DRAFT SURVEY AND EVALUATION OF IN VITRO

         TOXICITY TEST METHODS


            AUGUST  1976
             PREPARED FOR
      OFFICE OF TOXIC SUBSTANCES
 U.S. ENVIRONMENTAL PROTECTION AGENCY
         WASHINGTON, DC  20460

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EPA 560/5-75-007
                 DRAFT . SURVEY AND EVALUATION, PF__IN__VITRO_

                          TOXICITY TEST METHODS
                                 AUTHOR

                         GEOFFREY WOODARD,  Ph.D.


                            PROJECT OFFICERS

                         Dr. Lawrence Roslinski
                         Dr. Michael J. Prival
                        CONTRACT NO.  68-01-1895
                              August  1976
                              PREPARED FOR

                       OFFICE OF TOXIC SUBSTANCES

                     ENVIRONMENTAL PROTECTION AGENCY

                          WASHINGTON, DC  20460

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This document is available to the public through the
      National Technical Information Service
           Springfield, Virginia  22151

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This draft report has been reviewed by the Office
of Toxic Substances, EPA, and approved .for publi-
cation.  Approval does not signify that the con-
tents necessarily reflect the views and policies
of the Environmental Protection Agency, nor does
mention of trade names or commercial products
constitute endorsement or recommendation for use.

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                          TABLE OF CONTENTS
     ABSTRACT	.	   v

     ACKNOWLEDGMENTS	vii

  I. CONCLUSIONS	   1

 II. INTRODUCTION 	   3

III. METHODS EMPLOYED, FOR SURVEY	11

 IV. REVIEW EVALUATIONS	13

     A.  USE OF FERTILIZED EGGS IN STUDIES ON CHEMICALS . .  13

         Introduction 	  13

         Sources	13

           Avian Eggs	13

   !      Temperature and Humidity 	  14

         Turning of Eggs	14

         Time and Route of Administration	14

         Candling Procedures	14

         Stage of Development	15

         Site of Injection	16

         End Points	17
                                                            0
           Mortality
           Growth and Development
           Physiological Activity

         Correlation of In Vitro With In Vivo Methods ...  17

           Invertebrate Eggs	18

     B.  USE OF ISOLATED ORGANS AND TISSUE IN STUDIES ON
         CHEMICALS	.' . 21
                               -1-

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                     TABLE OF CONTENTS (cont.)
    Introduction	21

    Liver	21

    Brain	22

    Trachea	23

    Lung	<	25

    Kidney	26

    Intestine	28

    Adrenal Gland 	 31

    Heart	.(	32

    Bone	:  . 36

    Skin	37

    Striated Muscle 	 38

    Smooth Muscle 	 40

    Nerve	42

    Epididymal Fat Pad	43

    Discussion	44
                                   /
B.  USE OF MAMMALIAN AND AVIAN CELL CULTURE SYSTEMS
    IN STUDIES ON CHEMICALS	 45

    Introduction	45

      Cell Culture Methodology	45

    Human Cell Culture Systems	45

      Human Carcinoma Cell Culture Systems	45

      Human Lung Cell Systems	48

      Human Leukocyte Cell Systems	49

      Human Erythrocyte Cell Culture Systems	51
                         -ii-

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                     TABLE OF CONTENTS (cont.)


                                                       Page
      Human Liver Cell Systems	51


    Non-Human Cell Culture Systems	52


      Hamster Carcinoma Cell Systems	52


      Mouse Cell Culture Systems	60

      Rat Cell Culture Systems	69


      Rabbit Cell Culture Systems	76

      Monkey Cell Culture Systems	78


      Chicken Embryo Cell Culture Systems 	 79

    Miscellaneous Cell Culture Systems	81


      Rat Kangaroo	; . 81

      Guinea Pig	81

      Pig	81


    Discussion	82

D.  USE OF BACTERIA, FUNGI, PROTOZOA, AND PLANT CELLS
    IN STUDIES ON CHEMICALS	85

    Introduction	85

    Bacteria	85

      Growth Inhibition 	 85

      Potentiators	87

      Antitumor Compound Screens	88


    Fungi.	89

      Growth Inhibition 	 90


      Genetic Damage in Saccharomyces 	 90
                                         <
      Genetic Damage in Other Fungi 	 92


    Protozoa	93


                        -iii-

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                          TABLE OF CONTENTS (cont.)

                                                            Page

         Plants	96

           Growth Inhibition 	 96

           Effects On Chromosomal Material 	 97
                i
           Algae Cells	98

         Discussion	99

APPENDIX	A-l

     REFERENCES
                               -iv-

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                               ABSTRACT
The English language literature for the period 1954 to .May 1974 has been
searched by several methods including a hand search, a computer title
search, contacts with scientists currently engaged in related research,
and bibliographic references contained in individual papers for articles
in which in vitro methods were used to detect, define, and/or quantitate
biological effects of chemicals.  Copies of these articles were obtained
and reviewed under the following groupings:

(1)  Use of Fertilized Eggs in Studies on Chemicals

(2)  Use of Isolated Organs and Tissue in Studies on
     Chemicals

(3)  Use of Mammalian and Avian Cell Culture in Studies
     on Chemicals

(4)  Use of Bacteria, Fungi, Protozoa, and Plant Cells
     in Studies on Chemicals

An attempt has been made to include all systems that have been used
within these headings to assess the biological activity of chemicals.
Where such information was available, the applicability of those in
vitro test systems has been evaluated.
                               -v-

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                            ACKNOWLEDGMENTS
The following individuals have contributed to the early direction of
this review and/or to a critique of the four draft monographs:

John Autian, Ph.D.
Director, Materials Science Toxicology Laboratories
University of Tennessee

James R. Gillette, Ph.D.
Chief, Laboratory of Chemical Pharmacology
National Heart and Lung Institute

A. Wallace Hayes, Ph.D.
Associate Professor of Biochemistry and Microbiology
The University of Alabama

Frank M. Hetrick, Ph.D.
Professor, Department of Microbiology
University of Maryland

Roland M. Nardone, Ph.D.
Professor of Biology
The Catholic University of America

M. Jacqueline Verrett, Ph.D.
Biochemist
Food and Drug Administration

The contributions of these individuals have been invaluable.

The following personnel from Woodard Research Corporation were involved
in the preparation of the draft monograph:

	Name   	     Scientific Discipline

Marvin J. Bleiberg, Ph.D.      Pharmacologist
James F. Novotny, Ph.D.        Microbiologist
Vera M. Piper, M.S.            Microbiologist
Richard R. Thacker, B.S.       Etholegist
Marie W. Woodard, M.S.         Toxicologist
                               -vii-

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                                   I

                              CONCLUSIONS
In vitro methods have been reported for the assessment of nearly every
known manifestation of toxicity of a chemical.  The endpoint in the in
vitro system, however, is rarely descriptive of the toxic signs noted in
the whole animal.  Thus, in vitro systems are valuable tools for screen-
ing large numbers of chemicals for a single toxic manifestation, e.g., a
carcinogen.  On the other hand, to define the toxic properties of a
single chemical substance would require so many in vitro systems as to
be impractical.

In_ vitro test systems have proven very valuable tools in the study of
mechanisms of action, in drug metabolism, and in defining nutritional
and hormonal requirements.

In vitro systems have served as useful analytical methods for the con-
trol of potency of antibiotics and for detection of antibiotic residues
or antibiotic contamination.

In the field of toxicology, limited uses of in vitro methods have been
employed in areas such as evaluation of plasticizers and other com-
ponents of medical devices fabricated from polymeric materials.  The use
of fertilized eggs has enjoyed some attention in studies of teratogenic
potential of chemicals, but this method is still regarded by toxico-
logists as of marginal usefulness. The many methods that are being
proposed for evaluation of both mutagenic and carcinogenic potential
have yet to be shown to be sufficiently predictive to be recommended as
routine screening procedures.

A serious problem arises in the use of in vitro systems to arrive at any
reliable estimate of a quantitative expression of toxicity.  Concentra-
tions shown to be effective in in vitro systems in producing a measur-
able endpoint are often two or three orders of magnitude higher than can
be shown to be effective in whole animal systems.  A cytotoxic effect of
a chemical at these exaggerated concentrations compared with real life
is viewed by the toxicologist with considerable reservation.

Iri vitro systems results, for the most part, reflect the acute or single
dose effects of a given chemical.  Aside from the possible utilization
of in vitro systems for mutagenic and carcinogenic screening, such
systems have not been shown to be very useful in predicting the results
of the long-term administration of a chemical.  The usefulness of such
systems in predicting such toxic manifestations as eczematous sensitiza-
tion, hypertension, chronic kidney disease, cardiovascular disease,
blood lipid deposition in the vascular system, cataracts and other eye
defects, eighth cranial nerve damage, reproductive disorders, and cir-
rhosis of the liver is yet to be demonstrated.
                                 — 1 —

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                                  II

                             INTRODUCTION
A number of events in the past several years has alerted first the
scientific community and subsequently concerned citizens'  groups to
possible hazards associated with the widespread distribution of syn-
thetic and industrial chemicals in the environment.  Although Rachel
Carson's book "Silent Spring" (1) contains a number of excessive and
exaggerated statements, it did serve to alert the general public to the
potential hazards that could arise from the continued heavy and unre-
stricted use of DDT.  Since the publication of this book,  accounts of
other chemicals that may be producing long-term subtle effects have
appeared in the news media and include such chemicals as the polychlor-
inated biphenyls, the dioxins, the halogenated insecticides such as
aldrin, dieldrin, mirex, and hexachlorobenzene, arsenic, cadmium,
mercury, and most recently a widely used industrial chemical, vinyl
chloride.  Less publicized have been adverse findings with bischloro-
methyl ether, benzidine, and ethylene dibromide.

The above-mentioned chemicals are not newly synthesized chemicals.  They
have been around for years and some biological data on each of them were
available long before the chemical became notorious.  How then did the
"toxicological surprise" arise?  The answer in each case is that the
growth in the use of the chemical far surpassed the growth in the
understanding of potential toxicological and environmental hazards.
Increased use levels place many more populations at risk.   In a few
instances, use of the chemical has become so worldwide that all existing
animal and plant life are at risk.  Thus a very sensitive organism may
disappear forever.

Why does the understanding of the toxicology of a compounds lag so far
behind its expanded use?  Among the answers to this question are:

        Costs to conduct valid long-term experiments in animals
        are prohibitive.

     -  Time required to conduct such experiments is excessive.

     -  The sensitivity of the animal model test system is poor.

        In a test system as complicated as a whole animal, a single
        non-lethal effect may be obscured by massive amounts of
        extraneous normal biological variation.

     -  Unique expressions of toxicity continue to appear that
        previously developed protocols failed to detect.
                                 -3-

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An alternative approach to the study of the toxic properties of a
chemical that has occurred to many investigators is to make use of in
vitro test systems.  Thus, conventional animal model test systems could
be eliminated in whole or in part by the substitution of, or supple-
mentation by, in_ vitro test systems.  The desired goals of reduction in
costs, more rapid evaluation, improved sensitivity, increased test
population numbers, and diversification in endpoints are believed to be
achievable using in vitro systems.

The ,purpose of this survey is to determine, based upon a review of the
published literature and of ongoing research efforts, the strengths and
weaknesses of in_ vitro systems as test methods for the assessment of
toxicological hazards.

For purposes of this review, in vitro test systems have been confined to
the following:

     -  Fertilized Eggs

     -  Isolated Organs and Tissues

     -  Mammalian and Avian Cell Culture
                                 i

     -  Bacteria, Fungi, Protozoa, Plant Cells

Historically, investigators have been attracted to in vitro test systems
because of one or more of the following considerations:

  !   -  Apparent simplicity of an in vitro system.

     -  The possibility of using cells or tissues from the
        species potentially at risk, thus avoiding the uncer-
        tainties in extrapolating from.one species or strain
        to another.

     -  The possibility of conducting experiments on vast
        numbers of organisms as opposed to small numbers
        of whole animals.

        The possibility of taking advantage of rapid cell
        turnover rates so that many successive generations
        can be observed over a relatively short period of
        time.

     -  The ease of investigating a large number of bio-
        chemical reactions taking place in pure cell cul-
        tures or in isolated organs or tissues composed
        of relatively few cell types.

However, the characteristics of in vitro systems that have attracted
investigators have also been responsible for the limited value that
toxicologists have placed upon the findings in such systems.  In more
                                 -4-

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recent years, the investigators using in vitro systems are finding that
their apparent simplicity is deceptive.   It is now realized that the
cell at risk as a result of exposure to a chemical is often a cell which
is affected by a metabolite of the chemical or by secretions of other
cells in response to chemical exposure.   The in vitro cell system under
study may not be affected in any measurable way by the parent chemical.
It is also being realized that in the intact animal,  the concentration
of a chemical at a particular site is determined by the interaction of a
large number of dynamic processes, along with the physiochemical prop-
erties of the compound.  These include the following:

     -  Rate of absorption from the site of entry.

     -  Protein binding, binding to formed elements in
        the blood.

     -  Locus and rate of metabolic conversion.

     -  Routes and rates of excretion from the body of
        both the parent compound and its metabolites.

     -  Oil/water partition coefficient.

     -  pKa value for parent compound and its metabolites.

        Membrane diffusion characteristics.

     -  Site of tissue specific binding.

In any given in vitro test system, only one or two of the above factors
are operative so that effects that may be observed in vitro may not
reflect accurately the effects that will be observed in the intact
animal.  On the other hand, the absence of these interacting factors can
be taken advantage of in the development of in vitro bioassay systems
for the comparison of large numbers of closely related chemicals.

In any review of in vitro test systems,  it should be remembered that the
classical work of Otto Loewi beginning in 1921 (2) demonstrating that
acetylcholine is a neurotransmitter was carried out using isolated
perfused frog hearts.  The production of abnormalities in the developing
embryo as a result of the administration of thallium was first demon-
strated by Karnofsky in 1950 using the fertile chicken egg  (3).

There is an urgent current need for valid, reliable,  and simple in vitro
methods that will be predictive not only of teratological, mutagenic,
and carcinogenic effects but also of irreversible organ damage such as
emphysema, cardiovascular disease, nephritis, and myelin degeneration.
Chronic collagen disease and the auto-immune manifestations should be
subject to early detection also.  Effects of chemicals on mineral metab-
olism, endocrine secretion, and cellular uptake of essential substrates
have received some study in in vitro systems  (Section IV).  Increased
                                  -5-

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efforts in this area should improve materially our understanding of the
potential environmental hazards of widely dispensed chemical,  particularly
on the reproductive processes.

Three recent communications (4, 5, 6) provide some insight on the complex
considerations involved in the design, conduct, and most importantly the
interpretation of the results of in vitro systems which attempt to
predict carcinogenicity.  The following quotes seem of particular impor-
tance :

1.   "... as chairman of the AAAS Committee on Scientific
     Freedom and Responsibility, John Edsall is in a position
     to influence public policy ...  He notes in his letter
     of 18 July 1975 the finding that some carcinogens are
     mutagenic in bacteria.  This has been interpreted to
     mean that those carcinogens cause cancer by somatic
     mutation and has been taken by many as support for the
     venerable hypothesis that the malignant transformation
     of cells is a mutational event.  In addition, the screen-
     ing of compounds for their capacity to cause bacterial
     mutations has been adopted by a number of laboratories
     as a means of indicating carcinogenic potential.  The
     implied relation between mutagenesis and carcinogenesis
     still needs careful scrutiny with regard to its scien-
     tific validity and also because of its implications for
     public policy ...

     "Acceptance of screening for carcinogenicity by deter-
     mining mutagenicity lends tacit support to the hypothesis
     that malignant transformation of cells is caused by so-
     matic mutation.  This hypothesis has been tested expli-
     citly in several experiments and has been found wanting
     in each case." (4)

2.   "On the basis of our work, and the lines of evidence noted
     below, I find compelling the theory that chemical carcino-
     gens cause cancer through damage to DNA (somatic mutation).

     1)   It is known that cell regulation can be easily
          altered by mutation and that a heritable change
          in cell regulation is a characteristic property
          of a cancer cell.

     2)   The theory is simple and accounts for what is
          known about the molecular biology of cancer and
          chemical and radiation carcinogenesis.

     3}   Studies on the genetics of cancer suggest this
          idea.

     4)   Human mutants exist who are extremely prone to
          cancer and who lack DNA repair systems.
                                  -6-

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     5)    There is a correlation between capacity for
          repair of DNA damage and the occurrence of
          organ specific cancer.

     6)    Active forms of many carcinogens are electro-
          philes capable of interacting with DNA.

     7)    Ninety percent of carcinogens are mutagens;
          it is our hypothesis that the aromatic part
          of aromatic carcinogens is involved with
          stacking interactions in DNA, causing frame-
          shift mutations.

     8)    The well-known human carcinogen asbestos has
          recently been shown to be an efficient breaker
          of chromosomes . . .

     "It is likely that mutation (imitation) is not the only
     cause of cancer and that a few environmental chemicals may
     well work through other mechanisms; however, there is not
     much evidence that such environmental chemicals (or even
     viruses) are contributing in a major way to human cancer.
     Rather, the evidence indicates that chemicals are radia-
     tions in the environment (cigarette smoke, ultraviolet
     light, nitrosamines, and so forth) damage DNA and that
     this damage, incurred throughout our lifetimes, is the
     initiator of most of human cancer.  DNA damage is quite
     likely to be a major contributor to birth defects, aging,
     and heart disease as well." (5)

3.   "... The most subtle of these genetic changes, and yet
     the one likely to have the most far-reaching consequences
     for man, is point mutation, an alteration in one of the
     nucleotide bases of the DNA .  . .

     "In vivo testing in mammals probably has the greatest rel-
     evance to the human situation, but tests such as the spe-
     cific locus test in the mouse require enormous numbers of
     animals and are consequently prohibitively expensive and
     time-consuming for the routine testing of drugs and chemi-
     cals.  In addition they are less sensitive than microbial
     tests.  Other mammalian tests, including the dominant lethal
     assay, and tests using Drosophils detect gross chromosomal
     damage rather than point mutations.

     "Bacterial tests offer many advantages over mammalian sys-
     tems in that they are far cheaper, very rapid  (the results
     are obtained usually within two days) and extremely sensitive.
     Indeed microbial tests probably represent the only system
     suitable for screening large numbers of chemicals for muta-
     genic activity.  It has been suggested that the wide phylo-
     genetic difference between bacterial and mammals precludes
                                 -7-

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the use of the former in mutagen testing, but this objection
may be countered by the fact that the target molecule of
mutagens is DNA, which is substantially the same whether the
cell is eukaryotic or prokaryotic.

"Most of the bacterial tests for detecting mutagens utilize
bacteria bearing a mutation in a gene involved in the syn-
thesis of a particular amino acid; hence the bacteria (called
auxotrophs) require an external supply of the amino acid
for growth.  Spontaneously, and at low frequency, some of
the bacteria revert, or back-mutate, to the wild-type, able
to grow on media lacking the amino acid.  Mutagens can in-
duce such back mutations and so an increase in the number
of revertants over the number of spontaneous back mutations
can be used as an index of the mutagenicity of the compound
under test.  The best known of these back-mutation systems
are the histidine-requiring mutants of Salmonella tiphimurium
developed by Ames and the tryptophan mutants of Escherichia
coli described by Bridges.  In both systems the sensitivity
of the test has been increased by utilizing derivatives of
the tryptophan or histidine auxotrophs deficient in one or
more of the mechanisms by which the bacterial cells repair
damage to DNA.  The most useful of these are uvr strains.

"As it stands, this test has several disadvantages, not
the least of which is the considerable difference in per-
meability between bacterial and the mammalian cells.  In
the bacterial test, a negative result may be due not to a
lack of mutagenic activity on the part of a chemical but
to its inability to penetrate the cell wall and membrane
and reach the DNA within the bacterium.  To combat this,
Ames e_t al. obtained deep rough derivatives of S_. typhi-
murium strains, the cell walls of which were deficient in
lipopolysaccharide components and were thus more permeable
to various mutagens, including dibenz(a,h)anthracene.

"Perhaps, the most important drawback of the bacterial test
described above is that it does not take into account the
major differences in metabolism between mammalian cells and
bacteria  (e.g., the cytochrome P-450 drug-metabolizing sys-
tem of mammalian liver, a system which has no counterpart
in the bacterial cell).  Thus a non-mutagenic chemical me-
tabolized by such enzymes to an active mutagenic species
will not be detected by the simple bacterial system.  (Ob-
viously the converse is also true —a mutagen inactivated
by mammalian enzymes will give a  'false positive1 when
tested with bacteria.)  Thus mutagens such as acetylamino-
fluorene and dimethyInitrosamine, which are not mutagenic
per se but require activation by liver enzymes, do not revert
the histidine or tryptophan auxotrophs.  This problem has
been partially overcome by two methods, the host-mediated
assay and the activation of mutagens by liver homogenates
                             -8-

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"The second approach to the problem of mutagen activation,
the use of liver homogenates or microsomal fractions, looks
more promising.  This group (Ames group)  showed that sev-
eral compounds that do not normally mutate £. typhimurium
in_ vitro, will do so when a rat-liver homogenate is incor-
porated into the agar plate.  Unfortunately, this investi-
gation was confined to a very restricted range of mutagens,
mostly polycyclic aromatic hydrocarbons,  so it is not cer-
tain how well the system responds to other types of mutagen,
although dimethyl- and diethylnitrosamines are known to be
genetically active by this method.  One of the major prob-
lems with this method is the choice of animal for use as the
source of the liver extract.  Mailing & Frantz have shown
that for dimethylnitrosamine there is a remarkable differ-
ence in metabolic activity not only between liver homogenates
derived from the rat and mouse but also between those from
different strains of mouse.  Furthermore, the method does
not take into account compounds activated by enzymes other
than those associated with the liver.  To be certain of
detecting such mutagens, homogenates of other organs, such
as the lung, intestinal epithelium and kidney, would also
have to be used ...

". . .to ensure that all environmental mutagens are de-
tected, we may have to face the daunting task of using
many different strains of bacteria in combination with
liver homogenates from a variety of animals — besides
the possible use of homogenates of other organs . . .

"In spite of the limitations discussed in this article,
microbial mutagenicity tests are obviously the only means
available for screening large numbers of chemicals and
drugs for mutagenic activity, since in terms of speed and
sensitivity they are vastly superior to in vivo tests in
mammals ...

"Obviously, for a full definition of the mutagenic poten-
tial of a chemical it is necessary to determine many more
facts, such as the mutagen concentration at the site of
action, the active metabolic products and their rate of
absorption, metabolism and elimination."  (6)
                             -9-

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                                  Ill

                      METHODS EMPLOYED FOR SURVEY
In general, the survey covers the English language literature from 1954
through May 1974.  A few key publications have also been included that
have appeared since that time.

The literature review was accomplished through a hand search of more
than 20 of the most widely used journals in toxicology, pharmacology,
and cell biology; a computer title search; contacts with those having
ongoing research listed with the Smithsonian; and attendance of a number
of scientific meetings during 1973-1974 at which in, vitro methodology
was discussed.  Additional references were reviewed based upon biblio-
graphic citations in individual papers.  Finally, the outside experts
that were involved either in the early direction of the review or in
the review of the draft monographs provided a few references that had
escaped the attention of the survey team.
                                 -11-

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TEMPERATURE AND HUMIDITY

Environmental variables need to be standardized.  Temperatures of
incubation are stated in papers as  F or  C and range from 35 C (17) to
38 C (18).   In most instances incubation temperatures range from 37 C to
38 C.  Abbott and Craig (19) have emphasized optimal conditions required
for incubation of chick eggs for maximum hatchability.  They state that
differences in temperatures are required in still-air (102-103 F)  versus
forced draft incubators (99.8 F).

The rate of development may be dependent on environmental temperatures.

Humidity is a variable which is seldom recorded.  Reported values range
from 65% (20) to 86-90%.
TURNING OF EGGS

Another condition of incubation not generally explicitly stated in
methods section of papers is turning eggs several times daily.  Abbott
and Craig state that eggs must be separately hand ..turned or the rack may
allow for turning entire trays at once.  Among the few studies listing
this parameter specifically are Walker (7), Pagnini, et^ al_. (21), and
Mawhinney, et al. (22).  This is important for the proper hatching of
the chick.  However, shaking or jarring eggs before incubation is stated
to produce 60% mortality between the second and third days (Hamilton,
23),  This is said to result from a failure of the vitelline vessels to
organize out of the blood islands.
TIME AND ROUTE OF ADMINISTRATION

Time and route of administration of test materials are important vari-
ables.  Time of administration ranges from pre-incubation injection (8,
9, 14, 16, 24, 25, 26, and 27; McLaughlin, e_t al. 1963 (28) to injection
at mid-incubation (15 days).  Injection before incubation has been
stated to yield excess mortality.  Khera and Lyon (16) had an average
survival of 48% during the first four days of incubation after pre-
incubation injection, as compared with 75-95% survival of eggs injected
after four or more days of incubation via the yolk sac using propylene
glycol as a solvent.  The high mortality noted by Khera and Lyon at pre-
incubation injection contrasts with the low mortality observed by Verrett
(personal communication).  The high mortality at pre-incubation injection
reported by some may be a result of too vigorous twisting or turning of
the egg with a disturbance of the blood islands as described in the
preceding section.  Khera and Lyon (16) attribute this to differences
between strains of eggs.

CANDLING PROCEDURES

Candling  (transillumination) of eggs at regular intervals is done to
evaluate the viability of the embryo.  Intervals practiced vary as
follows:


                                 -14-

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     .  Daily (7, 14, 29)

     .  Fifth day and daily thereafter
        (8, 9, McLaughlin)

        Regularly, interval not stated
        (20, 30, 31)

     .  On days 4 and 6  (24)

        Day after injection, days 5-6, and
        every 1-4 days (16)

     .  On days 7, 14, 18  (32)

     .  On day 5 and 10  (33)

Several investigators prepared "Scotch Tape" (12, 34, 35) or coverslip
windows (36, 37) for direct observation of the embryo at injection and
afterward.
STAGE OF DEVELOPMENT

Injections given at the pre-incubation stage may interfere with the
initial processes of embryogenesis.  Studies which use eggs pre-incubated
for various periods of time therefore avoid chemical effects on initial
stages of embryonic development.  It is very common to inject at 3-4
days of development (30, 24, 16, 32, 20, 21, 38, 39, 33, 40, 41, 11, and
22).

The state of the embryonic development of the chick at the time of
injection may be described by reference to the 44 stages as listed by
Hamberger and Hamilton  (42).  Reference to number of somites (43) or a
feature of development  (44) at the time of injection is infrequent.  At
four days the vascular system and internal organs have formed in the
chick; however, formation of bone has not yet begun.  Embryogenesis of
the chick is much more rapid than of the rat at corresponding gestation
age, although the gestation and incubation periods are comparable.  Thus
the closing of the neural fold in the chick occurs at one day, as com-
pared to 10 days in the rat.  Liver and pancreas appear in three days as
compared with 12 days in the rat.

Many studies have used the 8-15 day incubation period (25, 45, 20, 46,
14, 29, 47, 17, 15, 40, 11, and 48), in most instances as part of a
series (16, 25, 20, 14, 17, 49, 40, 11, and 12) to compare toxicity.

Other pre-incubation time intervals prior to injection that have been
used in studies include 24-48 hours (24, 20, 34, 36, 33, and 50); 49-72
hours  (7 and 35), and 5-7 days  (51 and 22).
                                 -15-

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SITE OF INJECTION

The site of injection is a variable that has received some attention.
In eggs that have not been pre-incubated, only the yolk sac, air cell or
albumin are available.  The most frequent site of injection is the yolk
sac (30, 24, 31, 16, 25, 26, 32, 51, 21, 20, 29, 46, 52, 39, 33, 40, and
41).  This method has been subject to the criticism that material admin-
istered is unevenly distributed in the yolk because of solubility
problems.  According to Walker  (7) injected material may rise toward the
embryonic disc or never expose the embryo according to the specific
gravity of the material.  Walker  (7) describes the technique and use of
a yolk replacement method to insure uniform distribution of material.
He found a lower mortality rate with high doses of pesticides than by
the conventional yolk sac injection method, but higher mortality rate
with low dose levels.  There was also a higher rate of malformations
with the yolk replacement method.  Verrett  (personal communication,
1974)  states that the technique of Walker is not representative of the
art.  In her experience the technique results in almost 100% of abnormal
birds even with controls.  A technique was described by Klein et a_l.
(39) for perfusion of four-day embryos with dilute yolk solutions in
artificial medium containing salts, vitamins, amino acids and 8% yolk.
This permitted survival until 96 hours when an experiment was termi-
nated.  No foreign chemicals were studied in .this system.

The air sac is a route favored by some experimenters (14, 8, 9, 41, 53,
22, 54, and 55) .  In a comparative study, Verrett e_t al. (54) found that
doses of aflatoxin showed a greater toxicity (mortality) when injected
into the air cell than by yolk injection.  Material deposited in the air
sac gains access to the embryo by the egg membrane and albumin.

The chorioallantoic membrane develops during the second 24 hours of
incubation when the head fold appears.  This membrane has been utilized
by many for studies of embryonated eggs pre-incubated for 4-8 days.
Ridgeway and Karnovsky  (56) had shown that heavy metals are, in general,
more toxic when administered into chorioallantoic membrane than by yolk
sac.  Others have also utilized this route of administration (12, 15,
17, 35, 37, 38, 47, 49, 57, and 58).

Extra-ovo  (tissue culture) methods have been utilized for the study of
potentially toxic materials at special target sites.  This is generally
done after the tissue or organ has differentiated from its anlage.  Hay
(59) has reviewed the dependence of each stage of differentiation of a
given tissue or organ upon a preceding stage and type of tissue by means
of induction.  This problem has also been attacked by means of tissue
culture techniques sometimes with several types of tissue together in
the culture flask (59).  Chemicals  (drugs) and tissues used for such
studies include LSD, whole mount at one day  (43), cortison, growth of
femur of seven-day embryo  (13), Vit D , intestine, 20 days  (60); insu-
lin, tibia, 10 days  (61), antimetabolites whole mount connective tissue
(lathyrism), eight days (62); actihomycin D, early axial development,
11-13 somite stage  (63), propranolol, pre-neural heart, 50-55 hours,
80-85 hours  (64); and various drugs on heart primordia  (65).
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END POINTS

Mortality

Most studies have evaluated toxicity by mortality noted, or have recorded
this as ancillary data.  Studies in which mortality was compared at
several dosage levels or with an attempt made to calculate the LD,-ri have
been performed on phosphate and carbamate pesticides (7), a variety of
pesticides (16), solanine (32), alkylating agents (20), vaccine (29),
dinitrophenol  (14), benzimidazoles  (8, 9), azaserine (36), chlorproma-
zine (33), beta-aminopropionitrile  (58), volatile industrial chemicals
(66), and a variety of chemicals (28).

Growth and Development

The incidence of teratological effects and effects on gross development
was the major emphasis of a number of studies (7, 31, 32, 20, 43, 34,
14, 38, 52, 9, 36, 35, 37, 33, 40, 41, 11, 62, 67, 68,  69, 70, 71, 58,
55, 66, and 28).

The effect of test compounds on the development of a special target site
was emphasized in some studies.  Such studies included the effects of
LSD on development of neural tube (43), dinitrophenol on lens (14),
beta-aminopropionitrile on connective tissue  (38) and on skeleton  (58),
semicarbazide and insulin on skeleton  (58, 69, 70) and cardiovascular
drugs on heart (65).

Tritiated thymidine uptake has been used as a general measure of growth
and development  (72 and 22).

Physiological Activity

The effect of test compounds on physiological activity was also tested
in a number of studies:  heart - heart rate (64); bones - growth rate
(13), collagen content (49, 61), histochemistry  (58); hematopoetic
system - tritiated thymidine uptake  (30), ALA-synthetase induction  (53);
immune system - gamma globulin effect  (47), antibody production (15);
intesting - induction of calcium binding protein  (60),  and motor junc-
tion as a measure of muscle strength  (25).
CORRELATION OF IN VITRO WITH IN VIVO METHODS

In general fertile chicken eggs may have a similar or enhanced sensi-
tivity to chemicals as compared to rats depending on technique employed
and chemical under study  (24, 73).  McLaughlin et al. (66) state that
the toxicity of the industrial solvents as determined with their method
correlates with threshold limit values, guides used in industrial hygiene
practice.  Verrett (personal communication) states that she has compiled
a list of 30 drugs in which mammalian and chick teratology were studied
and in which there was teratological information in the human.  She
states that there is only one case where the chick and mammals did not
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show a teratologic response when it was found in the human.   In all
cases chick and mammal showed responses which, however,  were sometimes
different.

