Proceedings of the
Symposium on Analytical
Methodology for
Determining Immunotoxicity of
Chemicals, Including Pesticides

-------
    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

-------
                            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

-------
                            '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

-------
                       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

-------
                       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

-------
                        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

-------
        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

-------
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,

-------
                                                                             -  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.

-------
              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

-------
                                    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

-------
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).

-------
     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.

-------
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
-------
  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

-------
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

-------
                                     -14-
1.   Lischner, H.W., H.W. Punnett, and A.M. Digeorge. 1967.  Lymphocytes  in  congenital
     absence of the thymus. Nature (London) 214:580-582.
2.   Oppenhelm, J.J., R.M. Blaese, and T.A. Waldmann. 1970.  Defective lymphocyte trans-
     formation and delayed hypersensitivity in Wiskott-Aldrich syndrome. J.  Immunol.
     104:835-844.
3.   Penn, I., and I.E. Starzl. 1972. A summary of status of de novo cancer in  transpla
     recipients. Transplant. Proc. 4:719-732.
4.   Gatti, R.A., and R.A. Good. 1971. Occurrence of malignancy in immunodeficiency
     disease: a literature review. Cancer 28:98.
5.   Burnet, P.M. 1970. The concept of immunologic surveillance. Prog. Exp.  Tumor Res.
     13:1-27-
6.   Vos. J. 1977. Immune suppression as related to toxicology. CRC Crit. Rev.  Toxicol.
     5:67-101.
7.   Holt, P.G., and D. Keast. 1977. Environmentally induced changes in immunological
     function: acute and chronic effects of inhalation of tobacco smoke and other
     atmospheric contaminants  in man and experimental animals. Bacteriological  Reviews
     41:205-216.
8.   Koll^r, L.D. 1979. Some immunological effect? of lead,  candmium and methyl mercury!
     Drug and Chemical Tox. 2:99-10.
9.   Faith, R.E., M.I. Luster, and J.G. Vos. 1979. Effects on immunocompetence  by
     chemicals uf environmental concern. Ann. Rev. Biochem.  Toxicol. In press.
10.  Vos, J.G., J.A. Moore, and J.G. Zinkl. 1973. Effect of 2,3,7,8-tetrachlorodibenzo
     p-dioxin on the immune system of laboratory animals. Environ. Health Perspect.'
     5:149.
11.  Vos, J.G., and J.A. Moore. 1974. Suppression of cellular immunity in rats  and mici
     by maternal treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Int. Arch. Allerg)
     Appl.  Immunol. 47:777.
                                                                                17

-------
                                    -15-
-12.  Moore,  J.A., and R.E. Faith. 1976. Immune!ogic response and factors affecting
      its  assessment. Environ. Health Perspect. 18:125.
 13.  Faith,  R.E., and J.A. Moore. 1977. Impairment of thymus-dependent immune function
     by exposure of the developing immune system to 2,3,7,8-tetrachlorodibenzo-p-dioxin
     (TCDD). J. Toxicol. Environ. Health 3:451.
 14.  Faith,  R.E., M.I. Luster, and J.A. Moore. 1978. Chemical separation of helper cell
     function and delayed hypersensitivity responses. Cell. Immunol. 40:275-284.
 15.  Friend, M. and D.O. Trainer. 1970. Polychlorinated biphenyl: interaction with duck
     hepatitis virus. Science 170:1314.
 16.  Vos, J.G., and R.B. Beems. 1971. Dermal toxicity studies of technical  poly-
     chlorinated biphenyls and fractions thereof in rabbits. Toxicol. Appl. Pharmacol.
     19:617.
 17.  Vos, J.G., and T.H. De Roij. 1972. Immunosuppressive activity of a polychlorinated
     biphenyl preparation on the humoral immune response in guinea pigs.  Toxicol. Appl.
     Pharmacol. 21:549.
 18.  Vos, J.6., and L.  Van Driel-Grootenhuis. 1972. PCB-induced suppression of the
     humoral and cell-mediated immunity in guinea pigs.  Sci. Total  Environ. 1:289.
 19.  Vos. J.G., and H.  Genderen. 1973.  Toxicological  aspects of immunosuppression.
     J_n Pesticides and the Environment: A Continuing Controversy.  Edited  by W.B. Deichnu
     International Medical Book.  New York.
 20.  Koller, L.D., and J.E. Thigpen.  1973. Reduction of antibody to pseudorabies virus
     in polychlorinated biphneyl exposed rabbits. Am.  J.  Vet.  Res.  34:1605.
 21.  Loose, L.D., K.A.  Pittman, K.F.  Benitz, and  J.B.  Sillsworth.  1977. Polychlorinated
     biphenyl and hepachlorobenzene induced humoral Immunosuppression.  J. Reticuloendoth
     Soc. 22:253-271.
 22.  Loose, L.D., J.B.  Silkworth, K.A.  Pittman, K.F.  Benitz, and W.  Mueller.  1978.
     Impaired host resistance to endotoxin and malaria in polychlorinated biphenyl-
     and hexachlorobenzene-treated mice. Infect.  Immunity.  20:30-35.
                                                                                18

