DISTRIBUTION AND HISTOPATHOLOGICAI.
EFFECTS OF CIS-PLATINUMIII)
DIAMMINODICHLORIDE ON NON-TUMORED
AND TUMORED (SARCOM'A 180)
SWISS WHITE MICE

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
JEAN TOTH-ALLEN
1970

 

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This is to certify that the -
thesis entitled . '
DISTRIBUTION AND HISTOPATHOLOGICAL EFFECTS OF

CIS~PLATINUM(II)DIAMWINODICHLORIDE 0N
NON~TUMORED AND TUMORED (SARCOMA 180) SWISS WHITE MICE

presented by

Jean Toth—Allen

has been accepted towards fulfillment
of the requirements for . -

Ph.D. degree in BIORhYSiCS

SWRMM

Major professor

 

 

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ABSTRACT
DISTRIBUTION AND HISTOPATHOLOGICAL EFFECTS OF
CIS-PLATINUM(II)DIAMMINODICHLORIDE ON
NON-TUMORED AND TUMORED (SARCOMA 180) SWISS WHITE MICE
by
Jean Toth-Allen

Cis-platinum(II)diamminodichloride (cis-Pt(II)(NH 12) has been

3)2C
shown to be an effective cancer chemotherapeutic agent in laboratory
animals. As a first step toward understanding the metabolism and
mode of action of this compound, studies were undertaken to determine
the relative lifetime of platinum in the Swiss white mouse (both non-
tumored and tumored), as well as its distribution, with time, in the
major organs. Neutron activation analysis---with gamma-ray Spectro-
metry---was the analytical technique employed for this purpose. Three
animals were sacrificed at l, 6, 12 and 24 hours after injection of a
therapeutic dose (8 mg/kg), and then every 12 hours for 144 hours.
The major organs were removed and placed in pre-weighed polyethylene
irradiation vials, which were then weighed and heat-sealed. (Blood
samples were likewise collected, by heart puncture from 3 animals per
time, at various intervals for 24 hours.) A fourth animal was simul-
taneously sacrificed at each time interval, for histopathological studies,
and its major organs or representative sections thereof immediately
placed in buffered formalin for fixation.

The samples for platinum determination were irradiated for 8 hours

in a Triga Mark I reactor at a flux of approximately 2 x 1012

neutrons/cmZ/second. 199Pt, the radioactive isotope most abundantly

formed, has a relatively short half-life (31 minutes) and its charac-

teristic‘Y-rays are low energy. In a biological matrix, even

Jean Toth—Allen
qualitative detection of such an isotope is practically impossible.
However, 199Pt decays to another radionuclide, 199Au, whose half-life
is 3.15 days. Its characteristic Y-rays are, likewise, of low energy,
and high concentrations of 24Na tend to interfere with accurate
detection. Thus, after a lapse of approximately 60 hours after
irradiation—--for efficient formation of 199Au and radioactive decay of
matrix elements--a radiochemical separation was undertaken. After
complete digestion of the samples in a 1:1 solution of HNO3 and
H2804, the solutions were brought to volume with 6N HCl and passed over
a Dowex 1-X8 anion-exchange column. The hexachloro salt of the gold
remained bound to the surface of the resin, while most interfering
ions passed through. After a suitable wash with 6N HCl, the column
was placed in a 3" by 3" well-type NaI(T1) crystal and the area under
the 158keV photOpeak of 199Au determined and compared with that of a
control. Results were expressed as ng Pt/gm tissue and pg Pt/cc blood.

The samples for histopathological study were dehydrated, cleared,
infiltrated and embedded according to the paraffin method. 6H
sections were cut and a simple hematoxylin—eosin stain used for all
sections. The slides were then studied for evidence of pathological
alterations and their time course.

The distribution data revealed a low recovery of platinum at all
times measured. (Although the entire animal was not assayed for
platinum content, the level is still suspiciously low.) This and the
relatively high amounts consistently present in filtering (liver and
spleen) and excretory (kidney and intestines) organs indicates a
possible rapid excretion of large quantities, primarily via the urine.

Although a therapeutic dose was employed, the amount of platinum

present in the tumor is relatively low. This eliminates the possibility

Jean Toth—Allen

that the potent anti-tumor properties result from specific uptake into
neoplastic tissue. However, platinum is present as early as 1 hour
following treatment, and a greater sensitivity of neoplastic, as
compared to normal, tissue is a plausible alternative.

The total absence of platinum from brain samples suggests that the
original neutral molecule is rapidly metabolized to form a charged
molecule, incapable of passing the blood-brain barrier. The extreme
reactivity of the compound, as a result of the lability of its chloride
ions, supports such a suggestion.

The fall and subsequent rise in the amount of platinum present in
the major organs, during the first 24 hours following treatment,
suggests a temporary storage elsewhere in the body. Blood studies
revealed a similar pattern, ruling out combination with and retention
in the blood elements. However, peripheral shunting, as a temporary
edema, or merely temporary distribution into muscle, etc., are possible,
and present data are insufficient to resolve the problem.

