 1967. Immunization of mouse spleen
cell cultures from normal mice. J. Exp. Med. 126:423.
6. Cunningham, A. J., and A. Szenberg. 1968. Further improvements in
the plaque technique for detecting single antibody-forming cells.
Immunology 14:599.
7. Smith, B. 6., and H. M. Johnson. 1975. The effect of staphylococcal
enterotoxins on the primary in vitro immune response. J. Immunol.
115:575.
8. Ishidate, M., and D. Metcalf. 1963. The pattern of lymphopoiesis
in the mouse thymus after cortisone administration or adrenalectomy.
Aust. J. Exp. Biol. Med. Sci. 41:637.
9. Eardley, D. D., J. Hugenberger, L. McVay-Boudreau, F. W. Shen, R. K.
Gershon, and H. Cantor. 1978. Immunoregulatory circuits among T-
cell sets. I. T-helper cells induce other T-cell sets to exert
feedback inhibition. J. Exp. Med. 147:1106.
176
-------
TABLE I
Summary of the effects of ^n vitro exposure of mouse spleen
cells to gallic add on various Immunologlc tests
Treatment
2ME SEA
Direct antl-SRBC IIFC units/ ^-thymldlne upta
PFC/culture (mean ± SEM) ml (Log1Q) (mean CPM ± SEM)
NONE
+
+
+ +
GA (10 yg/culture)
+
+
* +
3850 ± 29
4917 ± 770
383 ± 192
150 ± 150
<50
5217 ± 235
<50
425 ± 25
<.05
<.05
1.85
2.45
<0.5
<0.5
<0.5
2.25
18700 ± 1300
30600 ± 1500
81400 ± 7000
68300 ± 4200
9800 ± 500
27500 ± 900
9000 ± 700
50700 ± 1900
3 2ME added to 10-5 M.
b
SEA added to 0.2 yg/ml.
c IIP units based on NIH Reference Mouse Fibroblast Interferon.
177
-------
TABLE II
A comparison and sumnary of the effects of 1n vivo
exposure of mouse spleen cells to gallic acTd and
cortisone acetate on various immunologic tests
. Direct anti-SRBC IIFC units/
Treatment 2MEa SEAD RFC/culture (mean ± SEM) ml (Log )
Saline (pH 2.5) - - 2640 ± 241
+ - 1880 ± 120
+ ND
GAd - - 4400 ± 321
+ - 5280 ± 241
+ ND
CAf - - <40
+ 960 ± 80
+ ND
<0.5
ND
2.93
<0.5
ND
i.ge
<0.5
ND0
1.4e
3
H-thymidine uptake
(mean CPM ± SEM)
2225 ± 46
ND
9579 ± 30
2730 ± 234
ND
12283 ± 157
693 ± 158
ND
3841 ± 23
a 2ME to 10'5 M.
b SEA to 0.5 yg/ml.
IIP units based on NIH Reference Mouse Fibroblast Interferon.
GA - 10 rig injected subcutaneously.
e Significant difference from control P = .05 by a Duncan Multiple Range Test.
^ CA - 5 mg injected subcutaneously.
178
-------
Chemical Carcinogenesis and Xmrunity
Effect of Methylnitrosourea on the.
Normal Imnunologic Function of Rats
2
Bruce S. Zwilling , Frank W. Chorpenning,
Adalbert Koestner and Nelvin S. Rheins
Departments of Microbiology, College of
Biological Sciences; Veterinary Pathobiology
and Conprehensive Cancer Center
The Chio State University
Columbus, Chio 43210
179
-------
Footnotes
1. Supported by Public Health Service contract N01-CP-53329 from the
Division of Cancer Cause and Prevention, National Cancer Institute.
2. Send correspondence to: Bruce S. Zwilling, Department of Microbiology,
College of Biological Sciences, The Ohio State University, U8U West 12th
Avenue, Columbus, Ohio, U3210.
180
- i -
-------
Abstract
Rats were treated with several doses of methylnltrosourea (MNU) or
the noncarcinogenic analog dlphenylnltrosamlne. Natural antibody levels
to several antigens as well as several parameters of lymphocyte and
macrophage function were assessed. Treatment with MNU did not appear
to alter most parameters studied. Changes noted in the splenic index,
pheripheral blood T cells and macrophage response to zymosan activated
serum were all correlated with the appearance of tumor. We concluded
that levels of chemical carcinogens that were capable of inducing tumors
did not directly suppress the immune response.
181
- 2 -
-------
Introduction
The biological evaluation of environmental toxicants has lead to the
realization that these substances could have a profound effect on host
defense mechanisms (1). Protocols for evaluating the immunotoxic effects
of environmental chemicals have been proposed (2). Studies, during the
past twenty years, concerning the effect of chemical carcinogens initially
led investigators to conclude that carcinogens may not only transform a
potential tumor cell but also, suppress the hosts immune response, thus
allowing the tumor to grow. The majority of literature describing these
immunosuppressive effects of carcinogens refers to carcinogenic doses
calculated to produce close to 10O& tumor incidence in a relatively short
period of time (3,^,5). Further, these studies evaluated active immune
responses. More recently, however, we and others have reported that
carcinogens, at tumorigenic doses, do not necessarily lead to a suppression
of immunological reactivity (6,7,8,9). In this report we describe our
findings that a carcinogenic nitroso compound MNU does not result in
the suppression of normal imraunologic function of rats.
182
- 3 -
-------
Materials and Methods
Animals; Outbred male Sprague Dawley (CD) rats, weighing 110-150 g were
obtained from Charles River, Wilmington, Massachusetts. The animals were
randomly separated and housed in groups of k per cage initially, then 2
per cage and given food and water ad libitum.
Carcinogen Treatment and Experimental Design: MNU and diphenylnitrosoamine,
a noncarcinogenic nitroso compound were provided by the Carcinogen Reference
Bank, NCI Frederick Cancer Research Center, Jrederick, Maryland. The
carcinogen was dissolved and administered as described in (10). Groups
of animals received 18 treatments of 9.0 mg/kg MNU or 0.5 mg/kg MNU every
other week for 36 weeks. Diphenylnitrosoamine was dissolved in 50$> ethyl
alcohol and administered at 9.0 mg/kg. The total dose of carcinogen did not
exceed 100.0 mg for the high dose group. On alternate weeks for the first
three months then monthly, for a total of 10 months, three animals from
each group were killed to determine the effects of carcinogen treatment.
Additionally, groups of animals were followed serially throughout the course
of the investigation. Analysis of these animals was limited to studies using
peripheral blood mononuclear cells. Lesions and specified tissue samples
from all organs were fixed in formalin at necropsy and prepared for histologic
examination.
Immunologlcal Assessment; To determine the effect of MNU treatment on normal
immunological function the parameters listed in Table 1 were evaluated.
Statistical Analysis; A mixed model analysis of varience with repeated measures on
the time deminsion was used to analyse the data. The main effects tested
included treatment and time; treatment by time interactions were also evaluated.
Analysis was computed using the BMOF2V package on an Amdahl 1*70/V6 system.