-------
                                   -16-

23.  Luster, M.I., R.E. Faith, and J.A. Moore. 1978. Effects of polybrominated
     biphenyls (PBB) on immune response in rodents.  Environ. Health Perspect.  23:30-35
24.  Archer, D.L., E.G. Smith, and J.A. Bukovic-Wess. 1978.  Use of an in vitro antibod
     producing system for recognizing potential immunosuppressive compounds.   Int.  Arc
     AllergyAppl. Immunol. 56:90-93.
25.  Vos. J.6., M.J. Van Logten, J.6. Kreeftenberg,  and W. Kruizinga. 1979. Hexechloro
     benzene-induced stimulation of the humoral immune response in rats. N.Y.  Acad.
     Sci. 320:535-550.
26.  Koller, L.D. 1973. Immunosuppression produced by lead,  cadmium and mercury.   Am.
     J. Vet. Res. 34:1457.
27.  Koller, L.D., J.H. Exon, and J.G. Roan. 1976. Humoral antibody response in mice
     after single dose exposure to lead or cadmium.  Proc. Soc. Exp. Biol. Med. 151:335'
28.  Koller, L.D., J.H. Exon, and B. Arbogast. 1977. Methylmercury effect on serum
     enzymes and humoral antibody. J. Toxicol. Environ. Health 2:1115.
29.  Seinen, W., J.G. Vos, R. Van Krieken, A. Penninks, R. Brands, and H. Hooykas.
     1977. Toxicity of organotin compounds. III. Suppression of thymus-dependent
     immunity in rats by di-n-buty Hindi chloride and di-n-octyltindichloride.
     Toxicol. Appl. Pharmacol. 42:213.
30.  Seinen, VI. and A. Penninks. 1979. Immune suppression as a cunseqje.ice of a
     selective cytotoxic activity of certain organometallic compounds on thymus and
     thymus-dependent lymphocytes. N.Y. Acad. Sci. 320:449-517.
31.  Santos, G.W., and A.H. Owens, Jr. 1964. A comparison of the effects of selected
     cytoxic agents on the primary agglutinin response in rats injected with sheep
     erythrocytes. Bull. Johns Hopkins Hospital 114:384.
32.  Turk. J.L., and L.W. Poulter. 1972. Selective depeltion of lymphoid tissue by
     cyclophosphamide. Clin.  Exp. Immunol. 10:285.
                                                                               19

-------
33.  Mansour, A., and D.S. Nelson. 1977. Effect of cyclophosphamide treatment on the
     response of rat peripheral blood lymphocytes to phytohemagglutinin. Cell. Immunol
     30:272.
34.  Speirs, R.S., R.W. Benson, B.J. Knowles, and D.  Roberts. 1979. Models for
     assessing the effect of toxicants on imnsunocompetence 1n mice. II. Effects of
     cyclophosphamide on the antibody responses to Type III pneumococcal poly-
     saccharide and tetanus toxoid in BALB/c female mice.  J. Environ.  Path. Tox.
     1:791-812.
35.  Dean, J.H., M.L. Padarathsingh, T.R. Jerrells, L.  Keys and J.W.  Northing. 1979.
     Assessment of immunobiological effects Induced by  chemicals,  drugs or food
     additives. II. Studies with cyclophosphamide.  Drug Chem. Toxicol.  2:133-153.
36.  Bekesi, J.G., J.F.  Holland, H.A. Anderson, A.S.  Fischbein, W. Rom, M.S.  Wolff,
     and I.J. Selikoff.  1978.  Lymphocyte function of  Michigan dairy farmers exposed
     to polybrominated biphenyls.  Science 199:1207.
37.  Thigpen, J.E., R.E. Faith. E.E.  McConnell, and J.A. Moore.  1975.  Increased
     susceptibility to bacterial infections as a sequela of exposure  to 2,3,7,8-
     tetrachlorodibenzo-p-dioxin.  Infect.  Immun.  12:1319-1324.
38.  Gainer, J.J., and T.W.  Pry. 1972.  Effect of arsenicals on  viral  infections in
     mice. Am.  J.  Vet.  Res.  33:2299-2307.
39.  Festing, M.F.W.  1979.  Properties of inbred strains and outbread stocks,  with
     special reference to toxicity testing.  J. Toxicol. Environ. Health.  5:53-68.
40.  Luster, M.I., J.H.  Dean,  and  J.A.  Moore.  1979. Evaluation  of  immune  functions  in
     toxicology. _In:  Methods  in Toxicology.  Edited  by W. Hays.  Raven Press. New York.
     In press.
41.  Vos, J.G., J.S.  Kreettenberg, H.W.B.  Engel,  A. Minderhouel and L.M.  Van  Noorle
     Jansen. 1978.  Studies  on  2,3,7,8-tetrachlorodibenzo-p-dioxin  induced immune
     suppression and  decreased resistance to infection: endotoxin  hypersensitivity,
     serum zinc concentrations and effects  of thymosin  treatment.  Toxicology  9:75-86.