Recently the chick embryo was adapted for the purpose of bioassay of
aflatoxin by the Association of Official Analytical Chemists because of
its great sensitivity to the toxin.

The chick embryo and other fertilized egg systems have a special utility
in evaluating possible effects of chemicals as environmental pollutants.
For example, there have been several reports regarding agricultural
chemicals which inhibit hatchability of bird's eggs (74).  The use of an
appropriate technique to evaluate this potential hazard thus assumes
economic significance.

One of the significant factors to be considered in correlating environ-
mental effects with experimental data is the deposition of a toxic
chemical in the egg.  In nature, contamination of the egg would occur
via the oviduct of the hen.  The yolk and albumin then serve as reser-
voirs for the toxic agent.  If deposited in the egg via the oviduct,
trace amounts of a toxic chemical would be uniformly distributed.
Chemicals intentionally injected into the yolk sac may not distribute
uniformly or be available at early stages of embryogenesis to produce a
toxic effect.  Guthrie and Donaldson (26) have shown that 40% of the
total radioactivity of the one-day old chick which develops from an egg
injected with radiolabeled DDT by the technique of McLaughlin  (28) is
found in the yolk sac at hatching.  The yolk sac is incorporated in the
body of the chick before hatching and provides a source of nutrition.
Thus the toxic effect of a chemical injected in the yolk sac may only
result after absorption at the time of hatching.  It would appear that
injection at 4-10 days into the chorioallantoic.membrane is a preferable
technique for conducting toxicity tests.  It has been shown that a low
incidence of mortality occurs in saline injected controls .at this age as
compared with pre-incubation treatment.  Moreover, the toxic material
gains immediate access to embryonic tissues.

The concentration of dieldrin has been measured in blood of embryos and
chicks hatching from eggs treated with this compound (Koeman et al.,
1965).  Dieldrin was injected by the method of McLaughlin et al.  He
also found that the concentration of dieldrin in the blood of the 0-6
hour old chick was little greater than that in the 14 day old embryo,
which indicated that the absorption process of the yolk sac did not
cause an important increase in the concentration of dieldrin in the
blood.  They state that a sufficient period after hatching is required
to determine whether the chick is susceptible to the test material.

Invertebrate Eggs

Toxicity tests have been conducted using invertebrate eggs as test
subjects.  Two papers are of particular interest in this respect:

1.   Various types of carcinogens were tested using the inhib-
     itory effect of hatching of Artemia Saline  (brine shrimp)

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     as the criterion for an effect (Buu Hoi and Pham-huu Chang,
     75).  Fourteen compounds belonging to seven different chem-
     ical groups were selected.  Thirteen of these compounds
     were confirmed as carcinogens in animal experiments; the
     other compound was structurally related to a carcinogen.
     In general, the inhibitory or stimulatory effect on hatch-
     ing did not correlate with the carcinogenic effect of a
     test compound.  Within the epoxide series, however, a clear
     cut difference was found which correlated with skin tumors
     produced by painting on mice.

2.   The fertilized sea urchin egg was the test object for a
     study on the influence of fractions of a tobacco smoke
     condensate on early development (76).  The stage of devel-
     opment attained by treated eggs was the criterion for a
     toxic effect.  The eggs were sensitive to fractions con-
     taining unsaturated hydrocarbons and other substances.
     They were not, however, sensitive to nicotine or other
     polycyclic hydrocarbons still present in these fractions.
     The sea urchin egg was therefore considered to be useful
     for subfractionation studies in the isolation of toxic
     materials found in tobacco smoke condensate.

Other papers which have appeared concerned special classes of chemicals
and special effects on the eggs and/or larvae of lower organisms.  Thus
the sea urchin was the subject of study of inhibitors of oxidative
phosphorylation (77) which induce birefringence of the mitotic appa-
ratus, inhibition of glucose metabolism  (78), inhibition of synthesis of
RNA and gene expression by actinomycin (79), inhibition of cleavage by
puromycin and inhibitors of cyclic AMP (80) and bizarre development
termed an animalizing effect after exposure to Evans Blue  (81).  Insect
larvae  (attagenue piceus) were test objects for inhibitors of choles-
terol synthesis (82).  In this work test compounds inhibited growth
(weight gain) of the larvae.  Zebra fish embryos (Brachydanio, rerio,
Hamilton) were test objects for a large number of steroidal estrogens
(83).  Compounds were evaluated by an ED_  determination based on the
cytostatic effect.  No relationship was found between estrogenic and
cytostatic potency.

In experienced hands, the use of fertilized chicken eggs for teratolo-
gical screening provides useful information.  The record would indicate
that the technique has limited usefulness in screening for other types
of toxic effects.

The use of the sea urchin egg in working out a mechanism of action of a
toxicant is at the present the best place for this technique.
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B.  USE OF ISOLATED ORGANS AND TISSUE IN STUDIES ON CHEMICALS
INTRODUCTION

In order to bridge the gap between the effects of a chemical on the cell
and the effect on the whole animal, isolated organs or organized tissues
are used.  The chemical is introduced by one of three methods, perfusion
through the vascular system, adding it to the bathing fluid in which the
tissue is suspended, and adding the material to tissue slices in an
apparatus such as the Warburg.  Among the parameters commonly measured
as indices of organ function are rhythmic contractions, fluid transport
and flow, organ secretion, oxygen uptake, carbon dioxide production, and
incorporation of radiolabeled substrates.
LIVER

The liver is a large glandular organ which converts most sugars into
glycogen and is concerned with storage of fat and excretion of-chemi-
cals.  For the purposes of this survey on isolated organs, liver homo-
genates and enzyme induction studies were not considered pertinent.
Because of the role of the liver in detoxification and metabolic fate of
materials, the isolated perfused liver and liver slices are important in
in vivo  test systems.

The isolated perfused liver is useful for studying the metabolism of
chlorinated hydrocarbons (84).  Cole e_t al. (49) used radiolabeled
endrin and dieldrin in the perfusate and compared the fat storage of
these two compounds in the intact rat and the isolated perfused rat
liver.  A modified method of Miller (85) was used.  The radiolabeled
insecticide dissolved in 0.1 ml acetone was added to the circulating
perfusate to provide an initial concentration of 0.003 mg/ml or 0.0003
mg/ml insecticide in the perfusate; 0.003 mg/ml approximated the initial
in vivo concentration in the blood.  The results obtained in the per-
fusion experiments were very similar to those in vivo, indicating that
the liver was the major controlling factor in different rates of excre-
tion of radioactivity from endrin 14  and dieldrin   C.

Klevay (86), using the method described by Cole et al. (84), confirmed
the study of Kunze and Laug  (87) that dieldrin is stored to a greater
degree in the adipose tissue of female rats than of male rats.  He found
that the greater ability of male livers to excrete dieldrin is consistent
with the greater storage by females and lesser toxicity to males.

The isolated perfused rat liver was used to study the metabolism of
phthalate ester plasticizers used in plastic tubing and bags for blood
storage (88).  The method of Miller et al. (85) was used.  The system
perfused an isolated liver for four hours.  The authors concluded that
the plasticizer butyl glycolylbutyl phthalate is extracted from plastic
tubing by the liver, and its product glycolyl phthalate is secreted into
the perfusion media.
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The metabolism of drugs which are eliminated solely by bibtransformation
in the liver show good correlation in intact animals and in isolated
perfused liver systems (89).  Metabolism studies using the isolated
perfused rat liver, included studies on the rate of pentabarbital dis-
appearance from the medium of livers isolated from normal and Walker 256
tumor bearing rats (90), and the protective effect of oleate on metabolic
changes produced by Halothane in rat liver (91).  Livers of 48 hour fed
or starved rats were perfused and exposed to Halothane (2.5% v/v).
Solutions of oleate (0.1 M) were prepared in albumin (10% v/v) and added
to the medium.  The inhibitory effects of Halothane on 0  consumption
and urea synthesis are conteracted when oleate is added to the perfusion
medium.  Oleate also counteracts Halothane induced inhibition of urea
synthesis and changes in liver tissue metabolite concentration.  The
degree of halogen induced inhibition of gluconeogenesis from lactate is
also decreased by oleate.
BRAIN

The inhibitory effect of several narcotic analygesics and other psycho-
tropic drugs on the active uptake of  H-norepinephrine was studied in
mouse brain slices and synaptosomes (92).  Codeine, hydromorphone,
levorphanol, meperidine, methodone, morphine and naloxone inhibited the
uptake of norepinephrine.  The study suggests that the inhibition of the
uptake of norepinephrine is related to the lipid solubility rather than
the specific structures of narcotic analygesics.

Nonbarbituric hypnotics were studied in the Warburg manometric apparatus
using brain slices of female Wistar rats  (93).  "Valmed"  (ethinylcyclo-
hexyl carbamic acid) was compared with "Seconal Sodium"  (sodium allyl,
1-methylbutyl barbiturate) in its ability to inhibit the potassium-
stimulated respiration of slices of brain cortex.  In concentrations
equivalent to those obtained during anesthesia "Seconal Sodium" depressed
the oxygen uptake of normal tissue whereas both Seconal Sodium and
Valmed inhibited the respiration of KCl-stimulated tissue.

Rat brain homogenates and whole brain tissue slices were used to study
the toxic effect of 6-hydroxydopamine (2.4,5-trihydroxyphenyltetylamine)
on nerve terminals  (94).  The uptake of  H-labeled dopamine, norepineph-
rine and serotonin into rat brain homogenates was measured.  The data
showed that 6-hydroxydopamine generated hydrogen peroxide and that
hydrogen peroxide can damage the biogenic amine uptake system.  Hydrogen
peroxide generated from 6-hydroxydopamine that accumulated in catechol-
amine terminals may be the cause of the long-lasting catecholamine
depletion that accompanies the destruction of nerve terminals.  Tissue
slice experiments performed with  ( H)-5-hydroxytryptamine produced the
same results.

Soman  (pinacolyl methyl phosphonofluoridate) and DFP  (diiopropyl phos-
phorofluoridate), potent inhibitors of esterases and particularly cho-
linesterase, were studied in slices of cerebral cortex  (95).  Respiratory
, rates were measured by the conventional Warburg technique.  The compound
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was added to the incubate in 0.2 ml isopropyl alcohol.  Respiratory
rates were depressed by Soman at concentrations between 10   M and
10~  M and at concentrations of DFP between 5 x 10   M and 5 x 10   M.

The effect of hyperbaric oxygen on oxygen uptake in guinea pig cerebral
cortex slices incubated in Krebs-Ringer glucose saline solution or in
the presence of various substrates was studied (96).   The concentrations
of glycogen inorganic phosphate, phosphocreatinine, adenosine triphos-
phate, K , and Na  were studied with glucose as the oxicizable substrate.
Formation of lipid peroxides, thought to be connected with oxygen
toxicity, were measured.  Tissue oxidative reactions, phosphocreatinine,
adenosine triphosphate and intracellular ions were diminished and lipid
peroxides increased.  The increase in lipid peroxides is attributed to
the toxic effects of hyperbaric oxygen.

Myelination was inhibited by 5-bromodeoxyuridine in sections of newborn
rat cerebellum cultured in the Maximow double coverslip assembly (97).
Explanted pieces of newborn rat cerebellum maintained in organ culture
show similar morphological and biochemical maturation to in vivo devel-
opment.  Myelinated axons appear in the cultures at 10 to 11 days.  When
5-bromodeoxyuridine at a concentration of 1.5 x 10   M was in the cul-
ture from explant, the cultures looked as healthy as controls except
that few or no axons became myelinated.  Thymidine at 2,5, or 7.5 times
the concentration of 5-bromodeoxyuridine when added to the cultures
simultaneously with 5-bromodeoxyuridine prevented the inhibition of
myelination.
TRACHEA

Ciliary activity, mucous flow and muscle contractions are important
mechanisms in the trachea for removing and clearing foreign particulate
matter from the upper respiratory tract.  Ciliary depressant action of
components of cigarette smoke was studied by Kensler and Battista (98).
The trachea tissue was placed in Tyrode's solution aerated with 95% 0 -
5% C0_, and sutured to a tracheal holder which raised the center portion
of the trachea.  Tracer particles were a mixture of finely powdered soot
and lycopodium spores.  The effect of materials on ciliary transport was
determined by measuring the time required for the particles to move a
distance of 5 mm.  Bleiberg (99) in studying the combined effects of
metaproterenol and cigarette smoke on ciliary activity in the rabbit
trachea used a slightly different method.  A humidified, temperature-
controlled tissue chamber was inclined at an angle of 15°C from the
horizontal with the cranial end of the tissue at the upper end of the
chamber.  The uphill axial movement of glass microbeads (0.03 mm in
diameter) placed on the mucous surface of a section of trachea was
measured as an index of the mucociliary transport activity.  Readings
were obtained by measuring the time in seconds for a single bead to
travel a known distance as viewed against the microscope reticle at
five-minute intervals.
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Bleiberg (100) reported that Freon-propelled metaproterenol aerosols
produced a sustained increase in muco-ciliary transport rate in rabbit
tracheas.  Tracheas were exposed to puffs of cigarette smoke prior to
and after metaproterenol application.  Pretreatment with metaproterenol
increased the number of puffs of cigarette smoke required for complete
inhibition of muco-ciliary transport activity.  When the trachea was
exposed to aerosol metaproterenol after ciliastasis from cigarette
smoke, there was some recovery of transport activity.

Isolated segments of hamster and rat trachea in organ culture are used
to study the effects of carcinogenic compounds on respiratory tissue.  A
detailed examination of the bioepithelium maintained in culture was done
by Kaufman (101).  Maintenance of tracheas in vitro allows for more
extensive incorporation of labeled precursors by the tracheal epithelial
cells than is possible by the administration of comparable amounts of
labeled precursors in vivo (101).  The respiratory tracts were removed
in toto from young adult male Syrian golden hamsters 2-4 months old, and
the tracheas separated from other tissue.  Tracheas incubated in Lerbouitz
L-15 medium, containing L-glutamine and   H-5-uridine, were examined
microscopically and biochemically.  The authors suggest that the inter-
pretation of biochemical changes during and after carcinogen administra-
tion needs to be correlated with the morphologic evaluation because of
the changes in cellular populations occurring in the trachea.

Crocker and Sanders  (102) reported on the influence of Vitamin A and
3,7-dimethyl-2,6-octadrenal  (Citral) on the toxic effect of benzo(a)-
pyrene on hamster trachea in culture.  They used tracheas from Syrian
hamsters 2 to 4 days old.  Two strips of mesh bearing explanted tracheas
were laid on the surface of clotted medium in the center of an organ
culture dish provided with an outer circumferential well containing a
moistened filter paper.  The concentration of benzo(a)pyrene was about
10.5 yg/ml (0.041 mM) in medium.  The clot surrounding the mesh was
removed and 0.05 ml Tyrode's solution containing tritiated thymidine was
dropped on each explant at 8 or 15 days, and finally fixed in Bouin's  .
fixative.  The study showed that benzo(a)pyrene and Vitamin A act direct-
ly on the respiratory epithelium in a competitive fashion and on carti-
lege with an additive effect.  The authors state "the organ culture
method thus appears applicable to the study of the mechanisms of action
of Vitamin A and a carcinogenic polycyclic hydrocarbon on trachea-
bronchial tissues."

Crocker  (103) exposed suckling rat trachea in organ culture to three
known carcinogens:  9,10-dimethyl-l,2-benzanthracene, 3,4-benzypyrene
and 20(3)-methylcholanthrene.  The effects of these three compounds were
similar, in that all produced an increase in the proportions of basal
cells undergoing DNA synthesis.  The authors compared three systems,
human fetal lung, adult mouse prostate and suckling rat trachea and
concluded that the unifying similarities in the three systems are
suppression of mesenchyme, stimulation of basal cell replication, and
induction of metaplasia.
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LUNG

Piper and Vane (104) describe a method for the perfusion of isolated
guinea pig lungs using Krebs solution.  This new assay method was used
to detect the release of active substances during anaphylaxsis in guinea
pig lung.  Piper and Vane found histamine "slow reacting substance-A,
prostaglandins E  and F  a, and other substances not previously described
released into the perfusate."  The heart and lungs were removed and the
pulmonary artery and trachea cannulated.  The lungs were suspended in a
chamber and perfused through the pulmonary artery with Krebs bicarbonate
solution gassed with 95% 0_-5% CO  at 37 C.  Lungs were taken from
guinea pigs sensitized 28 days previously with ovalbumen or from unsen-
sitized guinea pigs.  The effluent from the lungs was taken to superfuse
a series of isolated assay tissues; stomach strip, duodenum and colon of
the rat, longitudinal strips of jejunum and terminal ileum of the cat,
spirally cut strips of thoracic aorta of the rabbit, rectum of the
chick, ileum and trachea of the guinea pig.  The authors conclude "the
antagonism by aspirin-like drugs of the release of RCS  (new undescribed
substance) could well provide a basis for new and relatively simple in
vitro screening tests for anti-inflammatory compounds."

Shabad e_t al. (105) state that "the organ culture method of lungs can be
used to detect precancerous conditions and to test for rapid determina-
tions of the oncogenic activity of some chemical compounds."  A modified
method of Chen (113) was used for the organ culture of embryonic lungs
of mice and rats.  Strain A and C3HA mice and BD-lX rats were used.
Strain A mice have a high incidence of mammary gland carcinomas and
adenomas of lungs.  Strain C3HA had few lungs adenomas.  The trans-
pla'cental effects of urethan, dimethylnitrosamine and nitrosomethylurea
were shown.  Pregnant animals were treated with these agents 15-18 days
after copulation.  Females were killed on days 19 and 20 and lungs of
embryos removed.  Pieces of embryonic lungs was explanted on cellulose
plates and floated on the surface of a liquid nutrient medium.  These
organ cultures from intact mice and rats were maintained for 30-33 days.
The ef fect of administration of urethane in vivo was compared with that
seen in culture.  Pregnant mice were treated with doses of urethane  (30,
60, 90-100 mg) on days 15-18 of gestation and their offspring were
examined for lung adenomas on the same days that the organ cultures were
examined.  Lung adenomas and precancerous changes were seen in offspring
of mice treated with urethane depending on size of dose and time of
observation.  Similar changes in organ cultures were observed depending
on size of dose except that lung adenomas developed more rapidly in_
vitro than in vivo.  With urethane the first adenoma was recorded as
early as 4 days of explantation and at 14 days adenomas were observed in
two-thirds of the explants.  The authors conclude "these data should be
taken into consideration in working out prophylactic and hygienic meas-
ures for protection of populations from air pollution, cigarette smoke,
etc.  The increased sensitivity of embryonic tissue to oncogenic agents
makes it necessary to give the highest priority to the protection of the
health of pregnant women and newborn."
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Fetal human lung tissue from 3-5 months foetuses was exposed in culture
to 3:4 benzpyrene in concentrations of 1,4 and 6 yg/ml of medium (106).
The addition of the carcinogen induced epithelial hyperplasia of bron-
chioli and pneumonomeres and inhibition of stromal growth at all concen-
trations.  The percentage of explants showing hyperplasia was the same
(84-89%) for all three concentrations of 3:4 benzpyrene but the first
appearance of hyperplastic change was speeded up and the number of
hyperplastic foci per explant and the degree of hyperplasia in them
increased with rising concentration of the carcinogen.

O'Donnell e_t al. (107) reported on the maintenance of normal metaplastic
and diplastic states of human bronchial mucosa in organ culture and this
work was furthered (108) to study the toxicity of benzo(a)pyrene and air
pollution composite for adult human bronchial mucosa in organ culture.
Bronchial mucosal patches from adult humans were maintained in organ
culture for 5 to 11 days.  (Benzo(a)pyrene (15 yg/ml) or an air pollution
composite (700 yg/ml) were present in culture media during incubation of
explants.  All samples were incubated with tritiated thymidine before
fixation and each tissue piece was examined by histological and auto-
radiographic methods.  Benzo(a)pyrene and air pollution composite pro-
duced toxic destruction of all cell types or as a less marked effect,
suppression of DNA synthesis and distortion of morphological states of
columnar but not of regenerative epithelia.  Toxicity due to air pollu-
tion composite cannot be identified with either a single component of
air pollution composite nor any particular cell type.  Toxicity of
benzo(a)pyrene was interpreted as evidence that microsomal mixed-function
oxidases are present and active in metabolic conversion of benzo(a)pyrene.

Raj an e_t al. (109) studied the response of human pleura in organ culture
to asbestos fibers.  Human parietal pleura were dissected into 2-mm
square pieces and maintained in organ culture in a synthetic medium
containing calf serum.  Blue asbestos was suspended in the medium at a
concentration of 0.01%.  The culture vessels were maintained in an
atmosphere of 5% CO  , 50% O_ and 45% N , for up to 8 days.  Explants in
the presence of asbestos showed marked proliferation of mesothelial
cells when compared with controls in normal medium.  In some areas there
was invasion of the underlying tissue by the cells and the cells had
larger nuclei than controls.  There was also an increased amount of
collagen in the underlying tissue.
KIDNEY

Cortex slices, isolated perfused kidneys, entire papilla and isolated
renal arteries have been used to study the effects of vasoactive sub-
stances, diuretics, cardiac glycosides and other compounds on renal
function.  Hysell and Bohr (110) used the isolated perfused rat kidney
to determine renal vascular response to rat plasma, a vasoactive fraction
of hog plasma, epinephrine, angiotensin, KC1, serotonin, and vasopressin.
A perfusion pump maintained a constant rate of 3 ml/min of perfusate
through polyethylene tubing leading to a cannula in the renal artery.
An injection site just prior to the cannula in the renal artery made
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possible the rapid administration of small volumes of test substance.
Calcium concentrations in the perfusion solution were varied—alternate
perfusion fluid was whole rat plasma.  Addition of rat plasma or a
vasoactive fraction of hog plasma caused an increase in renal vascular
resistance.  The time course of the responses caused by these two agents
was the same but differed from that of the pressor responses caused by
epinephrine, angiotensin, KC1, serotonin or vasopressin.

Rosenfeld e_t al. (Ill) studied the effect of ouabain and potassium on
the isolated perfused rabbit kidney.  They found that plasma potassium
levels as high as 12.0 meq/liter did not inhibit ouabain induced natri-
uresis and diuresis, whereas in animals loaded with potassium a marked
inhibition did occur.

Krahe et al. (112)  studied the action of exogenous angiotension on
glomerular filtration rate and filtration faction in the isolated per-
fused rabbit kidney.  The kidneys were perfused at a constant pressure
of 97.1 +_ 3.0 mm/kg with a suspension of 40% volume rabbit erythrocytes
in Tyrode's solution to which 4 g % bovine albumin was added.  When
angiotensin II-amide in a dose of .1 ng*m  »min   was infused into the
perfusion system for 10 minutes, the glomerular filtration rate and
filtration factor increased during the infusion and fell to former
levels after the infusion.

The method for growing embryonic kidneys in tissue culture preparations
has been described by Chen (113).

Shabad e_t -al_. (114), using the method of Chen, studied the transpla-
cental effect of 7/12-dimethylbenz(a)anthracene, benz(a)pyrene, and
their analogs anthracene and pyrene, o-tolidine, 3,3'-dichlorobenzidine,
nitrosamine o-aminoazotoluene and p-aminoazotoluene.  Kidneys were
obtained from 19 to 21 day old mouse embryos.  The embryos were obtained
from mice that during the last third of their gestation received the
above compounds.  Explants were fixed with Bouins fixation at 4, 7, 11,
14, 18, 22, 26 and 30 days.  They showed that mouse kidney embryonic
tissue can be explanted in organ culture for 3 to 4 weeks.  Embryonic
tissue which was subjected to transplacental treatment of the different
chemicals, revealed a more intense growth of epithelium and survived
longer than did control cultures.

Crocker and Vernier  (115), using embryonic mouse kidneys, showed that
alterations of potassium concentration was one of the most likely causes
of renal maldevelopment.  In order to correlate animal data with human
disease, Crocker (116) used human embryonic kidneys in organ culture.
Kidneys were obtained from embryos 5 to 12 weeks of gestation and cul-
tures were grown for 2 to 5 days.  Medium 199 prepared virtually free of
potassium was used.  Control kidneys were grown in a medium with a
potassium concentration of 6.5 to 10 meq/liter while the'other kidney
was grown in a medium with potassium of 3 to 6 meq/liter.  In 38 embry-
onic kidneys grown with a potassium concentration under 6 meq/liter, the
following defects were seen:  (1) decreased number of branches of the
uretera^bud; (2) failure of nephron induction at the site of branching;
and (3) occasional dilatation of the ureteral bud.

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INTESTINE

Isolated segments of gastrointestinal tract provide a valuable test
system for studying intestinal hydrolysis, conjugation, metabolism and
transport.  Pekas and Paulson (117) incubated everted sacs of rat small
intestine in a pH 7.4 media containing either the insecticide 1-(1-  C)
naphthyl N-methylcarbamate (carbaryl), or 1-(1-  C) naphthol, and iso-
lated from mucosal and serosal fluids and the metabolite 1-(1  C) naph- •
thyl glucuronide.  The hydrolysis of carbaryl and conjugation of naphthol
indicated some degree of metabolism by the intestine before absorption.
Pekas (118, 117) used the method of Wilson and Wiseman  (119).  The small
intestine of young rats extending from the bile duct to the cecum was
divided into approximately equal,-parts and each section was everted and
incubated for two hours with 10   M carbaryl at 37 C under an atmosphere
of 95% 0-5% CO  .  The   C labeled constituents were chromatographed on
silica gel thin-layer plates/ and the radiospots scraped and counted by
liquid scintillation.

Shaw and Guthrie  (120) studied penetration through isolated sections of
the mouse gastrointestinal tract of five insecticides; malathion, car-
baryl, dimethoate, DDT and dieldrin.  To study the effect of age on
penetration, sections of small intestine and sections from the colon and
anterior region  of the rectum were obtained from mice 12 days, 6 weeks
and 12 weeks old.  Solutions of radioactive insecticide  (0.25 ml) were
introduced into  the lumen.  After 80 minutes the amount of radioactivity
in the serosal and luminal solutions and that remaining in the tissue
were determined.  The extent of penetration for mice of different ages
showed only trivial differences for carbaryl, DDT and dieldrin but both
malathion and dimethoate penetrated more rapidly through tissues of 12
day old 'mice".  Generally the amount of insecticide bound in gut tissues
was greater in 6 and 12 week old mice than in 12 day old mice.  DDT and
dieldrin were exceptions.  The authors found that differences in absorp-
tion of insecticide may occur in specific regions of the digestive tract.
Phosphate and carbamate insecticides, but not chlorinated hydrocarbons,
showed greater penetration into the colon.

Stookey e_t al_.  (121), using the method of Wilson and Crane  (122), stud-
ied fluoride absorption using intestinal segments from young rats which
were maintained  on low fluoride diet and fluorine-free water.  When four
different levels of fluoride were placed inside the intestinal segments
and rate of diffusion of fluoride through the intestinal wall measured,
comparable values were obtained, suggesting that the amount of fluoride
present is not a major factor governing the rate of diffusion.  The
authors found that the rate of absorption through the intestinal wall
was about twice  as great as in the stomach and that the  rate of absorp-
tion through the intestinal wall is related to the surface  area of the
intestine^  They presented evidence that an active transport system is
not involved in  absorption of fluoride in the rat.

Moore e_t  al_.  (123) studied the effect of  synthetic surfactants, alkyl-
benzenesulfonate-t linear alkyl sulfonate, cetyltrimethylammonium bromide
and Triton X-100 on intestinal permeability to glucose  in  the absence


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of a  functioning active transport mechanism.  Phloridzin was  used  to
block the active transport of glucose.  Segments of  the small intestine
of the golden hamster were used.  The surfactants  increased intestinal
permeability to glucose in a dose-related manner with  linear  alkyl
sulfonate producing  this  effect at  a lower  concentration than any  of  the
others tested.  Microscopic examination of  the  intestinal  segments
.showed that the mucosal epithelium  was not  altered by  low  surfactant
concentrations which increased intestinal permeability.

Using everted segments of rat stomach and small intestine, Lasagna's
group studied the metabolism of L-3,4-dihydroxyphenylalanine  (L-dopa)
 (124).   The tissues  were  separately incubated with  C L-dopa in Krebs-
Ringer phosphate buffer.  Metabolites in the tissue  and in the mucosal
and serosal fluids were separated by ion-exchange  chromatography and  the
radioactivity determined  by liquid  scintillation counting.  The metab-
olites found were phenylcarboxylic  acid, dopamine, and other  catechol-
amines.  The authors suggested that L-dopa  could be  significantly
metabolized in the gastric mucosa prior to  absorption.  The authors  .
conclude "the everted sac preparation is simple and  gives  reproducible
results.  It not only allows the simultaneous study  of drug metabolism
and drug transport across gastric and intestinal mucosa, but  can also
provide  information  on the effect of drug metabolites  on membrane  trans-
port  of  the drug."

Boass and Wilson 1125) used one gram sacs of monkey  intestine to study
absorption of Co  -labeled Vitamin  B   .  They found  that intrinsic
factor was necessary for  the uptake of B    and  that  the site  of maximal
uptake was in the low ileum.

Using everted jejunal intestinal segments of young rats, Feldman and
Gibalei  (126) showed that physiologic concentrations of the conjugated
bile  salt, sodium taurodeoxycholate markedly increased the permeability
of the everted rat small  intestine  to salicylate ion.  Addition of egg
lecithin or oleic acid and glyceryl monooleate  to  the  medium  diminished
this  effect and prevented changes in gross  appearance  of the  mucosa
which occurred when  the isolated intestine  was  exposed to  the bile salts
alone.

The frog gastric mucosa  (commonly that of Rana  pipiens) is used to study
gastric  acid secretion isolated from the influences  of blood  flow,
nervous  factors and  humoral substances  (127).   The method  of  Rehm  (128)
allows the study of  electrophysical parameters  such  as H   secretory
rate, transmucosal potential difference and the transmucosal  resistance.
The stomach is removed from a frog  and opened along  the lesser curvature,
the fundic mucosa separated from the muscular layer  of the stomach, and
the intact mucosal membrane mounted between two Lucite chambers.  The H
secretory rate is measured by a recording pH-stat  autoburette titrator.

Nakajima (127) determined the effects of nicotine  on the H secretion,
transmucosal potential difference and resistance using this system.   The
author found that nicotine produced a reversible dose-related inhibition
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of H  secretion with an increase in transmembrane potential difference
and calculated short-circuit current.  He suggests that nicotine may
selectively inhibit H  secretory mechanism.

Schwart and MacKrell (129) studied the potency of an anaesthesia agent
compound 347 (Ethrane - CHF -0-CF -CHF Cl) to inhibit frog gastric
secretion.  It was previously shown that the percentage of anaesthetic
required to produce a decrease in the H  secretory rate in frog is
proportional to the minimal anaesthetic concentration in man  (the amount
required to prevent a muscular response to a skin incision in 50% of
subjects to whom the anaesthetic is administered).  Ethrane was compared
with methoxyflurane, chloroform, halothane, and flurooxene.  Their
results suggest lipid solubility as a factor influencing the potency of
anaesthetics in inhibiting acid secretion.

Similar methods and measurements were made by Dinno ejt al. to study the
potency of barbiturates to inhibit frog gastric section  (13).  Bar-
biturates were added to the nutrient solution to yield 1 mM concentra-
tion of barbital, diallylbarbituric acid, phenobarbital, pentobarbital,
secobarbital or thiamytal.  Insofar as the hypnotic activity of narcotics
such as barbiturates is closely related to their relative lipophilic
character as defined by the logarithm of the octanol water partition
coefficient log P, the authors determined to what extent lipid solubil-
ity of barbiturates, as defined by log P, is a factor in inhibiting frog
gastric secretion.  They found that the decreases in H  secretory rate
in frog gastric mucosa was almost directly proportional to log P, up to
an 80% decrease in H  secretion rate.

Embryonic chick intestine maintained in organ culture has been used to
study the effects of Vitamin D  on calcium transport  (60, 131) and the
effect of hydrocortisone on enzyme induction  (132, N.Y. Academy ref.,
PSEB, Dec. 1966).

The method for preparing embryonic chick duodenum in tissue culture was
described by McCarty ejb al.  (133).  The duodenal loop of 14 to 20 day
old embryonic chicks was removed, sectioned transversely into 1 to 1.5
mm fragments, and the fragments arranged with the villi upward and the
serosal surface contacting a Millipore membrane.  The tissue and mem-
brane were floated over Eagle's medium and incubated under a drop of
medium in a humidified chamber.