-4- 183
-------
Table 1. Iramunological parameters used to evaluate the effect of MKU
Reference
I. Natural Antibody
a) Sheep red blood cells 11
b) Teichoic acid 12
c) Kilham rat virus 13
II. Lymphocyte Function
a) Peripheral blood
i. Differential
ii. T cells 1U
iii. B cells 15
iv. PHA cells 16
b) ThymuE
i. Thyraic index
ii. T cells Ifc
iii. B cells 15
iv. PHA response 16
c) Lymph nodes
i. T cells 1U
ii. B Cells P5
iii. PHA response 16
d) Spleen
i. Splenic index
ii. T cells ib
iii. B cells 15
iv. PHA response ^
III. Maerophage Function
a) Adherence 17
b) Phagocytosi s 18
c) Bactericidal capacity 19
d) Response to migration
inhibitory factor 20
e) Chemotaxi s
184
- 5 -
-------
Results
Natural Antibody; The teichoic acid EP50 hemolysin titers varied from
15 to 180 in animals treated with 9 mg/kg MNU (data not shown). Similar
observations were also made with rats from other treatment and control groups.
No alteration of the EP^Q hemolysin titers to SRBC or the hemagglutination
Inhibition titer to Kilham rat virus were noted.
Lymphocyte Function:
a) Thymus. No alteration in normal thymic involution occurred as
a result of carcinogen administration (data not shown).
b) Spleen. The splenic indices of animals receiving the low
carcinogen dose as well as the control groups receiving diphenylnitrosoamine or
no treatment (Table 3) gradually decreased with time. The splenic index of
animals receiving 9.0 mg/kg MNU underwent a dramatic increase beginning at
about the 2Uth week. Although no significant effects were observed in the
percentages of T cells or B cells within the spleen nor in the splenic T cell
response to PHA it should be pointed out that there appeared to be a slight,
but not significant, decrease in T cells and the PHA response occurring at
about the 2l»th to 32nd weeks (Table 2).
c) Lymph nodes: No significant changes in lymph node T cells or
B cells were noted during the course of the investigation.
d) Peripheral blood; A significant decrease in peripheral blood
lymphocytes was noted beginning at the 2Uth week in animals receiving 9.0 mg/kg
MNU (Table 2). A concomitant increase in neutrophils during this period was
also noted. The decrease in lymphocytes appears to be due to a decrease
in peripheral blood T lymphocytes. No difference in the T cell response to
PHA was observed nor was any alteration in peripheral blood B cells attributable
to carcinogen.
-6- 185
-------
e) Serial studies; Peripheral blood lymphocytes declined with a
concomitant increase in peripheral blood neutrophils in animals sampled serially.
Further analysis indicated that the lymphocyte decrease was due to a decrease in
circulating T cells (Table 2).
Macrophage Function; No alteration in macrophage adherence, phagocytic or
bactericidal activity was noted during the course of this investigation.
Similarily no alteration in the response to migration inhibitory factor was
observed.
The response of macrophages from carcinogen treated and control
animals to zymosan activated serum and to lymphocyte derived chemotactic
factor was evaluated. Macrophages from animals receiving 9.0 mg/kg MNU
exhibited significantly increased chemotactic responsiveness to zymosan
activated serum beginning at the 2l»th week of the investigation (Table 2).
No alteration in the response of the macrophages to lymphocyte derived
chemotactic factor was noted. Similar observations were made concerning
the response of peripheral blood monocytes in serial studies.
Histopathology; Lesions associated with the administration of MNU were
found only in the stomach and in the lyraphoid organs.
Stomach; In animals receiving the highest dose of MNU (9.0 mg/kg), 100<£ of
the rats surviving 16 weeks or longer developed neoplastic changes of the
nonglandular stomach, which ranged from early neoplastic proliferation to
invasive carcinoma. All animals surviving 2k weeks had gastric tumors.
Only 6 rats receiving 0.5 mg/kg MNU developed hyperplasia of the gastric
mucosa, none developed gastric tumors. No neoplastic changes were detected
in the stomachs of the control animals.
- 7 -
186
-------
Lymphoid organs; The thymus glands during the last 6 months were characterized
by lymphoid hyperplasia that was not characteristic of the control groups. The
spleens were also hyperplastic. No lesions of the spleen or thymus gland were
observed in control animals. One thymic tumor was noted in one rat receiving
9.0 mg/kg MNU at the 2Uth week.
187
-------
Table 2. Alterations in lammolotical Reactivity of Rats Receiving 9.0 Pi/fcg Methylnitroaeurea
Tin* Pathology
2
4
SO 6
8
10
12
16 4
20 *
24 ***•
28 **•
32 ***
36 *•*
40 ***
44 ***
Analysis of variance
Splenic
Index
.263*. 02
.245*. 03
.197*. 03
.207*. 03
.180*. 01
.175*.01
.142*. 01
.1S2*.02
.280*. 07
.6S7*.61
,577*.47
.335*. 33
.336* .28
.353*. 18
<.001
Spleen
PHA
7.5*1.2
8.1 *2.2
3.0*0.5
9.1*2.9
10.3*1.6
5.4*2.7
4.5*1.2
32.9*11.3
6.6*3.4
6.6*2.5
10.3*2.7
8.1*5.0
1.2*0.3
1.1*0
p-0?246
T Calls
Spleen (%)
86*2.7
94*2.2
91*0.5
84
82*1.1
90*4.1
89*1.2
89*3.5
84*6.0
76*7.2
69*7.4
78*7.5
72*2.8
79*. 6
p-0.121
Lymphocytes
Peripheral Blood (%)
Sacrifice
89*0.5
83*1.4
71*2.1
71*4.0
80*4.0
75*3.8
75*4.0
77*2.3
49*12.1
60*5.3
56*10.2
25*2.1
31*5.9
35*1.0
p(.001
Serial
..
88*2.9
85*3.4
—
85*4.1
81*5.8
76*7.4
65*12.1
49*9
44
57
p<.001
T Cells
Peripheral Blood (%)
Sacrifice
94*2.3
95*0.6
93*2.3
94
90*1.7
92*2.4
89*0.6
87*5.8
81*6.4
47*16.2
71*13.9
71*8.6
78*12.8
84*5.9
p-0.158
Serial
..
91*8.9
89*8.7
--
92*5.5
91*3.7
89*4.6
89
59
40
80
80
pCOOl
Nout roph i 1 9
Peripheral Blood (%)
Sacrifice
10*0.4
14*1.5
26*1.6
25*2.6
17*3.3
22*4.0
23*4.2
20*2.1
46*11.5
35*5.1
36*9.0
71*1.9
62*6.4
63*1.5
P<7ooi
Serial
..
10*3.2
13*3.4
~
13*3.8
16*4.2
21*6.3
32
48
54
41
K.ooi
Cheaxttactlc
Sacrifice
Peritoneal
309*205
193*177
407*154
115*95
446*194
168*84
71*25
84*33
462*346
519*113
165*209
710*669
432*118
311*100
p-.OOl
Response
Serial
Monbcyte
..