                                                                             20

-------
42.  Lefford, M.J. 1973. The measurement of tuberculin hypersensitivity in rats.  Int.
     Arch. Allergy Appl. Immupol. 74:570-585.
43.  Dean, J.H., R. Connor, R.B. Herberman, J.L. McCoy, and J.S.  Silva. 1977.
     The relative proliferation index as a more sensitive parameter for evaluating
     lymphoproliferative responses of cancer patients to mitogens and alloantigens.
     Int. J. Cancer. 20:370.
44.  Padarathsingh, M.L., J.L. McCoy, J.H. Dean, D.D. Lewis, J.W. Northing, and
     L.W. Law. 1977. Examination of general and tumor-specific cell-mediated immune
     responses in mice bearing progressively growing plasmacytomas. J. Natl. Cancer
     Inst. 58:1702.
45.  Mancini, G., A.D. Carbonara, and J.E. Heremans. 1965. Immunochemical quantitatit
     of antigens by single radial immunodiffusion. Immunochemistry 2:235-254.
46.  Dean, J.H., M.L. Padarathsingh, and T.R. Jerrells. 1979. Application of immuno-
     competence assays for defining immunosuppression. Ann. N.Y.  Acad. Sci. 320:
     579-590.
47.  Mantoani, A., T.R. Jerrells, J.H. Dean, and R.B. Herberman.  1979. Cytolytic
     and cytostatic activity of tumor cells of circulating human monocytes. Int.
     J. Cancer 23:18-27.
48.  Luster, M.I., G.A. Boormar., M.W. Herris, and J.A. Muore. 1979. Laboratory studi
     on polybrominated biphenyl-induced immune alterations following low-level enrol
     or pre/postnatal exposure. Immunopharmacology, in press.
49.  Luster, M.I., G.A. Boorman, J.H. Dean, and R. Luebke. 1980.  Effects of DES en
     inmune  responses in mice.  I_n_: "The Biological Relevance of Immune Suppression
     as Induced by Genetic, Therapeutic and Environmental Factors," Edited by
     J.H. Dean and M. Padarathsingh, in press.
50.  Luster, M.I., G.A. Boorman, J.H. Dean, M.W. Harris, R.W. Luebke, J.E. Thigpen,
     M.L. Padarathsingh, and  J.A. Moore. 1979. Examination of bone marrow immunolog
     and  host susceptibility  parameters following pre- and postnatal exposure to TC
     Int. J. Immunopharmacol,  in press.
                                                                               21

-------
51.  MacClean. 1979. Host resistance in surgical patients.  J. Trauma 19:297-304.
52.  Cunningham, A.J. 1965. A method of increased sensitivity for detecting single
     antibody-forming cells. Nature 207:1106.
53.  Katz, D.H., M. Faulkner, L.R. Katz, E. Lindh, C.C. Leonhardt, K. Herr, and
     A.S. Tung. 1977. A rapid, semi-automated counting procedure for enumeration of
     antibody-forming cells in gel and nucleated cells in suspension. J. Irrmunol.
     Methods 17:285.
54.  Luster, M.I., R.E. Faith, J.A. McLachlan, and 6.C. Clark. 1979. Effect of in
     utero exposure to diethylstilbestrol  on the immune response in mice.  Tox. Appl.
     Pharmacol. 47:279-285.
55.  McCulloch, E.A. 1979.  Abnormal myelopoietic clones in man.  J.  Nat!. Cancer
     Inst. 63:883-891.
56.  Cline, M.J., and D.W.  Golde. 1979.  Controlling the production  of blood cells.
     Blood. 53:157-165.
57.  Qvesenberry, P. and L. Levitt. 1979.  Hematopoietic stem cells  (Part 3).  N.  Eng.
     J. Med.  301:868-872.
58.  Sensenbrenner, L.L.,  J.J.  Marin,  and  M.  Colvin.  1979.  Comparative effects of
     cyclophosphamide,  isophosphamide, 4-methylcyclophosphamide, and phosphoromide
     mustard on marine  hematopoietic and immunocompetent cells.  J.  Nat.  Cancer Inst.
     62:975-981.
59.  Vistica, D.T., A.  Rabon,  and M.  Robinovitz.  1979.  Amino acid conferred protection
     against melpholon:  comparison of amino acids which reduce melpholon toxicity to
     marine bone marrow precursor cells  (CFU-C)  and murine  L1210 leukemic  cells.  Ros.
     Comm. Chem.  Path.  Pharmacol. 23:171-183.
60.  Delmonte, L. 1978.  Effects of myleron on  murine  hemopoiesis I  and I cell  tissue.
     Kinet. 11: 347-367.
                                                                               22