Histopathological studies revealed little if any relationship
between platinum concentration and amount of damage. Although large
amounts of platinum are consistently found in liver and kidney tissue,
minimal damage is evidenced. Most pathological alterations are seen
in rapidly proliferating tissue---gastrointestinal mucosa and lymphoid
elements of thymus and Spleen. Thus, as with most cancer chemother-
apeutic drugs and X—radiation, tissues with high mitotic activity are
primary targets. However, the general damage resulting from platinum
---at least as evidenced in mice and rats--—is considerably less than

that found for other such agents.

DISTRIBUTION AND HISTOPATHOIDGICAL EFFECTS OF
CIS-PLATINUM(II)DIAMMINODICHLORIDE ON
NON-TUMORED AND TUMORED (SARCOMA 180) SWISS WHITE MICE

by

Jean Toth-Allen

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biophysics

1970

a... — w- «J
/" 1.1;! F '//

Copyright by
Jean Toth-Allen
1970

To my husband

11

ACKNOWLEDGEMENT

The author wishes to acknowledge Dr. Barnett Rosenberg, as her
major professor, and to thank Dr. Bruce Wilkinson for his untiring aid,
John Jones (University of Michigan) for invaluable advice, Dr. Richard
Kociba for the pathological analysis, Mae Sunderland for histological
advice, Loretta Van Camp for varied assistance, and the numerous other
people who so often lent a hand. This investigation was supported by

PHS Training Grant No. GM-01422 from NIH and Grant No. CA-11349 from

the National Cancer Institute.

iii

TABLE OF CONTENTS

INTRODUCTION. . . . . . . . . . .

EXPERIMENTAL METHODS. . . . . . .

General Procedures . . . . .
LDSO Studies . . . . . . . .
Weight Studies . . . . . . .
Neutron Activation Analysi .
Histopathology . . . . . . .

RESUIJTS l O O O O O O O O O O O

LDsoTest..........
Gross Changes Resulting from
Neutron Activation Analysis.
Histopathology . .

DISCUSSION. . . . . . . . . . . .

Platinum Distribution.
Histopathology . . . . .
General Considerations . .

LIST OF REFERENCES. . . . . . . .

APPENDIX. . . . . . . . . .

Neutron Activation Analysis.

Page

0 O O O O O 1

O O 16

O O O O O O O O 16
O O O O 0 O O O O O O O O O 17
O O O O O O O O O O O O 18

O O O O O O O O O O O O 33

O O O O O 33
Treatment 0 O O O O O O O 33
O O O O 39
O 0 O O O O O 55
O O O O O O O O 73
O O O O O O O 73

. 87

O O O O 0 O O O O 97
O O O O O O O O O O O O 99

. . . . . . . . . . . . . . . . 104

. . . . . . . . . . . . . . . . 104

iv

Table

10.

ll.

12.

13.

14.

LIST OF TABLES

Page
Results with various platinum compounds. . . . . . . . . . . 12
Weights (in grams) of various tissues which show changes
following injection with 8 mg/kg of cis-Pt(II)(NH3)2C12.
(Three tissues pooled per measurement.). . . . . . . . . . . 37
Platinum distribution (D8 Pt/gm tissue) in the major
organs of the tumored animals. . . . . . . . . . . . . . . 42
Platinum distribution (”8 Pt/gm tissue) in the major
organs of non-tumored animals. . . . . . . . . . . . . . . 43
Amoung of platinum (pg Pt/cc blood) measured in the
blood samples from both tumored and non-tumored animals... 53
Comparative representation of pertinent
cis-Pt(II)(NH3)2C12 distribution data. . . . . . . . . . . 79
Distribution of triethylenephOSphoramide-BZP (TEPA),
a potent alkylating agent, in non-tumored and
several tumored animal systems . . . . . . . . . . . . . . 80
Distribution data following an intraperitoneal
injection of S-fluorouracil-2114C into mice
containing 10 day growths of Sarcoma 180 . . . . . . . . . 82
Distribution of I.V.-injected amethopterin in
non-tmored Akin mice a o a o o o o o o o o o o o o o o o o 83
Distribution of the tritiated methyl ester of
streptonigrin, an antibiotic, in mice bearing
sarcoma 180. O O O C O O O . O C C C O O Q C O O O O O O O 85
Distribution of radiomanganese in normal mouse
tissue 24 hours after I.V. injection . . . . . . . . . . . 86
Mitotic activity of body tissue. (After Bond, et.a1.46) . 89
Summary of the early lesions of acute radiation
illness in various organs of mice in order of
increasing radioresistance . . . . . . . . . . . . . . . . 94
Elements of biological media activated by neutron
bombardment./. ._. . . . . . . . . . . . . . . . . . . . . 116

V

Figure

10.

11.

12.
l3.
14.
15.
16.
17.

18a.