61.4*314
77.2*82.1
—
75.4*39.1
118.4*93.6
59*67.3
105.7*87
73.6*57
145.8*75
292.1*142
290.5*69
pt.001
cx>
CO
-------
Table 3. Ii
logical Reactivity of Rats Receiving 9.0 »iAg Diphenylnitrosaaine
flaw Pathology
2
4
• 6
s •
I 10
12
16
20
24
28
32
36
40
44
mm *
00
vO
Splenic
Index
.257*. 04
.385*. 29
.212*. 03
.2204.01
.192*. 03
.192*. 04
.187*. 03
.170*. 01
.1SS*.03
.160^.02
.1604.01
.1454.01
.15040
.ISO
Spleen
PHA
4.341.3
7.241.5
6.142.2
9.542.3
7.741.5
8.342.5
9.543.3
29.248.0
23.84136
27.4417.4
15.444.6
23.1410.3
21.4412.0
--
T Cells
Splem (%)
9442.8
8842.8
8142.6
80
8443.7
8441.7
9141.0
9044.9
9440
8145.0
78410.0
81411.0
8941.0
86
Lymphocytes T Cells
Peripheral Blood (%) Peripheral Blood (%)
Sacrifice
9241.7
8343.7
8542.8
8642.3
8342.5
7844.2
8941.8
8742.6
8442.2
83410.2
86
75
6945.2
61
Serial Sacrifice
8942.4
9043.6 8843.3
8942.8
8R43.9 94
9046.0
9141.8 9341.7
87*1.8 8942.3
8543.3 8745.8
8544.2 9446.0
82*2.5 9040
83 8042.0
75 81415
8547.0
90
Serial
..
9147.3
--
82411.2
--
55435.2
9444.5
9543.5
92
83
79
83
90
--
NeutropMIs
Peripheral Blood (%)
Sacrifice
641.7
1443.7
1342.8
1342.3
1441.6
1944.3
1041.6
1142.7
1341.7
1548.7
11
24
2745.0
38
Serial
..
844.8
—
1043.2
--
742. S
641.4
1043.1
12
12
15
16
22
••
Cheewtactic
Sacrifice
Peritoneal
2494125
4034269
1554132
994114
3444325
132487
1754170
1614182
1154122
4494313
97463
1624159
105435
106486
Response
Serial
Monocyte
..
61.9451.2
—
83.8452.4
--
101.6484.6
84.8474.6
92.1456.1
8.9452.2
71.6480.7
77.6475.1
46.9447.
65.5438.1
--
-------
Discussion
This investigation was designed to test the hypothesis that chemical
carcinogens nay act, not only by transforming a potential tumor cell, but
also by suppressing the immune response and allowing the tumor cell to grow.
The results confirm and extend our previous observations made using 1».5 mg/kg
MNU (6).
No effect of carcinogen treatment on natural antibody levels was observed.
This observation is in contrast to our previously reported observation that
teichoic acid antibody levels in animals that developed tumor were significantly
higher then those from control groups (6). Inspection of the data (not
shown) revealed that an upward trend appeared to be developing but was not
significant. This may be because animals in the high dose group failed to
gain weight and may have been nutritionally unbalanced during the latter course
of the investigation. Stomachs in some were partially blocked by tumor.
We have reported previously that lymphocyte changes occurred primarily
in the peripheral blood and spleen (6). It appears additionally that the
decrease in peripheral blood lymphocytes was primarily due to a decrease
in T cells. Although the decrease in peripheral blood T cells was not
significant in the sacrifice study, the significance of the serial studies
reinforced this interpretation.
The decrease in peripheral blood lymphocytes, decrease in percent T cells
in the peripheral blood, the increase in spleen weight and a trend toward
decreased splenic T cells and PKA responsiveness all occurred at the ?l*th
week of this investigation. This time coincided with the appearance of
malignant tumors. Earlier studies, using U.5 mg/kg MNU, indicated that an
alteration in immunologic parameters also seemed to occur when tumor appeared
at the 36th week. Coincidently, this coincided with the cessation of carcinogen
190
- 11 -
-------
treatment. Tumor appeared at the 2Uth week in studies using 9 mg/kg MNU
and carcinogen treatment was continued until the 36th week. Alteration of
innnunologic parameters coincided with the appearance of tumor and not with
the cessation of carcinogen.
The responsiveness of monocytes and macrophages to zymosan activated
rat serum increased dramatically in animals treated with 9.0 mg/kg MNU.
Increased macrophage responsiveness also coincided with the appearance of
tumor. This observation would appear to be in contrast to reports that
indicate that macrophage responsiveness to chemotactic stimuli decreases
when macrophages are derived from tumor bearing animals (22). It should be
pointed out however that translational movement and chemokinetic responses of
macrophages increase in the presence of tumor (23,23) and may account for our
findings.
The results of this investigation indicate that low levels of MNU
administered to rats by gastric intubation did not suppress normal
immunologic function. This conclusion was also made by Stutman (7)
using low levels of 3-methylcholanthrene, by Scherf (8) after studies with
four carcinogenic nitroso compounds and by Norbury et al (9) following
UV carcinogenesis. These results collectively indicate that carcinogenesis
may not necessarily result in a suppression of an immune surveillance mechanism.
Recent reports concerning the immunological consequences of toxic sub-
stances such as heavy metals (20,22,25,27) leads one to conclude that suppression
or stimulation may occur depending on the dose, the route of exposure and the
immunological parameter under investigation. Since changes in immunologic
function may occur indirectly as a result of exposure to carcinogens or
toxicants caution must be exercised in interpretation. Exposure should be
limited to environmental levels approximating normal routes. Direct contact
191
- 12 -
-------
with cells of the immune system should be avoided except in cases where
direct contact occurs normally such as in the lung. In this regard pre-
%
liminary investigations in our laboratory evaluating the direct effects of
MNU, cadmium and fly ash on hamster alveolar macrophage function indicate
that these substances suppress the chemotactic response and Fc receptor
activity of BCG activated alveolar macrophages. Only cadmium however
suppressed the tumoricidal capacity of the alveolar macrophages (Zwilling,
Panke, Somers, and Campolito, unpublished observations).
Earlier studies during the 10 year period from the mid 1960's to
mid 1970's, from numerous laboratories (3,^5) using high levels of carcinogens
indicated that carcinogens were immunosuppressive. These studies evaluated
active immune responses and the carcinogen treatments may have affected the
specifically iramune cells responding to antigenic stimulation rather then
causing a generalized immunosuppression. This may indicate that rapidly
dividing cell populations may be more susceptable to the effects of a carcinogen
or an environmental toxicant.
192
- 13 -
-------
References
1. Moore, J. A. 1979. The imraunotoxioology phenomenon. Drug and Chemical
Toxicology. 2:1-U.
2. Dean, J. H., M. L. Padarathsingh and T. R. Jerrells. 1979. Assessment
of inraunobiological effects induced by chemicals, drugs or food
additives. I Tier Testing and Screening Appoach. Drug and Chemical
Toxicology. 2:5-17.
3. Prehn, R. T. 19^3. Function ofdepressed inmunologic reactivity during
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1*. Rubin, B. A. Carcinogen-induced tolerance to homo transplantation.
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5. Stjernsward, J. 19&9. Immunosuppression by carcinogens. Antibiot.
Chemother. 15:213-233.
6. Zwilling, B. S., J. A. Filppi, F. W. Chorpenning et. al. 1978.
Chemical Carcinogenesis and Immunity: Immunologic Status of Rats
Treated With Methylnitrosourea. J. Nat'l Cancer Inst. 61:731:735.