-------
61.  Botnick.  I.E., E.G.  Hannon,  and S.  Hellman.  1978. Multisystem stem cell failure
     after apparent recovery from alkylating  agents.  Cancer  Res. 38:1942-1947.
62.  Golde, D.W., N. Bersch, and S.G. Quan. 1978.  Trimethoprim and sulphamethoxazole
     inhibition of haematopoiesis in vitro. Br.  0. Haematol. 40:363-367.
63.  Maurer H.R., and R. Henry. 1978. Drug evaluation on haemopoietic cells in vitr
     Arzn-Fors. Drug Res. 28:601-605.
                                                                           23

-------
          Modification of  Cell-Mediated  Immunity by




Polychlorinated Biphenyl  (AroclorR  1016) and Hexachlorobenzene^
                       Jay  B. Silkworth2



                        Leland D. Loose3
                                                                     24

-------
  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

-------
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

-------
                              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

-------
                                           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  (57C, 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 37C (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 37C 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 37C.  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 7C.



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

-------
 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 p0.05.
                                                         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

-------
                                    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
                  p0.05.
                                                                                                   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

-------
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

-------
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

-------
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* elli by cplM* Mils ftc
C57BL/6 aic* fed lthr  control dice, PCI 1016 or 1C! (or 3, 6. 13.
24 md 40 wk 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

-------
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

-------
                                           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

-------
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

-------
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

-------
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

-------
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

-------
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

-------
     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

-------
                              REFERENCES
                                                    t
1.  Friend, M., and D. 0. Trainer.  1970.  Polychlorinated biphenyl:
    Interaction with duck hepatitis virus.  Science.  170:1314.
2.  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. Immun.  12:1319.
3.  Loose, L. D., J. B.  Silkworth, K. A. Fittman, K. F. Benitz, and
    W. Mueller.  1978.   Impaired host resistance to endotoxin and
    malaria in polychlorinated biphenyl  (PCB) and hexachlorobenzene
    (HCB)  treated mice.  Infect. Immun.  20:30.
4.  Loose, L. D., K. A.  Pittman, K. F. Benitz, J. B. Silkworth, W.
    Mueller,  and F. Coulston.   1978.  Environmental chemical-induced
    immune dysfunction.  Ecotox. Environ. Safety.   2:173.  '
5.  Roller, L. D., and J. E. Thigpen.  1973.  Biphenyl-exposed
    rabbits.  Am. J. Vet. Res.  34:1605.
6.  Vos, J. G., and T. De Roij.  1972.   Immunosuppressive  activity of
    a polychlorinated biphenyl preparation on the humoral  immune
    response  in guinea pigs.  Toxicol. Appl. Pharmacol.  21:549.
7.  Vos, J. G., and L. van Dreil-Grootenhuis.  1972.  PCB-induced
    suppression of the humoral  and cell-mediated immunity  in guinea
    pigs.  Sci. Total Environ.  1:289.
8.  Loose, L. D., K. A.  Pittman, K. F. Benitz, and  J. B. Silkworth.
    1977.  Polychlorinated biphenyl and  hexachlorobenzene  induced
    humoral immunosuppression.  J. Reticuloendothel. Soc.   22:253.
                                   -34-
                                                                        67

-------
 9.  Vos, J. G., and H. van Genderen.  1973.  In: Pesticides and the




     Environment:  A Continuing Controversy.  New York, Intercontinental




     Medical Book Corp., P. 527.




10.  Vos, J. G., and J. A. Moore.  1974.  Suppression of cellular




     immunity in rats and mice by maternal treatment with 2,3,7,8




     tetrachlorodibenzo-para-dioxin.  Int. Arch. Allergy.   47:777.




11.  Vos, J. G-, M. J. van Logten, J. G. Kreeftenberg, P.  A. Steerenberg,



     and W. Kruizinga.  1979.  Effect of hexachlorobenzene on the immune




     system of rats following pre- and postnatal exposure.  Drug Chem.



     Toxicol.  2:61.