LIST OF FIGURES

Page
Schematic representation of the proposed stages
of drug effectiveness. (After Connors6). . . . . . . . . 4
Cut-away view of the Triga Mark I reactor . . . . . . . . 21

View of the lazy susan-type sample holder of
the Triga Mark I. O O O O C O C O O O O O I O O O O O O I 22

Staining series employed for all slide preparations . . . 31
Results of LD50 study . . . . . . . . . . . . . . . . . . 34
Average daily weights of various groups of mice . . . . . 35
Y-ray spectrum of 199Au . . . . . . . . . . . . . . . . . 41

Platinum recovery in both non-tumored and tumored
animal tissues. I O O O O O O O O O O O O 0 O O O O O O O 44

Platinum distribution data for liver + gall
bladder smples O O O O O O O O O O O O O O O O O O O O O 45

Platinum distribution data for kidney + bladder
samples 0 O O O O C O O O O O O O O O O O O O O O O o O O 46

Platinum distribution data for intestines (left) and

stomach + esophagus (right) samples . . . . . . . . . . . 47
Platinum distribution data for spleen samples . . . . . . 48
Platinum distribution data for lung samples . . . . . . . 49
Platinum distribution data for endocrine samples. . . . . 50
Platinum distribution data for heart samples. . . . . . . 51
Platinum distribution data for tumor samples. . . . . . . 52

Platinum distribution in the blood. . . . . . . . . . . . 54

Villi of control small intestine section.
Magnification - 60x . . . . . . . . . . . . . . . . . . . 57

vi

Figure

18b.

19.

20.

21.

223.

22b.

23.

24.

25.

26.

27.

28a.

28b.

29.

30.

31.

32a.

32b.

Page

Crypt region of control small intestine section.
Magnification . 240XI I I I I I I I I I I I I I I I I I I 57

Crypt region of treated small intestines. Tumored
animal, 48 hours after treatment. Magnification = 240x . 58

Squared villi of treated small intestines. NOn-

tumored animal, 12 hours after treatment.

Magnification = 60x I I I I I I I I I I I I I I I I I I I 59
Sloughing villi of treated small intestines.

Tumored animal, 12 hours after treatment.

Magnification = 60x . . . . . . . . . . . . . . . . . . . 59
Control section of stomach. Magnification = 60x. . . . . 60

Section of treated stomach. Non-tumored animal,
12 hours after treatment. Magnification = 60x. . . . . . 60

Control section of thymus. Magnification = 24x . . . . . 61

Section of treated thymus. Non-tumored animal,
36 hours after treatment. Magnification = 24x. . . . . . 61

Apparent reversal of thymus regions. Tumored animal,
108 hours after treatment. Magnification = 24x . . . . . 62

Control Spleen. Magnification = 60x. . . . . . . . . . . 64

Control spleen of tumored animal. Magnification = 240x.
(Arrows indicate neoplastic cells.) . . . . . . . . . . . 64

Section of treated spleen. Non-tumored animal, 12 hours
after treatment. Magnification = 60x . . . . . . . . . . 65

Section of treated spleen. Tumored animal, 84 hours
after treatment. Magnification = 120x. . . . . . . . . . 65

Control section of kidney. Magnification 60x . . . . . 68
Capillary congestion in medullary region of kidney.

Non-tumored animal, 24 hours after treatment.

Magnification = 60x . . . . . . . . . . . . . . . . . . . 68

Tubular Sloughing in treated section of kidney. Non—
tumored animal, 48 hours after treatment. Magnification
= 120x. (Arrows indicate slouthing tubular epithelium.). 69

Control section of tumor. Magnification 60x. . . . . . 70

Control section of tumor. Magnification 120x . . . . . 70

vii

Figure

33.

34.

35a.

35b.

Page

Section of tumor, 48 hours after treatment.
Magnification a IZOXI I I I I I I I I I I I I I I I I I I 71

Section of tumor, 72 hours after treatment.
Magnification = 120x. . . . . . . . . . . . . . . . . . . 71

Section of tumor, 120 hours after treatment, showing
formation of a capsule. Magnification = 60x. . . . . . . 72

Section of tumor, 120 hours after treatment.
Magnification a lZOXI I I I I I I I I I I I I I I I I I I 72

viii

INTRODUCTION

Cancer may be defined as a ”disease of multicellular organisms
which is characterized by the seemingly uncontrolled multiplication
and spread within the organism of apparently abnormal forms of the
organism's own cells."1 As such, it differs from most diseases
afflicting man in that the obvious stages of the disease are
represented by invasion by his own (albeit abnormally transformed)
cells, rather than those of a foreign organism. It represents an
infringement on the normal homeostasis of the body and may arise from
the breakdown of a normal control mechanism or the loss of response to
such control in a target tissue or cell. The causative agent--virus,
chemical or physical carcinogen, or some as yet unknown agent~~~may
produce an alteration in the affected cells which directly or
indirectly results in a malignant transformation. Cytological studies
of the chromosome constitution of various tumors revealed that
abnormalities, both in number and structure, are a common occurrence
However, no characteristic and exclusive chromosome pattern emerges
(except for chronic myeloid leukemia) and, thus, it is proposed that
such abnormalities, in general, represent a predisposition of the cell
to malignancy, rather than a cause thereof.2 Theories concerning
deletion of an essential cellular activity, quantitative or qualitative
alterations in DNA or RNA, variations in the nature or function of the
electron transport system, changes in hormone levels or interrelation-

ships, alteration of the normal electrolyte balance and/or of an ion
1

2
essential to this balance, or changes in membrane properties of the
cells affected have been proposed to explain the cause---or at least
the initial phase——-of malignant growth. Any and all such alterations,
however, may merely represent a symptom, or even a result, of the
disease rather than its cause.