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Res. 22:2f 1-1*22.
8. Scherf, H. R. 1972. Untersuchungen an mannlichen Sprague-Dawley- Ratten
uher Zirsammenhange Zvischen der immunodepressiven and der carcinogenen
Wirkung ber vier N-Ntroso-Verbin-dungen.Z Kiebsforsch. 77:189-193.
9. Norbury, K. C., M. L. Kirpke and M. B. Budmen. 1977. In Vitro reactivity
of Macrophages and Lymphocytes from Ultraviolet Irradiated Mice. J. Nat'l.
Cancer Inst. 59:1231-1235.
10. Swenberg, J. A., A. Koestner, W. Wechsler, et. al. 1975. Differential
Oncogen!c Effects of Methylnitrosourea. J. Nat'l. Cancer Inst.
5U:89-96.
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11. Rabat, E. A, and M. M. Mayer. Experimental Immunochemistry. Springfield,
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ll*. Golub , E. 1972. The Distribution of Brain-Associated%rtigen Cross
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I09i 168-170.
15. Bianco, C., R. Patrick and V. Niessenzweg. 1970. A population of
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16. Oppenheim, J. J. and R> senstreich, D. L. 1976. Lymphocyte Transformation:
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and Tumor Immunity. (B. R. Bloom and J. R. David, eds.) New York,Academic
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17. Nathan, C. F. , M. L. Karnowsky and J. R. David. 1971. Alterations of
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18. Schmidtke, J. R. and R. L. Simmons. 1975. Augmented Uptake of
neuraminidase Treated Sheep Red Blood Cells. Participation of
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macrophage bactericidal function. Infect. Immun. 6:101-103.
20. Bloom, B. and B. Bennett. 1971. The assay of Inhibition of macrophage
migration an* the production of migration inhibitory factor (MIF) an* skin
reactive factor (SRF) in the guinea pig. In Vitro Methods in Cell Mediate*
Immunity. (Bloom, BR, Gla*e PR e*s.) New York: Academic Press, p. 235-2U8^
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21. Snyderman, R. and M. Pike. 1976. Chemotaxis of Mononuclear Cells in
In Vitro Methods in Cell-Mediated and Tumor Immunity. (B. R. Bloom
and J. R. David, eds.) New York: Academic Press, p. 651-661.
22. Snyderman, R. and M. C. Pike. 1976. Defective macrophage migration
produced by neoplasms; Identification of an Inhibitor of macrophage
chemotaxis. The Macrophage in Neoplasm (Fink, M. Ed.) New York, Academic
Press, p. 49-65.
23. Meltzer, M. S., R. V. Tucker, and A. C. Brever. 1975. Interaction of
BCG-Activated Macrophages with Neoplastic and Nonneoplastic Cell lines
in vitro cinematographic Analysis. Cellular Immunol. 17:30-42.
24. Snodgrass, M. J., T. M. Harris and A. M. Kaplan. 1978. Chemokinetic
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Cancer Res. 38:2925-2929.
25. Roller, L. D., J. G. Roan and N. I. Kerkvliet. 1979. Mitogen Stimulation
of Lymphocytes in CBA Mice exposed TO Lead and Cadmium. Environ. Res.
19:177-188.
26. Graham, J. A., D. E. Gardner, M. D. Waters and D. L. Coffin. 1975. Effect
of Trace Metals on Phagocytosis of Alveolar Macrophages. Infect. Imraun.
11:1278-1283.
27. Hadley, J. G., D. E. Gardner, D. L. Caffin and D. B. Menjel. 1977. Inhibition
of antibody-mediated rosette formation by alveolar macrophages a sensitive assay
for metal toxicity. J. Reticuloendothel. Soc. 22:417-425.
195
- 16 -
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MITOGENIC STUDIES OF THE EFFECTS OF 1,3-BIS(2-CHLOROETHYL)-
1-NITROSOUREA (BCNU) AND INDOMETHACIN ON THE LYMPHOCYTES
FROM NORMAL AND TUMOR-BEARING MICE
William A. Stylos^and Thomas S. S. Mao2
1 2
.National Cancer Institute*'* , National Institutes of Health,
U.S. Public Health Services, Bethesda, Maryland 20205 and
The Waksman Institute2, Rutgers - The State University of
New Jersey, New Brunswick Campus, New Jersey 08903
At Present;
ilmmunobiology Study Section, Division of Research Grants,
National Institutes of Health, U.S. Public Health Services,
Bethesda, Maryland 20205
2
Graduate Programs, Department of Environmental Sciences,
Rutgers - The State University of New Jersey, New Brunswick
Campus, New Jersey 08903
196
-------
Abstract
In our previous work of the effect of Prostaglandin E^or E£ on the in Vitro
blastogenic response of lymphocytes from normal and tumor-bearing mice,
Indomethacin, a Prostaglandin-synthetase inhibitor and also a cancer drug, was
exogenously introduced into the test system to see its influence on the
mitogenic effect of Prostaglandins (5).
Since we are interested in the basal effect, if any, of the cancer drugs
which are currently in the clinical investigations, so we designed this
experiment and studied the direct effect of two cancer treatment chemicals,
namely, Indomethacin and BCNU [(l,3-bis-(2-chloroethyl)-l-nitrosourea] to
evaluate the direct influence on the mitogenic responses of lymphocytes from
normal and tumor-bearing mice. Ve found that Indomethacin is strongly
inhibitory to two T-cell mitogens, Con-A and PHA, and one B-cell mitogen, LPS,
when the lymphocytes from normal and tumor-bearing mice were used. Further-
more, BCNU was found to profoundly inhibit the blastogenesis of both normal
splenic lymphocytes and thymic lymphocytes at the higher concentrations and
Con-A, PHA and LPS were employed. Some weakly stimulative influence was also
detected but only at lower concentrations of BCNU tested.
197
-1 -
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Introduction
l,3-bis(2-chloroethyl)-l-nitrosourea and other therapeutic agents, such as
Indoraethacen are currently in the various stages of clinical trial for cancer
chemotherapy (1). Since the depressed.cell-mediated immunity has been observed
in the cancer patients and tumor-bearing animals and that tumor cells often do
possess a subversive activity which enables these cells to escape from the host's
immune surveillance, and that T cells are a primary target of these subversive
tumor cells (2).
The BCNU treatment of the tumored mice did result in significant derease in
T- and B-cell populations (3). Indomethacin, a prostaglandin-synthetase inhibiter.,
was studied for the effect in vivo on humoral and cellular immunity in humans(4)
and its mitogenic effect when protaglandins were present in the lymphocyte culture:
(5). In the present study, we are simply interested in the mitogenic effects of
BCNU and Indomethacin on the splenic and thymic lymphocytes from normal and tumor-
bearing mice without other compounds involved in the cell cultures.
Materials and Methods
I. Animals BALB/c and CD2Fj male mice. 6-8 weeks old, were supplied by the
Mammalian Genetics and Animal Production Section, Division of Cancer Treatment,
National Cancer Institute, NIH, Bethesda, Md. The animals were housed in plastic
cages and fed Purina laboratory chow with water ad libitum. All animals weighed
2L23 g before use
II. Drugs
(1) BCNU was used as a cytoreductive therapeutic agent and was supplied by
the Division of Cancer Treatment, National Cancer Institute (Drug Synthesis
and Chemistry Branch, DTP). This a Ikyla ting chemical was dissolved in a
steroid-suspending vehicle and administered sp in a constant volume of
0.01 ml/g of body weight in this in vivo Experiment (7).