12.  Luster, M. I., R. E.  Faith, and L.  D. Lawson.   1979.   Effects  of




     2,3,7,8-tetrachlorodibenzofuran (TCDF) on the immune  system in




     guinea pigs.  Drug Chem. Toxicol.  2:49.




13.  Cohen, S., P.  A.  Ward, and P. E. Bigazzi.  1974.   "Cell cooperation




     in cell-mediated immunity."  In:  Mechanism of Cell-Mediated




     Immunity.   McCluskey, R. T., and S.  Conn (Eds.).  New  York,  John




     Wiley and Sons, P. 338.




14.  Cerottini, J.  C., A.  A.  Nordin,  and K. T. Brunner.   1970.   Specific




     in vitro cytotoxicity of thymus-derived lymphocytes sensitized to




     alloantigens.   Nature (London).   228:1308.




15.  Cerottini, J.  C., A.  A.  Nordin,  and K. T. Brunner.   1971.   Cellular




     and humoral response  to transplantation antigens.  1. Development




     of alloantibody forming cells and cytotoxic lymphocytes in  graft-




     versus-host reaction.  J.  Exp.  Med.   134:553.




16.  Katz, D. H.   1977.  In:   Lymphocyte Differentiation,  Recognition,



     and Regulation.  New  York, Academic Press.




                                   -35-




                                                                         68

-------
17.   Cantor, H.,  and R. Asofsky.  1970.  Synergy among lymphoid-cells




     mediating graft-versus-host response.  2. Synergy in graft-versus-




     host reactions produced by BALB/c lymphoid cells of differing




     anatomic origin.  J. Exp. Med.  131:235.




18.   Tigelaar, R. E., and R. Asofsky.  1973.  Synergy among lymphoid-




     cells mediating graft-versus-host response.  5. Derivation by



     migration in lethally irradiated recipients of 2 interacting sub-




     populations of thymus-derived cells from normal spleen.  J. Exp.




     Med.   137:239.




19.  Klein, J.   1975 .  In:  Biology of the Mouse Histocompatibility-2




     Complex.  New York, Springer-Verlag, P. 474.




20.  Bach,  F. H., H. Bock, K. Graupner, E. Day, and H. Klostermann.




     1969.  Cell kinetic studies in mixed lymphocyte cultures - an in




     vitro  model of homograft reactivity.  Proc. Natl. Acad. Sci. USA.




     62:377.




21.  Wilson, D.  B., J. L. Blyth, and P. C. Novell.  1968.  Quantitative




     studies on  the mixed lymphocyte interaction in rats.  J. Exp.




     Med.   128:1157.




22.  Edelman, G. M.  1973.  Antibody structure  and molecular immunology.




     Science.  180:830.




23.  Coutinho, A., E.  Gronowicz, G. Moller,  and H. Lemke.  1976.  "Poly-



     clonal B  cell activators  (PBA)."  In:   Mitogens and Immunobiology.




     Oppenheim,  J. J., and D. L. Rosenstreich  (Eds.), New York, Academic




     Press, P. 173.




24.  Hayry, P.,  L. C.  Andersson, S. Nordling,  and M. Viralainen.  1972.




     Allograft responses Jjj vitro.  Transplant. Rev.  12:91.




                                   -36-





                                                                          69

-------
25.  Michie, D.   1973.  In:  Handbook of Experimental Immunology.




     Weir, D. M.,  (Ed.), Oxford, Blackwell Scientific Publications,



     P.  30.1.




26.  Simonsen, M., and E. Jensen.  1959.  In:  Biological Problems of




     Grafting.  Albert, F.,  (Ed.), Oxford, Blackwell  Scientific



     Publications, P. 214.




27.  Rich, S. S.,  and R. R. Rich.  1974.  Regulatory mechanisms in cell-




     mediated immune responses.  J. Exp. Med.  140:1588.




28.  Asofsky, R., H  Cantor, and R. E. Tigelaar.  1971.   Cell inter-




     actions in the graft-versus-host response.  Prog. Immunol.  1:369.




29.  Brunner, K. T., J. Mauel, J. C. Cerottini, and B. Chapius.  1968.




     Quantitative assay of the lytic action of immune lymphoid cells




     on  ^Cr-labelled allogeneic target cells in vitro;   Inhibition by




     isoantibody and by drugs.  Immunology.  14:181.




30.  Holden, A. V., and K. Marsden.  1969.  Single-stage clean-up of




     animal tissue extracts for organochlorine residue analysis.  J.




     Chromatogr.  44:481.




31.  Hosier, D. E.  1967.  A requirement for two cell types for antibody




     formation in vitro.  Science.   158:1573.




32.  Jannosy, G., and M. F. Greaves.  1972.  Lymphocyte  activation.  I.




     Response of T and B lymphocytes to phytomitogens.  Clin. Exp.