Hijmans and McCarty  (132) used half of a duodenum as control and added
0.5 yg hydrocortisone to the other half in a culture vessel.  They found
that embryonic duodenal cultures repsond to hydrocortisone with an
increase in the specific activity of invertase, and that this response
is dependent on the age of the initial tissue explant, duration of
exposure to hydrocortisone and the pretreatment of the tissue before
hydrocortisone application.

In further studying the effect of hydrocortisone on 16 and 19 day old
chick duodenum, Hijmans and McCarty  (132) used a chemically defined
medium.  They found that embryonic chick duodenum could be cultured for
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at least three days in a chemically defined serum-free medium.  Cells
in mitosis were present in cultures with or without serum.  Invertase
activity increased in cultures without 'serum and the induction of
invertase by hydrocortisone is greater in serum-free medium.

Corradino and Wasserman (60) used embryonic chick duodenum to study
Vitamin D induction of a calcium-binding protein in the intestine.
Crystalline Vitamin D  added to the culture medium  (400 IU per milli-
liter of medium) induced the formation of a substance immunologically
identical to chick intestinal calcium binding protein, and enhanced  the
uptake of radiocalcium by the intestine.  The chick intestine does not
normally produce calcium binding protein until after hatching.

Corradino (131) studied the response of the chick duodenum in organ
culture to the metabolites of Vitamin D , 25-hydroxycholecalciferol  and
1,25-dihydroxycholecalciferol.  The authors state that there is a marked
similarity between responses to Vitamin D iri vivo and in this system,
and that the mode of action can validly be studied in organ culture.
They suggest that it might be possible to utilize the extremely high
potency of 1,25-dihydroxycholecalciferol in this culture system as the
basis for a simple yet highly sensitive bioassay which might be used in
studying normal and disordered states of calcium metabolism.
ADRENAL GLAND

Systems for investigating the action of chemicals upon adrenals have
included isolated perfused adrenals  (cow 134, 135; dog 136),  cortex
slices  (cow 137), and portions of whole adrenal  (rat  138,  139, 140).

Hart arid Straw  (136) studied the effect of p_,pJ_-DDD on ACTH-induced
steroid production.  The isolated dog adrenal was retrogradely perfused
for a one hour period with 2,2'bis-(2-chlorophenyl, 4-chlorophenyl)-!,!-
dichloroethane  (p_,p_^-DDD).  In adrenals perfused with plasma  containing
a maximum stimulating concentration of ACTH and p_,p_^_-DDD,  the response
to ACTH was almost completely blocked.  Steroid production fell at
approximately the same rate as in the intact dog receiving an intra-
venous injection of 60 mg/kg of p_,p_^_-DDD.  p_,pJ_-DDD inhibited ACTH-
induced steroid production by greater than 90% within 2 hours.  A 30-
minute control perfusion with Krebs-Ringer bicarbonate measured baseline
steroid production.  This was followed by a 3-5 minute perfusion  with
ACTH and another 30-minute control period when the steroid response to
ACTH was monitored.  The adrenal was then perfused for 2 hours with
either Krebs-Ringer bicarbonate or control plasma containing  either drug
solvent or o,p'-DDD.

By this method a statistically significant correlation of  the in  vivo
action 'of o_,p_^-DDD in the intact dog and in the isolated perfused dog
adrenal was demonstrated.  When Krebs-Ringer bicarbonate glucose  was
used as the perfusion medium instead of plasma, ACTH-induced  steroid
production was not inhibited.  However, when p_,p_^-DDD is introduced into
adrenal slice preparations, the response to ACTH is not blocked and this
is attributed to lack of penetration to the interior  of the slice.

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Perfusion of cow adrenals with acetaldehyde (100 yg/ml) resulted in
1,3,3,4-tetrahydroisoquinoline alkaloids being formed  (135).  Acetal-
dehyde reacts with tissue catecholamines, epinephrine, and norepineph-
rine to form these alkaloids.  Insofar as ethanol is oxidized to acet-
aldehyde and is known to stimulate adrenocortical secretions in both man
and animals, the authors suggest that these alkaloids are either secreted
or leaked from nerve termini and contribute to behavioral changes caused
by ethanol.  The metabolites were measured through TLC of tissue homo-
genate supernatants.

The lowest acetaldehyde concentration at which 1,2,3,4-tetrahydroiso-
quinolines occurred in perfused cow adrenals was over 100 times that
which results in the blood of man ingesting a moderate amount of etha-
nol.  The authors feel that this limitation was due to current technol-
ogy related to merging of catecholamines on chromatograms.

Beef adrenal cortex slices were used to further characterize the effects
of ethanol on steroid synthesis (137).  One and 2% ethanol inhibited
aldosterone synthesis presumably at the site of conversion of corti-
costerone to aldosterone.  The authors suggest that this method provides
a means of examining specific actions of various agents directly upon
the adrenal without the modifying effects of pituitary and other sys-
tems.  However, the level of ethanol used was much higher than that
reached in severe intoxication in man.
HEART

Isolated atria, perfused hearts, ventricles, aortic strips and embryonic
hearts have been used to study the effects of chemicals on the cardio-
vascular system.  Increased contractile force (a positive inotropic
action) rate, atrial fibrillation, metabolism, and ion transport are
among the effects studied, using cardiac glycosides, steroids, prosta-
glandins and other drugs.

To study the effect of aldosterone on ouabain-induced potassium loss,
left atrial tissue, obtained from the hearts of young rabbits was per-
fused with a Ringer solution (142).  The atria was electrically stimu-
lated at a contract rate of 200 beats per minute and measurements made
of isometric contractile tension, effective refractory period, contrac-
ture and electrical excitability.  After a period of equilibration, drug
effects were studied by changing over to a perfusion medium containing a
known concentration of ouabain, d-aldosterone or a combination of both;
d-aldosterone did not prevent potassium loss caused by a high concentra-
tion of ouabain.

Levy et^ al.  (142, 143 and 144) used isolated rabbit left atria to study
the effect of d-aldosterone on ouabain-induced potassium loss  (142) and
the Na or K content of stimulated cardiac tissue  (142) and the effects
of Prostaglandin E  on isolated cardiac tissue  (144).
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D-aldosterone was found to be ineffective in preventing K  loss from
isolated atrial tissue caused by high concentrations of ouabain.  Also
there was a lack of antagonism of ouabain-induced changes in contractile
and electrical properties of atrial muscle.  This effect is in contrast
to the intact animal.  In vivo aldosterone enhances K accumulation in
skeletal muscle.  In the isolated heart d-aldosterone did not promote K
accumulation.

Prostaglandin E  was studied for its effects on the contractile force of
electrically driven or spontaneously beating rabbit atrial preparations
(144).  Prostaglandin E  produced a reproducible biphasic inotropic
action on rabbit atrial tissue.  Other prostaglandins were reported to
produce a positive inotropic effect in_ vivo and in vitro.

Isolated rat atrium was used to study myocardial utilization of meta-
bolic substrates required for tissue energy production and muscle con-
traction.  The influence of the medium which is in contact with the
atrium has been studied by Paradise et al. (145, 146, 147), Venturi
(148), and Lacuara (149).  Citrate bicarbonate-free medium and halothane
produced rapid depression of atrial contractility.  Glucose added to
depressed atria produced a marked increase in the force of contraction
(147).

Talesnik and Sunahara (150) used the Langendorff method for isolated
perfused rat hearts to study the effect of aspirin like substances as
suppressors of prostaglandin inhibition of metabolically-induced coro-
nary vasodilation.  The authors postulated that an inhibitor of prosta-
glandin synthesis would be useful in preventing coronary insufficiency
in conditions of cardiac stress.  They investigated the action of indo-
methacin and aspirin on the metabolically induced coronary vasodilation
which follows the increase of cardiac activity produced by noradrenaline
Ca   and tachycardia.  Young male rats were anaesthetized with ether and
the hearts isolated and perfused with Krebs-Henseleit bicarbonate per-
fusate.  The perfusate contained one half the usual calcium concentra-
tion.  Phentolamine was added to the perfusate to inhibit noradrenaline.
The force of contraction and coronary flow were measured continuously.
Aspirin and indomethacin enhanced metabolically induced coronoary vaso-
dilation.  Cardiac hyperactivity produced by noradrenaline, Ca   or
tachycardia was not stopped.

Isolated rat heart treated with 100 ug of the antibiotic chlortetra-
cycline followed by 1 yg epinephrine developed both inotropic and
chronotropic irregularities (151).  The authors postulated that this
effect is caused by chelation of the co-factor of cholinesterase which
inactivates the enzyme and permits acetycholine to accumulate and exert
its inhibitory effect on the heart.

Vahouny et al. studied factors affecting fatty acid synthesis, oxidation
and esterification using the intact perfused rat heart (152, 153, 154,
155).  Puramycin, an inhibitor of protein synthesis, was shown to have a
direct effect on the Krebs cycle activity by using aspartate 1   C an
immediate precursor of oxalacetate, an intermediate in the Krebs cycle
(155).

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An isolated perfused working heart from young female rats was used by
Hamberger and Isaksson to study the effect of chloramphenicol on mito-
chondrial and extramitochondrial systems  (156).  The authors believed
the isolated perfused working heart is advantageous for investigations
of metabolic inhibitors on protein synthesis in the cell, insofar as
increased load on the heart causes higher rates of protein synthesis. •
In this method developed by Morgan, in contrast to the Langendorff
method, the heart does the work.  The perfusate is introduced into the
left atrium and pumped by the left ventricle against a hydrostatic
pressure head.  Chloramphenicol was introduced into the perfusate at
concentrations of 50, 100, 250, and 500 yg/ml perfusate.  Chloramphenicol
decreased mechanical performance of the heart and caused a marked re-
duction in glucose uptake and lactate production.  Chloramphenicol
inhibition of mitochondrial protein synthesis in the isolated heart was
approximately 70%.

Isolated perfused rabbit hearts, using various modifications of the
Langendorff technique, were used to further investigate ionic altera-
tions after irradiation  (157, 158) and effects of fatty acids (159) on
the cardiovascular system.  Isolated rabbit hearts were irradiated at
the rate of 500 r per minute and compared with dog hearts irradiated at
150 r per minute.  The magnitude of the loss of potassium and the radia-
tion dose producing it were approximately the same for both systems.
The loss of potassium from hearts following cardiac arrest and further
loss occurring after resuscitation may be responsible for arrhythmias
noted in some human hearts following revival  (157).  The loss of potas-
sium from arrested and resuscitated rabbit hearts paralleled the potas-
sium loss observed in congestive heart failure in man  (157).  Tissue
calcium was increased in the isolated rabbit heart after resuscitation
and this was in agreement with work reported in the dog.

Connor et al.  (159) studied the effect of both saturated and unsaturated
fatty acids in the isolated rabbit heart.  0.1% stearic acid or 0.1%
oleic acid solution was added to the perfusate to the coronary vessels.
These fatty acids were very toxic; the coronary flow and the rate and
amplitude of contractions progressively deteriorated until there was
death of the heart.  Equimolar albumin incubated with the fatty acid
solutions bound the fatty acids and prevented this toxic effect.  Un-
bound fatty acids, saturated or unsaturated, were extremely toxic to the
heart.

Mir et al.  (160) used the isolated perfused rabbit heart to study the
pharmacological and toxicological effects of methacrylate monomers on
cardiac function.  They recorded the effects on the cardiac rate per
minute, force of contraction and coronary flow  (ml/min), of 13 methac-
rylate monomers each at dilutions of 1:1,000, 1:10,000, and 1:100,000 in
Locke's solution.  These compounds showed marked effects upon the iso-
lated heart.  Dose-response data are presented based upon both molar
concentrations and volume dilutions of these compounds.

The perfusion method of Langendorff with  various modifications has also
been applied to the isolated feline heart.  Swaine  (161) studied the
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 effect of phenoxybenzamine on Tyramine-induced catecholamine release in
 the isolated perfused cat heart.   After perfusing hearts for one-half
 hour with Krebs-Henseleit solution containing phenoxybenzamine,  they
 were perfused with phenoxybenzamine-free Krebs-Henseleit solution before
 addition of Tyramine to the perfusate.   The effect on the isolated cat
 heart by a muscle relaxant 2-amino-5-phenyl-l,3,4-oxadiazole hydrochloride
 caused no significant effect (162).

 Tanz e_t al. (163) studied 9-alpha-fluorohydro-cortisone in the isolated
 feline heart as well as the cat papillary muscle.   The findings of a
 positive inotropic effect in low  concentrations (0.5-1.0 ug/ml)  and a
 negative effect in higher concentrations (10-20 yg ml) correlated with
 histologic changes in cardiac muscle and with intramuscular ion exchange
 (163).

 Embryonic hearts are a valuable system for studying oxygen consumption,
 cell permeability and drug effects (164, 165).  Chick embryo hearts have
 been used at varying ages of the  embryo and sensitivity to drug effects
 at the different ages of development compared.  The effects of tetrodo-
 toxin on chick embryo hearts, single chick heart cells and aggregates of
 chick heart cells were compared (165).   Hearts were dissected from chick
 embryos aged 2 to 12 days.   Tetrodotoxin is reported to abolish spon-
 taneous activity in cells whose action potential is dependent on a
 transient increase in sodium conductance and in the chick heart this
 mechanism functions.  Sensitivity to tetrodotoxin increased with increas-
 ing embryo age.  All hearts from  2-4 day old embryos continued beating;
 by day 7, 43% of the hearts stopped beating.  Only a small fraction of
 isolated cells are sensitive to tetrodotoxin whereas aggregates formed
 from single cells are similar to  intact heart in both age-related and
 dose-related sensitivity (165).  Chick embryo hearts are useful for
 studies of glucose transport and  insulin action (164).  It was found
 that 2-deoxyglucose inhibits glucose uptake by 5 and 9 day old chick
 hearts and this inhibition is not modified by insulin action.  The
 mechanism regulating glucose uptake develops between the seventh and
 tenth day of embryologic development in the chick heart and at this
 stage glucose transport limits glucose uptake (164).

 Recent developments in maintaining fetal hearts in organ culture provide
 a system for studying the toxicity of compounds at various stages of
 fetal 'development as well as a potential bioassay for cardioactive
 drugs.  Both intact fetal hearts  and slabs of fetal hearts have been
 maintained in "culture.  Hughes and Longmore (166)  studied stages of
 development of mouse fetal hearts to survival time.  Heart rates were
 measured at different intervals during the culture period.  The authors
 also studied the beating and survival time for human, dog, cat and
 rabbit fetal hearts.  Mouse fetal hearts were cultured, on steel grids,
 from newborn, and 12-21 day old mice.

. Nineteen-day old fetal mouse hearts were beating and survived for 152
 days.  In comparing survival time of different species of different
 ages, it was found that younger and smaller fetal hearts survived the
 longest time.  Slabs of tissue survived for shorter times than did
 intact hearts.

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The performance of composite fetal hearts was studied (165).   Hearts
from litter mates, from different species and at different stages of
development-were used.  After 144 hours composite hearts beat synchro-
nously.  The beating rate of the composite was the rate of the faster
part.  Intact hearts from 38% term fetal cats survived for 39 days; 66%
term fetal rabbits survived 15 days.
BONE

A test system using fetal rat bones in tissue culture was used to study
agents that stimulate bone resorption.  25-hydroxycholecalciferol (25-
HCC)  (168), 1,25-dihydroxycholecalciferol (1,25-DHCC), and endotoxin
(169) were among the agents tested.

In this method  (170) bone shafts from 19-day rat fetuses labeled with
  Ca in vivo were incubated for 24 hours in a chemically defined medium
supplemented with 5% human serum inactivated at 60 C for 30 minutes.
Paired bones were transferred to vessels containing the same medium with
or without the test material.

Vitamin D  is hydroxylated in the liver to produce 25-hydroxycholecal-
ciferol  (25-HCC) which then enters the circulation and is taken up in
target organs  (176).  25-HCC is further converted to other active metab-
olites by a second hydroxylation largely in the kidney.  One of these
metabolites is 1,25-DHCC which is more potent than 25-HCC in mobilizing
calcium in vitro but not ill vivo.  The efficacy of these materials in
treating richetic animals was determined.  In the in_ vitro system the
release of previously incorporated 45 calcium from bones was measured.
Endotoxins  (169) were also used to stimulate release of 45 calcium from
fetal rat bone in tissue culture.  Endotoxins may play a role in bone
loss characteristic of human periodontal disease.

A test system utilizing-3 to 6 day old mouse calvaria in tissue culture
was used to study parathyroid  (172) as a powerful inducer of bone resorp-
tion, and diphosphonates  (173) and 2-thiophenecarboxylic acid  (174) as
inhibitors of bone resorption.  To study the inhibitory effect of 2-
thiophene-carboxylic acid, Fang e^t al.  (174), used cavaria of Swiss
albino mice of the Webster strain aged 4 to 6 days.  The frontal and
parietal bones were removed aseptically and cultured by two different
procedures.  In one method, the bone was attached to a coverslip with a
mixture of chicken plasma and chicken embryo extract  (2:1).  Control
medium was Gey's balanced salt solution and heated horse serum  (1:4).
In this control medium, a net uptake of calcium into bone was observed.
Bones were cultured in this medium containing parathyroid hormone alone
and also in combination with 2-thiophenecarboxylic acid.  In the second
culture method, bones were first labeled with   Ca by injection of
neonatal mice  2 days after birth; the bones were used 4 days later.
Each calvarium' was divided in half; one half served as an experimental
bone, the other as the control in a paired system.  The bones were
cultured in a  synthetic medium containing 5% heated rabbit serum.  The
release of   Ca from bones was measured in samples of medium by means of
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liquid scintillation spectrometry.   The bones were dissolved in 0.5 ml
formic acid and analyzed for the remaining isotope.  The author con-
cludes "2-thiophenecarboxylic acid is a simple compound, and therefore
may provide a useful new pharmacological tool for studies of bone metab-
olism and calcium transfer."

The effect on rat bone marrow metabolism by anaesthetic agents, nitrous
oxide, halothane, and cyclopropane (175) in the Warburg apparatus was
studied.  These anaesthetic agents are known to depress bone marrow
hematopoiesis in vivo and comparable doses to that used in man were
tested in the in, vitro system.  The measurement of the effect of these
agents on rat bone marrow oxygen consumption and anaerobic glycosis
showed no demonstrable inhibition at clinical concentrations.  At higher
concentration halothane depressed oxygen consumption.
SKIN

The skin is one of the largest organs of the body and assumes many
complex functions.  Among the functions studied using isolated segments
of skin from different species include transport characteristics  (176),
endogenous respiration (177), regulatory mechanism of lipid synthesis
(178), tensile strength (179), desquamation (180), and morphogenesis in
organ culture (181).

Early studies utilized skin pouches from skin removed from the hind legs
of frogs (176, 182).  The influence of steroids upon osmotic pressure
changes in skin pouches was studied (182).  Ringer's solution was intro-
duced into the pouch and the pouches were immersed in a bath of Ringer's
solution.  Steroids, dl-aldosterone, 2-methyl-9-oc-fluorohydrocortone,
prednisone, and prednisolone were added to the contents of one pouch,
the other pouch served as control.  dl-Aldosterone and 2-methyl-9-ac-
fluorohydrocortone produced an increase of osmolarity and increase in
total fluid volume inside the pouch.  Helman and Miller (182) reported
an in vitro method for the study of frog skins that does not produce
edge damage, a problem associated with skin studies.  By their technique
the tissue adhesive isobutyl-2-cyanocrylate (ethicon, Inc.) was used to
glue frog abdominal skin to Lucite gaskets which were sealed in Lucite
chambers with liquid Sylgard 184  (Dow Corning).  After the tissue adhe-
sive made contact with the skin it polymerized to form a bond between
the skin and the gaskets.  To determine the electrical resistance of the
skin, constant-current pulses, several hundred milliseconds in duration,
were passed through skins bathed on both sides with Ringer's solution.
Polyethylene bridges filled with 3M NaCl-agar connected the bathing
solutions to the external electronics and the tips of the voltage probes
were permanently fixed 0.5 mm from the surface of the skin.

Voute et al.  (180) found that aldosterone promotes a moult in isolated
frog skin and this moult was associated with bioelectric changes.  After
addition of aldosterone, the mitochondria-rich cells become pear shaped
and separate from the stratum corneum by a space filled with amorphous
material called a "lake."  The average number of mitochondria-rich cells
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with a "lake" in the outermost layer of the stratum granulosum is 9.3 in
aldosterone-treated skins, whereas it is 4.0 in controls.

Ziboii and Bradi (178) used isolated segments of rat skin to study the
effect of tetrolyl-pantetheine on lipid synthesis.  Skin specimens (60-
80 mg) were removed from the shaved and clipped back of a Sprague-Dawley
rat.  The specimens were incubated with a tracer amount of sodium ace-
tate 1-  C in Krebs-Ringer phosphate buffer and various concentrations
of tetrol-pantetheine at 37 C.  To estimate the total   C incorporated
into lipids, the incubation mixture was extracted four times and the
radioactive acetate in the extract was removed by chromatography.  The
incorporation of   C into lipids from acetate 1-  C was approximately
70% inhibited when tetrylyl-pantetheine was added to the incubation
medium.  Incubation of skin specimens with sulfhydryl compounds such as
L-cysteine and 2-mercaptoethanol failed to reverse the inhibition of
lipid synthesis by tetrolyl-pantetheine.  The authors conclude "this
study has further demonstrated the usefulness of whole skin preparations
for the study of regulatory mechanisms of lipid synthesis ..."

The morphogenetic effects of high levels of Vitamin A on adult mammalian
epidermis in culture was reported by Barnett and Szabo (181).  Explants
of skin consisting of the superficial dermis and whole epidermis were
obtained from the dorsum of the ear of adult guinea pigs and cultured
with the epidermis upward using a modified raft technique.  The cultures
contain Eagles or BGJ medium and 22-30. IU Vitamin A alcohol dissolved in
ethanol.  At the time of explantation and after 3, 6, and 10 days of
culture, explants were fixed and studied with an electron microscope.
Marked differences from control cultures were observed.  Among the
changes observed were no signs of overall keratinization by 6 days in
vitro, infrequent and short desmosomes and formation of numerous regu-
larly spaced micro-villi on both the free and internal cell surfaces.
Intercellular spaces were widened and canaliculi-like structures were
formed.  The authors compare their findings with those on in vivo
squamous metalasia of adult tracheal epithelium in Vitamin A deficient
rats and conclude "our epithelial cells, transforming from a kerateniz-
ing type to a mucous type, share a great many features with tracheal
cells which are midway along the path of squamous metaplasia from a
respiratory type."
STRIATED MUSCLE

A striated frog muscle, the sartorius, has proved to be a useful tool
for studying the effects of drugs on muscle contracture, metabolism,
structure, and action of the neuromuscular junction.  Caffeine pene-
trates muscle fiber membranes and activates contractions by producing an
increase in the intracellular Ca  concentration  (184) and is a useful
reference drug for studying muscle contractions and kinetics.

Using the single neuromuscular junctions of the frog sartorius muscle,
Longnecker et al. found that black widow spider venom causes exhaustion
of miniature end plate activity and depletes the nerve terminal of
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vesicles (185).  A sartorius muscle, with its nerve intact, was dis-
sected from Rana pipiens and mounted in a "lucite" chamber at 20-25 C.
Muscle twitch was blocked by bathing the preparation in a solution
containing 0.5 M CaCl , 4 mM MgCl , 2.5 mM KC1, 110 mM NaCl and 5 mM
Tris-buffer (pH 7.4).  Intracellular records of end plate potential and
miniature end plate potentials were obtained by impaling the end plate
region of surface muscle fibers with glass micropipettes filled with 3 M
KC1.  Extracellular records of the nerve terminal spike and miniature
end plate potentials were made with micropipettes filled with 4 M NaCl.

Marco and Nastuk (184) found that caffeine (0.25 to 2 mM/liter) produced
sarcomeric oxcillations which appeared at both the neuromuscular junc-
tion and non-junctional regions of frog skeletal muscle fibers.

Feinstein and Paimre  (186) present a discussion of the action of local
anaesthetics on excitation-contraction coupling in both striated and
smooth muscle.  They report that respiratory stimulation accompanies the
action of caffeine in frog muscle and is abolished by anaesthetics when
the associated intracellular calcium release is blocked.

Single muscle fibers, isolated from the semitendinosus muscle of the
frog, were used to study the relationship between the length of a frog
striated muscle fiber and the force it can produce (187).  The authors
report that one of the factors responsible for decreasing the force of
muscle contraction with shortening is inactivation of the myofibrils in
the core of a fiber, and that caffeine antagonizes this inactivation and
changes the length-force relationship at short muscle lengths.

Mouse or rat diaphragm tissue is a. striated muscle which has been used
to study the effects of drugs on carbohydrate metabolism and on muscle
tension.

A useful modification is the stimulatory effect of insulin on glucose
uptake in diaphragm tissue.  Oyama and Grant (188, 189) used pooled
mouse hemidiaphragms of male albino mice weighing 25-30 grams.  The
tissues were incubated in a Warburg apparatus in Krebs-Ringer bicar-
bonate,, with insulin and glucose.  Glucose uptake was calculated as mg
glucose per 10 grams of dry weight of tissue.

In comparing the effects of phenethyldiguanide, a hypoglycemic agent, in
the total guinea pig, in guinea pig liver slices and in the isolated rat
diaphragm, Tyberghun and Williams  (190) showed consistent results with
increased glucose uptake, decreased glycogen deposition and oxygen
consumption and increased lactic acid formation.  The rat diaphragm was
used to study cellular enzyme loss from muscle, by inducing such loss
with disodium dihydrogen ethylene diamine tetraacetate  (Na H EDTA)
(191).  The effects of ouabain on the isometric twitch tension in muscle
was studied with the isolated phrenic nerve diaphragm preparation  (192).
Ouabain increased the isometric twitch tension of the indirectly stimu-
lated rat diaphragm.  The diaphragm was analyzed for sodium, potassium
and calcium following exposure to ouabain plus indirect stimulation,
indirect stimulation alone, and to ouabain alone.  The greatest shifts
in Na, K, and Ca occurred in stimulated muscle in the presence of oua-
bain (192) .
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A striated muscle, the chick skeletal muscle, was used to study the
effects of chloroquine in tissue culture (193).   Long-term use of
chloroquine, in humans, for the treatment of malaria, has been asso-
ciated with retinopathy and myopathy.  Concentrations greater than
5 yg/ml in the culture medium interfered with myogenesis, the degree of
damage varying with drug concentration and duration of exposure.  The
method of Nameroff and Holtzer was used (194).  Mononuclear cell prep-
arations were made, through trypsinization, centrifugation, and disso-
ciation, from 11 day old chick embryo breast muscle tissue.  Chloroquine
phosphate was added to the experimental cultures before the cells were
placed in petri dishes.  The authors related their findings to human and
animal studies and concluded that long-term therapy in which chloroquine
concentrations in muscle may reach 40-80 times plasma levels would far
exceed the concentrations that produced tissue culture morphologic
changes.

Cohen and Fischbach (19,5) , using muscle fibers grown from myoblasts
obtained from embryonic chick breast tissue, showed that muscle fibers
in 7 to 10 day cultures generate action potentials and twitch in re-
sponse to depolarizing stimulus.  Many fibers twitch spontaneously but
the amount of activity varies from culture to culture and within a
single culture over long periods of time.  They demonstrated that the
activity of muscle fibers that develop in vitro from isolated myoblasts,
is sufficient to reduce the sensitivity of the surface membrane to
acetylcholine.
SMOOTH MUSCLE

The isolated rabbit aortic strip is a classical assay method used to
study the effects of various agents against epinephrin-induced contrac-
tions on vascular smooth muscle.  The assay method of Furthgott and
Bhadrakom and a modification by Wurzel et al. . (196) uses spirally cut
strips of rabbit aorta 2.0-2.5 cm in length and 2.0-4.0 mm wide.  The
strips are suspended in an organ bath at 37 C, in Krebs-bicarbonate
solution containing glucose and continually gassed at 95% 0-5% CO .
The drug under test is added to the tissue bath.  Tension is measured by
a force-displacement trnsducer and recorded on a polygraph.

Lyons and Swain (197) used a tension of 4 g on aorta strips and 1 g on
vena cava strips, to study the effects of catecholamines and the response
of blocking agents to these catecholamines on both aorta and vena cava
strips.  Epinephrine, norepinephrine and isoproterenol contract isolated
strip of both aorta and vena cava.  Isoproterenol is the least 'potent.
Phenoxybenzamine reduced contractions but they were not significantly
reduced by pronethanol.

Shemano and Fallen (198) studied the effects of L-3,3'5-Triiodothyronine
on epinephrine-induced contractions of isolated rabbit aorta, and found
potentiation when copper acetate or trace metal contaminants were present.
EDTA similarly potentiated epinephrine contractions.
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St. Clair and Loflana (199) describe an organ culture method for main-
taining arterial tissue in a metabolically active state for up to nine
days.  Segments of aorta, weighing approximately 25 mg, were obtained
from normal and atherosclerotic white carneau pigeons.  These segments
were placed intimal surface down on grids in organ culture dishes with
medium.  The culture medium contained Eagle's MEM (Earles), 20-50% serum
to which has been added the radioactive lipids, cholesterol 1,2,- 14 and
oleic acid 1-  C, penicillin, 1-glutamine, and streptomycin sulfate.

The amount of free and esterified cholesterol in individual segments was
determined by gas-liquid chromatography following separation on TLC.
Using this technique the .authors demonstrated the esterification by
arterial tissue of radioactive cholesterol added to the culture medium,
and the effect of atherosclerosis on cholesterol esterification.

The isolated rabbit or guinea pig ileum is used to study the spontaneous
activity of the intestine and the effect of measurable amounts of drugs
against this activity.  Mir et al. (200) studied the response of methac-
rylate monomers on guinea pig ileum and upon acetylcholine and barium
chloride induced contractions.  The spontaneous contractile activity of
a 1.5-2 cm section of guinea pig ileum in Tyrode's solution was recorded,
and then the test compound was added to the bath.  Eight of nine methac-
rylate monomers tested produced inhibition of pendular movements and
relaxation of the muscle within 15-30 seconds.  The effect of each
compound could be terminated by promptly washing with fresh ,Tyrode's
solution.  The isolated guinea pig ileum has been used widely in study-
ing drug effect for antispasmodics, analygesics, vasoconstrictors, etc.
It was used to determine the antiacetylcholine activity of d-, 1- and
dl-piperidyl methadone.

Ross et al. (202) found that atropine and morphine effectively block
angiotensin and nicotine-induced spasm on the isolated guinea pig ileum.
Hershberger and Hansen (203) found that hydrocortisone at 1.25 rag/ml
inhibited effects on activated anaphylatoxin on isolated guinea pig
ileum.

The uterus or oviduct is used to study effects on smooth muscle outside
the gastrointestinal tract.  Costa (204) studied the reactivity of uteri
of spayed rats brought into oestrus by injection of ovarian hormone, to
seratonin, creatinine sulfate, acetylcholine and oxytocin before and
after treatment with Frenquel (alpha-4 piperidyl diphenyl carbinol
hydrochloride), chlorpromazine (.10-(3-diethylaminopropyl), 2-chlorophe-
notenbthiazine hydrochloride), reserpine, demethoxy reserpine, Lergigan
(N-t2-dimethylamino-2-methyl-l-ethyl) phenothiazine hydrochloride),
mescaline  (3,4,5-trimethoxyphenyl ethylamine sulfate) and LSD  (d-lyser-
gic acid diethylamide).  They determined the uterine reactivity to a
series of increasing concentrations of serotonin, acetylcholine and
oxytocin starting from a minimally active concentration and increasing
until the maximal uterine contraction was reached.  Then the reactivity
of the uterus to the contractile agents was again tested after the
hallucinogenic or tranquilizing drugs had been left in contact with the
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uterus for three to five minutes.  Frenquel, chlorpromazine, and reser-
pine antagonized serotonin-induced contractions but not acetylcholine or
oxytocin contractions.

Mescaline facilitates serotonin activity and at high concentration
causes contractions.  A concentration of 1 mg/1 of LSD antagonizes both
serotonin and mescaline-induced contractions whereas low concentrations
of LSD facilitate the effects of serotonin and mescaline on the uterus.

Lener et al. (205) studied the relative effects of prostaglandins PGE
and PGF  on adenyl cyclase activity of oviductal ampulla and isthmus,
and uterotubal junction and the uterus of the immature rabbit.  They
found that the uterus responded to 10 yg/ml PGE  by doubling the percent
conversion of labeled nucleotides into cyclic AMP but synthesis was
unaffected by a similar concentration of PGF .  Isoproterenol incubated
with immature rabbit uteri increased and oxytocin decreased adenyl
cyclase activity in the immature rabbit uterus.