(2) Indomethacin was purchased from Sigma Chemical Co., St. Louis, Mo. For use
in the _in vitro experiments indomethacin was dissolved in 95% ethyl alcohol,
pervaporated under N£ and brought to a concentration of 1 x 10"° M with
sterile RPMI 1640 medium containing 107. heat-inactivated fetal bovine serum
containing 2 mM glutamine and 1% penicillin-streptomycin (5).
III. Tumor The Madison lung 109 carcinoma, which was derived from a spontaneous
neoplasm in a BALB/c mouse, was kindly supplied by Dr. Ruth L. Geran, Drug
Research and Development at National Cancer Institute, NIH, and has been main-
tained as a transplantable line in BALB/c mice(3).For these studies in this
investigation, 5.0 x 10 viable tumor cells were injected subcutaneously
in the right inguinal region of CD2F1 «ice.The experimental animals attained
palpable tumors 11 days after inculation. Then, BCNU is injected sp (See section
IV. Lymphocte Preparation Lymphocytes were obtained from normal and tumor-bearing
CD?Fi mice's spleens as well as from normal BALB/c mice's spleens and thymuses
by aseptic removing techniques. The spleens and thymuses were triturated in
10 ml od sterile RPMI 1640 medium supplemented with 107. heat-inactivated fetal
bovine serum, 2 mM glutamine and 1% penicillin-streptomycin in sterile 15 x 150
mm Petri dishes (Falcon, Oxnard, Calif.). The dishes were incubated in a
humidified atmosphere of 57. C02 and 95% air at 37 C for one hour in order to
remove adherent macrophages. The nonadherent cells were collected and incubated
at room temperature for approximately 5 min. with 0.11 M ammonium chloride to
deplete RBCs. The nonadherent, RBC-depleted cells, taken up in 4 ml of RPMI 164(
were layered over 5 ml of Ficoll-Hypaque separation medium containing 12 parts
of 14% Ficoll 400 (Pharmacia, Piscataway, N.J.), and 5 parts 32.87. Hypaque
Sodium (Diatrizoate sodium, Sterling Winthrop Research Institute, Rensselaer,
New Yorkk) specific gravity * 1.09. The gradient was centrifuged at 300
- 2 -
-------
for 30 min. at room temperature. The interface, containing the separated
lymphocytes were collected, washed three times with RPMI 1640, checked for
viability with Trypan Blue, and brought to a concentration of 1.25 x 106
viable cells/ml.
V. Injection of BCNU The experimental (n>2Fi attained palpable tumors of 5-7 mm
in size by the llth day after tumor inoculation. On the 12th day. animals
which were to receive BCNU were distributed to the appropriate groups and were
injected BCNU sp in a constant voloume of 0.01 ml/g of body weight. The day
of BCNU treatment was considered Day 0 and the in vitro assays ( Mitogenic tests
were started 1 day later (8) •
VI. Respbnse to mitogens In order to study the effect of exogenously added chemical
namely Indomethacen, on the in vitro blastogenesis of lymphocytes from either
normal or tumor-bearing mice, each component of the assay was adjusted either to
the desired cell number or desired concentration of chemical to the timing of the
assays. First, 50 yul of Indomethacen at a final concentration of 1 x 10"° M
or RPMI 1640 medium were added to each well of a Micro-test II Tissue Culture
Plate (3040, Falcon, Oxnard, Calif.). Next, 50 ;il of Phytohemagglutinin P
(Difco, Detroit, Mich.) 10 ul final cone., Con ca naval in A (Pharmacia Fine
Chemicals, Piscataway, N.J.) 5 ug/ml final cone., or RPMI 1640 medium were added.
These concentrations were found to be optimal by wide range titrations of the
different mitogens titrated versus both splenic or thymic lymphocytes. Then,
1.25 x 10 5 viable splenic or thymic lymphocytes contained in 100 jil RPMI medium
were finally added to each well. When all three components were added stepwise
and in order to microtiter wells, the timing of the experiment was initiated.
The cultures were incubated for 48 hrs. at 37" C in a 57. C02- 957. humidified
air atmosphere. After that, 50 pi of a 1 to 10 dilution of %-methyl thymidine
(NET-027 Z, Specific Activity 40-60 Ci/mM, New England Nuclear, Boston, Mass.)
were added to each well. The cultureswere incubated for an additional 18 hrs.
The cells were washed with deionized water by use of a Mash II cell harvester
(Microbiological Associates, Bethesda , Md.). All radioactive filters were counte
in a liquid scintillation spectrometer ( Packard Model 3385 Tri Carb, Downer's
Grove. 111.) with Aquasol (New England Nuclear, Boston, Mass.) as the scintillati
fluid (5, 6, 7).
VII. Experimental Design and Presentation of the Blastogenic Data For determining
the effect of Indomethacin on the In Vitro blastogenic responses of lymphocytes
from normal and tumor-bearing mice, the lymphocytes from 40 CD2F1 normal and 40
tumor ed mice were pooled. After isolation and purification of the splenic
lymphocyte* by Ficoll Hypaque gradient centrifugation, the lymphocytes were reactet
with a physiological concentration of 1 x 10'° M Indomethacen or RPMI 1640 mediui
A concentration of Indomethacen as well as the medium control were conducted in
triplicate(In the presence of the lymphocytes and mitogens) . These results are
reported as cpm, that is, the mean of triplicate cultures ith mitogens added
minus the mean of triplicate cultures no mitogens added ± SE. In addition to
Acpm, a percent inhibition was calculated in order to assess the degree of
inhibition(or stimulation) for a concentration of chemical added (5) •
VIII. Statistical Analys s Statistical treatment of the data from the mitogenic
studies in this investigation was performed using Student's t test ( Two-way ).
199
- 3 -
-------
Results
I. Inhibitory Effects of BCNU on in Vitro Blastogenic Response of Normal
BALB/c Mouse Lymphocytes
(1) Normal Splenic Lvphocytes in Table 1
The BCNU-injection of normal BALB/c mice was found on Day-1, Day-3
and Day-5 to profoundly inhibit the blastogenesis of splenic lymphocytes
which were exogenously treated with PHA, Con A and LPS, and indicated
that the mitogenic response was weakly inhibited on Day-10 and Day-12 by
LPS and weakly stimulated on Day-10 by PHA and ConAand also weakly stimu-
lated on Day-12 by Con A. It has been clearly shown that the same kind of
splenic lymphocytes failed to be stimulated by LPS during the whole period
from Day-1 to Day-12 tested.
(2) Normal Thymic Lymphocytes in Table 2
The BCNU-injection of normal BALB/c mice was also found to stongly inhibit
the blastogenesis of thymic lymphocytes on Day-1, Day-3 and Day-5 which
were treated with Con A. However, in the case of exogenous treatment with
PHA, the blastogenesis of thymic lymphocytes was initially/weakly inihibited
on Day-1 and then weakly stiaulated on Day-3 and Day-5.