     Immunol.  9:483.



33.  Goldberg, L.  1966.  Liver enlargement produced by  drugs:   Its




     significance.  Proc. Eur. Soc. Drug Toxicity.   7:171.
                                   -37-
                                                                           70

-------
34.  Poland, A., W. F. Greenlee, and A. S. Kende.  1979.  Studies on the




     mechanism of action of the chlorinated dibenzo-p-dioxins and




     related compounds.  In: "Health effects of halogenated aromatic




     hydrocarbons."  Nicolson, V. J., and J. A. Moore (Eds.), NY




     Acad Sci.  320:214.




35.  latropoulos, M. J., A. Milling, W. F. Mueller, G. Nohynek, K.




     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-

-------
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

-------
                            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

-------
                                  - 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  37C 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

-------
                                -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

-------
                               -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

-------
                                 - 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

-------
                              -  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

-------
                                  -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

-------
                                - 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

-------
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

-------
                         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

-------
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

-------
                            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 @37C 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

-------
                              -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 37C, 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-labeld 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 37C-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

-------
                                  -3-
26162                 28564                      30691
26233                 29039                      32254
27034                 29298                      30931
26476+280             28967+215                  31292+496

          t4.71               t4.38
         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 - Jfc ,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 - 37C - 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
          t79.8
         df-4
          P 0.001
         3326+58

         t7.68
        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 +I1697-923  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 37C 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

-------
                              -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 37C - 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 37C-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
                      37C - 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 37c 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 37c - 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 = 12446

CONTROL: (CPM IN SUPERNATANT)

TOTAL COUNT: 7 - 13061 .80

MAXIMUM RELEASE: JT - 1132214

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
34,3',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-3,78-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

-------
                             -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.
                                                                   123

-------
                           -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.
                                                                124

-------
                                              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

-------
             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

-------
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.
                                                                           127

-------
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
                                                                       128

-------
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.
                                                                       129

-------
     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 37C 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
                                                                         130

-------
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 37C 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 4C.
     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

-------
later, 1 ml of organism suspension was given intravenously.   Rabbits
were bled 2 weeks after the intravenous injection.  Serum was prepared
and stored at -20C.  Before use, the serum was heat-inactivated at 56C
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 -4C 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 37C 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 37C 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
37C.  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
                                                                          132

-------
                                   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
                                                                        133

-------
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 37C. 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

-------
                                   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 4C 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

-------
                                   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 -70C 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

-------
                                   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

-------
                                   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

-------
                                   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

-------
                                   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.                                                              '

-------
                                   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

-------
                                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

-------
    106 r
UJ
-
ID
z
ui
o.
i
o
o
io5
   10-5
                                             10-2
                   DILUTION OF  IST ANTIBODY
                           Figure 1

-------
   10'
UJ
CC
UJ
Q.
z  10
D
O
o
   I03
                            1/10
                        x>  I/100
      10
       .-5
                         ^  i/ipoo
                                      - NEW PLATE


                                      -O" AGED PLATE
10"*   .      10"'          10

DILUTION  OF  IST  ANTIBODY
                           -2
                           Figure 2

-------
           I05
         UJ
         i-
         D
         Z
         (O

         Z

         o
         o
           I0
              M
                 \
                     \,
\1
 \
    \
*\
      LI
               I/I     1/2    1/4

                   ANTIGEN GRADIENT
           1/32
           1/64
                         Figure 3
LD

-------
     10*
            LUNG LAVAGES
                                   V
   o
   o
   m

   in
   s
   E
     to2
                 234567


                   WE.EKS, POST-.INFECTION
                        Fioure 4
o

i
o>
o
o
m
-I  tO*"
a
u
             p
   I03
               234567

                 WEEKS,   POST-INFECTION
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

-------
                       TABl3
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)
	(xSE)	

   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

-------
                           Literature Cited
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
the lung on immunity in lung-associated lymph nodes.  Drug  Chem.
Toxicol. 2_: 35-47.

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
test for detection of My co plasma pneumoniae antibody.  Proc. Soc.  Exp.
Biol. Med. 143:97-105.