Easley et al. (206) compared the effects of pentagastrin, a pentapep-
tide/ on isolated uterine smooth muscle of virgin female rats with
isolated guinea pig ileum and in situ uterine segments of the rat, dog
and baboon.  Pentagastrin stimulated uterine contractions in vitro and
in situ.  The uterus was not as responsive to pentagastrin as was the
ileum.

Wessels et al. (207) review the role of microfilaments in cellular and
developmental processes, and cytochalsin B as a tool to investigate
these filaments.  When the oviduct is removed from a five day old chick
36 hours after estradipl injection and placed in organ culture in the
presence of cytochalsin B, all new gland formation ceases, glands al-
ready present regress by sinking back into the oviduct wall and the
microfilament bundles are dispersed.  The authors believe that this
shows a positive correlation between integrity of the filaments and the
morphogenetic process and that the critical role of calcium and high
energy compounds in muscle contracting may be part of a primative,
cytochalsin-sensitive system.
NERVE

Isolated nerves have been used to study the effects of compounds such as
epileptogenic agents (208, 209), hallucinogenic agents  (210), and nar-
cotic analgesics  (211) on nerve function.  Ayala et al.  (208) used the
crayfish muscle stretch receptor to study the effect of penicillin as an
epileptogenic and stellate ganglion of the squid to study the effect of
penicillin on an isolated synapse (209).

A desheathed cervical vagus nerve from a rabbit was used to demonstrate
the effect of A  tetrahydrocannabinol on nonmyelinated nerve fibers
(200).  For monophasic electrical recording a sucrose gap apparatus was
used.  For diphasic recording the nerve was mounted in a glass capillary
chamber in which five annular platinum electrodes for stimulation and
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recording were embedded.  The compound action potential of the non-
myelinated fiber was elicited each minute.  The perfusion fluid con-
tained Pluronic (a surfactant) in Lock solution, and 500 yM of A  tetra-
hydrocannabinol.  The compound action potential fell by 7.5, 13.3 and
18.6% of its initial vjalue after 15, 30 and 45 minutes of drug exposure.
Threshold effects of A  tetrahydrocannabinol in this method were obtained
at much higher doses than produce psychoactive effects in humans.  How-
ever, it was demonstrated that A  tetrahydrocannabinol directly affects
nerve fibers.
EPIDIDYMAL FAT PAD

The epididymal fat pad method has proven to be a sensitive and simple
method for the bioassay of insulin.  Addition of insulin to rat epidi-
dymal fat pads in vitro causes increases in glucose uptake, in CO-
evolution with glucose as substrate in fat synthesis and incorporation
of amino acids into protein  (212).  In this simple method epididymal fat
pads from young rats are placed in an incubation medium containing
Krebs-Henseleit or Krebs-Ringer buffer, test material, a substrate, and
are equilibrated with 95% 0-5% CO .  Wide use of this method is made
for studying glucose metabolism and lipolytic activity.

A glucose analog 6-deoxy-6-fluoroglucose was studied on both epididymal
adipose tissue and rat diaphragm (213).  6-Deoxy-6-fluoroglucose was
found to be an effective inhibitor of the oxidation of glucose.  The
stimulatory effect of insulin on glucose metabolism was blocked in fat
tissue by N-ethyl maleimide, a sulfhydryl blocking agent, indicating a
surface; action on the cell rather than an intracellular effect  (214).
Ethylene diamine tetraacetate was found to potentiate insulin activity
using the epididymal fat pad method (215).  EDTA is known to chelate
zinc and crystalline insulin contains 0.3 to 0.6% zinc  (215).

Epinephrine and non-epinephrine were shown to stimulate production of
nonesterified fatty acids and glycerol from rat adipose tissue when
incubated in rat plasma  (216, 217).  The effect of eight amino acids
upon glucose uptake of rat epididymal tissue showed significant inhibi-
tion of glucose uptake by arginine, glutamic acid, histidine, leucine,
lysine and tyrosine  (218).  Colchicine, which produces fatty infiltra-
tion in mice and rats, did not show a. direct stimulation of lipolysis in
the in vitro fat pad test  (219).  Pyrazole is a potential inhibitor of
alcohol dihydrogenase.  Because of its possible effect on ethanol-
induced fatty liver, pyrazole was studied using rat epididymal adipose
tissue in vitro both on basal and theophylline stimulated lipolysis
(220).  The authors found that pyrazole does not reduce basal lipolysis
but significantly decreases glycerol and free fatty acid release when
lipolysis is stimulated  (220).

The antilipolytic action of guanine derivatives on free fatty acid
esterification was studied by measuring the incorporation of palmitate-
1-C   into the lipid fraction of rat epidydimal adipose tissue  (221).
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DISCUSSION

Isolated organs are useful and work well as a bioassay system for screen-
ing chemicals for specific organ directed activity.  Three classical
assay systems, the epididymal fat pad, the guinea pig ileum, and the
rabbit aortic strip have been used for more than 20 years.

The epididymal fat pad is a simple and sensitive method for the bioassay
of insulin (212).  Wide use of this method is made for studying glucose
metabolism and lipolytic activity.  The guinea pig or rabbit ileum is
used to study the effect of measurable amounts of drugs on intestinal
activity.  It has been widely used for antispasmodics, analgesics, and
vasoconstrictors.  Isolated rabbit aorta strips have been used success-
fully as a test system for studying the effects of various agents against
epinephrine-induced contractions on vascular smooth muscle.

The isolated perfused liver appears to be a valuable system for studying
the metabolic fate of materials and good correlation with the intact
animal is frequently seen.  This system has been used to study plasti-
cizers, pesticides, and anaesthetic agents.

Piper and Vane  (104) described a method using isolated guinea pig lungs
which could provide a simple screening test for anti-inflammatory drugs.

Isolated segments of trachea have been used to study effects of ciga-
rette smoke, aerosols, and air pollutants on mucus flow, muscle contrac-
tions, and ciliary activity.  The results have been reproducible and
correlate well with in vivo studies.

Everted segments of rat stomach and small intestine have given repro-
ducible results.  Lasagna (124) states that this method "not only allows
the simultaneous study of drug metabolism and drug transport across
gastric and intestinal mucosa, but can also provide information on the
effect of drug metabolites on membrane transport of the drug."  A stri-
ated muscle, the frog sartorius, has been useful for studying the effects
of drugs on muscle contracture, metabolism structure and action at the
neuromuscular junction.  Caffeine and local anaesthetics have been
studied using this method.

Isolated organs and tissues, including organ culture systems, have been
very useful in getting at mechanisms of toxic action and providing an
understanding of how a chemical exerts its toxic effect.  In this role,
such systems should become increasingly important.  However, the use of
isolated organs has serious limitations for studying toxicological
effects, due in part to the modulating systems existing in the whole
animal which can either increase or decrease an effect.  The usefulness
of these systems for toxicological screening also remains limited,
primarily because such systems are most useful for screening large
numbers of chemicals for a specific effect as opposed to screening a
chemical for multiple biological effects.  Additionally, in most cases,
isolated organ systems use almost as many animals as an in_ vitro test
would require.  Consequently there is little saving in total animal use.
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C.   USE OF MAMMALIAN AND AVIAN CELL CULTURE SYSTEMS IN STUDIES ON
     CHEMICALS
INTRODUCTION

Cell Culture Methodology

Within the past 20 years, the techniques of culturing cells in vitro
have been simplified so that their use is no longer restricted to highly
specialized research projects.  Techniques have become well defined and
media and equipment are readily available to biochemists, microbiologists,
pharmacologists, and other biological scientists.  Perlman  (222) has
stated that there are many uses of animal cell cultures other than those
related directly to virus and cancer research.  These include  (i) study
of mechanisms of cytotoxicity and correlation of the cytotoxicity of
drugs with other pharmacological attributes;  (ii) study of the biogene-
sis of hormones and other "vital" products at the cellular level; (iii)
determination of nutritional requirements of mammalian cells from "spe-
cialized" tissues or cells grown' under unusual stresses; and (iv) study
of host-parasite relationships at the cellular level.  In fact, this
list could now be expanded to include virtually any aspect of cell
biology including aging, intermediary metabolism, cytogenetics, and cell
cycle kinetics.

Smith et al. (223) reported that a statistical analysis on the correla-
tion between tissue culture cytotoxicity and whole animal toxicity
demonstrated a significant correlation for seven chemical classes of
compounds.  However, the correlations were not predictive for all com-
pounds as markedly cytotoxic agents had relatively low acute animal
toxicities and vice versa.

Although in vitro and in vivo correlations may not be absolute, cell
culture techniques have created new approaches for assessment of chemi-
cal activities.  Rapid bioassays for evaluation of primary and early
events in carcinogenesis may be conducted in an environment free of host
modifications and at the cellular level (224).  The extensive testing,
validation, and comparison with whole animal results can provide a
useful additional method of screening for toxicity in spite of problems
of relating cellular level results to their effects in the whole animal.
It is of prime importance to note that potential toxicity may only be
expressed as any significant change in the growth and function of treated
cells when compared with untreated cultures.
HUMAN CELL CULTURE SYSTEMS

Human Carcinoma Cell Culture Systems

Human carcinoma cells have been used frequently in toxicological studies
which include nucleic acid analyses, karotype analyses, as well as
metabolic and cytological properties of cultures.  HeLa cells  (human
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cervical carcinoma),  KB cells (Human nasopharynx carcinoma), and HEp 2
(human larynx carcinoma)  are routinely used in such studies.

Kolodny (225) studied the inhibition of cell division by sodium chloro-
iridate on a variety of cells including HeLa cells.  Confluent cultures
exposed to 3 x 10   M sodium chloroiridate were pulsed at appropriate
intervals with  H-leucine,  H-uridine, or  H-thymidine.•  Protein and
nucleic acids were isolated following a 20-minute pulse at 37 C and
counted.  Results indicated that the compound inhibited synthesis of
both DNA and RNA but not protein.

A potent carcinogen,  4-nitroquinoline 1-oxide (4NQO) was found to inac-
tivate HeLa cells and Sendai Virus-carrier HeLa cultures (HeLa-HVJ)
(226).  Incubation periods in the presence of 10 yM 4NQO produced 10 to
100 fold inactivation of HeLa as measured by 30  to 60% inactivation of
HeLa-HVJ cells.

The demonstration of an increased susceptibility of cells to the effects
of ionizing radiation by combination with potentiating chemicals could
be employed as both a biological radiation assay and as a practical
application of radiation therapy.  Hadacidin was found to potentiate the
lethal action of X-radiation on a number of tumor cell lines, including
HeLa, HEp 2, and KB cells (227).  Cloning experiments were conducted in
order to demonstrate additive cytotoxicity.

The cytotoxicity of Triton WR-1339 for HeLa cells was studied by Zimmer-
man e_t al_. (228).  The respiration rates, levels of Krebs cycle enzymes,
and,the morphology of mitochondria were altered by non-ionic detergent
with this treatment.

Brookes and Duncan (229)  conducted experiments on human embryo cells and
compared their results with the effects produced on HeLa cells.  The
fates of benzo(a)pyrene (BP) and dimethylbenz(a)anthracene, (DMBA) in
cell culture was followed by use of tritiated hydrocarbons.  The binding
of BP to DNA, RNA, and protein was significantly less in HeLa cells than
in human lung cells while the binding of DMBA was similar in both cases.
Neither BP nor DMBA affected the plating efficiency over a range of 0.05
to 50 yM/ml of medium.  The authors suggest that BP possesses transform-
ing potential for human lung cells.

Toxicity studies using mycotoxins in HeLa cells have been reported
(230).  Crude extracts at levels of 5 to 20 yg/ml were observed to pro-
duce lysis of cells after 18-hour exposures.  Monolayers of cells were
trypsinized and counts of suspensions demonstrated cell cytotoxicity.

Chabbert and Vial (231) described a diffusion method for the study of
the cytotoxicity of antitumor agents on HeLa and KB cells grown as
monolayers (231).   The monolayer is covered with agar medium and paper
discs impregnated with the cytotoxic agent are deposited on the surface
of the medium.  After 16 hours, the agar medium is replaced by liquid
medium and the culture is allowed to grow.  A clear zone, indicative of
toxicity, develops in the area where the toxic substance was deposited.
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The action of 2,6 diaminopurine (DAP), a purine analog effective against
transplantable tumors, was studied in KB cells.  The compound was shown
to stimulate glucose utilization and lactic acid porduction.  Cytotoxic-
ity of hadacidin and 2-deoxy-D-glucose was potentiated in KB cells by
diaminopurine.  The compound was administered in growth medium and
cultures were observed for four to five days for cytopathology and for
48 hours for biochemical determinations.

An agar bioautographic system for analysis of cytotoxic agents was
developed by Grady e_t al. (232) .  KB cell monolayers under agar are used
as a support for developing paper chromatograms of actinomycin D, mito-
mycin D, streptovitacin, echinomycin, and fermentation liquors contain-
ing unidentified cytotoxic agents.  After a 24-hour incubation, the agar
and papergram were floated from the Petri dishes with Earle's salt
solution.  Zones of inhibition were observed directly, or by formalin
fixation followed by Giemsa staining.  The technique allows detection of
multiple component cytotoxic activities and identification of components
during purification.

The inhibitory effects of hadacid on KB cell cultures was studied by
Neuman and Tytell (233).  The growth-inhibitory substance which is
active against human derived tumors was found to cause interference with
purine metabolism.

The synthesis of DNA by HeLa cells as affected by dihydroxyphenylethyl-
amine (Dopamine) was studied by Prasad and Kallmorgen (234) .  Several
concentrations of dopamine (5-50 Vg/ml) were added to culture media and
radioautographic preparations using  H-thymidine were made as a function
of time.  .The compound was found to reduce  H-thymidine incorporation
into newly synthesized DNA and therefore inhibited growth of the cells.

The effect of methyl methanesulphonate  (MMS) on replication of newly
synthesized DNA in non-synchronized cultures of HEp 2 cells was studied
by Coyle et ajl. (235) .  Cells were treated with MMS for one hour.  The
MMS was washed out and the cells were incubated in medium for various
periods -prior to addition of tritiated thymidine.  MMS-treated HEp 2
cells were found to make DNA by semiconservative synthesis but that a
portion of this new DNA is not itself replicated.

The effect of Dilantin on cell proliferation in cell cultures of human
fibroblasts was studied by Shafer (236) and Houck ejb al. (237).  Gingival
fibroblasts and cutaneous fibroblasts were used respectively to deter-
mine if Dilantin did indeed stimulate cell proliferation in vitro which
would be analogous to observations of decreased wound healing times
observed in Dilantin-treated humans and animals.  Dilantin at a con-
centration of 200 yg/ml was found to stimulate proliferation of gingival
fibroblasts by cell couting procedures and a concentration of 2 Ug/ml
increased the rate of human diploid fibroblast proliferation.  Tritiated
proline analyses and total protein analyses did not demonstrate any
effect on cellular collagen synthesis by Dilantin-treated fibroblasts.
Cell doubling time, total protein, and collagen analyses could be em-
ployed to study chemical effects in other cell systems.
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Human Lung Cell Systems

Human lung cell cultures have been employed as host systems for toxicity
studies of chemicals, aerosols, and gases using lung explants, primary
fetal cultures, and diploid fibroblastic cultures.

Leuchtenberger et_ al. (238) exposed human lung explants to puffs of
fresh marijuana or tobacco smoke and performed chromosome and DNA anal-
yses on treated cells.  Feulgen microfluorometry and karyotype analyses
showed that marijuana and tobacco smoke evoked abnormalities in DNA
synthesis, mitosis, and growth which were observed early and persisted
for prolonged periods after exposure.  The use of in vitro systems for
such studies provides rapid results and are more economical than when
whole animals are employed.

Stockton et al. (239) developed a system in which human embryonic lung
cells could be exposed to ozone in order to evaluate its effects on
cellular proliferation and morphology.  Cell culture monolayers, washed
with protein-free BSS and inverted to insure removal of BSS and more
direct contact with the gas, were exposed to several concentrations of
ozone in air plus 5% CO .  During long exposures, cultures were rinsed
with BSs at frequent intervals to prevent cell damage due to dehydra-
tion.  Ozone concentrations as low as 4 ppm appeared to have an inhib-
itory effect on cell proliferation possibly caused by alteration of cell
membranes.

Barile and Hardegree (240) studied the effects of the oil-adjuvant
emulsifying agent Arlacel A (AA) on a series of cell culture systems
which included WI-38 huinan embryonic lung cell cultures.  Cell culture
monolayers were inoculated with AA by layering the monooleate onto the
medium.  After a three hour incubation, the cells were fed fresh medium.
Cytotoxic effect (CTE) was scored at three days.  Four of 19 lots of AA
assayed by cell culture procedures were found to produce CTE.  The 19
lots were also assayed by the Berlin test procedure to determine mouse
toxicity.  Observations of percentage of weight change and presence of
peritoneal adhesions at sacrifice showed that 3 of 4 lots toxic by cell
culture assay were toxic by the mouse assay.  The findings showed that
the cell culture assay is equally if not more sensitive than the routine
mouse assay.

Aflatoxins, a mixture of toxic metabolites produced by Aspergillus
flavus, the primary biological assay of which is conducted in Peking
White Ducks, were studied in embryonic lung cells (L-132) by Legator and
Withrow  (241).  The aflatoxins were dissolved in either propylene glycol
or chloroform and added to culture bottles into which cell suspensions
in medium were added.  The chloroform, however, was removed prior to
addition of cells.  A 43-55% reduction in mitotic frequency was observed
using slide preparations.  The results of the study furnish a biological
parameter of aflatoxin toxicity in which 0.01 yg of toxicant can be
detected within 24 hours.  The duckling and embryonated egg procedures
detect levels of 0.25 ug within 10-21 days.
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Carr and Legator (242) studied the effect of hexachlorophene on the
metabolism of human embryonic lung cells.  Through the use of pulsing
studies employing tritiated thymidine, urine, and lysine during one cell
generation, the authors observed a slight inhibitory effect on cell
growth at a concentration of 1 yg/ml.  DNA, RNA, and protein synthesis
was not inhibited at hexachlorophene levels of 1 yg/ml but inhibition of
the synthesis of these macromolecules was noted at concentrations of 5,
7.5, and 10 Ug/ml.  The adverse effects were thought to be associated
with the uncoupling of oxidative phosphorylation.

Human Leukocyte Cell Systems

Human leukocytes cultured in_ vitro have been demonstrated to be extremely
important research systems to evaluate the toxicological effects of
drugs on cells of human origin.  Procedures for culturing leukocytes are
readily available and the method by which cells are obtained for study
is not traumatic to donors.  The indices used for leukocyte studies
include chromosome analyses, phagocytic activity, DNA, RNA, and protein
synthesis, and viability evaluations through use of counting procedures
and vital dye exclusion observations.
      i
The production of chromosomal aberrations following exposure of human
lymphocytes to adriamycin  (243) , chlorpromazine, and meprobamate (244),
ICRF 159 (1,2 bis dioxopiperazine 1-yl) propane  (245), Busulfan (246),
cyclamates (247), and acetyl salicyclic acid (248) have been reported.
Routine orcein or Giemsa staining procedures were used to stain meta-
phase spreads prepared with colcemid, colchicine, or vinblastine sulfate
following drug treatment.

Adriamycin was found to produce chromosome aberrations in leukocytes
treated before or simultaneously with deoxyribose cytosine at levels of
0.05 to 0.25 yg/ml and 1 to 4- x 10   M/ml.  Significant chromosome
abnormalities were not observed when chlorpromazine (CPZ) was added to
leukocyte cultures at concentrations of 5 x 10   to 2 x 10   M. _How-
ever, blast transformation was inhibited by concentrations of 10   to
10   M.  Meprobamate in concentrations from 10   to 10   M did not
influence the rate of blast transformation as in the case with the CPZ-
treated cultures.

The cytostatic agent ICRF 159 at concentrations of 1 to 10 yg/ml was
found to block the entry of cultured human lymphocites into mitosis and
stop dividing cells in prophase and early metaphase (G.-M).  Dose levels
of less than 1 yg/ml of Busulfan produced no observable chromosomal
alterations but doses of 1 to 20 yg/ml produced secondary constrictions
and breaks.  Cyclamate at a concentration of 200 yg/ml (15 g/75 kg)
stimulated chromosome breakage in human leukocytes.

Human leukocyte cultures and leukocytes isolated from human volunteers
were exposed to acetylsalicyclic acid in a dose range of 0.1 to 300 yg/ml.
Leukocytes also were cultured from volunteers who had been treated with
2400 mg per day for 30 days.  No significant chromosomal aberrations
were observed in either system.
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Savel e_t al_. (249) and Lieberman e_t ad. (250)  studied the uptake of
labeled thymidine to determine the effects of aflatoxin, proximate
carcinogens, and alkylating agents on cultured human lymphocites.  The
addition of aflatoxin to phytohemagglutin-stimulated peripheral blood
lymphocytes resulted in inhibition of tritiated thymidine uptake when
compared to control cultures.  In addition,, aflatoxin suppressed thymi-
dine uptake in lymphocyte cultures obtained from PPD and mumps reactive
donors when the cultures were challenged with PPD and mumps antigens.
In these assay systems, nitrogen mustard, methyl methanesulfonate, and
ethyl methanesulfonate increased thymidine incorporation 7 to 8 fold
above that noted in the controls.  B-propiolactone and N-acetoxy-2-
acetylaminofluorene stimulated incorporation of 4 to 9 times as much as
controls.  The demonstrations of increased thymidine incorporation
suggests that DNA is being repaired following drug-induced breakdown.
Whether the repair is functional is not known and studies at the molec-
ular level must be conducted in order to determine functional repair and
to identify cell damage and/or malignant transformation.

Lieberman (250) and MacKinney and Vyas (251) used dye exclusion tests to
measure lymphocyte viability when the cells were exposed to carcinogens,
alkylating agents and diphenylhydrantoin, respectively.  Either trypan
blue (0.1%)  or erythrocin B  (0.4%) in 0.15 M phosphate buffered saline
was used to identify cytotoxic effects produced by the test compounds.

Hirshaut et al. (252) examined the effects of four clinically active
antileukemic drugs in six long-term human leukocyte cultures.  The drug
concentration-time relationships  (G x T)  and dose response curves for
6-mercaptopurine, methotrexate, vincristine, and prednisolone were
evaluated in cell lines RPMI 6410, AL-2,  RPMI 8205, P J, RPMI 2217, and
L 1210.  All four drugs inhibited cell growth of long-term leukocyte
cultures with the level of inhibition depending on the drug and the
concentrations employed.  The findings generally agreed with clinical
observations in humans.  The authors stated that in vitro procedures
will not replace clinical trials but will, however, provide a tool in
which C x T relationships might be predicted more rapidly.

Kvarstein and Stormorken (253) demonstrated that acetylsalicyclic acid,
butazolidine, colchicine, hydrocortisone, chlorpromazine, and imipramine
inhibited the uptake of polystyrene latex particles and oxygen consump-
tion by human leucocytes.  The drugs were added to cell suspensions,
incubated for five minutes at 37 C using varying concentrations and then
exposed to 1.1 nm diameter polystyrene latex particles  (PLx).  The
phagocytosis of PLx was quantitated and oxygen consumption of cultures
during phagosytosis was measured using a Clark electrode.  All the drugs
tested inhibited both the uptake of PLx and oxygen consumption.  The
authors postulated that the phenomena may be caused by drug influence on
lysosomal membranes or cell plasma membranes.

The effect of hycanthone (an antischistosomal drug) on phytohemagglutinin
(PHA) stimulated human lymphocytes was studied by Sieber et al.  (254).
Five ml volumes of cells were exposed to hycanthone concentrations of
0.05 to 50 yg/ml.  Labeled precursors,  H-thymidine,   C-uridine, and
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14
  C-leucine, were added to cell suspensions two hours before harvesting
at 48 hours.  The cells were washed, macromolecules harvested and radio-
activity in DNA, RNA, and protein measured by liquid scintillation
spectrometry.  The morphology and mitotic indices of PHA stimulated
leukocytes was also observed.  Protein synthesis was not inhibited at
any time interval, whereas both DNA and RNA synthesis were inhibited at
various times with maximum inhibition at 30 hours.  When hycanthone was
added at the beginning of the 48 hour incubation period, the mitotic
index (MI) decreased to 50% of the control.  The MI was reduced to 0 at
concentrations of 20 yg/ml or higher.  At concentrations of 50 yg/ml,
chromosomal analysis could not be made due to extensive damage.  In
addition to in vitro studies, the authors conducted embryotoxicity
studies in pregnant mice.  Hycanthone was administered on days 6-11 of
gestation by the subcutaneous route.  The drug did not produce terato-
genic activity although body weights of fetuses were depressed.  Doses
of 25-50 yg/kg increased fetus resorption.

Human Erythrocyte Cell Systems

Although specific cell culturing techniques are not available for eryth-
rocyte studies, techniques are available to detect physiological changes
produced by exposure to toxic compounds.  Cell deformability, measured
by changes in filterability, has been used to determine the effects of
prostaglandin E , epinephrine, and isoproterenol  (255).  Osmotic fragil-
ity and hemolysis (256 and 257) have been used to evaluate cellular
damage produced by p-chloromercuribenzoic acid, lead and mercury.  The
inhibition of erythrocyte agglutination by homologous antisera has been
used to study the physiological effects of phenothiazines and anti-
histamines  (258).  Test compounds were added to the antisera and slide
agglutination tests were performed.

Human Liver Cell Systems

Human liver cell cultures have been used for in vitro hepatotoxicity
studies involving metabolic and cytotoxic parameters.  Savchuck et al.
(259) used a cell culture system to investigate the action of arsenicals
on living systems.  Chang's human liver cells were exposed to sodium
arsenite, sodium arsenate, arsenobenzene, 4-nitrophenyl arsonic acid, 3
nitro-4-hydroxyphenyl arsonic acid, and arsonelic acid.  The growth
response of mammalian cells was determined after a period of continuous
exposure to the arsenicals using DNA content as an index of size of cell
populations.  The DNA assay used was a colorimetric test for deoxyribose
using trichloroacetic acid hydrolysis, p-nitrophenylhydrazine, n-butyl
acetate extraction, and NaOH colorimetric development.  Deoxyribose
content was detected at 560 my.  The cells were observed to tolerate all
the arsenic compounds tested except arsenobenzene.  The value of this
study was to present a DNA assay which is useful in estimating cell
populations in cell culture.

Dujovne and Zimmerman  (260) amd Zimmerman and Kendler  (261) evaluated
the effects of two phenothiazine compounds, chlorpromazine  (CPZ) and
promazine (PZ), as well as structurally related analogues on the enzyme
content of Chang's human liver cells.  Exposure of Chang cells to each

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of the phenothiazines led to leakage of lactate dehydrogenase, malate
dehydrogenase, asparatate aminotransferase and glutamic oxalacetic
transaminase.  The demonstration of enzyme leakage from tissue culture
cells of hepatic origin exposed to phenothiazines in vitro was taken as
presumptive evidence that these drugs alter or damage the permeability
of cell membranes.  It should be noted, however, that this class of
drugs exert potent antihistaminic and anticholinergic effects by block-
ing cell receptor sites.
NON-HUMAN CELL CULTURE SYSTEMS

Hamster Carcinoma Cell Systems

The transformation of Chinese hamster and Syrian hamster embryo cells in
vitro has been reported by a number of investigators including DiPaolo
e_t al. (262, 263, 264), Casto e_t al_. (265), Huberman et al^. (266), and
Sivak and VanDuuren (267, 268, 269).

The research presented in a major paper by DiPaolo (264), describes the
morphological, oncogenic and karyological characteristics of Syrian
hamster embryo cells transformed in vitro by certain carcinogenic poly-
cyclic hydrocarbons and continues to explore the possibilities and
results presented in previous papers (262, 263).  The in vitro results
are correlated with other in vivo experimentation demonstrating the
efficacy of the in_ vitro transformation assay of hamster embryo cells to
elucidate and predict carcinogenic compounds.  Carcinogenic hydrocarbons
induced a quantitative transformation on cells as detected by the pro-
duction of discrete colonies after plating.  This transformation was not
observed in studies using pyrene (noncarcinogen) or solvents alone.
Transformation was defined as a criss-cross pattern of both light and
dense clones producing altered colonies which were not observed in the
controls.  Altered and normal colonies were then developed into estab-
lished cell lines which were analyzed in terms of their morphology,
oncogenicity and karyology.  Cells of the established lines derived from
each transformed culture formed tumors when implanted subcutaneously
into hamsters.  No tumors were produced when control cells were rein-
oculated into irradiated animals.  The authors conclude that since the
colonies were transformed only with the carcinogens and developed cell
lines which produced tumors when injected into hamsters while the con-
trol untransformed lines did not, that the iri vitro model is reliable
for studies of carcinogenesis and is relevant to in vivo cancers  (264).
It was noted that the frequency of appearance of altered clones was
related to the known carcinogenic potency of the compounds tested and
that the toxicity increase with the amount of the compound was also.
related to its potency as a carcinogen.  At a constant carcinogen con-
centration, the number of altered clones increased with the number of
cells exposed (263).

DiPaolo, Donovan, and Nelson have also demonstrated that an in_ vitro
transformation in Syrian hamster embryo cells using polycyclic hydro-
carbons can occur with no concomitant cytotoxicity (270).
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The transformation of hamster cells that were seeded to form colonies
was enhanced when the cells were treated with either a flavone or benz-
(a)anthracene before the addition of a potent carcinogen, benzo(a)pyrene
or 3-methylcholanthrene.  7,8-Benzoflavone and benz(a)pyrene prevented
the cytotoxicity by the carcinogens while 5,6-benzoflavone did not.  The
authors thus showed that it is possible to disassociate the transforming
and toxic properties of benzo(a)pyrene and 3-methylcholanthrene.

Umeda and lype describe an improved system of comparison for in vitro
transformation rates based on the cytotoxicity which is produce by
chemical carcinogens (271).  Almost all chemical carcinogens tested have
cytotoxic as well as transforming effects on cells.  Usually the trans-
formation rate and survival curves are plotted independently against the
concentrations of the carcinogen used.  This makes it difficult to
compare the transformation rate at equitoxic levels of different car-
cinogens.  The authors overcame this problem by plotting the transforma-
tion rate against the relative plating efficiency of the carcinogen-
treated cells.  They used this technique in presenting their own data on
studies of the effects of pH variation in the culture medium of Syrian
hamster embryo secondary cells plated over lethally X-irradiated rat
embryo feeder cells.  These cultures with pH values of 7.8 and 7.4, were
then treated with 9,10-dimethyl-l,2-benzanthracene (DMBA).  Seven to
eight days after the carcinogen treatment they were fixed in methanol
and stained with Giemsa.  The authors plot the data generated by the
conventional methods previously described and by their own.  Their
procedure graphically depicted the correlation between the cytotoxicity
and transformation rate induced by DMBA, although the different metab-
olites of DMBA may differ in their relative capacities for transforamton
and toxicity.

Syrian fetal hamster cells were exposed to transforming doses of certain
polycyclic hydrocarbons (7,12-dimethyl benz(a)anthracene, 0.05 yg/ml;
benzo(a)pyrene, 10 yg/ml;  3-hydroxy benzo(a)pyrene, 10 yg/ml; benz(a)-
anthracene, 6 yg/mlj and analyzed to show the early changes in chromo-
some number and structure after treatment  (272).  Metaphases were found
having abnormalities in chromosome number and structure within 24-74
hours after exposure.

Cocarcinogenesis in hamster embryo cells, a concept previously described
with the Rauscher leukemia virus-infected rat embryo cell lines is
explored by Casto et al.  (265).  Pretreatment of hamster cells in. vitro
with carcinogenic polycyclic hydrocarbons markedly enhanced the trans-
formation of these cells by an oncogenic adenovirus (SA7).  Eighteen
hours prior to addition of the virus treatment with benzo(a)pyrene, 3-
methylcholanthrene, 7,12-dimethyl benz(a)anthracene, dibenz(a,h)anthra-
cene, or dibenz(a,c)anthracene enhanced transformation but was inhibited
when the cells were treated five hours after the viral addition.  Non-
carcinogenic hydrocarbons did not stimulate SA7 transformation.  Trans-
formation was enhanced in direct proportion to chemical concentration up
to a point.  Higher concentrations resulted in either a decrease or
complete inhibition of the viral transformation.  The data indicated
that the stimulation of viral transformation resulted from a direct
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effect of the chemicals on the cells which increased their sensitivity
to the adenovirus.transformation.

Gasto and DiPaolo (273) produced a major state-of-the-art review of
cocarcinogenesis iri vitro (273).  However, in this paper, X-ray enhance-
ment of chemical transformation was included as well as viral enhance-
ment.