II. Inhibitory Effects of Indomethacin on in Vitro Blastogenic Response of
Normal and Immune CD2Fi Mouse Splenic Lymphocytes
(l)Datantnormal Splenic Lymphocytes in Table 3
The exogenous addition of Indomethacin to the lymphocyte cell culture
strongly exercised the inhibitory effects on both T-cell mitogens (Con A
and PHA)- and B-cell mitogen (LPS)- sensitive responses. No stimulative
effect what-so-ever was detected with all mitogens tested in this study.
(2) Data on Immune (Tumor-bearing) Splenic Lymphocytes in Table 4
Indomethacin has showed inhibitory effects on the tumor ( Madison lung
109 carcinoma )-bearing CD2Fimouse splenic lymphocytes incubated with
both T-cell and B-cell mitogens tested in this investigation. However,
the data indicated that there are some different degree of inhibition
with different mitogens, namely,weak inhibition with PHA, profound
inhibitory effect with Con A and strong inhibition with LPS.
Discussions
I. The data presented,concerning the mitogenic effect of BCNU-injection in
normal BALB/c mice, indicated that the blastogenesis of splenic lymphoctes
in the In Vitro was not stimulated by both T- and B-cell mitogens tested
on Day-1 - Day-5 and that it was only slightly stimulated later on Day-10
by Con A and PHA and on Day-12 by ConA alone. However, there was no any
stimulating mitogenic response was observed by LPS during the whole perid of
Day-1 to Day-12 in the same test.
In our previous study also with BCNU but in the tumored control ( Tumor-
bearing mice with only BCNU injected ip ) of CD2ri mice, the splenic lymphocytes
were not stimulated at all by Con A from Day-1 to Day-2l and rather severely
inhibited instead. As for the B-cell mitogen, LPS, is concerned in the same test,
the mitogenic response was considerably inhibited but rather markedly constant
and decidedly un-stimulated from Day-1 to Day-2l testing period. Further-more.
200
- 4 -
-------
in the case of another T-cell mitogen, PHA, is concerned also in the same test,
che BCNU-treated BALB/c mice splenic lymphocytes were not stimulated, but resulted
in negative mean cpm[(->H-thymidine(3H-tdR)3 on Day-15 and Day-2l, indicating a
severe depletion of PHA-sensitive splenic lymphocytes by BCNU ( 3).
The a fore-mentioned different characteristics in mitogenic responses between
the normal and tumored splenic lymphocytes, although both were BCNU-treated,
are suggested due to some factors, including the difference in the strain of mice
used, concentrations of BCNU administered by ip or sp and other somewhat different
experimental procedures applied to these two separate investigations performed in t\
different times. More-over, in fact that an "Iramunofluorescece technique" was
used by applying anti-T or anti-B serum to the partially purified splenic lym-
phocytes isolated from the various groups of tumored mice for the indirect imrauno-
fluorescent studies in our previous investigation (3). The results from this study
showed that the relative percentages of the T-cells not the same. There was a
definite decrease in the percentage of the splenic T-cells of the tumored control
mice ( With no BCNU injected ) relative to the T-cells detected for the untumored
controls( Also no BCNU injected ). The BCNU-treated mice had the different.T-cell
percentages, which are not in a very wide range, from those detected for the
tumored controls (3).
II. Indomethacen, a prostaglandin-synthetase inhibitor, was tested for its mitogenic
effect on the splenic lymphocytes of both normal and tumor-bearing CD£Fi mice.
The data indicated that this compound exhibited inhibitory effects on all mitogens
tested for the lymphocytes derived from both normal anf tumor-bearing mice. How-
ever, the inhibitory effects appeared^different with different mitogens used in
the same test system. That is that the inhibitory effect on the individual mitogens
is in the order of LPS>ConA>PHA in both cultures of the normal and tumor-bearing
CD2F^ mice. More-over, the percent inhibitions (£) of three individual mitogens
for normal and tumor-bearing splenic lymphocytre are not the same. But in general
speaking, the inhibitory effects of Indomethacen on all mitogens in the normal
lymphocyte cultures are relatively more severer than that in the case of lymphocytes
derived from the tumor-bearing mice.
201
- 5 -
-------
<\J
•Table I o
C\J
INHIBITORY EFFECT OF BCNU ON NORMAL BALB/c
SPLENIC LYMPHOCYTES
MITOOENS DAY 1
NORMAL SPLENIC LYMPHOCYTES NORMAL SPLENIC LVMPHO. FROM SCNU TREATED MICE
cptn *H TdR cpm *H TdR PERCENT* Pc
INCORP Ji K 10* CELLS* INCORP./O * 10* CELLS INHIBITION
PHA 3610*300 1000*02 »t <001
CONA 37787*1438 3113 ±128 W <0.001
LPS 47SM±1S27 10074 ±34t 71 <0001
NONE 247 ±17 110*32 - -
DAY 3
PHA 2110*100 1017*140 S2
CONA 24071*3004 3007±2M 00 <0.01
LPS 20031 ±122t 3031 ±431 00 <0.001
NONE 208*21 03 ±10 - -
DAYS
PHA 007±01 407±43 40
CONA 7210*412 1042*177 74 <0.001
LPS 12470*2077 1241*00 00 0.10
CONA 420*01 001*147 -20 >0.10
LPS 33000*1022 11714 ±041 40 >010
NONE 330*43 377*04
DAY 12
PHA 107*04 on* 31 10 >0.10
CONA OM*110 1000*200 -21 >S.10
LPS 7620*004 61391474 32
-------
o
CM
Table 2
INHIBITORY EFFECT OF BCNU ON NORMAL BALB/c
THYMIC LYMPHOCYTES
MITOOENS
PHA
CONA
NONE
NORMAL THYMIC LYMPHOCYTES
cpm »H TdR
INCORP Ax 10* CELLS'
1400*249
617*7 ±417t
140 ±a
DAV1
NORMAL THVMIC LVMPHO
cpm *H TdR
INCORP.AX 10* CELLS
1229 ±136
12M9t1M1
247 1 17
FROM BCNU TREATED MICE
PERCENT*
INHIBITION
13
7%
P«
>010
< 0.001
DAY 3
PHA
CONA
NONE
1720133
440M±13M
299 ±§7
132917V
571ft 241
147 ±BS
— •
n
>t.M
<0.001
DAVE
PHA
CONA
NONE
1033 ±132
314M±2609
274 ±90
1221 ill*
10S73 ±407
194 1 12
— it
•c
>0.10
<0.002
DAY 10
PHA
CONA
NONE
7M7t10t7
HO*
M3±§7
2239 ±196
NO
3Mt17
72
<001
•MEAN USEMI OF TRIPLICATE CULTURES.