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
Series 43, pp. 1-21.
                                                                     152

-------
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.
22:1119-1122.
                                                                     153

-------
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.
CRC Rev. Toxic. 5:67-101.
                                                                         154

-------
       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

-------
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

-------
                                -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

-------
                                -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

-------
                                -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

-------
                                -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

-------
                                -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

-------
                                -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

-------
                                 -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-
REFERENCES
      1.  Koller, L.D.  (1979).  Effects of Environmental Contami-
nants on the Immune System.  In:  Advances in Veterinary Science
and  Comparative Medicine.  Vol. 23, edited by B.I. Osburn and
R.D.  Schultz.
      2.  Gainer, J.H., and Pry, T.W. (1973).  Effects of Arsenicals
on Viral Infections in Mice.  Am J Vet Res  33: 2299.
      3.  Koller, L.D.  (1975).  Methylmercury:   Effect on Oncogenic
and Nononcogenic Virus in Mice.  Am J Vet Res   36: 1501.
     4.  Cook, J.A., Marconi, E.A., and DiLuzio, N.R. (1974).
Lead, Cadmium, Endotoxin Interaction:   Effect  on Mortality and
Hepatic Function.  Toxicol Appl Pharmacol  28: 292.
     5.  Cook, J.A., Hoffman, E.O., and DiLuzio, N.R. (1975).
Enfluence of Lead or Cadmium on the Susceptibility of Rats to
Bacterial Challenge.  Proc Soc Exptl Biol Med   150:  741.
     6.  Rippe, D.F., and Barry, L.J.  (1973).   Metabolic Manifesta-
tions of Lead Acetate Sensitization to Endotoxin in Mice.   J
Reticuloendothel Soc  13: 527.
     7.  Hemphill,  R.E., Kaeberle, M.L.,  and Buck, W.B.  (1971).
Lead Suppression of Mouse Resistance to Salmonella typhinurium.
Science  173: 1031.
     8.  Exon, J.H., Koller, L.D., and Kerkvliet,  N.I.  (1979).
Lead-Cadmium Interaction:  Effects on  Viral-Induced  Mortality  and
Tissue Residues in Mice.  Arch Environ Health.  In press.
                                                               165

-------
                               -11-
     9.  Gainer, J.H.  (1972).  Increased Mortality in Encephalo-
myocarditis Virus-Infected Mice Consuming Cobalt Sulfate:  Tissue
Concentrations of Cobalt.  Am J Vet Res  33: 2067.
     10.  Roller, L.D.  (1973).  Immunosuppression Produced by
Lead, Cadmium and Mercury.  Am J Vet Res  34: 1457.
     11.  Koller, L.D., and Exon, J.H. (1977).  Methylmercury:
Effect on Serum Enzymes and Humoral Antibody.  J Toxicol Environ
Health  2: 1115.
     12.  Koller, L.D., and Kovacic, S. (1974).  Decreased Anti-
body Formation in Mice Exposed to Lead.  Nature  250: 148.
     13.  Luster, N.I., Faith, R.E., and Kimmel, C.A. (1978).
Depression of Humoral Immunity in Rats Following Chronic Develop-
mental Lead Exposure.  J Environ Path Toxicol  1: 397.
     14.  Koller, L.D., Exon, J.H., and Roan, J.G. (1975).  Anti-
body Suppression by Cadmium.  Arch Environ Health  30: 598.
     15.  Bozelka, B.E., Burkholder, P.M., and Chang, L.W. (1978).
Cadmium, a Metallic Inhibitor of Antibody-Mediated Immunity in
Mice.  Environ Res  17: 390.
     16.  Ohi, G., Fukuda, M., Seto, H., and Yagyu, H. (1976).
Effect of Methylmercury on Humoral Immune Responses in Mice under
Conditions Stimulated to Practical Situations.  Bull Environ
Contamin Toxicol  15: 175.
     17.  Koller, L.D., Exon, J.H., and Brauner, J.A. (1977).
Methylmercury:  Decreased Antibody Formation in Mice.  Proc Soc
Exp Biol Med  155: 602.
                                                                166

-------
                              -12-
     18.  Koller, L.D., and Brauner, J.A. (1978).  Decreased B-
Lymphocyte Response after Exposure to Lead and Cadmium.  Toxicol
Appl Pharmacol  42: 621.
     19.  Koller, L.D., and Roan, J.G. (1979).  Effects of Lead,
Cadmium and Methylmercury on Immunological Memory.  Submitted for
publication.
     20.  Garworski, C.L., and Sharma, R.P.  (1978).  The Effects
of Heavy Metals on  H Thymidine Uptake in Lymphocytes.  Toxicol
Appl Pharmacol  46: 305.
     21.  Koller, L.D., Roan, J.G., and Kerkvliet, N.I. (1979).
Mitogen Stimulation of Lymphocytes in CBA Mice Exposed to Lead
and Cadmium.  Environ Res  19:  177.
     22.  Faith, R.E., Luster,  N.I., and Kimmel,  C.A. (1979).
Effect of Chronic Developmental Lead Exposure on Cell Mediated
Immune Function.  Clin Exp Immunol  35: 413.
     23.  Trejo, R.A., DiLuzio, N.R., Loose,  L.D., and Hoffman,
E. (1972).  Reticuloendothelial and Hepatic  Functional Alterations
Following Lead Acetate Administration.  Exp  Mol Path  17:  145.
     24.  Koller, L.D., and Roan, J.G. (1977).  Effects of Lead
and Cadmium on Mouse Peritoneal Macrophages.   J Reticuloendothel
Soc  21: 7.
     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

-------
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
IMSJSSI^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 2lth 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 2lth 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



     carcinogenesis.  J. Nat'l. Cancer Inst.  31:791-805.