Transformation of hamster embryo fibroblasts by benzo(a)pyrene and 7/12-
dimethylbenzCa)anthracene has been reported (269).  Cells of the trans-
formed lines had altered karyotypes and the benzo(a)pyrene induced line
was tumorigenic in_ vivo.

The effect of phorbol myristate acetate on RNA synthesis in benzo(a)-
pyrene transformed hamster embryo lines along with 3T3 and SV40*3T3
mouse cultures was studied (268).  While the chemical enhanced RNA
synthesis in the stationary culture of 3T3 cells, it did hot affect the
virally transformed mouse cell line or the chemically transformed
hamster embryo line.

Syrian hamster embryo cells were included along with C3H mouse prostate
cells as the only two systems wherein chemical carcinogenesis had been
firmly established in 1971 (274).  The binding of labeled carcinogenic
polycyclic hydrocarbons to the DNA, RNA and proteins of the transformable
cells in culture was demonstrated and certain correlations to in vivo
work were described.

Sivak and VanDuuren (275) utilized a mixed culture system in order to
assess the tumor promoting activity of certain tobacco leaf extracts and
cigarette smoke condensate (phorbol myristate acetate).  This system was
described in previous papers with mouse 3T3 and SV40-3T3 cell lines     N
(268, 276, 269).  Briefly, the growth of clones of either virally or
chemically transformed cells when mixed with an excess of contact inhib-
ited (untransformed) cells is enhanced on exposure to tobacco leaf
extracts, phorbol esters from croton oil and other chemicals.

N-2-fluorenylacetamide  (FAA)  and its two metabolic derivatives, N-
hydroxy-N-2-fluorenylacetamide and N-acetoxy-N-2-fluoroenylacetamide,
were studied with Chinese hamster cells for toxicity and mutagenicity
and with Syrian hamster cell lines for toxicity and transformation.  The
frequency of mutations as measured by the production of 8 azaguanine-
resistant colonies in Chinese hamster cells was calculated per 10
survivors taking into account the number of cells at the time of treat-
ment and the percent survivors.  Cytotoxicity, in the Chinese hamster
cells which were plated, treated with the acetone-dissolved compound,
incubated 6-8 days, methanol-fixed and stained with Giemsa, was expressed
as the percent of the number of colonies in the treated dishes to those
in the untreated dishes.  Cytotoxicity and transformation was performed
in the Syrian hamster embryo cells.  The colonies, after plating, were
fixed, stained and scored for transformation and percent survivors 7-8
days later.  Cytotoxicity was calculated the same as for Chinese hamster
cells and percent transformation was determined as the percent of the
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number of transformed colonies found as compared to the total number of
colonies scored.  Cytotoxicity, mutagenicity and transformation frequency
increased with the concentration and degree of reactivity of the deriva-
tive FAA.

The most commonly reported alterations in malignant cells are an increased
aerobic glycolysis and an inhibition of respiration by glucose addition
(277).  Aerobic glycolysis of hamster embryonic cells (HE) transformed
with 4-nitroquinoline-l-oxide and its derivative, 4-hydroxyaminoquinoline-
1-oxide HC1 increased.  Respiration was inhibited by glucose addition in
the presence of pyruvate.

An investigation was performed to elucidate the mode of incorporation
and binding of the carcinogen, 4-nitroquinoline-l-oxide (4-NQO) to
hamster embryo cell (HE) cultures (278).  This carcinogen is incorpo-
rated within a short time after introduction to the growth medium and is
bound to the macromolecules of the HE cells.  This binding persisted for
72 hours after treatment.  The rate of incorporation of N-4QO is directly
proportional to its concentration and inversely proportional to cell
number.

Rhim and Huebner depict an in vitro transformation assay of 12 major
fractions of cigarette smoke condensate using mouse and hamster embryo
cell lines  (279).  Transformation to numerous fractions correlated well
with the corresponding development of tumors in vivo.

The flavone, 7,8-benzoflavone  (7,8-BF), at concentrations 10 to 20 times
greater than that of the hydrocarbons, almost completely inhibited the
metabolism of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene to their
water-soluble derivatives in hamster embryo cell cultures (280).  The
isomer 5,6-b'enzof lavone  (5,6-BF) induced some aryl hydrocarbon hydroxyl-
ase activity (AHH) in the cells, while 7,8-BF inhibited normal enzyme
activity as well as induction of AHH by benz(a)anthracene.  7,8-BF
protected hamster embryo cells against cytotoxicity induced by 7,12-
dimethylbenz (a) anthracene (DMBA), benzo(a)pyrene, and 3-methylcholan-
threne while 5,6-BF gave a slight protection at high concentrations.
This data was compared to the effects of 5,6-BF and 7,8-BF on DMBA-
induced adrenal necrosis and lung tumorigenesis in vivo, and certain
mechanisms of action and inhibition were proposed.

The transitions between the stationary, or confluent, state and the
active, or cycling, state were examined for hamster cells in vitro
(281).  Values of a number of cell parameters  (fraction of cells syn-
thesizing DNA, rate of DNA synthesis, amount of DNA per cell, growth
rate) indicated that the cells entered the stationary phase in G  and
then doubled their DNA content without further division.  While Che
sensitivity to sulfur mustard or 1,3-bis(2-chloroethyl)-l-nitrosourea
(BCNU) was not significantly different for active or confluent cells,
the sensitivity to actinomycin D was greater for the confluent cells.
The conclusion drawn is that the "stationary phase" of confluent hamster
embryo cells does not correspond to the G  of .hemopoietic stem cells,
and that a state of "no cell cycle" is not by itself sufficient to give
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resistance to the drugs studied.  Also, the post-treatment ability of
the cells to repair the damage caused by sulfur mustard or BCNU was not
enhanced by a state of "no cell cycle."

Diamond studied the metabolism of some polycyclic hydrocarbons in mam-
malian cell cultures to determine the relationship between carcinogen-
induced cytotoxicity and metabolism of the carcinogen (282).  The metab-
olism of 3,4^-benzpyrene (BP) and 7,12-dimethylbenz(a)anthracene (DMBA)
was studied in Syrian hamster, rat, and mouse embryo, mouse fibroblast,
human fetal lung and kidney and HeLa cell cultures.   Two extraction
procedures were employed to measure the metabolism of the initiated
hydrocarbons in the cell cultures.  One measured the metabolism of the
hydrocarbon to "alkali-extractable derivatives" and the other to "water-
soluble derivatives."  The kinetics of hydrocarbon metabolism indicate
that there may be a sequential conversion of the parent compound first
to alkali-extractable derivatives and then to water-soluble derivatives.
Each of the cell cultures, which was sensitive to the growth-inhibitory
effects BP or DMBA was able to metabolize the respective hydrocarbon to
water-soluble derivatives.  Little or no ability to metabolize the
hydrocarbons was detected in cells resistant to the cytotoxic effects.
Two. resistant hamster cell lines were exceptional in that they did
metabolize the hydrocarbons to water-soluble derivatives.  However, no
alkali-extractable derivatives were recovered from the medium of these
cultures.

In order to investigate whether or not the development of carcinogen-
induced cytotoxicity is due to selection of resistant cells or to the
carcinogenic properties of dimethylbenz(a)anthracene (DMBA), Diamond et_
al^. studied the carcinogen-binding capacity to DNA,  RNA, and protein,
and carcinogen-induced cytotoxicity by Syrian hamster embryo cell cul-
tures -at various passage levels (283).  The results suggest that primary
cultures include many sensitive cells with a high binding capacity for
DMBA.  With successive passages the sensitive and/or transformed cells
(high binding) are lost and the population becomes characterized by
resistant, low binding cells.

Kihlman et al. describe an experiment demonstrating the ability of
caffeine to increase the frequencies of chromosomal aberrations produced
by various physical and chemical agents including UV and x-irradiation,
4-NQO, alkylating agents, and mitomycin in root tips of the broad bean,
vicia faba, and in cell cultures of the Chinese hamster  (CL-1) (284).
Caffeine post-treatments strongly potentiated the frequencies of chromo-
somal aberrations from U-V, mitomycin C, thio-TEPA,  and 4-NQO while not
affecting the aberration frequency of x-irradiation.  The data also
indicate a correlation between the effect of caffeine on the production
of chromosomal aberration and on the loss of proliferative capacity of
the cells.  The authors hypothesize that the same molecular mechanisms
may be responsible for both types of effect.

While normal hamster cells were1 either susceptible or resistant to the
cytotoxic effects of dimethylnitrosamine  (DMNA), transformed hamster
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cells were always resistant (285).  Normal hamster cells remained sus-
ceptible to DMNA when tested four months after the increase in cellular
lifespan which was induced by the DMNA;  however, at a later time in
culture there was a selection of resistant cells showing a growth advan-
tage in both in vitro and in vivo systems.  N-nitro-somethylurea (NMU),
a related nitroso compound, was cytotoxic to normal cells but even more
cytotoxic to the transformed hamster cells.  The differential effect of
these chemicals appears related to the difference in the growth rate of
the normal and transformed cells.  DMNA has to be converted enzymatically
to its cytotoxic intermediate while NMU apparently is nonenzymatically
converted to a cytotoxic intermediate.  DMNA contrasting with benzo(a)-
pyrene (BP) was cytotoxic to normal human cells but like BP was not
cytotoxic to transformed human cells.  The authors believe this dif-
ference indicated that the enzyme required to convert DMNA to its cyto-
toxic derivative is not identical with BP hydroxylase.

A comparison was made of the biological activities and chemical prop-
erties of chlorambucil and Trenimon, alkylating agents used to treat
malignancies, on V78-1 Chinese hamster cells growing in vitro (286).
The cells were grown in a culture chamber in a temperature-controlled
box under a microscope fitted with a flash and camera for time-lapse
photography.  Pictures were taken at 1.5 minute intervals for one gen-
eration before the chemical was added to the medium and for two genera-
tions afterwards.  The study was directed at measuring the division
delay and loss of colony-forming ability of the cells.  The qualitative
response of the two compounds was similar in respect to division delay;
however, the ratio of doses to get the same quantitative effect is 4,000
chlorambucil to 1 of Trenimon.  A similar large difference was observed
in the sensitivity of the cells to the two compounds concerning colony-
forming ability.  The large quantitative differences between their
biological activities is a reflection of the relative ability of the two
compounds to penetrate the cell under the conditions of the cell culture.

In order to ascertain the nuclear alterations in mammalian cells induced
by L-canavanine  (CANA), hamster tumor cells from polyoma virus (PV) were
grown in an arginine-deficient medium, treated with 2.2 mM CANA, tryp-
sinized, centrifuged, dehydrated in ethanol, embedded in Epon and sec-
tioned.  They were then stained with uranylacetate and examined with an
electron microscope.  After four hours, the cells were observed to have
nuclear alterations characterized by the formation of irregular aggre-
gates of the nucleoplasmic contents which were frequently attached to
the nuclear membrane.  It was postulated that the formation of canavanyl
protein-DNA aggregate interferes with DNA replication.

Freeman et_al.  (287) studied the activation and isolation of hamster-
specific C-type RNA viruses from tumors induced by hamster-embryo fibro-
blast cell cultures, which were transformed by 3-methylcholanthrene and
cigarette-smoke condensate.  Although these cell lines were negative for
infectious virus before inoculation into animals, the hamster-specific
C-type RNA viruses were isolated from tumors or from cell lines derived
from the tumors.  The authors conclude that since the infectious C-type
viruses are not usually shown in hamster tissues of normal or tumor
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origin, the chemical treatment and the activation of the viruses are
positively correlated.

Lossio (288) and Lossio and Wigler (289)  exposed synchronized Chinese
hamster cells to chemicals in order to elucidate the stage of the cell
cycle in which cytotoxic effects occurred.  The technique of cell syn-
chronization was by reversal of colcemid inhibition.  A mitotic index of
95-98% was obtained and an almost pure culture of cells in metaphase was
plated at time zero.  The lethal damage induced by the exposure of these
cells to various concentrations of 5-fluoro-2'deoxyuridine (FUdR) was
not restricted to cells exposed during the period of DNA synthesis
(288).  The colony survival fraction observed after treatment for one
hour with 5 x 10~  M FUdR was extremely low (0.0001-0.0003%)  whether the
drug was administered during early G, late G, early S or middle S
phase.  Since the survival of cells treated with FUdR during mitosis was
significantly higher  (0.62), it seems that the mitotic cells were less
sensitive.  Depending onhow long after the cells were removed from the
FUdR, the addition of 10   M thymidine or conditioned medium either
reversed the lethal effects of the chemical or increased the survival
rate of the cells.

Certain thiopyrimidines were studied for sytotoxic effect on a syn-
chronized clone of Chinese hamster cells with a generation time of 16
hours  (289).  All four analogues tested induced cytotoxic effects, as
measured by colony-forming ability, which increased with the concentra-
tion of the chemical and the length of the exposure time.  Short periods
of treatment (one hour) produced little effect at low concentration;
however, they affected the survival of the cells differently when admin-
istered at different stages of the cell cycle.  Two peaks of maximum
sensitivity, one at late H  and the other at H , correspond to the peaks
of maximum RNA synthesis in mammalian cells.  Thus, the cytotoxic! effects
of thiopyrimidine analogues are probably related to their interference
with RNA synthesis.
The effects of ouabain on baby hamster kidney (BHK) cells were studied
including cell growth, macromolecular synthesis, ATP content, intra-
cellular concentrations of sodium and potassium, and the cell membrane
potential (290).  For the growth experiments, the cells were trypsinized.
from dense cultures and seeded on Falcon tissue culture dishes.  The
density determined by counting in a hemocytometer and the results were
the mean of two replicate plates. ..Ouabain inhibited growth, reduced the
incorporation of  H-thymidine and   C-amino acids, promoted a loss of
cell potassium and a gain of cell sodium, and reduced the membrane
potential at a level of 4 x 10   M.  The recovery from ouabain treatment
was dependent on the concentration of the drug and the period of exposure.

Baby hamster kidney fibroblasts and a polyoma virus transformed variant
(PyY) were grown as monolayer cultures and treated with tritium-labeled
samples of phenanthrene, benz(a)anthracene, 7-methyl benz(a)anthracene,
dibenz(a)anthracene (291).  After 24 hours, DNA, RNA, and protein was
isolated from these cells.  Similar experiments were performed with the
tritiated K-region epoxides, dihydrodiols, and phenols prepared from
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these polycyclic hydrocarbons.  The four K-region epoxides were clearly
more reactive to the constituents of both cell lines than the parent
hydrocarbons, or the corresponding dihydrodiols and phenols.  The reac-
tivity of these epoxides within a biological system is discussed with
particular reference to polycyclic hydrocarbon binding, cytotoxicity,
and carcinogenesis.  A correlation appears between the extent of hydro-
carbon metabolism and the amount of binding to cellular macromolecules.
This fact plus knowledge of in vitro microsomal systems indicate that
reactive intermediates are formed from polycyclic hydrocarbons by the
action of certain microsomal enzymes which oxidatively metabolize aro-
matic double bands.

Latner and Longstaff showed that a baby hamster kidney cell line (BHK 21),
when maintained in the presence of crude histone preparations for three
days, underwent morphological and behavioral transformations similar to
transformations obtainable with viruses and mycpplasmas (292).  Many of
the cells were multinucleated giants with serrated margins, which show
some loss of contact inhibition.  A marked tendency toward centripetal
aggregation and multilayering was also envinced.

Mayorca et al. describe a conditional state of transformed phenotype on •
BHK   Clone   cells transformed by dimethylnitrosamine or nitrosomethyl-
urea, and on a single "spontaneously" transformed clone (293).  The
transformed phenotype (clonal morphology and the ability to plate in
soft agar) is exhibited when the cells are grown at 38.5°C, while the
phenotype is normal when cultivation occurs at 32 C.  The conditional
state 'of these cells does not extend to their growth characteristics in
that their plating efficiency at the two temperatures in liquid medium
is similar.  Conversion from the normal to the conditionally transformed
phenotype and vice-versa is produced by shifting the temperature.

Kao and Puck examined the behavior of four known carcinogenic nitroso
compounds to determine whether they were mutagenic and, if so, whether
there was any pattern in their ability to produce single gene mutations
and chromosomal aberrations which could be correlated with their carci-
nogenic activity (294).  Actively-growing Chinese hamster ovary cell
monolayers were treated with these water-soluble compounds and then
analyzed for survival of colony formation, induction of chromatid breaks
and exchanges, and production of auxotrophic mutations.  All the com-
pounds were effective in producing cell-killing, chromatid breaks and
mutagenesis.  There was a 50,000 fold difference in the mean lethal dose
values (Do) as shown by single cell survival curves; however, all the
compounds yielded constant values for their efficiencies of production
of chromatid breaks and single gene mutagenesis, but not chromatid
rearrangements, when these were expressed in terms of the Do values.  N-
nitrosomethylurea, N-nitrosomethylurethane, and N-methyl-N'-nitro-N-
nitrosoguinadine all produced single-hit survival curves while N-nitro-
sodimethylamine produced a multiple-hit survival curve.  This system may
offer distinct advantages for studying the interrelationships between  '
mutagenic and carcinogenic actions in mammalian cells.
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Kao and Puck have demonstrated a methodology to quantitate mutagenesis
of mammalian somatic cells by physical and chemical agents (295).  Using
Chinese hamster ovary cell cultures (CHO), the authors have developed a
system to induce not only single gene mutations but also additional
auxotrophic mutants.  They have refined a method for quantitating the
efficiency of single gene mutations to specific auxotrophies.  Muta-
genesis in the forward direction has been measured after treatment of
the CHO cells with ethyl methane-sulfonate, N-methylN'-nitro-N-nitroso-
guanidine, hydroxylamine, an acridine mustard (ICR-191), caffeine, and
ultraviolet and x-irradiation.  The single cell survival curves and the
efficiency of chromatid breakage and rearrangement were measured for
each chemical agent.  Similar measurements were also performed with a
water-soluble carcinogen N-nitrosomethylurea which was shown to be
effective in producing auxotrophic, somatic mutations.  The authors
believe these results offer promise of illuminating the relationships
between cell killing, chromosomal aberration, single gene mutations and
carcinogenesis produced by the various agents.  The methods elaborated
can be used in the routine testing of drugs, food additives, and environ-
mental pollutants for mutagenic action in mammalian cells in vitro.

The neoplastic transformation of hamster lung cells after their exposure
to cigarette smoke condensate and the effects of tobacco tar on untrans-
formed cells was performed in_ vitro (296).  The hamster lung fibroblasts
were transformed into malignant cells after three hours of exposure to
crude cigarette tar dissolved in ethanol (10 or 100 Vg/ml).  Primary
injuries to the cells included nuclear pyknosis, cell necrosis, and an
enlarged, vacuolated cytoplasm.  These were observed between 2 and 48
hours post-treatment.  In one instance, giant cells were noticed at
about 48 hours after treatment.  Transformation manifested by random
orientation of the cells  (piling-up and criss-crossing) and continuous
growth in vitro for over 300 days, occurred 100 days following treat-
ment.  The transformed cells cultured for 100 to 160 days produced
tumors when transplanted into the cheek pouch of hamsters iii vivo.  Five
of the nine animals which were inoculated with 100 yg/ml of the tar-
treated cells  (HT-100 strains) over 160 days iri vitro, died from the
tumors while the others, along with one implanted with cells of a dif-
ferent strain  (HT-10), were sacrificed for histological confirmation.
The tumors, histologically, were pleomorphic fibrosarcomas.  Low doses
 (.1 x 10  or less) of control cells did not produce tumors after 270 days
in culture while higher doses of 10  control cells or more produced
tumors when injected into the animals.

Mouse Cell Culture Systems

Mouse cells and tissues have been extensively cultured and used for iii
vitro toxicity studies.

The L-929 clone of mouse fibroblasts have been employed as an assay
medium for a number of purposes including analyzing plastic and rubber
toxicity  (297, 298, 299, 300, 301), alcohol toxicity  (302), and the
toxicity of various drugs and compounds  (303, 304, 240, 228, 305, 306,
307, 308, 309).
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Investigators from the Drug Plastic Research and Toxicology Laboratory
of the University of Texas analyzed the toxic effects of plastic and
rubber materials on L-929 fibroblasts (297, 298, 299, 300).  The toxic-
ity of a rubber accelerator was analyzed by incorporating the rubber
chemicals on dyed cell monolayers and by perfusing chemical solutions
onto asynchronous cell cultures in a Sykes-Moore tissue culture chamber
(297).  A phase-contrast microscope was used to record the toxic effects
on the cell cultures.  A tissue culture technique was demonstrated for
screening the toxicity of plastic materials via direct contact of the
plastic with L-929 cell monolayers in Eagle's medium (298).

Prior to this methodology, the standard procedures used in medical
practice was an in vivo implantation technique originated by Brewer and
Bryant (310).  This consists of implanting small strips of a plastic
sample into the paravertebral muscle of rabbits.  After a period of 3 to
7 days, the animals are sacrificed and the implant sites are examined by
both microscopic and histopathological methods.  The in vitro contact
technique is less expensive and more sensitive than the in vivo implant-
ation method.  An adaptation of the above method was required for those
plastic samples having low density or odd shapes (299).  An agar dif-
fusion method to determine not only plastic sample but also solid and .
liquid extract toxicity was designed.  Replicating L cells and non-
replicating chick embryo cell monolayers were covered with an agar/calf
serum overlay and evenly dispersed neutral red stain.  Plastic samples
were implanted on the culture plate which was inverted and incubated.
After 24 hours, toxicity was indicated by clear, colorless zones of dead
cells around the sample.

Growth inhibition studies were used to depict the changes in mammalian
cell cultures caused by the plastic additive, triethylcitrate (300).
Cell populations were determined using the methods of Mclntire and Smith
modified by Hori (311).  Then, after a simple extraction procedures,
nucleic acids (total purines and pyrimidines) were measured by their
absorbance at 268'my.  It was noted that the inhibitory action of tri-
ethylcitrate was independent of the inoculum sizes used.

Wallace L. Guess produced a general paper on the state-of-the-art of
tissue testing of polymers in 1970 (301).  The acute effects on tissues
of pure polymers such as polyethylenes and polypropylenes, etc., and of
compounded polymers and their additives, the plasticizers  (dioctyl
phthalate, citric acid esters), stabilizers  (organometallic compounds),
colorants, ultraviolet screening agents, and fillers are described.  The
primary test systems in use, the rabbit muscle implantation technique,
the agar diffusion cell culture technique using either mouse "L" fibro-
blasts or chick embryo cells, are compared.

The relationship of in_ vitro and in vivo toxicity of a series of methyl-
and halogen-substituted alcohols was examined with respect to their
octanol-water partition coefficients, charge and steric parameters
(302).  A high correlation was found between the tissue culture toxicity
and the hemolytic activity of the compounds.  The product of the intrinsic
toxicity  (slope of the dose-response curve in tissue culture) and the

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inverse of the octanol-water partition coefficient for aliphatic alco-
hols had a uniform relationship to acute in_ vivo toxicity (LD  -single
dose required to kill 50% of the mice in 7 days).   Other predicted
relationships between in vitro and in vivo toxicity were also confirmed.
The growth inhibitory response of the "L1 cells was employed for the
tissue culture assay.  Comparative analyses of the results were carried
out with the Free-Wilson (purely mathematical) and Hansch-Fujita (ther-
modynamic equilibrium) models.  These supported the conclusion that the
tissue culture system conforms closely to the equilibrium (time-independent)
model of structure activity proposed by Higuchi and Davis.

L-929 fibroblast monolayers were inoculated with Arlacel-A (AA), an
emulsifying agent for oil-adjuvant vaccines, in order to evaluate its
toxicity (240).  A characteristic cytotoxic effect consisting of intra-
cytoplasmic refractile bodies was produced and this cell culture system
was deemed a sensitive and reproducible assay for the evalution of AA
toxicity.
                                                                        •
Quinacrine, a drug used to treat malaria, produced nucleolar fragment-
ation in L-929 cells cultivated in monolayers.  Fedorkb and Hirsch  (307)
demonstrated this effect by the use of phase contrast and electron
microscopy and hypothesized that the structural changes in the "L" cell
nuclei are related to the known binding of quinacrine to DNA.

Two studies utilizing different methodologies to determine the effects
of antibiotics on L-fibroblasts have been reported (309, 312).  In the
first (309), the survival response of cultured L-cell monolayers treated
with bleomycin showed that the drug not only exerts a lethal effect but
also induces resistance in the cells that are not killed.  In the other
(312), 24 different antibiotics were tested for their effects on the
multiplying ability of "L" cells in suspension culture.

Growth inhibition studies with L-"fibroblasts, using 16 local anaesthetics
was compared with intravenous mouse toxicity, threshold tissue irritant
concentration and threshold intracutaneous local anaesthetic activity
(303).  Both mouse L-929 cells and liver cell cultures were used.
Toxicity was determined by measuring the growth inhibition caused by the
drugs.  Cell population counts were determined at the beginning and end
of each experiment by the "total purine and pyrimidine" method of Mclntire
and Smith  (311) with complete inhibition corroborated by microscopic
examination.  A significant logarithmic correlation was derived between
the threshold irritant concentration and the in vitro cell toxicity
only.

The scissions caused by 4-nitroquinoline 1-oxide of proteins liking DNA
in cultured "L" fibroblasts and the subsequent rejoining of this DNA
when removed from the 4 NQO have been reported.  The "L" fibroblasts
were cultured in a protein and lipid-free synthetic medium (DM-120).
Cell growth was then estimated by counting cells following the. simpli-
fied replicate tissue culture methods.  Colony-forming ability after
treatment was determined by plating  (fixing (methanol), and staining
(Giemsa) the cells), and counting the colonies formed (305, 306).  A
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sucrose density gradient centrifugation and a viscometric technique
which detects the DNase activity in the pronase were performed.  Strand
breakage increased with the increase in concentration of the carcinogen.
However, both single and double strand breaks became completely repaired
with a concommitant restoration of growth after a sufficient incubation
time in a medium without the carcinogen ("recovery incubation").

A number of cell lines including mouse L-929 fibroblasts were treated
with the nonionic detergent, Triton WR-1339, to ascertain its effect on
cellular respiration and mitochondrial morphology (228).  The inhibitory
effect on cellular respiration was related directly to the cytotoxic
response of the cells to the surfactant.  L-929 cells were conditionally
sensitive, while AV-3 and HeLa were markedly sensitive and primary rat
and chick embryo cells were insensitive.  The most striking character-
istic noted in the treated cells was mitochondrial damage.  The condi-
tionally sensitive L-929 cells appeared to repair the induced damage
following the removal of Triton WR-1339 from the media.

Horikawa et al. (313) investigated the dark reactivation of damage which
was induced by ultraviolet light in mouse "L" cells, porcine kidney
cells and mouse Ehrlich ascites tumor cells in_ vitro to determine the
difference in sensitivities of these cell lines to x-rays and ultra-
violet light and whether or not the reactivation processes after cel-
lular damage by these two forme of irradiation are identical.  The
induction of pyrimidine dimers into the cellular DNA is the primary form
of damage caused by ultraviolet irradiation.  Reactivation mechanisms
which split or excise these ultraviolet-induced dimers were measured by
the colony-forming ability of the cell lines.  There were no striking
differences to x-irradiation among the three cell lines, but there were
different sensitivities to UV light, porcine kidney cells being most
sensitive and Ehrlich cells least sensitive.

The transformation in vitro of C3H mouse prostate cells is considered by
some researchers (314, 315, 274) to be a reliable model for chemical
carcinogenesis.  Carcinogenic polycyclic hydrocarbons, though chemically
inert, undergo extensive metabolism in experimental animals as analyses
of bile, urine, feces, and liver homogenates have shown.  Since metab-
olism is necessary for the malignant transformation of cells, it is
important to follow the  kinetics of metabolite production in cells
under conditions where malignant transformation can be obtained.  Mouse
C3H prostate cells are one of the several in vitro system in which the
malignant transformation by polycyclic hydrocarbons can be studied
quantitatively (hamster embryo, rat embryo, AKR mouse) and so are ideal
models for this type of assay.  The metabolism of five polycyclic aro-
matic hydrocarbons to water-soluble and organic-soluble products was
studied in C3H cells under conditions suitable for their malignant
transformation (315).  Prostate cell monolayers were grown in BME, and
after a period of exposure  (8-12 days) to a medium containing the hydro-
carbon in question, were methanol-fixed and stained.  Cytotoxicity was
expressed as the percentage of the number of colonies in treated dishes
over the number of colonies in control dishes.  No correlation was found
between the carcinogenicity of the compounds in_ vivo or in_ vitro and
metabolism to water-soluble compounds in these cultures.

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After a transformation assay, the technique of utilizing the formation
of water-soluble products from polycyclic hydrocarbons as a measure of
metabolism in_ vitro was performed to investigate the influence of micro-
somal triphosphopyridine nucleotide-dependent enzymes on the malignant
transformation (314).  Since metabolic activation of chemically inert
carcinogens is considered to be essential for their biological activity,
this work pursues the hypothesis that the metabolic activation of cer-
tain carcinogenic hydrocarbons is carried out by microsomal mixed-
function oxidases and that epoxides are the carcinogenic substances.

Tritiated hydrocarbons were added to medium in which C3H monolayers were
growing (274).  After incubation, the cells were washed, harvested, and
frozen at -80 .   They were then fractionated into alcohol-soluble and
insoluble portions, and the cellular constituents were isolated by a
modification of the procedure of Diamond et al. (316).  In this manner,
the binding of carcinogenic polycyclic aromatic hydrocarbons to the DNA,
RNA, and proteins of the transformable cells was demonstrated.  Thus the
observation that the carcinogenic hydrocarbons are firmly bound in_ vivo
to the nucleic acids and proteins of mouse skin was confirmed and ex-
panded.  Also a good correlation was found between carcinogenic potency
in vivo and transforming activity in vitro.

C3H mouse embryo cells were cultured and grown in a medium suffused with
4-nitroquinoline 1-oxide (4NQO) (317).  At various passage levels, the
cultured cells were implanted subcutaneously into C3H mice for assay of
malignant transformation.  The tumor latency period in vivo of the 4NQO
treated cell lines was much longer than that of untreated controls; thus
the chemical delayed the spontaneous malignant transformation ill vitro
of the mouse embryo cells.

Smoke from tobacco and/or marijuana was used to expose mouse kidney
cells (318, 319), epithelioid cells of mouse lung (320, 321, 319), and
peritoneal macrophages (322) in order to evaluate its toxic character-
istics.  Leuchtenberger and Leuchtenberger (318, 319) demonstrated that,
while puffs of unfiltered cigarette smoke and its gas phase evoked rapid
destruction of mouse kidney and lung monolayer cultures, cultures ex-
posed to puffs of charcoal-filtered cigarette smoke did not undergo
significant alteration.  The fact that the gas phase alone contains
factors affecting cell proliferation is corroborated by in_ vivo studies
which showed that the incidence and spectrum of tumors in mice were
increased after inhalation of both whole smoke and the gas phase alone.

These experiments used a Filtrona CMS-12 smoking machine.  Lung cell
cultures were prepared on coverslips and exposed to puffs of fresh
cigarette tobacco smoke and tobacco mixed with marijuana smoke from a
Filtrona CSM-12 smoking machine (321).  Comparisons of effects on mor-
phology, mitotic index, and DNA synthesis in epithelioid cells of lung
explants were then made.

The effects of air pollutants, especially NaNO_, on mouse lung, rat
lung, and rabbit endothelium were described (320).  The cells were grown
in Rose multipurpose culture chambers and, after attachment, exposed to
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 the culture medium containing the NaNO .  All cell types tested in vitro
 exhibited partial but reversible inhibition in oxidative activity during
 treatment with NaNO?.

 Mouse peritoneal macrophage cell cultures and rabbit alveolar macrophage
 cells were exposed to whole cigarette smoke or to its vapor phase in a
 vertical Perspex chamber (322).  A radiotracer assay technique for the
 determinations of  H-RNA and protein synthesis was employed.  The derived
 data indicated that short-term exposure to cigarette smoke severely
 inhibits protein synthesis in macrophages.  However, if the macrophages
 are exposed to low levels of cigarette smoke for a longer time, both
 protein and RNA synthesis increase markedly.  These data support elec-
 tron microscopic evidence of both active protein synthesis in vivo in
 human smoker's lungs, and the apparently contradictory fact of a severe-
 ly reduced rate of protein synthesis in rabbit alveolar macrophages in
 vitro as reported by Yeager (323) .

 Mouse peritoneal macrophages were also used to examine the cytotoxic
 effects of silica, diamond dust, and carageenan (324).  Phase-contrast
 and electron microscopy, histochemical techniques for lysosomal enzymes,
 and measurements of the release of lysosomal enzymes into the culture
 medium were the methods of analyses.