MEAN OF TRIPLICATE DET'NS BCNU TREATED LYMPHO. \
~•
xlOO
I OF TRIPLICATE DET'NS UNTREATED LYMPHOCYTES/
'PROBABILITY is CALCULATED AGAINST NORMAL MICE FOR EACH DETERMINATION MADE. STUDENT'S T TEST
*ND,NOT DONE
-------
O
CM
Table 3
INHIBITORY EFFECT OF INDOMETHACIN
ON NORMAL CD2Fi SPLENIC LYMPHOCYTES
INDOMETHACIN
UNTREATED TREATED
CPM
*HTdRA.*x10* PERCENT0
MITOOEN SPLENIC tVMPHOCYTES INHIBITION
CONA IMOttiMT** 17I7812M B3
.PHA IIMItfW 3GM±47 77
tP8 B7744±2BB7 3M)±1M M
oo
NONE 2332i«a 317 ±1 — I
•MEANiSEM OF TRIPUCATE DETERMINATIONS.
i /MEAN OF TRIPUCATE DET'NS INDOMETHACIN TREATED LVMPHO\^.^
. 1 ^ M£AN Qf TR|p|JCATE DErNS UNTREATEO LVMPHOCm8 )***
-------
LO
o
Table 4 (VI
INHIBITORY EFFECT OF INDOMETHACIN
ON IMMUNE (TUMOR-BEARING) CDaFi
SPLENIC LYMPHOCYTES
INDOMETHACIN
UNTREATED TREATED
CPM
•HT4R/K.OX10P PERCENT*
MITOOEN SPLENIC LYMPHOCYTES INHIBITION
CONA HM2i7t»* 2SM4*400 62
PHA 3M2±232 2MSS±4M 21
LP8 34M±12S7
NONE «7D±3i 10M±40 - '
'MEANtSEM OF TRIPLICATE DETERMINATIONS.
^PERCENT INHIBITION - 1 - fMEAN OF TRIPLICATE DET'NS INDOMETHACIN TREATED LYMPHOlL t
MEAN OF TRIPLICATE DET'NS UNTREATED LYMPHOCYTES /
i
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References
(1) Carter, S. K., Schabel, F. M. et al. (1972) l,3-bis(2-chloroethyl) -1-nitro-
urea (BCNU) and other nitroureas in cancer treatment: A Review, Advances in
Cancer Research, 16:273-332.
(2) Plescia, 0. J., Grinwich, Z. and Plescia, A. M. (1976) Subversive activity
of syngeneic tumor cells as an escape mechanism from immune surveillance
and the role of prostaglandins. Ann. N. Y. Acad. Sci., 276:455.
(3) Stylos, W. A., Chirigos, M. A. and Lengel, C. R. (1978) Lymphocyte stimu-
latory effects of an ether-extracted preparation of Brucell abortus. Cancer
Treatment Reports, 62:1949-1954.
Goodwin, J. S., Selinger, I). S. et al. (1979) Effect of Indoraethacin in vivc
on humoral and cellular immunity in humans, Infection and Immuninity, 19:430-^
(5) Stylos, W. A., Lengel, C. R., Lyng, P. J. and Chirigos, M. A. (1979) The
effect of prostaglandin EI or E£ on the in vitro blastogenic response of lymp
cytes from normal and tumor-bearing mice, J. of Immunopharmacology , l(2):195-2
(6) Mao, T. S. S. and Chirigos, M. A. (1978, March) Mitogenic effects of pyran
copolymers on lymphocytes, Federation Proceedings. 37(3) :829.
(7) Mao, T. S. S., Stylos, W. A. and Chirigos, M. A. (1980) The stimulation of
raurine splenic lymphocytes by maleic anhydride-divinyl ether copolyners (DIVES'.
of different molecular weights, Abstract of Papers. Paper # 17.3.34 PW, 4th
International Congress of Immunology (Paris, France, July 1980)
(8) Chirigos, M. A., Schultz, R. M. et al. (1978) Comparative adjuvant effects
of levamisole and Brucella abortus in murine leukemia, Cancer Treatment
Reports, 62:1943-1947.
206
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PANEL DISCUSSION ON THE CURRENT STATUS OF THE DEVELOPING
DISCIPLINE OF IMMUNOTOXICOLOGY - Jack H. Dean* Presiding
Dr. Dean (NIEHS); There has been a vast Interest 1n Immunotoxicity
assessment the last couple of years which has served as a stimulus
for this symposium. I first became aware of this emerging field
at a Gordon Conference on Safety Assessment 1n 1978. Immunotoxicity
occupied about two days of discussion at that meeting. I was most
Impressed 1n the comprehensive approach taken by most workers at
that meeting for the Immunotoxicity assessment of chemicals and
drugs. The first question I would Hke to ask the panel 1s why
perform a comprehensive 1mmunotox1c1ty assessment 1f a simple WBC
and differential or lymphold organ histology would provide the
necessary information? I believe this question burns in the hearts
of a lot of people who are currently doing routine toxicity testing.
Dr. Luster (NIEHS): In jn utero studies with TCDD, thymus atrophy
and depressed spleen cellularlty were seen without other evidence
of acute clinical toxicity with the exception of anemia, although
rfe not-d effects on tumor and bacterial sjsceptibil1ty upon
challenging the animals. I think this point holds true for a lot
of other xenobiotics. The routine determination of lymphoid organ
weights and cellularity should be used as an adjunct to Immune
studies. Simple steroid stress, for example, can Induce lymphoid
and thymus atrophy, so I suspect that weight measurement alone are
not good parameter for an Immune assessment, except as adjuncts to
jjn vitro function studies and challenge experiments with Infectious
agents or t-umor cell studies.
At Present: Department of Toxicology, Sterling-Winthrop 207
Research Institute, Rensselaer, N5f 12144 u
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Dr. Bellantl (Georgetown): I'd like to just comment on the factors
that one evaluates to determine effects of an environmental agent
on the Immune system. One measures cell death and Immune dysfunction.
It seems to me simplistic to look for a quantitative decrease 1n
cell numbers and that the ultimate effect would be varying degrees
of dysfunction. Thus, functional measures would be more sensitive
probes. I think the reason why we want to do Immune function
assessment as part of a standard toxicology protocol would be to
have a more sensitive measurement of toxldty.
Dr. Plescla (Rutgers University!: I think that by starting out
looking for crude measures of gross effects that this particular
task of safety assessment could be achieved. Perhaps by using
a simple screening test. It will become necessary to do some
of the more refin-2'1 i.nin^rie function tests since I don't think
these gross tests for toxiclty will always be sensitive enough.
Dr. Keller (University of Idaho): Let me comment on this question
I think the problem 1s that if you have high levels of toxicity
and cell death other direct toxic effects will also be seen. In
that case, 1f we have direct toxldty, the effect on the immune
system Is Incidental. I think the effects we should be more
concerned with are the more subtle effects on other target
organs, like the Immune systen or endocrine system, as well as
Indirect effects where one does not see direct toxldty but
picks up some functional effect. This 1s the general assumption
for doing special target organ toxiclty studies.
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Dr. Dean: The next question Is how strongly do you all feel about trying to
correlate changes 1n immunologlc parameters with host resistance alterations?
Is this correlation already well documented from clinical experience or do we
need better correlation between ^n vitro immune function changes and
alterations In host resistance in rodents (e.g., bacterial, viral or tumor
susceptibility)?
Dr. Bellanti: It seems like most of us in Immunology believe that 1f there is
thymus atrophy or absence there will be increased susceptibility to infectious
agents. I wonder if the animal data documents this well enough and if there
are correlations between these parameters and functional responses in
experimental animals. I am going to try to answer the question, or at least
begin the discussion, from a clinical approach in the human. We can measure
gross immune deficiencies in patients some of which are inborn errors of the
Immune system. What these really represents is the tip of the iceberg in the
population. The majority of the patients for example that we've studied at
Georgetown Medical School present with recurrent infections and have subtle
defects that require more specialized studies, using methods which may not
been developed yet. Examples are the determination of T cell subpopulatlon.