 1*.  Rubin, B. A.  Carcinogen-induced tolerance to homo transplantation.



     Prog. Exp. Tumor Res. 5:217-292.



 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.



 7.  Stutman, 0.  1975.  Iramunodepression and Malignancy.  Adv. Cancer



     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.




                                                                             193




                                    - 14 -

-------
11.  Rabat, E. A, and M. M. Mayer.  Experimental Immunochemistry.  Springfield,



     111.  C. C. Thomas, 1961. p.133.



12.  Decker, G. P., F. W. Chorpenning and G. T. Frederick.  1972.  Naturally



     occurring antibodies to bacillary teichoic acid.  J. Immunol. 106:2lU-222.



13.  Kilham, L. and L. J. Oliver.  1959.  A latent virus of rats in tissue



     culture.  Virology 7:U28-l437.



ll*.  Golub , E.  1972.  The Distribution of Brain-Associated%rtigen Cross



     Reactive With  Mouse   in the Brain of Other Species.  J. Immunol.



     I09i 168-170.



15.  Bianco, C., R. Patrick and V. Niessenzweg.  1970.  A population of



     lymphocytes bearing a membrane receptor for antigen-antibody-complement



     complexes.  I.  Separation and Characterization.  J. Expt'l. Med.



     132:702-720.



16.  Oppenheim, J. J. and R> senstreich, D. L.  1976.  Lymphocyte Transformation:



     Utilization of Automatic Harvesters.in In Vitro Methods in Cell-Mediated



     and Tumor Immunity. (B. R. Bloom and J. R. David, eds.)  New York,Academic



     Press,  pp. 573-585.



17.  Nathan, C. F. , M. L. Karnowsky and J. R. David. 1971.  Alterations of



     Macrophage Functions by Mediators From Lymphocytes.  J. Expt'l. Med.



     133:1356-1376.



18.  Schmidtke, J. R. and R. L. Simmons.  1975.  Augmented Uptake of



     neuraminidase Treated Sheep Red Blood Cells.  Participation of



     Opsonic Factors.  J. Nat'l. Cancer Inst. 5U:1379-138U.



19.  Simon, H. and J. N Sheagren.  1972.  Migration inhibitory factor and



     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^



                                    -  15 -

-------
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




     response of activated macrophages to  soluble products of neoplastic cells.




     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 -

-------
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 -

-------
                                    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 CDFi 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                     47SM1S27                               10074 34t             71       <0001
NONE	247 17	110*32	-	-
                                                DAY 3
PHA                     2110*100                                1017*140             S2
CONA                   24071*3004                                30072M             00        <0.01
LPS                     20031 122t                                3031 431             00        <0.001
NONE	208*21	03 10	-	-
                                                DAYS
PHA                       00701                                  40743             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
440M13M
299 7
132917V
571ft 241
147 BS
 
n
>t.M
<0.001
DAVE
PHA
CONA
NONE
1033 132
314M2609
274 90
1221 ill*
10S73 407
194 1 12
 it
c
>0.10
<0.002
DAY 10
PHA
CONA
NONE
7M7t10t7
HO*
M37
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     3GM47        77

            tP8       B77442BB7     3M)1M        M
                                                                                                  oo
            NONE       2332ia      317 1                                                         I

MEANiSEM OF TRIPUCATE DETERMINATIONS.
                i /MEAN OF TRIPUCATE DET'NS INDOMETHACIN TREATED LVMPHO\^.^
               . 1 ^  MAN 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       3M2232     2MSS4M        21

  LP8       34M12S7
 NONE       7D3i      10M40        -                                                '
'MEANtSEM OF TRIPLICATE DETERMINATIONS.

^PERCENT INHIBITION - 1 - fMEAN OF TRIPLICATE DET'NS INDOMETHACIN TREATED LYMPHOlL t
                     MEAN OF TRIPLICATE DET'NS UNTREATED LYMPHOCYTES   /
                                                                                      i

-------
                                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
                                  -  10 -

-------
 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
                            -1-

-------
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.

                                                           208
                            -2-

-------
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?
                                      -3-
                                                                       209

-------
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
                                      -4-

-------
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.
                                     -5-
                                                                      211

-------
                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.
                                                             212

-------
                              -2-

     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.
                                                             213

-------
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.
                                                             214

-------
           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
T


-------