 Frei and Oliver (325, 326, 327) studied the action of the carcinogen
 methylnitrosourea  (MNUA) on primary mouse embryo cells in mass tissue
 culture.  In vivo methods have shown that tissue differences in the
 methyla;tion of DNA after a single carcinogenic dose of MNUA influence
 the,transformation of mouse embryo cells.  The results indicated that
 the chemical enhanced the malignant transformation of these embryo
 cells.  After recovery from the primary injury caused by MNUA, the cells
 undergo a stepwise transformation in which increased plating efficiency
 appears first, and is followed by the appearance of morphologically
 transformed colonies which are invasive when implanted into irradiated
 hosts.  It was also shown that in the early phases of the malignant
 transformation, MNUA prolongs the S phase of the cell cycle without
t killing cells (328).  These experiments failed to resolve the question
 of whether the acceleration of biological transformation was due to the
 induction of heritable change in the cells or to the selection of malig-
 nant cells present in the initial cell population.

 Rhim, Creasy, and Huebner described the altered cell foci found in
 mouse-embryo tissue cultures that had been previously infected with
 wild-type AKR (RNA tumor) viruses which were observed 9-14 days after
 treatment with the chemical carcinogen 3-methylcholanthrene (329).
 These foci from transformed cells consisted of randomly oriented,'piled-
 up, spindle-shaped cells, the colonies of which when heavily stained
 with Giemsa were grossly visible and countable.  These changes in mor-
 phology were not detected in uninfected cells treated with 3-methyl-
 cholanthrene or in untreated cells infected with virus under the same
 experimental procedures.  This cocarcinogenic system is suggested as a
 rapid, quantitative test for the measurement of the oncogenic potential
 of  certain carcinogens.  .It is possible that the infectious, but non-
 transforming RNA tumor viruses provides nascent oncogenic information

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which, after activation by 3-methylcholanthrene, serves as the specific
genetic determinant of transformation.

Mouse embryo cells have been compared with other cell lines (269, 279).
The toxic and transforming effects of phorbol esters, tobacco lead
extracts, and cigarette smoke condensate fractions on several popula-
tions of mouse embryo cell cultures were compared with effects on 3T3
cells.  An in vitro transformation assay of 12 major fractions of cig-
arette smoke condensate on rat and mouse cells infected with C-type RNA
tumor viruses and on uninfected hamster cells has been conducted also.
The combined effects of RNA tumor viruses and carcinogenic compounds in
producing transformation of various cell lines is a highly sensitive and
reproducible test system for qualitative in_ vitro work (330).  The same
condensate fractions producing statistically significant tumor-inducing
activity are also most active in transforming cells.

Since 7,12-dimethylbenz(a)anthracene  (DMBA) initiates skin tumors more
effectively during diestrus than during estrus and so may be inhibited
by estrogens, Nebert e_t a^. studied the effect of the steroids, 17-$-
estradiol and testosterone on aryl hydrocarbon hydroxylase, an enzyme
system which could be a common pathway in metabolizing both polycyclic
hydrocarbons and steroids  (331).  They showed that the hydroxylase
system is induced by the polycyclic hydrocarbons DMBA and 3-methyl-
cholanthrene in mice in vivo and noted that, in mouse fetal cell cul-
tures, 17-$-estradiol in the growth medium prevents hydroxylase induc-
tion only at concentrations 6 to 60 times greater than the' levels of the
polycyclic hydrocarbon inducer.

3T3 mouse fibroblasts and Simian virus 40-.3T3) are mammalian cell lines
popularly utilized alone and with other cell cultures to determine
chemical toxicity in vitro.  Sodium chloriridate produced irreversible
inhibition.of cell division of 3T3 and SV-40.3T3 cells in vitro at doses
of 3 x 10   M (225).  Although DNA synthesis was blocked, RNA and pro-
tein synthesis continued and resulted in cells of very large size.
Ammonia decreased cell multiplication and altered morphology to a greater
degree in the 3T3 line than in the transformed line and this occurred
with minimal changes in the pH of the culturing medium (332).  Sivak and
VanDuuren (268, 276, 269, 275) studied the effects of carcinogens and
tumor promoting agents on assay media consisting of separate and mixed
populations of 3T3 and SV-40.3T3 mouse fibroblasts.  A notable property
of the 3T3 (untransformed) cell line is its sensitivity to density-
dependent inhibition of cell division.  Thus, in mixed cultures of both
transformed and untransformed 3T3 cells, the untransformed cells inhibit
the outgrowth of the SV-40.3T3.  If non-toxic amounts of phorbol myris-
tate acetate (0.01 to 1.0 yg/ml), an active tumor promoter in mouse skin,
are added to the mixed culture, the density-dependent inhibition is
reversed and the growth of clones grom the transformed lines is enhanced.
This phenomenon is also true for a number of other tumor-promoting
agents including tobacco leaf extracts, phorbol esters from croton oil
and other chemicals (268, 275).

While it is generally considered that transformed populations are more
resistant to the toxic effects of the carcinogenic hydrocarbons than the


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comparable untransformed cells, this is not always the case.  Even after
a 13-19 day recovery period after exposure to benzo(a)pyrene, the trans-
formed 3T3 cells when cloned exhibited toxic effects for over 20 genera-
tions.  These results indicated a permanent change in cell sensitivity
had occurred, although certain properties characteristic of neoplastic
cells in culture were not observed.  This and other results suggest that
tumor promotion iri vivo does not involve a selection brought about by
some general toxicity of the promoting agent.  Also, the inhibition of
growth in mass cultures as an index of toxicity is a relatively insen-
sitive parameter when compared to cloning efficiency (269).

At subtoxic levels, the growth of the transformed clones is directly
related to the dose of the tumor agent and the density of the contact
inhibition overlay.  Since the loss of contact inhibition is a neo-
plastic characteristic, the method provides a short-term reproducible in
vitro technique to determine the ability of agents to promote tumorgen-
icity.  This method eventually may supplant the usual time-consuming
initiation promotion in vivo mouse skin assay especially when applied to
isolated chemical compounds or purified chemical mixtures.

Specific murine tumor cell lines are often cultured for use in assays.
Previous papers (305, 306) dealt with the scissions of protein linking
DNA by 4-nitroquinoline 1-oxide (4NQP) in mouse "L" fibroblasts.  It was
demonstrated that 4NQP and the carcinogen 4-hydroxylaminoquinoline 1-
oxide (4-HAQO) can induce single-strand breaks in the DNA of cultured
Ehrlich ascites and tumor cells (333).  The sedimentation behavior of
DNA from normal and carcinogen-treated cells was analyzed by the alka-
line sucrose gradient method and autoradiographs were prepared of un-
scheduled DNA synthesis in carcinogen-treated cells.  As in the previous
studies, when the cells are incubated after treatment, most of the DNA
fragments can be rejoined.

Murine L-5178Y lymphoma cells were cultured as a medium to investigate
the biochemical effects of acronycine  (334) and barbiturates (335) on
nucleic acid synthesis.  Both L-5184 cells and IRC rat monocytic leu-
kemia cells were grown in suspension culture and subjected to acrony-
cine, an antineoplastic alkaloid, at concentrations of 0.5-12 yg/ml.
The drug rapidly inhibits culture growth which agrees with the known
action of acronycine against the L-5178Y tumor in vivo.  DNA synthesis
is inhibited at higher concentrations, but is not a prerequisite of the
arrest of culture growth.  The L-5178Y cells were much more sensitive to
the actions of the drug than the IRC rat cells.

The inhibitory actions of barbiturates, especially pentobarbital, upon
the kinetics of cell growth and upon the synthesis of nucleic acids and
protein in L-5178Y lymphoma cells and P-815Y murine mastocytoma cells
grown in suspension culture was explored  (335).  Pentobarbital inhibited
the growth and synthesis of nucleic acids and protein in the P-815Y
mastocytoma cells.  The inhibition increased with an increase in the
concentration of drug and was also time-dependent with a high level of
drug.  Similar results were obtained in lymphoma cells synchronized by
sequential treatment with thymidine and deoxycytidine plus Colcemid.
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A population of DDT-resistant cells was selected from mouse L-5178Y
leukemic cells grown in suspension in a model G-25 Brunswick Gyrotory
incubator-shaker at 37 C by chronic treatment with sublethal concentra-
tions of DDT (336).   These cells also exhibited resistance to other DDT
pesticide analogues.  One clone of cells, after passing through a con-
tinuous culture of 300 generations in the absence of DDT treatment,
retained the resistant characteristics of the parent culture.

A Sarcoma-180 cell line (S-180) which was maintained in a monolayer
culture for 12 years and was sensitive to 4,4'-diacetyl-diphenyl-urea-
bis-guanylhydrazone (DDUG), was compared with a resistant subline with
respect to cross resistance to methylglyoxal-bis-guanylhydrazone (CH G),
2-chloro-4', 4'-bis(2-imidazolin-2-Y )terephtholanilide (NSC 3828), and
Vincristine (BCR) (337).  These lines were also compared in terms of the
nature and rate of cellular uptake of DDUG and its intracellular dis-
tribution and binding.  The cellular uptake rate for L-1210 leukemia
cells in_ vivo, a system showing resistance to DDUG, was unchanged in
relation to systems having no resistance.  No metabolic conversion of
DDUG was observed in sensitive and resistant L-1210 cells or rat liver
in vivo.  This observation may be correct for S-180 cells also.

Chloramphenicol  (50 yg/ml) inhibited the rate of protein synthesis in
mouse myeloma cells grown in suspension culture by 50% (338).  While
there is a decrease in the amount of globulin synthesized, the rate of
synthesis per cell is unchanged.  The observed decrease is traced to the
inhibition of cell, proliferation caused by chloramphenicol.

Takayama and Ojima investigated the photosensitizing activity of car-
cinogenic and noncarcinogenic polycyclic hydrocarbons on a mouse tumor
cell strain isolated from a mammary carcinoma (339).  These cells were
exposed to each of 8 polycyclic hydrocarbons and then illuminated with
white light from a tungsten lamp.  The carcinogenic hydrocarbons (benzo-
(a)pyrene, dimethylbenzacridine, methylcholanthrene, dimethylbenzanthra-
cene, and benz(a)anthracene) were found to be much stronger in photo-
sensitizing activity than the noncarcinogenic ones  (anthracene, pyrene,
and acridine).  Among the former compounds, benzopyrene is the strongest
and benzanthracene the weakest.  A positive association between photo-
dynamic activity and carcinogenicity was demonstrated.

The cell uptake of benzopyrene occurred in as fast as one second of
incubation.  When the cells were illuminated, lysosomal staining with
neutral red became markedly reduced.  This alteration in lysosomal
staining took place prior to the occurrence of any other detectable
abnormatlities.  The photodynamic effect produced by the combined use of
a photosensitizing substance and light is due primarily to the cell
autolysis by the hydrolytic enzymes which were released from lysosomes
as a result of photodynamic damage to their membranes.

Jackson made a study on the effects of polychlorinatted biphenyl com-
pounds  (PCB) on the growth and DNA synthesis of Krebs-2-murine ascites
cells grown in vivo and in vitro (345).  Agreement exists regarding
growth and DNA synthesis inhibition of cells grown in vivo and in vitro.
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Rat Cell Culture Systems

A number of iii vitro studies utilizing rat embryo cells in culture have
been reviewed (341, 279, 342, 228, 343, 344, 315, 345).  Rhim and Huebner
(343) treated monolayers from a continuous rat embryo cell line  (S-1193b)
derived from Fischer rats with various levels of polycyclic aromatic
1193b) derived from Fischer rats with various levels of polycyclic aromatic
hydrocarbons (3-methylcholanthrene, 7,12-dimethylbenz(a)anthracene, and
benzo(a)pyrene or dimethyl sulfoxide (control)).  The cells treated only
with the carcinogens underwent transformation in_ vitro.  Newborn rats
were then inoculated subcutaneously with freshly trypsinized cells in
order to determine the transplantability of the transformed cells, and
progressively growing transplantable tumors were, in fact, produced.

Gelboin, Kinoshita and Wiebel, in an important paper, explore the in-
duction and role of microsomal.hydroxylases in the toxicity and carci-
nogenesis of polycyclic hydrocarbons (346).  Utilizing primary and
secondary cultures of whole hamster, rat, and mouse embryos, 3T3, HeLa
and a variety of other cell cultures, they demonstrate the mechanism for
the induction of aryl hydrocarbon hydroxylase in vitro.

While the enzyme system of the liver generally converts polycyclic
hydrocarbon to either weakly carcinogenic or noncarcinogenic compounds,
it may also be involved in the activation of these compounds to either
carcinogenic or toxic metabolites.  Among the evidence for this dicho-
tomy, the authors demonstrate that the toxicity of the polycyclic hydro-
carbons to cells grown in culture is directly correlated with the pres-
ence of aryl hydrocarbon hydroxylase activity; that the inhibition of
hydroxylase activity in cells by 7,8-benzoflavone is paralleled by an
inhibition of the toxic effects of 7,12-dimethylbenz(a)anthracene  (DMBA);
and that 7,.8-benzoflavone inhibits the enzyme in mouse skin homogenates
and inhibits DMBA-induced skin tumorigenesis.

The benzine extraction of airborne particulate matter collected in Los
Angeles yeilded carcinogenic material  (374).  The residue from this
benzene extraction contains substances, some organic, which are soluble
in methanol.  The methanol extract and some of its component fractions
were tested in certain rodent cell culture systems which were developed
as assay methods for the screening of carcinogen compounds.  Both a
high-passage Fischer rat embryo cell line, and a low-passage cell line
derived from. NIH Swiss albino mouse embryos and infected with AKR leu-
kemia virus were used.  .In both systems the methanol extract demonstrated
cell transformation activity approaching that of 3-methylcholanthrene.
The major inorganic component of the extract fractions, ammonium nitrate,
showed no activity on the mouse cell system.  Neither the neutral alone
nor the recombined non-neutral fractions separately showed any activity
either, although they were active together.  The methanol extract com-
bined with the conventional benzene extract showed much lower activity
than either extract alone.

Rauscher C-type RNA murine leukemia virus infected rat embryo cells were
the primary assay vehicle in transformation studies for Freeman et al.
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(342, 344, 348), Rhim and Huebner (279, 349, 350), Hetrick and Kos (345)
and Price et^al. (351, 352).  This system was developed to study the
interrelated effects of murine leukemia viruses and exogenous chemical
agents.  The rationale for evaluating the C-type virus-infected embryo
line as an oncogenic inducer is that these viruses possess all the
characteristics necessary to implicate them as the basic determinants in
a wide variety of leukemic and non-leukemic neoplasias.

Secondary rat embryo cultures were treated simultaneously with 0.1. ug
diethylnitrosamine (DENA) and either CF-1 or Rauscher C-type RNA virus
(342).  Control cells were treated with either the chemical or virus
alone.  The virus-infected and DENA-treated cells alone remained basic-
ally unchanged.  However, those treated with both became overgrown with
randomly oriented spindle cells between the sixth and twelfth subcul-
tures.  Cultures treated with virus and DENA lost their normal and
diploid condition and became aneuploid after later subcultures.  This
evidence suggests that the C-type RNA oncogene provides specific infor-
mation for the transformation event.  Rat cells in culture are ideal for
in vitro transformation studies since they rarely, if ever, undergo
spontaneous morphological transformation.  The assumption that a malig-
nant transformation has occurred must be corroborated by producing
transplantable tumors.  Special in vivo techniques for producing these
in rats are required for a variety of reasons including inherent resist-
ance in the rats.

Over 30 polycyclic hydrocarbons, azo dyes, aromatic amines and other
chemicals were tested to see if in vitro transformation of high-passage
rat embryo cell cultures infected with Rauscher leukemia virus (RLV)
correlated with the known carcinogenic activity of the same compounds iii
vivo  (.3441.  The criteria for transformation included the development of
macroscopic foci of spindle cells, lack of polar orientation and contact
inhibition and the development of tumors when transplanted into newborn
Fischer rats.  Although certain exceptions were recorded, the in vitro
results generally indicate that transformations were induced by known
carcinogens but not by their noncarcinogenic analogues.

Freeman et al. also tested extracts of particulate matter from conden-
sates of city air for their ability to transform rat or hamster cell
cultures  C3481.  Rat embryo cultures chronically infected with Rauscher
leukemia virus  CRLB) were transformed by benzpyrene or by extracts of
city smog while uninfected rat embryo cultures were not.  The smog
extracts were 600 times more active than pure benzpyrene as transforming
agents.

Hamster embryo cell cultures infected with hamster leukemia virus  (HaLV)
were equally as sensitive as the RLV infected rat cultures to the trans-
forming effects of the smog; however, uninfected hamster cultures were
also transformed, although tenfold higher doses of city air extract were
required to do this.

The transformation of Rauscher leukemia-infected rat embryo cultures in
the presence of foreign compounds was reported in two other cases  (279,
345).  The first is a work which was previously described using mouse

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embryos which also used RLV-infected rat embryos (F-119 line) and un-
infected rat embryo cell lines (F-lll).   This was a transformation assay
for possible environmental carcinogens,  specifically on fractions of
cigarette smoke condensates.  The results correlated well with previously
performed in vivo work in that a smoke fraction showing high tumori-
genicity in vivo also had high transforming qualities in vitro.

Low levels of the antischistosomal drugs, hycanthone and lucanthone act
as cotransforming agents in RLV-infected rat embryo cell cultures (F-1706
line), while neither the RLV infection or the chemical alone produced a
transformation (345).  Foci of the transformed cells appeared on the
first subculture after chemical treatment and eventually became the
predominant cell type.  The transformed cells also exhibited the classic
criteria of loss of contact inhibition,  random oriented spindle cells,
and tumorigenicity when implanted subcutaneously into newborn rats.

The transforming effect of benzo(a)pyrene (BP) was compared in three
cell lines in vitro by Rhim et al. (349).  A rat embryo cell line in-
fected with Rauscher leukemia virus, a Swiss mouse line infected with
AKR wild-type C-type RNA virus, and an uninfected hamster-embryo line
when treated with BP were all transformed and produced tumors when
reintroduced into homologous hosts.  The infected mouse-cell cultures
had transformed foci 9 days after BP treatment, while uninfected mouse
and rat cells did not transform or cause tumors in_ vivo.  The infected
mouse-cell line was the most sensitive system for chemical transforma-
tion.  Oncogenes of the RNA tumor virus genomes were derepressed by the  ..
action of BP which induced the transformation in the cells.

Rhim e_t 'al_. continued their investigations into carcinogenesis by describ-
ing the malignant transformation which was induced in rat embryo cells
infected with Rauscher leukemia virus by 7,12-dimethylbenz(a)anthracene
CPMBA)  (350).  As in prior studies, no transformation occurred in cul-
tures either treated with the chemical alone or infected with the virus
alone.  The foci of the transformed cells had much more rapid replica-
tion rates than the untransformed and untreated embryo cells and were
randomly oriented and spindle-shaped.  While the transformed cells
produced local sarcomas when reintroduced subcutaneously into newborn
rats, the infected or DMBA-treated untransformed cells did not.  Cells
derived from these tumors were reestablished into tissue culture and
like the tumor tissue itself, contained group-specific complement-fixing
antigens characteristic of the murine leukemia-sarcoma virus complex and
the C-type RNA particles.

Price, Suk, and Freeman investigate the effect of the sequence of
treatment of Type C RNA tumor viruses on Fischer rat embryo cell cul-
tures as a determinant for chemical carcinogenesis (351).  These cul-
tures were treated with 3-methylcholanthrene before or after inoculation
with Rauscher murine leukemia virus.  Transformation was not observed in
the untreated control cultures, cultures given virus or 3-methylcholan-
threne alone, or cultures treated first with 3-methylcholanthrene fol-
lowed by inoculation with the virus after removal of the chemical.
Transformation was dependent on the presence of the Rauscher leukemia
virus at the time of chemical treatment.

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Price et al. studied the oncogenic potential of the four major canna-
binoids found in marijuana (352).  High passage Fischer rat embryo cells
were inoculated with Rauscher leukemia virus and then treated with 1.0 yg
or 0.01 yg per ml of each cannabinoid.  The data indicate that the can-
nab inoids, with one exception, are inactive as transforming agents in
this assay stem.  The one exception,  (-)-trans A -tetrahydrocannabinol,
produced a transformation only after extended subculture (13 passages)
of the cells.  The activity of this cannabinol is weak in relation to
that of 3-methylcholanthrene.

The antiviral and antitumor activity of hydroxyguanidine is described
relative to its use as an antitumor drug  (341).  Hydroxyguanidine was
examined for in vitro activity against the Moloney sarcoma virus, Rauscher
pseudo type M-MSV (RLV) by assaying for its effect on focus forming
units and/or in vitro cytotoxic effects (cell growth inhibition) in four
tumor lines including L-1210 and L-51784 leukemia, Novikoff hepatoma and
Walker 251 carcinosarcoma.  The drug was also assayed for its activity
in vivo against four experimental tumors.  The drug exhibited definite
antiviral and cytotoxic  (antitumor) effects in vitro as well as anti-
tumor activity  in_ vivo and shows that other compounds combining the
dual moieties of antiviral -agents and antitumor drugs would possibly
show potent chemotherapeutic activity.

Rat embryo cultures were employed, along with human cell lines, to
depict the sensitivity of cells treated with the detergent Triton WR-
1339 C228).  Inhibition of the respiration rate of cells and the amount
of mitochandrial damage was related to the cytotoxic response of the
cells to the compound.  While the respiration of certain sensitive cell
lines CAV-3; HeLa) was markedly inhibited at low doses, the respiration
of insensitive lines like primary rat and chick embryo cells was un-
affected at much higher levels.

The metabolism of polycyclic aromatic hydrocarbons in cell cultures was
studied in a number of cell lines under conditions for their malignant
transformation.  The primary line utilized was C3H mouse prostate cells;
however, rat embryo and hamster embryo cultures were also used.

Other rat cell lines employed to explore in vitro cytotoxicity are rat
heart (353, 354), rat fat cells  (355), rat lung  (320), rat thymus lymph-
ocytes  (356}, rat pulmonary and peritoneal macrophages (357), rat liver
C358, 359, 360, 361), and rat kidney  (362, 363).

The interaction of carbon monoxide and hypoxia on the growth and con-
tractile ability of cultured rat heart cells was described  (353).
Carbon monoxide sustained the contractile activity of the cells, reduced
their growth, and affected cell toxicity as measured by nonviable cell
count in a concentration-dependent manner when the oxygen tension was
maintained at 20%.  A simultaneous reduction in oxygen tension enhanced
the inhibition of cellular growth by CO  and produced some reduction in
the contractile rate.  Inhibition of growth was much faster in cultures
exposed to both reduced Q • tension and CO  than in those exposed to
reduced tension alone.
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Lechat £t al_. studied the effects of the tricyclic antidepressant,
imipramine, on cultured rat heart cells (354).   After five minutes of
contact at concentrations which were higher than 5 yg/ml, imipramine
stopped the beating of cultured rat myocardial cells.  This inhibitory
effect on beating was suppressed in a K -free medium.  After three days
of contact, imipramine induced a vacuolization of both the myoblast
(muscle-like) and fibroblast-like cells in.a medium with or without K .
Apparently this effect is nonspecific for cardiac cells as it was also
induced by the drug on HeLa cells.  Two different mechanisms to explain
the effect of imipramine on the cultured heart cells are proposed.  The
first mechanism, which concerns the immediate effect of vacuolization,
could be related to an effect on membrane permeability; the second,
which occurs later and includes cessation of cell beating, could result
from a general cytotoxic effect.

Pesmethylimpiramine  QOMI) blocks the lipolytic effect of norepinephrine,
ACTH, theophylline, and dibutyryl cyclic AMP in isolated rat fat cells
C355L.  Rat cells were prepared from epididymal fat, pads.  Various
biochemical tests were then performed in which the antilipolytic activ-
ity of DMI was quantified and mechanisms for its effect were discussed.

Isolated cells harvested from trypsinized rat and mouse lungs were
examined in tissue cultures to determine the effects of NaNO   (320).
All the cell types studied showed a partial but reversible inhibition in
oxidative activity during treatment with NaNO .  Electron microscopy
also revealed changes in the nuclear shape and mitochondrial ultra-
structure during NO  treatment.

The lymphocytolytic activity in_ vitro of one of the nitrogen mustards,
methylbis  (beta-chloroethyl) amine, was investigated  (356).  The exper-
iment was designed to study the influence of this chemical on aerobic
glycolysis in a suspension of rat thymus lymphocytes.  Incubation of the
suspension was carried out in a Dubnoff Metabolic Shaking Incubator, and
the protein was precipitated.  The lactic acid in the supernatant was
then determined and the results were expressed as the percent stimula-
tion of aerobic glycolysis.  MBA not only possesses lymphocytolytic
activity but also stimulates the aerobic glycolysis of thymus cells.

In vitro cell culture methods were used to measure the toxicity of
polypropylene using polyvinyl chloride with a known toxic additive  (5%
dibutyltin diacetate) as a positive control  (357).  The tests utilized
measured the viability, adhesiveness, phagocytic activity and structure
of rat pulmonary and peritoneal macrophages and the viability and mi-
totic activity of calf kidney and chick embryo fibroblasts cultured in
the presence of extracts of the plastics in plasma.  The tests were
showri to be sensitive indicators of the presence of toxic materials.
The jn vivo effects were also studied by infusing two dogs with quan-
tities of the polypropylene extract.

The malignant transformation of a diploid strain of rat liver paren-
chymal cells  (RLC-10) treated with 3.3 x 10~  M 4-nitroquinoline 1-oxide
 C4-NQP) in culture was explored  (359).  Rat  liver cells were used
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because they transform spontaneously in vitro less often than do mouse
or hamster cells.  In each of five experiments the treated cells became
malignant.  They were backtransplanted after the treatment intraperito-
neally into two suckling rats to confirm this malignancy.  Continuous
cinematography over a six-month period revealed no marked change in the
static morphology of the treated cells but did show a loss of inter-
cellular adhesiveness suggesting some change in the cell membrane.  The
untreated controls did not produce tumors on in vivo reimplantation.
However, they did undergo spontaneous transformation by the seventeenth
month.

A study was conducted to describe aryl hydrocarbon hydroxylase activity
inducible in primary fetal liver cell cultures by various microsomal
enzyme inducers  (358).  This experimental model in cell culture may be
beneficial for comparing the effects of certain drugs, insecticides, and
polycyclic hydrocarbons on cell oxidase activity.  The maximum net rate
at which the hydroxylase activity accumulates is approximately the same
when phenobarbital, 3-methyIcholanthrene, or benz(a)anthracene is in the
growth medium at optimum concentrations.  An additive effect is obtained
when either phenobarbital or p_,p_^-DDT is present with a polycyclic
hydrocarbon in the growth medium but not when the cells are treated with
phenobarbital plus p_,p_[_-DDT or with a combination of two polycyclic
hydrocarbons.

Bendict, Gielen, and Nebert describe a study demonstrating polycyclic
hydrocarbon-produced toxicity, transformation and chromosomal aberra-
tions as a function of aryl hydrocarbon hydroxylase activity in cell
cultures  (.361).

Phenobarbital prevents the cytotoxic effect of benzo(a)pyrene  (BP) in
fetal rat hepatocytes in vitro.  BP is not toxic to HTC,A9 or HeLa cells
in which the aryl hydrocarbon hydroxylase activity is either absent or
very low.  However, BP is cytotoxic to each of these lines when they are
grown together with the liver cells in the presence of phenobarbital.
While chromatid breaks are associated with polycyclic hydrocarbon-
produced cytotoxicity, aneuploidy is more closely correlated with the
malignant transformation of hamster secondary cultures.  Benz(a)anthra-
cene of oc-naphtho flavone competitively inhibit the hydroxylation of
other polycyclic hydrocarbons such as the carcinogens 7,12-dimethylbenz-
Calanthracene  CDMBA) or BP.  Also, the exposure of the fetal hamster
secondary cells to excessive amounts of benz(a)anthracene prior to,
during, and following treatment with DMBA prevents malignant cell trans-
formation from occurring.

NLW cells derived from the liver of a newborn Wistar rat were trans-
formed by aflatoxin B , a potent liver carcingoen, in vitro  (360).  The
cells were cultivated for 161 days  (12 subcultures) and then exposed to
aflatoxin B  for 5 to 7 days at concentrations ranging from 10 to 0.01
ppm.  They were then cultivated further in a maintenance medium.  A
delayed cytotoxic effect was observed for several weeks after exposure,
especially at the two-week period, after which the surviving cells
transformed morphologically.
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After these cells were cultured for 87 days after aflatoxin exposure,
they were transplanted into Wistar rats and produced recognizable fibro-
sarcoma.  The results indicate that aflatoxin B  is a potent carcinogen
in vitro and is able to produce malignancies in vitro at wide ranges of
concentration.

Felling et al. assessed a number of methods used in the safety testing
of medical plastics (363).  The toxicity of a variety of plastic medical
devices (catheters, tracheostomy tubes, syringes, etc.), heart valves,
and polyvinyl chloride (PVC) samples of known constitution were evalu-
ated.  The agar overlay tissue culture technique of Guess et al. (299)
using primary rat-kidney cells and mouse L-929 cultures, implantation
tests in female rabbit muscle and male rat subcutaneous tissue, exam-
ination of the systemic effects of administering extracts of plastics to
rats and mice, and red-cell osmotic fragility tests were compared for
the demonstration of toxic effects.  PVC samples which contained known
concentrations of an organotin 'compound were used as positive controls.
^?hile none of the test samples registered toxicities as high as those of
the positive control plastics, the tissue culture technique was the most
sensitive method used.  Implantation of the rabbit sacrospinalis muscle
for 7 days was the most sensitive of the two implant systems tested and
the results showed a good correlation with the tissue culture.  The
systemic tests on plastic extracts failed to demonstrate toxicity, even
with the positive control plastics.

Grasso, Gaydon and Hendy assessed lysosomal changes as an index of
toxicity in cell cultures as a screen for the safety testing of medical
plastics C362)..  Four samples of polyvinyl chloride (PVC) which con-
tained 0, 0.17, 0.50, and 1.4% of dibutyltin diacetate but were other-
wise identical were used to evaluate two tissue culture methods commonly
used in the safety testing of plastics.  In the agar overlay technique,
primary neonatal rat kidney cells were grown in petri dishes, covered
with 1% agar and stained with neutral red.  The pieces of the test
plastics were placed on the agar and the plates were examined after 24
hours.  The area of neutral red loss under the plastic samples was the
index of toxicity.  In the other technique, primary neonatal rat kidney
cells were maintained in a growth medium of serum which was previously
used to extract dibutyltin diacetate from the PVC samples.  Lysosomal
acid-phosphotase activity was used in addition to the loss of neutral
red to indicate cytotoxicity.  The extent of cell necrosis was in direct
proportion to the concentration of dibutyltin diacetate in the plastics
for both methods.  Nevertheless, the agar overlay method was the more
sensitive in detecting low concentrations of the cytotoxic agent in the
PVC samples.  The histochemical method did show cytotoxic changes in the
cells earlier than did the loss of neutral red but did not improve the
sensitivity of the serum extract technique.

A rapid in vitro technique was developed and used in determining the
effects of heavy metals on the cytotoxicity of thiosemicarbazone and
other antitumor agents (364, 365).  The method determined respiration-
related cytotoxicity and was a modification of the Arai-Suzuki agar-
dilution test system in which no serum is needed so that metal ion
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concentration can be rigidly controlled (366).  Serial dilutions of
potential antitumor agents were incubated in direct contact with freshly
isolated tumor cells, normal cells or cells from tissue culture lines.
The effect of the agent was measured by the decrease in dehydrogenase
activity as a direct reflection of decreased respiration, with methylene
blue as the indicator.  Because of the short time necessary to run the
test (<3 hours) no serum was required in the medium.  The rat Walker 251
carcinosarcoma was the primary assay culture although other lines were
also used including Jensen Sarcoma, Murphy-Sturm lymphosarcoma, carci-
noma 755, and C3H mammary sarcoma  (364).  The test proved quite consis-
tent statistically, and also elucidated the effects of copper and zinc
ions on the cytotoxicity of thiosemicarbazone.  The data indicated that
a nontoxic level of copper ion CO.4 yg/ml) could activate thiosemicarba-
zone which by itself has little activity.   This agrees with the iii vivo
observation that thiosemicarbazone is an effective antitumor agent in
animals fed a diet high in copper but inactive when the diet is defi-
cient in copper.