Present knowledge of the cause or causes of the diseases called
cancer is, at best, incomplete. A preventative approach is therefore
presently unfeasible. However, numerous approaches to cures are
possible. Surgery and radiation, alone or in combination, have had
good success in the early stages of a malignancy. However, the general
characteristic of invasiveness--—manifested as neoplastic metastasis---
makes such treatment of little use in later phases of the disease. In
this case, chemotherapy may play a vital role.

The concept of treating cancer by chemotherapy is by no means of
recent vintage. Such endeavors began more than a thousand years ago,
but efforts were sporadic and bore little fruit.3 However, since World
War II there has been a great upsurge in activity in the field of
cancer chemotherapy. The first major breakthrough came with the
discovery in 1946 of the chemotherapeutic activity of an alkylating
agent--—nitrogen mustard (HN-2)---against tumors in mice. This was
followed in 1948 by a similar discovery concerning the action of an
antimetabolite, amethopterin. In the decade that followed, the types
of agents which powerfully alter experimental neoplasms were expanded
to include narcotics (e.g. urethane), antibiotics (e.g. actinomycin D),
mitotic poisons (e.g. colchicine), and several as yet unclassified
agents.4

The success of cancer chemotherapy to date has been variable.

3

Numerous factors can influence the effectiveness of the individual
drugs. Response can vary widely according to route of administration,
as a result of activation or deactivation by various organs. Rate of
excretion of a drug can likewise affect its activity, although in many
cases the potency rather than the selectivity of the drug is most
affected in this way. For certain compounds the dosage schedule
employed is extremely critical in determining whether or not tumor
regression will occur. Moreover, extra-cellular deactivation by
compounds in the sera, and low permeability into neoplastic tissue can
present formidable problems.5 Even for those tumor systems which
initially prove amenable to drug therapy, drug resistance can fairly
readily develop. Despite such complications, various agents or
combinations of such have produced successful remissions, or, at least,
considerable extension of life.

Despite widely differing chemical structures, most cancer
chemotherapeutic agents (with the possible exception of hormones) act
by interfering with some stage of nucleic acid (mainly DNA) or protein
synthesis.6 Figure 1 shows schematically the stages where the various
agents are believed to exert their effects. Of these agents, the various
alkylating agents are perhaps the most widely known and used. These
biological alkylating agents add a compound radical to the molecules
with which they react, rather than merely an alkyl group as the name
implies.3 They are electrophilic reagents which react with nucleo-
philic centers of biological molecules, deactivating these molecules
in most cases. If the target molecules are essential for cell division,
mitosis is inhibited; if not the altered molecules are discarded after

variable delays and cell division proceeds.4 The effects on cells

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range from temporary blockade of division to a cessation of division
accompanied by continued growth (giant cell formation) to irreversible
toxicity.

There are four main types of alkylating agents: nitrogen and
sulfur mustards, ethylene imines, alkyl methane sulfonates and bis
epoxides.7 The most effective agents contain at least two reactive
groups---are bifunctiona1---, and all such agents are relatively
indiscriminate in their attack on cells, reacting with proteins, RNA
and DNA alike. At dose levels just sufficient to cause cell death,
evidence has accumulated that DNA is the most sensitive molecule to
alkylation.6 The exact nature of the interaction of these agents with
DNA is still uncertain but several possibilities exist. Cross-linkage
of the double helix by bifunctional agents, most probably involving
guanine residues, is very possible. Such a linkage would be relatively
stable and result in inhibition of cell division. Another possibility
is a linkage formed between the DNA molecule and its associated protein,
producing an abnormal connection between the two and thus preventing
separation and duplication, or possibly interfering with mitotic
separations of chromosomes.

Bifunctional alkylating agents have a number of characteristics
in common, many or all of which may be shared by other antitumor agents.
they interfere with cell mitosis; produce fragmentation and clumping of
chromosomes; are mutagenic and carcinogenic; usually have profound
effects on blood and bone marrow; damage the intestinal mucosa; inter—
fere with growth; have antifertility and teratogenic properties and

3,7,8

suppress the immune response mechanism. Central nervous system

stimulation with convulsions is also common with most of these agents,

with the exception of the ethylenimmouium.derivatives.9‘ They show
variable effects against numerous tumor systems, and resistance is fairly
readily developed, with cross-resistance among the various agents in

this class common.