I think that the more we learn about these mechanisms of dysfunction and the
more we study these subtle derangements the better off we will be.
Dr. Dean: From your clinical experience what measure seem to correlate best
with alterations of host resistance?
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Dr. Bellanti: Well classiflcally alterations 1n lymphocyte function or
numbers correlates best with increased sensitivity to bacterial challenge.
Similarly, T cell deficiency are associated more with viral or fungal
infections. One thing we have neglected to mention is autoimmune problems,
their association with inducible defects and the connection between selective
IgA deficiency and autoimmune disease. You could go on and on with these
sorts of correlation, but I think autoimmunity is a problem of xenobiotic
exposure that we haven't mentioned, yet it is equally as important, as altered
susceptibility to bacterial or viral or fungal infections.
Dr. Luster: I think ideally what we should do is take advantage of tests done
in humans (e.g., MLC or mitogen) to attempt to correlate the degree of immune
dysfunction in man with that seen animal exposured to xenobiotics. In animals
we can determine that, for example, that a 50% suppression in MLC indicates
that the animal is going to have an increased . tumor frequency or an
increase in host susceptibility to infection agents. From this information we
can extrapolate as to the potentially hazardous of this environmental chemical
to humans.
Dr. Ed Hu (USEPA): If we see any immune alteration or increased tumor
production following xenobiotic exposure in rodents the agent must be suspect
in humans and exposed workers should be monitored.
Dr. Dean: Another aspect is that immunologists interested in basic immunology
may find many of these xenobiotic agents more interesting as dissectional
probes to understand how the immune system works and in so doing we may
discover new drugs or chemicals producing more desirable effects.
210
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Dr. Dean: I think I must now conclude the panel discussion and thank each of
the panel members. We should all take a moment to thank Dr. Tom Mao for the
marvelous job he has done in coordinating this symposium.
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Immunotoxicology in Perspective
Otto J. Plescia, Ph.D.
Hakeman Institute of Microbiology
Rutgers, The State University of New Jersey
New Brunswick, New Jersey 08854
Immunology began to take shape in the mid-19th century as
a branch of bacteriology concerned with immunity to infectious
microorganisms. Today, immunology not only has assumed the
status of an independent scientific discipline but it has
developed into one with many branches of its own. During the
last 80 years have emerged immunochemistry, immunopathology,
immunogenetics, cellular immunology, immunopharmacology, and
most recently immunotoxicology.
Our concern is with immunotoxicology. What is it, and why
is there a need to carve yet another branch out of immunology?
In recent years we have come to regard our environment as a
potential threat to mankind, not because of the natural
infectious organisms that lurk in it but because of the
increasing number of potential toxic substances that have been
created by man and released into the environment. Our concern
with environmental pollution has resulted in the creation of
the Environmental Protection Agency to deal with this problem.
In order to cope with it we must be able to identify
environmental substances that are potentially toxic and to
establish tolerable levels of such toxic substances. And to
achieve this capability we must have rapid reliable screening
methods. Immunotoxicology can fill this need.
The immune system of man and animals is vulnerable to
toxic substances, including those that may be found in the
environment as pollutants. For example, cytotoxic drugs that
are used in the chemotherapy of cancer are immunosuppressive so
that their indiscriminate use is ill advised. Thus,
immunological tests that measure that toxicity of substances
against immune cells should serve well to identify toxic
environmental substances. Such tests are intrinsically rapid,
relatively simple, reproducible, and they can be carried out in
vitro at minimal cost compared with conventional animal
toxicological tests. This expectation of success is supported
by the preliminary results of studies that were presented at
this symposium. These tests should now be standardized for
uniformity. It should also be noted that as our knowledge of
the processes of activation, differentiation, development and
function of immune cells progresses we should do even better in
adapting immunological methods to assess environmental
substances for their toxicity.
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Immunotoxiclogy, in my opinion, should proceed on two
fronts. In addition to providing test methods useful for the
identification of toxic substances in our environment it can
also treat toxic substances as valuable probes in furthering
our basic knowledge of the immune system itself. In this
latter sense immunotoxicology overlaps with immunopharmacology-
Whether or not immunological methods are ultimately
adopted in the routine testing of environmental substances for
toxicity, any prevalent toxic substance, however it is
identified, should also be assessed for immunotoxicity. The
immune system is central to man's well-being, and any
toxicological study would be incomplete without including it as
a potential target.
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CLOSING REMARKS: IMMUNOTOXICITY SYMPOSIUM
by Joseph A. Bellanti, M.D.
Director, International Center
for Interdisciplinary Studies
of Immunology
Georgetown University
School of Medicine
Washington, D.C. 20007
Immunotoxicology may be defined as the study of the
harmful effects of environmental agents on the immunologic
system. These include the wide variety of responses resulting
from simple chemicals and trace elements to more complex
interactions involving pesticides/ insecticides, food products
and food additives. In addition, we may also begin to direct
our attention to the role of these substances in the
pathogenesis of allergic and autoimmune disorders and
malignancy, long suspected to have an environmental basis.
It is obvious from the discussions of this conference that
the diverse components which comprise immunotoxicology will
require the participation of an equally diverse group of a
variety of disciplines including the basic sciences of
chemistry, biology, pharmacology and toxicology as well as the
clinical disciplines of medicines, pediatrics, obstetrics and
veterinary medicine. There will also be a need for more
research with a particular emphasis on studies of the
developing host because of the great vulnerability of the
developing fetus to these toxic substances.
The Association of Official Analytical Chemists will play
an important role in the standardization of testing procedures
in order to assure accurate results which are comparable
between laboratories.
In this particular age when the emphasis is on
environmental health and disease prevention, immunotoxicology
will be paramount, not only in these next decades but also in
the 21st century. The interdisciplinary approach must also
involve an effective alliance between the federal agencies with
the scientific communities and the public. An effective
dialogue must be established which will demonstrate to the
nation that science and technology can progress simultaneously
under the energizing force of social commitment. Not only will
this involve the need for research but an expanded need for
innovative training programs for the training of young
investigators in this rapidly developing and important field.
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APPENDIX: An appeal from the syraposiun participants to the
Federal regulatory agencis
We, contributors of the Iicr.unotoxicity Methodology Symposium which was held
on October 16, 1979 in Washington, D. C-, are convinced that insnunotoxicity
is indeed another toxicology line which is distinctively different from the
current and official toxicity criteria being used for the Federal regulation
of the toxic substances, including the pesticides and other environmental
pollutants. A .few scientifically sound and mutually acceptable cethods for
measuring the inaounotcxicity of hazardous chemicals and biologicals are •
now available. Therefore, we feel strongly that isznaiotoxicity could be
considered as a new criterion for Federal regulatory application to themeasurenent
ofi.toxicity of toxic substances either now or in the near future. The research
efforts and regulatory guideline decision in this newly developed field, ^f
continuously supported.
Immunotoxicology should beencouraged
an
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