As was reported previously within the mouse section, IRC rat monocytic
leukemia cultures were used in conjunction with L-51784 mouse lymphoma
cells to test the effects of acronycine on nucleic acid synthesis and
population growth.  RNA syntheses was rapidly inhibited at concentra-
tions of 0.5-12.0 yg/ml but culture growth is only arrested at much
higher levels.

Rabbit Cell Culture Systems

Many studies have used primary rabbit alveolar macrophages to investi-
gate the specific action of various pollutants on the lungs.  Several
reports deal with cigarette smoke as either a vapor or aqueous extract.
Among its many actions on lung function, cigarette smoke interferes with
the phagocytic activity of alveolar macrophages.  Green  (367) studied
various reducing agents for their possible protective action on the
cells against cigarette smoke.  Freshly harvested alveolar macrophages
were placed in tissue culture flasks with Staphylococcus albus bacteria
and appropriate chemicals.  Six ml of freshly drawn cigarette smoke was
introduced by syringe and the cells were incubated for two hours after
which the bacterial counts were made on the cultures, the counts serving
as an index of macrophage function.  It was observed that both gluta-
thione and cysteine prevented the toxic effect of smoke in a dose-
related response.  This protective role of sulfhydryl agents suggests an
pxidant action of cigarette smoke on these pulmonary cells.  An effect
on the cell membrane is suggested by the observation that coincident
w,ith the loss of phagocytic activity, cells exposed to cigarette smoke
separated from the flask surface although they did not lose their
ability to exclude vital dyes.

Green and Carolin studied the depressant effect of cigarette smoke on
the in vitro antibacterial activity of rabbit alveolar macrophages  (368).
They developed an in vitro quantitative phagocytic system in which
cigarette smoke was added to a mixture of rabbit alveolar macrophages
and Staphylococcus albus in plastic tissue-culture flasks.  The subse-
quent changes in the numbers of cultivable bacteria were determined by

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quantitative bacterial culture.  The smoke had a marked depressant
effect on the phagocytic activity of the alveolar macrophages.  This
inhibiting action varied quantitatively with the volume of smoke used,
the type or brand of cigarette, and the kind of filtration (mechanical
or aqueous) employed.  The active component of the smoke was largely
contained in the gaseous and filterable phase.  Nicotine, acetaldehyde,
formaldehyde, and cyanide did not, by themselves, affect the alveolar
macrophages in doses comparable to their content in smoke.  Cell via-
bility, as measured by the ability to exclude vital dye, was not affected
by the smoke.

Powell and Green (369) used aqueous extracts  (by agitating smoke with
water or Hanks' balanced salt solution) to investigate the enzymological
basis for impairment of phagocytic function resulting from the exposure
of alveolar cells to smoke.  Enzymatic (by histochemical methods) and
phagocytic (quantitative bacterial culture) determinations on the alve-
olar macrophages after two hours incubation with smoke extract were
made.  Supplementary experiments on the effect of smoke on cell-free
enzyme systems were also performed.  A' concomitant loss of phagocytic
competence and loss of enzyme activity after exposure to smoke extract
point to a relationship between macrophage phagocytic ability and the
activity of glyceraldehyde 3-phosphate dehydrogenase.

Again using an aqueous extract of cigarette smoke, Yeager (370) studied
its effect on protein synthesis in M. mgbiv induced alveolar macro-
phages.  Uniformaly labeled L-leucine-  C was used as the radioactive
substrate and the appearance of radioactivity, measured in a Beckman
liquid scintillation counter, in the washed trichloroacetic acid in-
soluble materials of alveolar cells is assumed to represent de novo
protein synthesis.   It was observed that the water soluble constituents
of cigarette smoke depress protein synthesis in rabbit alveolar cells in
vitro.  This effect is dose dependent, partly reversible and greater
than can be accounted for by the increase in acidity caused by the
addition of a smoke solution to cell suspensions.  It occurs at con-
centrations of smoke solution which do not affect cell viability or
protein turnover.  The major portion of this depressant activity is in
the gas phase.

                                                                 14
While Yeager had reported the reduced incorporation of L-leucine-  C
into prptein, other workers presented electron microscopic evidence of
increased protein synthesis in alveolar macrophages lavaged from human
smoker's lungs.

Therefore, Holt and Keast  (322) were prompted to reexamine protein
synthesis in macrophages in response to a number of tobacco smoke
regimes.  Both mouse peritoneal and rabbit alveolar macrophages were
investigated.  It was found that short-term exposure to cigarette smoke
(both vapor and aqueous extract) severely inhibits protein synthesis in
macrophages while low levels of smoke for long periods result in marked
increased synthesis of both protein and RNA.  These results suggest that
macrophages have the capacity to adapt to toxic changes in their envi-
ronment.
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Other than cigarette smoke, little is known about the effects of spe-
cific, gases on alveolar macrophages, the prime defense mechanism of the
alveolar areas.  Hence, Weissbecker et al. developed a simple, rapid
method for the determination of in_ vitro alveolar macrophage viability
after exposure to a variety of common air pollutants by the dye exclu-
sion test  (371).  A hanging drop of rabbit alveolar cell suspension was
exposed to gases in an air-tight chamber for one hour; an experimental
model which attempts to reproduce conditions of the lower respiratory
tract.  The effect of the various gases on the macrophage viability is
given in chart form.  Some gases were found to interfere effectively
with the.defense provided by the macrophages.

Because of the suspected action of lung irritants on pulmonary defense
mechanisms, studies were performed to assess the potential toxicity of
particulate forms of vanadium oxides on rabbit alveolar macrophages in
roller cultures  (372).  Cytotoxicity (using cell death as an end point)
was determined to be directly related to solubility.  Macrophage damage
from vanadium oxides and its relation to urban air concentrations of
this metal and acute pneuminities is discussed.

Allison used rabbit alveolar macrophages in parallel with primarily
mouse peritoneal macrophages to determine the mechanism by which silica
kills macrophages  (.324).  The toxicity of silica particles stems from
the fact that the particles are readily taken up by the macrophages and
react readily with lysosomal membranes causing secondary lysosome forma-
tion and release of lytic enzymes.  This study is an illustration of
selective  toxicity; the silica is selectively toxic to macrophages.

Kessel, Monaco and Marchisio designed an in vitro study to test the
specificity of the cytotoxic action of silica  (373).  In an attempt to
elucidate  the pathogenesis of silicosis, they inoculated monolayers of
guinea pig peritoneal macrophages, cultures of rabbit and rat macro-
phages, human cell  (macrophages, neutrophils, plasma cells, erythro-
cytes) and some primary tissue explants  Cskin, liver, heart, and muscle
of chicks), along with the continuously cultivated monkey kidney and KB
cell lines with media containing sterile, powdered  silica, hemalite and
carbon.  Cytotoxicity was measured by the ability of the cell cultures
to metabolize.  The supernatant culture fluids were analyzed for lactic
acid produced per unit of time by both chemical and enzymatic means.  In
general, the data indicate that an appropriate surface is necessary for
the characteristic Cytotoxicity of silica, and that this toxicity is
specific for the macrophage.

Monkey Cell Culture Systems

Toxicities of  15 analogous compounds  (374), related to either sterig-
matocystin or  aflatoxin B  , were evaluated on primary monkey  (Cerco-
pithecus aethiops) kidney epithelial cells grown on glass coverslips in
roller tubes.  They were exposed for 24 hours to the various compounds
which were dissolved in Hank's balanced salt solution to a final con-
centration of  2 mg/liter.  The effects of these compounds on nucleolar
morphology, mitosis, and incorporation of  H-thymidine and  H-uridine
were studied.   Structurally, these compounds were divided into  three

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 groups manifesting distinct toxic patterns which indicated a correlation
 between toxicity and structure.   Apparently an unsaturated A '  -furo-
 benzofuran system and the position of methoxy and hydroxy groups on the
 xanthone ring of the sterigmatocystin analogues affected the cytotoxic-
 ity in these cell cultures.

 Cultures of a monkey kidney (Cercopithecus aethiops)  cell line  (R-l CA;
 hypodiploid; 57-59 chromosomes),  containing reduced proportions of
 anisonucleolinar nuclei,  were treated for 8, 24 and 48 hours with var-
 ious doses of thioacetamide (TAA, a water-soluble carcinogen) and hy-
 droxylamine (HA, a mutagen) in the media (375).  It was tentatively
 assumed that the nucleoli may be  a critical site for the carcinogenic
 impact.  Thus, these cultures were observed for nucleolar and mitotic
 alterations.  These alterations were observed 24 and 48 hours after TAA
 administration.  A good correlation was found between the developmental
 patterns of anisonucleolinosis and abnormal mitoses in the TAA  treated
• cells.  HA failed to induce anisonucleolinosis but induced smaller
 nucleoli with no changes in their ability to incorporate  H-uridine.

 Monkey kidney cell cultures were  also used to study the effect  of bleo-
 mycin Can antibiotic) on cell survival.  From these results1, discussed
 in the mouse section, some principles of bleomycin chemotherapy are
 outlined C309).

 Chicken Embryo Cell Culture Systems

 Chick embryos have been used extensively for biological studies, both
 whple embryos and monolayer cell  cultures prepared from their tissues.
 Guess ejt al^  (.297, 299) have shown that chicken embryo cell monolayers
 overlaid with nutrient agar and stained with a vital dye can be used
 successfully to screen the toxicity of various chemicals and/or mate-
 rials.  In both of these studies  replicating mouse cells were used in
 parallel with the nonreplicating  chick embryo cells.   More details of
 these studies can be found in the section dealing with mouse cell cul-
 tures.

 In 1969, Wilson and Stinnett (376) studied the effect of two agricul-
 tural chemicals, malathion and malaoxon, on two primary cell systems,
 chick embryo heart cells and pectoral muscle cells, using a respiration
 chamber constructed specifically to measure oxygen consumption  of mono-
 layers of cells growing on coverslips.  Cells were exposed to insecti-
 cide containing media for periods up to 60 minutes and then counted.
 Both 3 x 10   M malathion and malaoxon are strongly toxic to the growth
 of pectoral and heart muscle cell cultures and malathion is more toxic
 than malaoxon.  An acute dose of  malathion, 3 x 10   M inhibits cell
 respiration; however, incubation of the cells in 3 x 10   M malaoxon
 does not.  The evidence presented suggests that malathion inhibits the
 energy metabolism of chick embryo cells and that the conversion of its
 P-»-S group to the p-K) group of malaoxon results in a disappearance of the
 inhibition.

 This paper also describes a respiration chamber suitable for the meas-
 urement of respiration of monolayers containing 200,000•or more cells.


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It consists of a polarographic system connected to a gas analyzer and
strip chart recorder.  Such a device could be very useful for the assess-
ment of toxicity of potential respiratory poisons.

Wilson, Stinnett, et^ al_. (377) studied malathion and malaoxon on res-
piration of chick embryo pectoral muscle cultures using the previously
mentioned respiration chamber.  Malathion and malaoxon inhibited growth
(cell number, protein, DNA) of the cells.  Low-protein phosphate-buffered
medium with pesticide was more toxic than high-protein, carbon dioxide-
buffered medium.  Growth was also inhibited by parathion and paraoxon.
Determinations of cholinesterase activities and isoenzymes of cell
cultures and respiration of tissue homogenates, cells and mitochondria
are included in this report.  The observations presented give a detailed
account of inhibition of pesticides of acetylcholinesterase in cells in
culture.  This study confirms the prievious study's finding that the
inhibition of respiration may play a role in the toxicity of malathion
to cultured cells.

For an evaluation of germicidal efficiencies and toxicities of a group
of antibiotics intended for clinical application, Salle and Amesur  (378)
used an embryonic chick heart tissue fragment-saline suspension.
Portions of the tissue suspension, bacterial suspension  (Micrococcus
pyogenes var. albus) , and antibiotic dilution were combined in a tube
and agitated in a 37 C water bath.  After two washings the fragments
were embedded in plasma in Carrel flasks and allowed to grow.  A toxic-
ity index for the antibiotics, a ratio of the highest dilution to pre-
vent growth of tissue fragments to the highest dilution to kill test
bacteria under test conditions, is obtained.

Dorsal root ganglia from chick embryos cultured by the hanging-drop
method on collagen-coated cover glass in media containing different
amounts of mercury compounds were studied by Kasuya to show the rela-
tionship between the structure of mercurial compounds and their toxicity
on nerve tissue in culture  (379).  The adverse appearance and/or inhi-
bition of migration and outgrowth of the cells were observed.  From the
results, it was postulated that the binding of mercury compounds by
their hydrophobia and hydrophilic moieties with specific binding sites
on membranes is a major factor in their toxic action.  The author fur-
ther suggests that by application of tissue culture methods, it may be
possible to investigate the neurotoxicity of mercury compounds more
accurately without considering such factors of absorption and distri-
bution.

Racz and Marks  (380), utilizing the intact chick embryo for the bulk of
their investigation, made supplementary use of monolayer chick embryo
liver cells to demonstrate the presence of active DDC and Ox-DDC,
porphyria-inducing drugs, in cell culture media 24 hours after incu-
bation.

Using chicken erythrocytes incubated with N  -glycine as a test system,
Abbott, Jr., and Gindin  (381) compared the effects on heme synthesis of
a series of benzimidazole derivatives having nitro or chloro groups
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instead of a methyl in the five position.  Similar studies with 5-
hydroxybenzimidazole are also included.  This investigation was prompted
by an earlier supposition that benzimidazole derivatives inhibit an
important metabolic reaction fundamental to more than one biosynthetic
process since they were,.found to^inhibit virus duplication and prevent
the incorporation of N   from N  -glycine into heme by chicken eryth-
rocytes during in vitro incubation.
MISCELLANEOUS CELL CULTURE SYSTEMS

Rat Kangaroo

Palmer e_t al^. (382) used an aneuploid cell line from the rat kangaroo
(Potorous tridactylis apicalis) to determine whether the persistent
presence of DDT or its major metabolites produced chromosomal abnormal-
ities in an iri vitr£ system.  The cytogenetic effects of £,pJ_-DDT, o,p'-
DDT, p_,p_^_-DDD, o_,p_^_-DDD, p_,p_^_-DDE, o_,p_|_-DDE, and p_,p_^_-DDA were  inves-
tigated by exposing various concentrations of the chemical, diluted  in
media, to the monolayers for 24 hours and then examining the cells for
CPE, mitotic index and chromosome damage.  It was found that the  iso-
meric configuration of the compounds is related to the production of
exchange figures; the p,p' isomers produced exchange figures and  the
o,p' isomers failed to produce exchange figures even though cells were
observed with multiple chromatid breaks.  These types of damage are
probably of great mutagenic significance since they are the cells most
likely to survive and carry an alteration of the genetic material.

Guinea Pig

Parazzi et 'al. (383) investigated the site of cellular damage induced by
silica particles in. .vitro to guinea pig peritoneal macrophages.   Tri-
dymite (50 y - 150 y) and coal dusts  (0.5 y - 5 y) were suspended in
Hank's media immediately before adidtion to the cell cultures and re-
mained in contact with the cells for various intervals up to seven
hours.  The early phases of phagocytosis were followed by measuring  the
release of. some cell enzymes  CLDH, ribonuclease, APH, SDH, GDH) into the
culture media and by observing the integrity of the cell membrane using
fluorochromatic methodology.  The evidence presented suggests that when
silica is added to macrophage cultures in vitro, it interacts with the
external cellular membrane and that the first cellular lesion occurs at
this site.  Further damage might then be inflicted by the liberation of
lysosomal"enzymes.  Coarse particles  (50 y to 100 y) which could  not be
phagocytized by the macrophages showed that mere contact of the external
cell membrane with the surface of the silica particles was sufficient to
damage the membrane which was evidenced in a very short time by the  loss
of fluorochromasia and some escape of cytoplasmic enzymes.  The coating
of the particles with 20% guinea pig serum resulted in a delayed  release-
of enzymes.

Pi£

4-Nitroquinoline N-oxide  (4-NQO), a potent carcinogen for mice  and rats,
has been found to be mutagenic as well as cytotoxic.  Horikawa  et al.

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(384) investigated the 4-NQO-induced damage in cultured mammalian cells
by comparing the response in three cell' lines:  mouse L cells, porcine
kidney stable (PS) cells, and Ehrlich ascites tumor cells.  The cells
were suspended in 5 ml of culture media containing different concentra-
tions of 4-NQO for various intervals and were then plated in chemical-
free media and observed for colony forming ability.  Toxicity was deter-
mined by directly plating cells in media containing various levels of
4-NQO.  For the analysis of 4-NQO incorporation, cells were grown for
various intervals in media containing  H-4-NQO and the radioactivity was
determined by a liquid scintillation counter.  It was concluded that PS
cells are more resistant to 4-NQO than the other two cell lines and
mouse L cells are the most sensitive to 4-rNQO.  In a previous paper the
authors showed that Ehrlich cells were more resistant to ultraviolet
light than the other two cell lines and PS cells were the most sensitive
to UV.  These results seem to indicate that the effect of UV on cells
differs from that of 4-NQO.  However, there are no differences in the
incorporation rate of  H-4-NQO into cells among the three cell lines.
The authors also suspect that the differences in ability among the three
cell lines to reduce the compound 4-NQO to a more carcinogenic but less
cytotoxic intermediate, 4-HAQO caused differences in sensitivity to 4-NQO.
The activity of the reduction pathway in the target cells, which yields
4-HAQO, is thought to be essential for its carcinogenic mechanism as
well as its cytotoxicity.
DISCUSSION

Cell cultures, particularly those of highly differentiated cell types,
are important tools for physiological, pharmacological, and toxicolo-
gical studies.  Difficulties observed in many studies, however, reveal
problems which have and might ultimately be encountered if in vitro
systems are to be employed for the primary toxicological screening of
pesticides and other pharmacological agents.  Cell cultures consist of
single cell types, in the case of established cell lines, or several
cell types in the case of primary cultures derived from a whole organ,
tissue,.-or embryo.  Hence their study will not directly detect events
which depend upon the integrative biological mechanisms of the animal.
Two aspects of the problem caused by working in vitro must be borne in
.mind.  In some instances metabolic activation must occur in order for a
compound to become toxic.

Should a test cell culture system not include cells with this metabolic
capability, a potentially toxic chemical may appear to be innocuous as a
result of in vitro testing.

The other side of the coin concerns detoxification.  The intact orga-
nism has a detoxification capability which may exceed that of an in_
vitro system.

Specific procedural problems which might affect the sensitivity and
results of in vitro cell culture toxicity testing are as follows:

(1)  Cell cultures may contain latent or occult viral agents
     which may produce false positive toxic effects or, in
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     carcinogenic studies, produce an unexpected cocarcino-
     genic effect.

(2)   The test sytems may be contaminated with Mycoplasma which
     perhaps will produce cell degeneration due to nutrient
     depletion or cell infection or profound alteration in cell
     metabolism.

(3)   Great care must be used in the selection of cell culture
     media and its constitutents, particularly the animal
     source of serum to be used, the supplier, and even the
     batch.  These factors, as well as the presence or absence
     of antibiotics, must be controlled because they may ad-
     versely influence cell metabolism or they may form inter-
     fering complexes with the test compound.

(.4)   Transformation of cells by chemicals is not an absolute
     indication of the tumorigenic activity of the same chem-
     icals in animal hosts.

(5)   When immunologically competent cells are used for trans-
     formation studies, specific care must be used to select
     compatible allotypic cells which will not be affected by
     graft-host responses, or will not transform spontaneously.

C6)   Specific toxic effects may not be manifested in early cell
     passages and a sufficient number of serial transfers must
     be conducted to allow observation of these responses.

The overall degree of correlation between in vitro and in vivo studies
indicates that in vitro toxicity testing of chemicals, drugs, and phys-
ical agents is scientifically practical within specific limitations.  In
vitro methods, which allow a more careful control of agent concentrations
and cell exposure times than is possible in animal systems, are simple
enough to permit a large number of samples to be evaluated in a short
period of time.  Other advantages which might be realized by using in
vitro systems are:

Cl)_  When studies concern expensive or rare compounds, micro-
     gram quantities can be tested, whereas larger quantities
     are needed for bioassay in animals.

(2}   The specific dose delivered can be closely controlled
     since the use of cell cultures circumvents physiological
     and metabolic effects which occur within the whole animal.

(3)   The relative cost per compound tested in a large scale
     screening program has been estimated to be $1500 to $2000,
     a cost considerably less than long-term animal studies.

C4)   In vitro toxicity testing procedures can be used to char-
     acterize concentration-time relationships which make it
     possible to estimate dose-time schedules for in vivo or
     clinical trials.

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D.    USE  OF BACTERIA, FUNGI,  PROTOZOA, AND  PLANT  CELLS  IN STUDIES
      ON CHEMICALS
 INTRODUCTION

 In  attempting to  develop  alternatives  to  long-term mammalian toxicity
 testing,  the use  of  nonmammalian  systems  must  be  considered.   The use of
 single  cell  organisms, which are  easily cultivated in large numbers has
 an  inherent  advantage.  Results can be obtained in a short span of time,
 and very  large numbers of individuals  monitored.

 It  is felt that extrapolation of  information obtained in one cellular
 system  to another more complex multicellular system is valid if a care-
 ful review of the affected subcellular systems is conducted.   All cells
 have structural and  metabolic properties  in common;  all cells contain
 DNA, are  enclosed by semipermeable membranes,  possess electron transport
 systems.

 In  reviewing the  literature, a search  was made for reports on the use of
 plant cells, protozoa, fungi, and bacteria to  assess the toxicity of any
 chemical. Some of these  reports  were  quite specific in detailing toxic
 effects at the subcellular and metabolic  levels—others were concerned
 only with a  plus-minus indication of toxicity. Correlations attempted
 by  the  authors between their cellular  data and that from mammalian
 systems have been emphasized.
 BACTERIA

 The use of bacteria as indicators of compound toxicity goes back to the
 early studies on antimicrobial agents,  and later antibiotic studies,
 where the bacteria themselves were the  end point of desired knowledge.
 In recent years, however,  more emphasis has been placed on the use of
 bacteria as indicators of  drug effects  in higher organisms and ulti-
 mately in humans.   As the  following discussions show,  in some cases
 these correlations can be  made, but in  other cases they cannot.
r
 Growth Inhibition;

 Cells are exposed to the chemical under test either in broth or on agar
 plates, with observations  on change in  turbidity or zones of inhibition
•as the end points.  These  are methods widely used in assaying antibiotics
 and antimicrobials, and yield only a plus-minus qualification of toxicity,
 and no information oh the  biological mechanism of toxicity is obtained.
 Escherichia coli (175, 385,  and 386) was tested in this manner against
 promazine and chloropromazine, 2,4-dinitrophenol, and various nitroso-
 guanidines.  In one of these studies £386) in which strains of E_. coli
 resistant to l-methyl-3,-nitro-l-nitrosoguanidine (NG)  are used,  it was
 found that these are resistant to a variety of "radiomimetic" compounds.
 In addition, one of the mutants seems to be specifically resistant to
 the precise chemical configuration of NG.  The authors question if such
 specific events occur in mammalian cells, and could they account for
 resistance of tumors to radiomimetic agents.
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In a study of the toxicity of a toxin produced by Alternaria mail  (230),
it was found that some gram-positive cocci and some fungi were inhibited
by the compound but Bacillus sp_. and some gram-negative bacteria were
unaffected.  Mice and human cells in tissue cultures were also tested
against the toxin, and the authors conclude that "the range of organisms-
mice, human cells in tissue culture, fungi, and bacteria—adversely
affected by the crude extract indicates that this isolate of Alternaria
mali produces a broad spectrum of toxicity."

Some studies have yielded specific information on the biological mech-
anism of toxicity.  IS. coli was treated with ethionine  (387) and the
effect on cell viability observed.  Cell extracts show that ethionine
inhibits specific enzymes leading to protein synthesis, and it was
stated that "ethionine is toxic to a broad spectrum of biological sys-
tems . . . from microbial growth inhibition to the genesis of a variety
of tissue aberrations in mammals."

In a study (388) to determine the toxicity of a series of tetrazolium
salts against seven bacterial species  (gram-positive and gram-negative),
it was determined that the ditetra salts were more toxic than the mono-
tetra salts and that the growth inhibition is probably due to inter-
ference with cellular enzyme systems.

A herbicide, paraquat (1,1'dimethyl'4,4'bipyridilium dichloride), was
assayed for toxicity against E^. coli  (389) .  It was found in this case
that the compound was only bacteriostatic, reversibly inhibiting the
syntHesis of DNA, RNA, mRNA, and protein through interference with
energy metabolism.

A group of food additives, all lipophilic acids, was tested against
Bacillus subtilis, E_. coli, and B_. pumilis  (390).  The resulting growth
inhibition was found to be due to the cell's inability to take up amino
acids and phosphate groups.

A group of Salmonella mutants were used to study the toxicity of B-
propiolactone (391).  It was found that the chemical acts directly upon
cell DNA.  Salmonella were also used to assay the toxicity of a group of
unsaturated carbonyl sugars produced during the irradiation of sugar
solutions  (392).  These compounds inhibited the growth of these bacteria
through irreversible reactions such as addition of sulphydryl groups and
substituted amines to cellular constituents.  Schubert and Sanders also
determined that these compounds inhibit glycolysis and respiration in
tumor cells, inhibit the growth of ascites cells and produce chromosomal
breakage in vitro.

Clostridium perfringens has been used to study the inhibitory effects of
a series of alcohols  (393).  The inhibitory effect increased with the
molecule chain length, and it was also noted that lecithinase production
by the cells was a more sensitive indicator of alcohol toxicity. than was
cell growth.  Specific DNA synthesis was inhibited in a large group of
bacterial species when treated with 2,3-dihydroxmethylquinoxaline-l,4-
di-N-oxide (394).  This compound has also been found to have activity in
protozoa and viruses.

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The studies discussed above used observations on the growth and/or
viability of the treated cells as an indicator'of toxicity.  In a unique
study (395), the ability of treated bacteria  (B_. megaterium) to convert
to viable protoplasts was used as the toxicity indicator.  Magnesium
chloride and a series of cationic and anionic surface active agents were
tested with this system.

Potentiators;

A great deal of interest has arisen in the effect of "potentiators" on
the toxicity of compounds in test systems.  In a study on the effect of
UV irradiation on  E_. coli B (396) chloroquine was found to reduce the
survival of irradiated cells, and appears to inhibit the dark repair
mechanism.  Hadaciden (N-formyl hydroxyamino acetate) was found to
potentiate the lethal effects of ionizing radiation in 12. coli  (397).

In a study to select possible potentiators (398), 36 pairs of chemo-
therapeutic agents were assayed against IS. coli.  Wheeler, Schobel and
Skipper state that the mode of action of chemotherapeutic agents appears
to be the same in bacterial and mammalian systems.  However, Guthrie et
al. (399), reporting on "collateral sensitivity" to chemotherapeutic
agents, found that the effects of a pair of antimetabolites on bacteria
cannot predict the effect in another biological system.  This group used
mutants of E_. coli and B_. subtilis which were resistant to purine antag-
onists.  They were exposed to the original drug plus another under test,
using agar diffusion, and observed for inhibition of growth.

A more specific assay for toxicity is the determination of effect on the
DNA of the treated organisms.  A group of nitrofuran derivatives was
used to treat E_. coli B and B/R cells (400) followed by analysis of the
lengths of the resulting DNA.  It was found that the active DNA damaging
compounds are reduction products of nitrofurans and that a direct correla-
tion exists between the number of DNA breaks a compound produced in E_.
coli and the degree of carcinogenicity in mammals.

In recent years, the use of bacteria as indicators of chemical muta-
genicity has gained importance.  Ames has stated that since mutagens
alter DNA, and the DNA of all organisms has the same structure and
contains the same nucleotides, any organism may be used as an indicator
system for mutagens  (401).  Bacteria offer many advantages over more
complex material.  It is possible to treat and screen a large number of
cells in a short time.  They have short generation times, small genomes,
are haploid, and can be grown on defined media.  Bacteria may be used to
detect forward, reverse, and suppressor mutations.  Both mutagenic and
inactivating DNA alterations can be detected.

Great care must be used, however, in transposing the interpretation of
data obtained in bacteria to what occurs in higher life forms.  Bacteria
lack some of the protective mechanisms of mammalian cells.  Metabolic
alterations of the chemical under test are different in bacteria and in
mammalian cells; however, the development of the host-mediated assay and
of the microsome activation preparations has overcome part of this
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difficulty.  The uptake and transport of the compound may differ.
Correlations are being made of data obtained in bacteria with data
obtained in mammalian systems and it is expected that the dichotomy will
be resolved.

Studies with mutagens in bacteria have been done mainly with the Sal-
monella tester strains of Ames (402, 403, 404, 405, 406).  Ames and his
coworkerg have developed Salmonella histidine mutants which detect base-
pair substitutions and frame shift mutations, through reversion to
prototrophy.  They are rendered highly sensitive to most mutagens through
loss of a lipopolysaccharide coat and through deletion of a gene in the
excision repair system.  The chemical is spotted on an agar layer lack-
ing histidine, containing the Salmonella and microsome preparation.
Mutated colonies are the only ones able to grow and are directly counted.
Ames has found that a wide variety of chemical carcinogens are detected
as mutagens using this system.  These carcinogens include polycyclic
hydrocarbons, aromatic amines, nitroquinolines, aflatoxins, fluorenes,
and can be detected at nanogram levels.

Hartmann et a!U (407) in earlier work has used Salmonella mutants for
rapid spot tests for mutagens.  They found that hycanthone was mutagenic
in this system, while miracil D,  a closely related compound, was not.  A
mutant strain of E_. coli which was deficient in DNA polymerase was
growth inhibited by some well documented carcinogens, including nitro-
somethyl- and ethyl-urea, the comparable urethans, MMS, EMS, hydroxy-
urethan, nitrosofluorene, and hydroxylaminofluorene.  Dichlorovos and
Captan, ingredients of several pesticides, were found to be mutagenic to
an,auxotrophic mutant of E_. coli (408).  A large number of pesticides
were not mutagenic in this system.

A review article by Stoltz e_t al. (409) includes the mutagen screen
system as one of the three offering promise in this area.

The development of microbial assay systems for mutagen detection is
progressing, as can be seen, to a high level of predictability for
mammalian systems.  The use of these assays in combination with others
in higher organisms can yield a great deal of information useful to
those concerned with human safety.

Antitumor Compound Screens;

A group of papers published in 1958 outlines the rationale for the use
of microorganisms in an ongoing screen of antitumor agents  (410, 411,
412).  The collaborative studies included the use of 16 microbial sys-
tems (bacterial, fungal, and protozoan) and human cell cultures of
normal and neoplastic origin.  The organisms for the multiple bioassay
system were selected in order to base the in vitro screening procedure
on a variety of diverse metabolic pathways.  In the work reported by
Foley, McCarthy and coworkers (412), the results obtained from microbial
bioassay of 200 selected compounds are reported.  In general, the
organisms were grown in appropriate broth containing the test compound,
and the concentration which allowed only half-maximal growth determined.
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The microorganisms used included Streptococcus faecalis, Excherichia
coli, five Lactobacillus species, Leuconostoc citrovorum, Candida
albicans, Neurospora crassa, Saccharomyces carlsbergensis,  Tetrahymena
pyriformis, Glaucoma scintillans, and Colpidium campylum.

Little direct correlation was found between the results obtained in the
different microbiological systems.  No single bioassay system detected
all of the known active compounds.  While £. pyriformis gave 82% identi-
fication only 39% were picked out by Candida albicans.  Certain combina-
tions of three or four of the bioassay systems can be selected which
give comparable positive percentages as the whole group.  The authors
make the point that microbiological assay systems can be readily devised
by choice of proper strain and medium to detect compounds exhibiting
antimetabolite activity against any specific essential metabolite.

The limitations of any in vitro screen are  (1) host metabolic-activation
of a compound with no corresponding activation by the test system;  (2)
occurrence of indirect action; (3) inability of in vitro systems to
handle as large a dosage as can be obtained in the in_ vivo situation;
(4) instability of compounds during the duration of an in_ vitro test.

In an accompanying paper by Foley, Eagle and coworkers  (410), the results
of the previous study are compared with data obtained with experimental
tumors in vivo.  They found that mammalian cell cultures appear to be
superior to microbiological systems in assessing the effectiveness of
antitumor agents.

In a report by Schabel  (411), another type of antitumor agent screening
program utilizing bacteria is described.

In this screen, a combination of drug-resistant and drug-sensitive
strains of Escherichia coli or Streptococcus faecalis are used to dis-
tinguish new agents with modes of action differing from known agents.
Inhibition of the parent drug A-sensitive line with failure to inhibit
the drug A-resistant line of the same organism suggest that compound X
has a mode of action similar to that of compound A.  These assays are
done as spot tests, with a filter disk containing the test compound
placed on a lawn of the suitable organism, and zones of inhibition
observed.  It is also possible to study the biochemical mode of action
of known anticancer agents with this system.