Another major class of chemotherapeutic agents contains the anti-
metabolites---basically folic acid antagonists, antipurines and anti-
pyrimidines. These agents have molecular structures similar to those
of the natural metabolites and interfere with the function of the latter.
The folic acid antagonists---main1y aminopterin and amethopterin
(methotrexate)---interfere with the reduction of folic acid to
tetrahydrofolic acid, the active form of this coenzyme. This inhibition
is mediated by the strong, specific binding of these agents with the
active site of the reducing enzyme. In tumor cells, it is believed that
these compounds mainly inhibit thymidine formation, resulting in
inhibition of DNA synthesis.6 Alone and in combination with other drugs
these antagonists have been used against a variety of cancers.
Unfortunately, as with the alkylating agents, resistance is fairly
readily developed. Moreover, these agents result in bone marrow
depression and anemia; ulcerations of oral and gastrointestinal mucosa;
hepatic dysfunction; alopecia; skin rashes; abortion and teratogenic
effects.7

6-Mercaptopurine is probably the most widely used purine antagonist
and 5-fluorouracil (S-FU) or its deoxyriboside (S-FUDR) the most common
antipyrimidine. The ribotide of 6-mercaptopurine is formed intra-
cellularly and appears to act by inhibiting purine synthesis at an
early stage in its biosynthetic pathway by so-called "pseudonegative

feedback".3 (Another well known purine analogue, 6-thioguanine is

believed to cause inhibition by direct incorporation into DNA, thus
resulting in the formation of an abnormal molecule.6) Resistance to
6-mercaptopurine is readily developed and severe bone marrow depression,
gastrointestinal disorders, hepatotoxicity and renal impairment are

fairly common side reactions.7’8

It has been successfully employed in
various cases, however, particularly those involving acute childhood
leukemia or chronic granulocytic leukemia.7

The catabolism and anabolism of uracil and its fluoro derivatives
are extremely similar. Thus, S—FU takes part in a multitude of bio-
chemical reactions in the cell. It is believed to block the methylation
of deoxyuridylic acid to form thymidylic acid, and it is incorporated
as an odd base in RNA.3 It has been found effective against various
carcinomas but, unfortunately, resistance is developed once again.

Among its toxic side—effects are gastrointestinal injury leading to
dehydration and electrolyte imbalance; bone marrow toxicity; mild
anemia; and alopecia and dermatitis.8 Its riboside, S-FUDR, is somewhat
less toxic though equally as active and has thus replaced S-FU, for the
most part, in clinical practice.

In general, the antimetabolites have not proved as successful
clinically as tests with laboratory animals seemed to predict. It
appears that the pharmacology of these drugs in humans is sufficiently
different from laboratory animals to prevent achievement of doses
effective on tumor cells without producing excessive toxicity in the
hosts. This appears to result from a greater instability of these
agents in humans as compared to animals.4

Although not included in the scheme in Figure 1, various sulfhydryl

inhibitors have been shown to have anti-tumor properties. Sulfhydryl

8

proteins are an essential portion of the nucleic protein, which is
indispensable for the structural integrity of the chromosomes. Moreover,
the mitotic apparatus---asters, centrioles, spindles, etc.-—— is
extremely rich in sulfhydryl proteins, and, in fact, such protein may
account for a large mass of the dividing cells—--perhaps up to 50%.

Such agents are in some sense selective for cancer cells, possibly
because the sulfhydryl-containing enzyme succinic dehydrogenase is
necessary for maintenance of cancer cell membranes. Also cancer cells
are streamlined for proliferation, implying that they may be mainly
sulfhydryl-protein in structure.7 However, like most anti-cancer agents
they are not highly selective for tumor cells per se but tend to be
selectively toxic to rapidly dividing cells.6 Thus numerous toxic side-
effects are likewise encountered with these agents.

Various antibiotics have been employed as cancer chemotherapeutic
drugs. Actinomycin D, possibly the most commonly used of these drugs,
is thought to combine physio-chemically with DNA. It does not interfere
with DNA synthesis but RNA formation is inhibited. It is believed that
the actinomycin molecule "fits" into the minor groove of the DNA helix,
thus inhibiting formation of the RNA synthetase.3 Mitomycin C, another
frequently used antibiotic, is believed to attack essential sulfhydryl
groups in vivo.7 Varied success has been attributed to these agents.
Toxic effects on bone marrow, liver, kidney and the gastrointestinal
tract, as well as depression of the immune response, are common side-
effects.

The Vinca alkaloids—--extracts of the Vinca rosea plant, the
periwinkle—-—have also found some success in cancer chemotherapy. The

principle agents are vinblastine and vincristine. Their mechanism of

9

action is presently unknown, but they produce metaphase arrest and
general mitotic inhibition.10 They have produced some success against a
variety of tumor systems and cross-resistance within the group is unknown.
Their toxic side-effects are those common to most anti-cancer agents,

with the addition of neurologic disorders, which may be severe.

Various other miscellaneous compounds, such as methylglyoxal
bisguanylhydrazone (MeGAG), hydrazines and various hormones, have been
used in cancer chemotherapy with varying degrees of success. Most com—
pounds attack fast growing tissue rather than just cancerous tissue and,
therefore, toxic side-effects are common and a balance between therapeutic
efficiency and host toxicity must be carefully maintained. The number
of agents of all classes both in clinical and preclinical testing, as
well as in use, is legion. Workers in the field are optimistic that
chemotherapy will supply a suitable control. In this regard, a new
class of chemotherapeutic compounds---inorganic platinum compounds---has
recently been discovered. Early animal studies indicate that these
compounds may surpass those presently available.

These agents were first discovered while investigating the possible
effects of an electric field on the growth processes of bacteria.11
Under the experimental conditions E. coli formed long filaments, up to
300 times the length of a normal cell. Further studies revealed that a
platinum salt, produced by electrode reactions, was the active agent for
this phenomenon. The complex formed was tentatively identified as
(Pt(IV) C16)-2. However, when bacterial chambers were inoculated with

fresh solutions of (NH (Pt(IV) 016), higher concentrations then

4’2

originally estimated were necessary to cause filamentous growth. It

was soon discovered that solutions of (NH (Pt(IV)Cl6) exposed to light

4)2

10

for some time were more efficient in inhibiting cell division than
freshly prepared solutions. UV-irradiated solutions were found to con-
tain three species of platinum compounds---the doubly negative species
which was found to be bacteriocidal; a singly negative species, initially
found to cause neither inhibition of growth nor cell division; and a
neutral species which inhibits cell division but not cell growth.12’l3
(Later studies showed that the singly negative species is extremely
unstable, even in the dry, crystalline form, and fresh solutions of this
compound were found to inhibit cell division.14) The active neutral
Species formed was eventually identified to be cis-Pt(IV)(NH3)2C12.

With further testing it was found that various group VIIIB transi-
tion metal ions were capable of inhibiting cell division in E. coli,
although not apparently interfering with growth. When various bacterial
species were similarly tested gram-negative bacilli were found most
sensitive, while gram-positive bacilli responded only at near toxic-
levels. No coccus tested showed any effect. With the removal or
decrease of platinum, the filamentous bacteria were seen to reinitiate
cross-septation and, in time, only normal sized bacteria were again
present.13

The distribution of an active neutral platinum species

(cis-Pt(IV)(NH C14):UIboth E. coli and gram-positive cell lines

3)2
(Bacillus cereus and Staphylococcus aureus), as well as the distribution

2

of the growth inhibitor Pt(IV)C16- {in E. coli, were investigated, using

radioactive 191Pt (half-life approximately 3 days). In the induced
filamentous forms of E. coli, platinum was found associated with metabolic

intermediates, nucleic acids and cytoplasmic proteins. For those E. coli

inhibited by Pt(IV)Cl6-2, the platinum was found only in association with

ll

cytoplasmic proteins. For the unaffected gram—positive cell line it was
found that the platinum did indeed enter the cell, combining predominantly
with metabolic intermediates.15

Various inorganic platinum compounds, with platinum in either the
two or four valence state, were either synthesized or commercially
obtained and purified. Tests for the effect of such compounds on experi-
mental tumor systems were subsequently carried out. Significant
regression of the mouse solid Crocker Sarcoma 180 was achieved with
several of these compounds. Subsequent tests on the L1210 strain of
mouse leukemia by Microbiological Associates, Inc., under contract to
the Cancer Chemotherapy National Screening Service (CCNSC), showed
several compounds to be fairly potent antileukemic agents, with signi-
ficant increases in mean survival time attained.16 In accordance with
the Protocols of CCNSC for tests with the Sarcoma 180, tumors were
implanted on day 0 and treatment started day 1. Table 1 gives the
results for various platinum compounds on Sarcoma 180 in both ICR and
random-bred Swiss mice.17 T/C x 100 is a measure of compound efficacy,
T/C being the ratio of treated tumor weight to control tumor weight at
times of sacrifice. The host toxicity varied tremendously from compound
to compound and such effects for a given compound were often inconsistent.
It is of interest to note that the (Pt(II)(NH3)4)++ salt, which had no
evident tumor effect, was tolerated as high as 400 mg/kg on a daily
dose schedule.

Subsequently, tests were undertaken with mice in which the Sarcoma
was allowed to attain a reasonably large size (8 day growths, typically
averaging 0.5-1.5g in weight) before treatment was initiated. One or

two injections, appropriately Spaced, of either cis-Pt(II)(NH3)2C12 or

12

Table 1: Results with various platinum compounds.

 

 

Compound Dose Schedule T/C x 100
-2 .