Although these methodologies were not specifically designed to assay for
toxicity in whole animals per se, they could be readily adapted to
become useful tools for toxicity testing.  A great deal of information
on many different groups of compounds has been amassed in anticancer
agent screens, and should be tapped for the more general purpose of in
vitro toxicity assessment.
FUNGI

Fungi and mammalian cells possess some common metabolic pathways and
enzyme systems, but it cannot be assumed that the entrance of a compound

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into a cell or the action of modifiers or "activators" on the molecule
is the same in both types of cells.  It has been found that certain
unusual side effects of drugs in mammals can be predicted through the
use of yeast, but it is not known if such inhibitors function in higher
eucaryotes as in the lower (413).  In addition, differences in structure,
degree of specialization and environmental modification preclude any
close correlation.  On the other hand, where effects of chemicals are
found to be upon structures or metabolic functions common to both
cells, or where provision is made for "activation" of a compound, then
such correlations can be usefully made.  This is certainly true in.the
case of mutagens where the toxic action is on chromosomal material, as
yeast cells and other fungi, being eucaryotes, have a closer relation-
ship to mammals than bacteria, which are prokaryotic.

Growth Inhibition;

The direct toxicity of a chemical on the organism can be assessed through
observations on growth and/or survival, or through analysis of specific
disruptions in functions of the cell.

Using observations on growth, nitroquinolines and nitropyridines were
assayed for toxic effects against S_. cerevisiae (414, 415), and it was
noted that there existed a correlation between the degree of inhibition
of yeast by a compound, its degree of carcinogenicity in mammals, and
its mutagenicity in lower organisms.  Epstein and St. Pierre (44) point
out the dynamic association found with this group of compounds:  muta-
genic in yeast <=>, carcinogenic in rodents <=>, photodynamic activity
in protozoa <=>, phage induction.

In contrast to the above correlation, when a varied group of cholesterol
synthesis inhibitors were studied, no correlation was found between the
ability of a compound to inhibit cholesterol synthesis in rat liver
homogenates, and to inhibit the growth of yeast cells (413, 416).  In
one of these studies (416), several species of yeast were used, includ-
ing Hansenula, Candida, Geotrichum, and Saccharomyces.

Inhibition of growth, sporulation and morphological changes are produced
in Aspergillus, Penicillium, Neurospora, Cladosporum, Mucor and Rhizopus
species during exposure to a mycotoxin, rubratoxin..  The work reported
by Reiss (417) indicated that a toxin level of 10 yg/ml was sufficient
to produce toxicity in some of the test species.

Genetic Damage in Saccharomyces;

The structural organization of chromosomal material in yeasts is com-
parable to that in mammalian cells.  Forward and reverse mutations and
reciprocal and non-reciprocal mitotic recombination events are easily
studied.

In a study by Epstein and St. Pierre  (414), respiratory deficient mutants
of Saccharomyces cerevisiae were produced with nitroquinolines and
detected on agar plates- containing triphenyltetrazolium chloride.  It
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was found that those compounds which were toxic to the cells were muta-
genic.  In a study (418) of the toxic effects of nitrous acid and alkyl-
ating nitrosamides on S_. cerevisiae, it was found that the frequency of
induced reverse mutations were dependent on the temperature of treat-
ment.

When nitroquinolines, nitropyridines and their derivatives were studied
(415), using the spot plate technique and DNA repair deficient Sac-
charomyces as the test organism, a correlation was found between yeast
growth inhibition (due to lethal mutations) and previously reported
carcinogenicity in mammals.  This type of assay has been proposed as a
screening method for carcinogens.

In a modified spot test system by Fink and Lowenstein  (419) the effect
of test conditions was shown.  When EMS, NG, and DBS were added to cells
on minimal agar, no increase revertants was seen, but when cells were
treated on complete media and then replicated to minimal, an increased
reversion frequency was evident.

Mitotic recombinations are induced by base-pair substitution frameshift
mutagens and carcinogens, and apparently occur at higher frequencies
than do "mutations."  In three papers (420, 421, 422), the induction of
non-reciprocal mitotic gene conversion in Saccharomyces was studied with
a group of pesticides and herbicides and their N-nitrosated derivatives
(Benzthiazuron, Propoxur, and Carbaryl).  A strain of Saccharomyces
which requires both adenine and tryptophan is used, and the cells, after
treatment, are plated on media containing only one of these compounds.
Gene conversion creates cells which no longer require both compounds.

The non-nitrosated compounds had no influence on conversion frequencies,
but the nitrosated derivatives displayed marked convertogenic activity.
N-nitroso compounds display a wide range of biological activity  (acute
and chronic toxicities, carcinogenicity, and teratogenicity).  The
authors point out the possibility of conversion of the inactive pesti-
cide in the human gut to these highly active forms.  The same group also
studied the genetic effects of a group of 32 herbicides in the same
system.  Only two of these herbicides showed activity  (Diquat and 2,4-D).

In a study by Vogel, Fahreg and Ohe  (420) a group of triazenes was
tested in four genetic test systems:  mitotic gene conversion in Sac-
charomyces ; host-mediated assay using mouse and Saccharomyces; genetic
abnormalities in Drosopyila; and chromosome aberrations in cultured
human leucocytes.  The triazenes have been described as "indirect muta-
gens," requiring metabolic activation for effectiveness.  The three
compounds tested were PDT  (l-phenyl-3,3-dimethyltriazene), PyDT  (1-
(pyridyl-3)-3,3-dimethyltriazene), and PyNDT  (1-(pyridyl-3-N-oxide)-3,3-
dimethyltriazene).

In Saccharomyces, both PDT and PyDT produced convertants.  The same was
true in the host-mediated assay, although PyDT showed more activity than
PDT, the reverse of the results iri vitro.  PyNDT was not active in
either system.  In Drosophila, recessive lethals were produced by PDT
and PyNDT, with PDT being more active.  Chromosome aberrations in human


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leucocytes were produced at very low levels by all three compounds.
The authors conclude that for the detection of "indirect" mutagens, the
host-mediated assay and Drosophila tests are clearly superior.

Another effect of mitotic recombination, reciprocal crossing over, is
the basis of a test system using Saccharomyces.  Mitotic crossing over
has been demonstrated in a wide range of organisms including Aspergillus,
Drosophila, soybean, and mouse cells.  In the Saccharomyces test system
the strain D5 carries two recessive alleles of the gene ade-2  (colored,
adenine requiring) which complement each other to yield white, non-
adenine requiring colonies.  Induced mitotic crossing over produces pink
and red sectored colonies, which can ben directly counted to yield
recombination frequency information.

Zimmerman  (423) used this system to verify ultraviolet irradiation and
EMS as mutagenic agents which induce crossing over in Saccharomyces.  In
a study (424) examining the activity of DMN (dimethylnitrosamine), DEN
(diethylnitrosamine), 1-NA  (1-naphthylamine) arid 2-NA (2-naphthylamine)
in inducing crossing over in yeast, Mayer found that these compounds
required conversion for activity.  The production of active breakdown
products on the Udenfriend hydroxylation medium was shown by the high
rate of recombinants in Saccharomyces.

A review by Brusick and Mayer (425) presents results of the testing of a
series of compounds for gene conversion, forward and reverse mutation in
Saccharomyces, and a discussion of the advantages and disadvantages of
using yeast cells for mutagen screening.  (See also "Mutation  Induction
in,Yeast," by R. K. Mortimer and L. R. Manney, Chemical Mutagens; Prin-
ciples and Methods for Their Detection, Vol. 1, A. Hollaender, Ed.,
Plenum Press, 1971.)

Genetic Damage in Other Fungi :

Other fungi besides Saccharomyces have been used to study the  toxic
effects of chemicals for both growth inhibition and genetic effects.
Synkavete  (426)  (Na -2-methyl-l,4-naphthohydroquinone diphosphric acid
ester) was assayed for toxicity against Histoplasma, Norcardia, Asper-
gillus, Tricophyton, and Microsporon species.  The fungi were  grown on
agar containing the compound.  Fungal growth was inhibited at  0.15 to
1.20 mg/ml; the compound was toxic to mice at 0.4 mg/g.

Maneb, a commercial fungicide, was reported (427) to exert a toxic
effect on Colletotrichum through alteration in amino acid, succinic
acid, and phosphorus metabolism and production.  The mycelial  dry weight
of Aspergillus niger was used to indicate that dinitrobenzene  is toxic
to that organism  (428) at 1.0 yM.  The toxicity was attributed to the
depression of citric acid cycle intermediates.

A group of benzimidazole and thiophanate fungicides were assayed for
toxicity through their ability to induce genetic segregation in Asper-
gillus nidulans grown on media containing the chemicals  (429).  This
effect, probably caused by nondisjunction events, is detected  by the
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appearance of sectors with white or yellow conidia from a treated het-
erozygous green strain.  Benzimidazole, which is mutagenic to other
organisms, was both nontoxic and noneffective in causing genetic segre-
gation in Aspergillus.  Five derivatives of benzimidazole, MBC, benomyl,
TBZ, DTFB, and TTFB were toxic at low concentrations.

The two carbonate derivatives (MBC and benomyl) increased the frequency
of sectoring; the two fluoromethyl derivatives (DTFB and TTFB) did not.
Thiophanate and two derivatives were also very active genetically.  The
authors state that the genetic activity may be the cause of the toxic-
ity, and that the widespread use of these fungicides may introduce a
genetic hazard.

Another nondisjunction detection system (430) was used to assay 110
pharmaceutical specialties.  A strain of Aspergillus containing reces-
sive genes for resistance to an antimetabolite yields, after crossing
over or nondisjunction, conidia which will grow on media containing the
compound.  The two types of events can be distinguished from the colony
color and other physiological properties.   Using agar diffusion plate
assays, the workers detected four groups of compounds which showed
"mutagenic" effects.  Quinolines, sulfa drugs, benzodiazepines, and
pyrazolidines contained active compound members.

Fungi are amenable to many different mutational tests, covering changes
in quality, quantity and arrangement of genetic material, and as such
may have more relevance to man than the bacterial test systems.
PROTOZOA

Free-living protozoa have been in use as "test animals" since the early
1900's.  In 1913, Woodruff and Underbill initiated the use of the term
"biological indicators" to describe this use.  More recently, Hutner et
al.  (431) have urged the use of particle-ingesting protozoa in cytotoxic
chemical screens because of their exceptional ability to utilize high-
molecular weight and fat-soluble materials.  The use of protozoans as
test organisms in toxicity studies would appeear to bridge the gap
between undifferentiated prokaryotic organisms such as bacteria, and the
more complex metazoa.  Protozoa possess highly developed and specialized
organelles for motility and reproduction.  They lend themselves to many
kinds of biological research since protozoan cell structure, metabolic
functions, and nutritional requirements are similar to that of mammalian
cells in many instances.  Some correlations have been made in terms of
response to agents, and protozoa have been effectively utilized in
testing the toxicity of various substances.

Tetrahymena pyriformis has been the object of most of these studies in
toxicity evaluations.  Observations have been made of this organism, and
others, in terms of growth and survival, metabolism, gross and ultra
morphology, and ciliastic action and movement.  The effects of chemicals
on the growth of protozoa is measured in terms of change in culture
turbidity, production of inhibition zones, and change in direct cell
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counts.  Death of the cells is ascertained by dilution subculturing,
staining characteristics and cessation of ciliary beat.  Studies of the
effects of chemicals on growth and survival are reviewed first.

In a study (432) on hypocholesteremic agents, protozoa, both sterol
synthesizing and nonsynthesizing, were used to compare inhibition of
growth with the inhibition of cholesterol synthesis as seen in mammals.
Ochromonas, Euglena, and Tetrahymena were inhibited by triparonal and
benzmalecene, and the authors feel the organisms may be useful in screen-
ing for such compounds.

Growth inhibition of Ochromonas sp. (433) was used to study the toxicity
of thalidomide and its breakdown products.  It was concluded that the
mechanism of toxicity of these compounds may be through interference of
cellular oxidation.  The authors state that "a protozoan test system is
useful for studying the potential 'side actions' of drugs in higher
animals . . . with some drugs they mimic man and other animals in their
response."  However, this interpretation cannot be applied indiscrim-
inately.  It was found, for example, that cycloheximid'e has no toxic
effect on Amoeba, even at a concentration of.3 mg/ml  (434).  This chem-
ical has been found to affect SOS ribosomes.  Amoeba have 70S ribosomes.

Tetrahymena pyriformis was used to assess the toxicity of mycotoxins.  A
60% inhibition of growth was produced by Rubratoxin B at 50 yg/ml.
Aflatoxin B and Ochratoxin (435) were not as effective in inhibiting
growth as was Rubratoxin and it was found that these  mycotoxins had
little effect on cell respiration.

A group of purine antagonists was tested for the inhibition of growth of
Tetrahymena (436).  Those compounds which were inhibitory were found to
affect acetate metabolism and sterol synthesis.  Some of the analogs
(protopine, phenylbutyric acid) cause "suicide" of the cells.  This
phenomenon does not fit the pattern of the other inhibitors.  Another
antimetagolite, 5,6-dimethylbenzimidazole, caused growth inhibition in
Endamoeba histolytica  (437), when the cells were treated with the com-
pound in the media.

In a series of papers on the use of microorganisms (endpoint = growth
inhibition) in antitumor agent screens (410, 412), it was reported that,
although mammalian cell cultures appear to be superior to microbiologi-
cal systems (fewer false positives), Tetrahymena pyriformis picked up
82% of the-positive compounds.  Poley ejb al^. felt that the compounds
missed by the protozoan might require physiological alteration by the•
host for activity.

Johnston e_t al. (438) have published data on the use of several species
of protozoa in a chemotherapeutic agent screen.  Euglena, Ochromonas,
and Tetrahymeria were compared to mammalian cell cultures.  The authors
consider these to be morphologically homologous.  Autobiographic and
turbidimetric measurements were used to measure toxic effects.  In agar
systems, the protozoa detected more known positives than did the mam-
malian cells.  In an assay conducted by Price et. ail.   (439), however,
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HeLa cells were more efficient and more selective in pinpointing anti-
tumor compounds than were the protozoans.  A close relationship was
found although, in degree of inhibition, between an agent's HeLa cyto-
toxicity and its antiprotozoan activity.

Jacob (440) has stated that the growth of Tetrahymena in pure culture
may be compared to the growth of somatic tissue cells.  She tested a
group of aminoazobenzene dyes and metabolic inhibitors against T_.
pyriformis for growth inhibition.

Another use for Tetrahymena as "test animals" has been in connection
with antimalarials.  Clancey describes the lethal and growth inhibitory
properties of five different compounds  (441).

Some investigators have focused on the structural changes effected by
toxicants.  In these studies, the treated cells are observed directly
for toxic effects.  In addition to changes in cell and organelle struc-
ture, observations on ciliary beat and motility of the cells have been
used to assess chemical toxicity.

When 4-nitroquinoline 1-oxide and 4-aminoquinoline 1-oxide were tested
against Tetrahymena, it was found that the effect of the active compound
(4NQO) was on protein synthesis in the cytoplasm, resulting from the
loss of ribosomes (442).  In an earlier study by the same group, it was
found that of a gfoup of 4NQO derivatives, the proven carcinogenic
substances produced anomalies in cell division, while those which were
noncarcinogenic did not.

Ethidium bromide, an antitryposomal compound, was assessed for its
effect on growth and production of cell changes in protozoa.  In Tetra-
hymena  (443), the drug produced changes in mitochondrial morphology and
appeared to arrest cell division via reversible effect on. DNA synthesis.
Both DNA and RNA replication were inhibited after treatment in Trypano-
soma mega and Crithidia luciliae, resulting in growth inhibition (444).

In a study with benzopyrene and other polynuclear aromatic hydrocarbons
in combination with ultraviolet irradiation (445), it was noted that a
correlation exists between the extent of photodynamic toxicity evidenced
by morphological changes and loss of motility in protozoa and carcino-
genicity in mammals.  The effects of metallic ions (Cu, Zn, and Ph) were
evaluated with Paramecium (446).  The organisms were so sensitive to a
plasma membrane injury caused by these ions that the author recommends
their use as indicators of low-level toxicity due to such ions in solu-
tions.

Metrazol, a pharmaceutical, was .used to treat Amoeba proteus cells which
were suspended in a chamber slide in media containing the drug (447).
Changes in locomotion and in membrane potential were observed, and it
was concluded that these changes were due to disruptions in potassium
transport.

The effect of some carcinogens, including aminofluorenes and naphthyl-
amines,  on the growth, respiration, and enzyme activity in Tetrahymena
was studied (448).  The data obtained were ambiguous;  different results

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were obtained in synthetic than in natural media.  Carcinogenic myco-
toxins were used to treat Tetrahymena pyriformis (435).   The three
compounds tested were weakly toxic to the cells, and only a marginal
effect on their respiration was noted.

The widest use of protozoans in toxicity testing has been in assaying
cigarette smoke.  These studies have employed observations on ciliary
beat of the cells and morphological changes.  Wang has suggested that
ciliated protozoa may be compared with human respiratory epithelium, in
their response to the effects of smoke.  In this type of study, the
cells are exposed to residue in broth media or to gaseous phase com-
ponent in perfusion chambers.  In tests using nontobacco cigarettes,
Kennedy and Elliott (449) reported that the collected residues produced
a reduction in motility and ciliary beat, reduction in 0_ consumption
and structural degradation of the mitochondria.  In an earlier study
(450), these effects were shown when the cells were exposed to tobacco
smoke.  The cilia toxic effects of tobacco smoke have been" demonstrated
in cells from a number of different phyla.  In a series of studies by
Weiss (451, 452), similar work was conducted with Paramecium aurelium.
Observations were made of motility and cellular disintegration.  It was
found that the gas phase contains the major portion of the toxic sub-
stances.

It is evident that the use of protozoa in toxicity screens should be
increased, and efforts made to utilize their unique properties.  Some
valuable information has already been gained and new assays can be built
upon those reported here.
PLANTS
The use of plant material to assay chemicals for toxicity with regard to
effects in mammalian systems is not well established.  Differences in
uptake mechanisms, permeability, repair systems, structure, and the
unique CO  fixation of plants preclude direct correlation.  Even so,
with the continuing development of undifferentiated plant cell cultures
utilizing defined synthetic media, it is possible to pinpoint the toxic
effects of chemicals on metabolic pathways common to plants and animals.

Growth Inhibition;

Plant tissues have been used to study the toxic and physiological effects
of herbicides.  The in vitro plant systems which can be employed include
whole plants, plant segments, isolated leaf cells, and cell cultures.
Whole plants may be grown in a solution or emulsion of the chemical and
the rate of growth through fresh weight measurements and/or survival
studied.  Rice plants were tested in this manner against propanil in
combination with parathion or paraoxon (453).  It was found that para-
thion and paraoxon block the hydrolysis of propanil by the plants.
Similar protective mechanisms occur in other systems.

To study the effect of compounds on the growth and survival of plant
cells in tissue culture, small plugs are cut from the cotyledons and

                                -96-

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placed on agar nutrient media or in a liquid nutrient media.  In two
studies  (454, 455), using Glycine max (soybean) and Populus deltoides
(cottonwood) in tissue culture, picloram, 2,4,5-T and dicamba were
assayed for their comparative toxic effects.  Growth of plant cells in
tissue culture on synthetic media makes it possible to isolate specific
enzymes, metabolic byproducts, total protein, etc., for study of the
physiological effects of chemicals (see H.E. Street, "Plant Cell Cul-
tures, Their Potential for Metabolic Studies," in Biosynthesis and Its
Control in Plants, B. V. Milborrow, ed.).

Tissue levels of nitrate and nitrate reductase in the detached leaves of
Hordeum vulgare L, after exposure to DCMU (l,l-dimethyl-3-(3,4-dichloro-
phenyl)-urea) and the triazines, Simazine and Atrazine, were studied by
Aslam and Huffaker (456).  These chemicals are known inhibitors of
photosynthesis (suppressing the fixation of C0_) and the effect on
nitrate reduction is a result of this inhibition.

Effects On Chromosomal Material;

In contrast to the preceding studies which seem to have little relation-
ship with nonphotosynthetic systems, the use of plant cells to study the
effects of chemicals on chromosomal material seems more pertinent.

B. K. Vig (457, 458)  has developed a method for assessing the occurrence
of chromosomal disturbances, including somatic crossing over, in soybean
(Glycine max).  The seeds are soaked in a solution of the compound under
study, allowed to germinate, and the compound and first simple leaves
ex,amined for "spots."  Ethylmethane sulfonate, daunomycin, and a group
of DNA synthesis inhibitors (caffeine, actinomycin, puromycin, cytosine
arabinoside, FDU) were tested for their effects in this system.  Through
comparison of their effects, Vig concluded that a specific event in DNA
repair is responsible for the complementary exchanges picked up in the
soybean system.

The excellent visualization of chromosomal material in plants make them
ideal for mutagenesis studies, and some correlations with human cells
can be made.  B. S. Kilman (459)  reviews the-use of root tips to study
the effects of chemicals on chromosomes and asserts that they should be
regarded as the ideal plant tissue for studying such effects due to ease
of handling, presence of a large number of dividing cells and potential-
ity for direct exposure.  A good correlation between the chromosome
breaking activity of chemicals in plant and in animal cells appears to
exist, although the type of effect and concentration necessary to pro-
duce that effect may be very different.   Chromosomal damages in studies
of this type are determined through microscopic examination of stained
chromosomal preparations of colchicine-treated or untreated plant cells
in metaphase or anaphase.

In a study by Sturelid (460)  the chromosome breakage caused by TEPA (an
aziridine derivative) and its analogues in Vicia faba and Chinese hamster
cells was compared.  The plant cell material for chromosomal analyses
was obtained from the lateral root tips of 9 day old seedlings which had
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been soaked in a solution of the compound for two hours.  The hamster
cells were grown and treated as monolayer cultures.  They found that the
mammalian cells were 23 times more sensitive than the plant cells to the
effect of TEPA, although this difference could have been due to the
differences in treatment temperature.  Although the types of aberrations
seen in the two preperations differed and was influenced by temperature,
both arose from lesions produced in the chromosomes followed by a period
of DNA synthesis with conversion to visible chromatid aberrations.
These alkylating agents induce mutations and/or chromosomal aberrations
in Drosophila, Neurospora, Allium, and in mouse and human cultured
cells.

In another study by Sturelid (461) on the differential reaction of
Allium root tips and Chinese hamster cells to treatment with caffeine,
3-ethoxycaffeine (EOC) and 6-methylcoumarin  (6-MC), it was found that,
again, temperature influenced the type and degree of damage.  At 17 C
both plant and animal cells contained subchromatid and chromatid ex-
changes,, although the animal cells suffered a greater degree of this
damage.  With 37 C treatment, chromatid fragmentation was produced in
the animal cells.  The effects of caffeine as a potentiator for damage
by other agents was studied in Vicia faba root tips (284).  It was found
that caffeine inhibits the DNA repair gap filling process, and therefore
increases chromosomal aberration frequencies.
                                                                  v

Phleomycin, a compound which blocks replication of HeLa cells, was
tested for its effect in cultured lily cells (462).  This compound was
found to affect Allium similarly, blocking the progression of the chro-
mosomes from one organizational form to another.  A study (463) using
onion root tip cells  (Allium cepa), treated with reserpine phosphate,
showed that the effect of this alkaloid was mitotic arrest in late
prophase.  The compound was toxic at 4 mg% to the germinating bulbs.

Algae Cells;

In addition to the use of plant tissue for toxicity testing, some work
has been done utilizing eucaryotic algae to study effects on cell growth.
Chlorella vulgaris has been used to assay the activity of soil herbicides
(464).  .01 ppm of diuron and 0.5 ppm of monuron caused 50% growth
inhibition.  Chlorella pyrenoidosa was treated with a group of quinone
derivatives in broth culture (465).  Observations were made of effects
on cell counts, chlorophyl concentration, oxygen evolution, and viabil-
ity.  A group of unicellular algae were assayed for the effects of
polychlorinated biphenyls (466).  The growth of diatoms was inhibited
more by PCB's than by DDT.  Two fresh water and one salt water algae
were found to be resistant to PCB's and DDT.

In attempting to use plant tissue for study of toxicity of chemicals and
to apply the results to mammalian tissues, the inherent biochemical and
structural differences must be considered.  Temperature, pH, oxygen,
tension and age of the material are also factors which greatly influence
the effects produced.  Obviously, more correlative data must be accumu-
lated before the feasibility of using these systems is decided.
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DISCUSSION

The use of nonmammalian cell systems for toxicity testing is now well
established.  The use of protozoa for studies of toxicity of smoke
elements, of bacteria in cancer chemotherapeutic screens, and of Sac-
charomyces and Salmonella in mutagen screens is well documented and
standardized.  Many investigators feel that the data obtained from use
of these microorganisms can be applied to what occurs in mammals.
                                               6
The advantages of using single cell toxicity test systems are many.  Of
most importance is time.  Toxic chemical assessment in whole animals is
a lengthy procedure, whereas the cellular systems yield results in 24-48
hours.  Large numbers of individuals can be screened concurrently, and
several different toxicity indicators observed in a single experiment.
Single cell systems can be tailored to give a higher probability of
toxicity detection; for example, nutritionally deficient strains are
used to examine specific antimetabolities.  The cost of running assays
is greatly reduced from that in whole animals.  Most of these tests can
be performed by laboratory technicians with little prior training.

The main obstacle to extrapolation of nonmammalian cell system screen
data to mammals lies in the lack of knowledge of what actually occurs in
the whole animal.  Uptake, transportation, detoxification, metabolic
alteration,'potentiation, and elimination of the compound are processes
which occur only in the whole animal and can profoundly alter the effect
of a chemical on the ultimate target cell or system.  Provisions must be
made for these events when using simplified systems; i.e., the use of
metabolic activators and of reactive compound derivatives in the mutagen
screens.

Further work on protozoan test systems would be advantageous.  These
organisms are more closely related to mammalian systems than bacteria,
have complex structural and metabolic properties, and yet are easy to
manipulate.  The development of bacterial general toxicity screens would
be useful since their metabolism has been so well studied.  Further
refinement of the mutagen screen system is in progress.

It is obvious that all of the enormous number of new chemicals entering
the human environment, in addition to the untested ones already with us,
cannot be tested in mammals.  The resources for such a task, in time, in
personnel, or in money, simply do not exist.  The work presented here
shows that usable information about chemicals can be obtained from
bacterial, fungal, plant cell, and protozoan test systems.
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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
1. REPORT NO.

  EPA  560/5-75-007
                              2.
                                                            3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
  Draft Survey and  Evaluation of In Vitro  Toxicity Test
  Methods
                                   5. REPORT DATE
                                      August    1976
                                   6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
                                                            8. PERFORMING ORGANIZATION REPORT NO.
           Geoffrey  Woodard
9. PERFORMING ORGANIZATION NAME AND ADDRESS
  Woodard Research  Corporation
  12310 Pi nearest Road
  Herndon, Virginia   22070
                                   10. PROGRAM ELEMENT NO.

                                      2LA328
                                   11. CONTRACT/GRANT NO.

                                    68-01-1895
12. SPONSORING AGENCY NAME AND ADDRESS
  Office of Toxic  Substances (WH-557)
  Environmental Protection Agency
  401  M Street, S.W.
  Washington, DC   20460
                                   13. TYPE OF REPORT AND PERIOD COVERED
                                        Final
                                   14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
  The English language  literature for the period  1954 to'May 1974 has  been searched.  A
  computer title search,  contacts with scientists  currently engaged  in related research,
  and bibliographic references contained in individual  papers were pursued.   Copies
  of articles were obtained  and reviewed under  the following groupings:   1)  Use of
  Fertilized Eggs in Studies on Chemicals, 2) Use  of Isolated Organs and  Tissue in
  Studies on Chemicals,  3) Use of Mammalian and Avian Cell Culture in  Studies on
  Chemicals, 4) Use of  Bacteria, Fungi, Protozoa,  and Plant Cells in Studies on
  Chemicals.  An attempt  has been made to include  all  systems within these headings.
  Where such information  was available, the applicability of those in  vitro  test
  systems has been evaluated.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                      b.lDENTIFIERS/OPEN ENDED TERMS  C. COSATI Field/Group
  In  Vitro Methods
  Toxicity Test Methods
  Toxicity
  Evaluation - In Vitro
  Fertilized Eggs
  Isolated Organ Tis.sue
  Mutagens
Test

Cell Culture
18. DISTRIBUTION STATEMENT
   Unlimited
                                              19. SECURITY CLASS (ThisReport/
                                                 Unclassified
                                                 21. NO. OF PAGES
                                                    110
                                              20. SECURITY CLASS (Thispage)
                                                 Unclassified
                                                                         22. PRICE
EPA Form 2220-1 (9-73)

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 !          1.   REPORT NUMBER
 .               Insert the EPA report number as it appears on the cover of the publication;

 ;          2.   LEAVE BLANK

••'          3.   RECIPIENTS ACCESSION NUMBER
'!',      .        Reserved for use by each report recipient.

           4.   TITLE AND SUBTITLE
 I               Title should indicate clearly and briefly the subject coverage of the report, and be displayed prominently.  Set subtitle, if used, in smaller
                type or otherwise subordinate it to main title. When a report is prepared in more than one volume, repeat the primary title, add volume
 ,               number and include subtitle for the specific title.

•:]          5.   REPORT DATE
•'               Each report shall carry a date indicating at least month and year. Indicate the basis on which it was selected (e.g., date of issue, date of
 :              approval, date of preparation, etc.).

           6.   PERFORMING ORGANIZATION CODE
•!               Leave blank.

           7.   AUTHOR(S)
'••]          .     Give name(s) in conventional order (John R. Doe, J. Robert Doe, etc.). List author's affiliation if it differs from the performing organi-
 i               zation.

 !          8.   PERFORMING ORGANIZATION REPORT NUMBER
 •               Insert if performing organization wishes to assign this number.

'          9.   PERFORMING ORGANIZATION NAME AND ADDRESS
 i               Give name, street, city, state, and ZIP code. List no more than two levels of an organizational hirearchy.

 !          10.  PROGRAM ELEMENT NUMBER
 ,               Use the program element number under which the report was prepared. Subordinate numbers may be included in parentheses.

 '•          11.  CONTRACT/GRANT NUMBER                   o                               •
                Insert contract or grant number under which report was prepared.

 •          12.  SPONSORING AGENCY NAME AND ADDRESS
                Include ZIP code.

           13.  TYPE OF REPORT AND PERIOD COVERED
.;               Indicate interim final, etc., and if applicable, dates covered.

;          14.  SPONSORING AGENCY CODE
,               Leave blank.

           15. SUPPLEMENTARY NOTES
                Enter information not included elsewhere but useful, such as: Prepared in cooperation with, Translation of, Presented at conference of,
                To be published in, Supersedes, Supplements, etc.

.-!          16. ABSTRACT
I               Include a brief (200 words or less) factual summary of the most significant information contained in the report. If the report contains a
•               significant bibliography or literature survey, mention it here.

i          17.  KEY WORDS AND DOCUMENT ANALYSIS
•               (a) DESCRIPTORS - Select from the Thesaurus of Engineering and Scientific Terms the proper authorized terms that identify the major *
'.              concept of the research and are sufficiently specific and precise to be used as index entries for cataloging.

'              (b) IDENTIFIERS AND OPEN-ENDED TERMS - Use identifiers for project names, code names, equipment designators, etc. Use open-
•               ended terms written in descriptor form for those subjects for which no descriptor exists.

"i               (c) COSATI FIELD GROUP - Field and group assignments are to be taken from the 1965 COSATI  Subject Category List. Since the ma-
               jority of documents are multidisciplinary in nature, the Primary Field/Group assignment(s) will be specific discipline, area of human
i               endeavor, or type of physical object.  The application(s) will be cross-referenced with secondary Field/Group assignments that will follow
]               the primary posting(s).
i
!           18. DISTRIBUTION STATEMENT
{               Denote releasability to the public or limitation for reasons other than security for example "Release Unlimited." Cite any availability to
j               the public, with address and price. /

           19.&20.  SECURITY CLASSIFICATION
               DO NOT submit classified reports to the National Technical Information service.                                           '  *

j           21. NUMBER OF PAGES
1               Insert the total number of pages, including this one and unnumbered pages, but exclude distribution list, if any.

1           22. PRICE
:               Insert the price set by the National Technical Information Service or the Government Printing Office, if known.
       EPA Form 2220-1 (9-73) (Reverse)

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