(Pt(II)C14) daily 60 or greater
(Pt(IV)Cl6)”2 daily 10-35
(Pt(II)NH3Cl3)- daily 16—20
(Pt(IV)NH3C15)_ daily 50 or greater
cis-Pt(II)(NH3)2C12 daily 0 2 (at 2mg/kg)

1 shot, day l 6.0 (at 8mg/kg)
cis-Pt(IV)(NH3)2Cl4 daily 30-82

1 shot, day 1 11 (at lng/kg)
cis-Pt(II)(NH2)2(CH2)2C12 daily 33 or greater

1 shot, day l 18 (at lémg/kg)
cis-Pt(IV)(NH2)2(CH2)2Cl4 daily not reproducible

1 shot, day l 48 (at 8mg/kg)
trans-Pt(II)(NH3)2Cl2 daily 70 or greater
trans-Pt(IV)(NH3)2Cl4 daily 75 or greater

1 shot, day 1 75-120
(Pt(IV)(NH3)3Cl3)+ daily 45-70
(Pt(II)(NH3)4)++ daily no tumor effect
(Pt(IV)(NH3)4C12)++ daily 70 or greater

 

Although most of the above data was collected using Swiss white mice, a

few values were obtained using ICR mice.

l3

cis-Pt(IV)(NH were quite efficacious. A stasis of growth was

3)2C14
initially evident and after several days the tumor became necrotic in
appearance, began to be "pinched off" externally and “fell out" quite
literally in many cases. For those animals in which a “fall out'I was
accomplished, the gaping hole closed and scarred and eventually no sign
of the tumor's presence was apparent. This type of tumor "cure" is
typical of that for a spontaneous regression, but the percentages of
cures are far greater for platinum treatment than for spontaneous
regression.l8

Animals successfully cured of Sarcoma 180, after treatment on day l
or day 8 after implant, were found to be immune to repeated implantation
---that is, a later implant either failed to grow or grew to a small
extent and was then reabsorbed. As far as presently tested, such
immunity appears to remain as long as one year after cure and quite
possibly considerably longer.19

One male and two female random-bred Swiss mice, which had been

cured by treatment with cis—Pt(II)(NH by day 8 treatment, were

3)2C12
mated to test for possible genetic mutations resulting from drug
activity. Both females delivered normal sized litters (IO and 11 young)
which appeared normal and were observed to remain so beyond weaning.

As a test for possible problems in pregnancy as well as teratogenic
effects, several pregnant female mice were obtained and administered a

therapeutic dose (8mg/kg) of cis—Pt(II)(NH at various stages of

3’2012
th th

gestation-——from the 14 to the 20 day. All offspring born appeared

grossly normal. However, some animals had smaller than normal litters

(possible reabsorption of abnormal young?) and several young died after

birth, with no reason for death readily apparent.21 It is postulated

14

that, since in most instances the mother mouse was herself ill from the
effects of the platinum compound (treated animals commonly remain
visibly ill 4 to 7 days after treatment), the death of her young was
possibly a result of her inability to nurse efficiently. However, such
tests were merely preliminary and further work is necessary along this
line.

Tissue culture tests of the effects of various platinum compounds,

using human amnionic AV cells, indicate a decrease in uptake of

3
tritiated thymidine, tritiated uridine and tritiated leucine after treat-
ment with various compounds. The extent of inhibition is greatest with
those compounds showing the greatest anti-tumor activity and at drug
levels comparable to tumor therapeutic levels. Moreover, the effect is
irreversible in this system, in the sense that inhibition is not
removed with removal of the platinum compound.22 Preliminary tests with
a primary tissue culture line reveal less inhibition with comparable
drug doses and apparent reversibility of effect.23 This may result from
a greater sensitivity of transformed or malignant cells, to the action
of the platinum compounds, as compared to normal cells.

From the above and similar studies it became evident that various
platinum compounds---in particular cis-platinum (II) diamminodichloride
(cis-Pt(II)(NH3)2C12), cis-platinum (IV) diamminotetrachloride

(cis-Pt(IV)(NH l4), and cis-platinum (II) ethylenediaminedichloride

3)2C

(cis—Pt(II)(NH (CH2)2C12)---are potent anti-tumor agents in test

2)2
animals. Moreover, it is quite evident that, as with all cancer
chemotherapeutic agents, they are toxic to the host. However, at

therapeutic doses, any injury to the host is evidently repairable, as

cured animals continue to survive with no apparent ill effects.

15

In an effort to gain some insight into the mode of action of these
compounds, the author undertook distribution studies of cis—Pt(II)(NH3)2C12
in both tumored and non-tumored animals, as well as histopathological

investigations. Cis-Pt(II)(NH Cl2 was chosen because of its tremendous

3)2
and consistent success in treating solid Sarcoma 180 (both small and
advanced tumors), as well as mouse L1210 leukemia and other mouse and
rat tumor systems. The thesis material presented herein thus includes
data concerning the distribution and histopathology of the drug in
laboratory animals, as well as several minor experiments with the same

system, all designed to provide some initial data for elucidation of a

mode and site of action for this drug.

EXPERIMENTAL METHODS

General Procedures

 

For all experiments random-bred, specific pathogen free, female
Swiss white mice were obtained from Spartan Research Laboratories
(Williamston, Michigan). The mice weighed 18 to 20 grams on arrival.
Translucent plastic, box-like mouse cages, with wire mesh lids, con-
taining a food bucket and through which a typical laboratory water
bottle could be inserted, served as animal housing. Corn husk litter,
supplied by Spartan Research Laboratories, was used for bedding. Six
to fourteen mice were housed per cage, depending upon the needs of the
particular experiment. Both food (Purina laboratory chow) and water
were continuously available, ad libitum, unless otherwise specified, and
their supply was checked daily.

Usually animals to be used in tumor tests received their tumor on
the day of arrival. The solid Sarcoma 180 used was originally obtained
from Mr. S. Poilly of NIH and had been successfully passed through
numerous generations of both ICR and random-bred Swiss white mice. A
six to eight day (rapidly growing) tumor growth was obtained from one
of the mice designated for transfer purposes. (Transfer of the tumor
line, every six to ten days, into six to twelve animals, is necessary
for maintenance of fresh, viable tissue for test purposes.) The
animal was sacrificed by cervical dislocation and the tumor excised.

(All instruments used for removal, preparation and implantation of the

16

17

tumor were sterilized before use.) The excised tumor was placed in a
sterile petri dish containing a 0.1% solution of chloramphenicol, with
0.85% NaCl. Tenxto twenty milligram pieces were cut from obviously
"healthy" portions of the tumor mass and were loaded into 13g stilleto-
type needles. Tumor implantation was into the left, forward axillary
region, with initial entrance of the needle at the lower region of the
animal's rib cage.

Cis—platinum(II) diamminodichloride (cis-Pt(II)(NH 12) used for

3)2C
all reported experiments was synthesized and purified by Thomas Krigas,
according to procedures described elsewhere.24 The compound was main-
tained in a dry, crystalline state and all solutions were prepared
immediately before use, with sterile physiological saline as the solvent.
A Mettler balance and volumetric flasks were used for the preparation of
all solutions. The solutions were prepared in such a way that 0.025cc
of the solution per gram of body weight was required to attain the
desired dosage. All animals were weighed immediately before injection
and received an injection amount, according to the individual weight to
the nearest gram, by the intraperitoneal (IP) route. (Animals were
individually coded and weighed in a 400ml beaker on a single pan, 16000
gram capacity, Dial-O-gram balance. The balance, with the beaker, was
initially balanced at 100 grams and all initial weights were recorded.)

A 1:200 solution of Amphyl was used as a disinfectant for all cages,

equipment and work surfaces.

LD50 Studies

 

As a preliminary estimate of the toxicity of the cis—Pt(II)(NH3)C12

18

a single—dose, LD50 study was undertaken, according to the procedures

outlined by the CCNSC.25 Ten female animals were employed per dose
level. All animals weighed 18 to 25 grams initially and were then
fasted overnight, prior to injection. Initial weights were recorded and
the mice were checked daily for deaths. Autopsies were performed as
deaths occurred, none later than one hour after death. The animals were
observed for 14 days after treatment. Final weights and total weight

changes were recorded for all survivors and approximately % of the

survivors were autopsied, following sacrifice by cervical dislocation.

Weight Studies

 

Early studies, in particular with tumored animals, indicated that
the treated animals are grossly ill for 4 to 6 days following injection

with a therapeutic dose (8mg/kg) of cis—Pt(II)(NH In order to

3)2012'
investigate more carefully the time course of the grossly-evident

effects of this platinum compound, as monitored by weight changes, 4 sets
of animals were prepared. Two cages containing 6 animals each were set
aside for each set---one set to receive neither tumor nor platinum; the
second to receive only platinum; the third to receive only a tumor; the
fourth to receive both a tumor and platinum. The animals were weighed
and tumors implanted the day after arrival. Individual weights were
measured at the same time of day, each day. Injection of the platinum
compound into the treated animals, according to individual weight, was
accomplished on the 8th day of tumor growth. Daily weight measurements
were continued for 6 days after treatment. All animals were then sacri—

ficed, by cervical dislocation, and autopsies performed on two, randomly

chosen animals from each set.

l9

Neutron Activation Analysis

 

The principle purpose of this thesis investigation was to discover
the time dependent distribution of the platinum compound in the labora-
tory mouse, as well as any concommitant histopathological effect. In
order to follow the distribution of the compound---or at least that
portion with which the element platinum remains attached---a tracing
method had to be established. The commonly used practice of employing
radioactively labeled tracers did not prove feasible at the time.
Platinum exists as but few convenient radioisotopes which are not readily

available. Although the isotope 195mPt did become readily available, no

195m

personnel or facility to produce cis— Pt(II)(NH C12 was available.

3)2
Moreover, the half-life of approximately 4 days presented a problem, in
that the time span of interest, after injection, was unknown and large
amounts of material would need be handled within considerably short
periods of time. To avoid these and other formidable problems, interest
was turned to neutron activation analysis. Activation analysis, in
general, involves the formation of a radioactive isotope by bombardment
with such sub-atomic particles as neutrons, protons, alpha particles.

(See the Appendix for details of the theory of neutron activation

analysis and associated y-ray spectroscopy.) Use of such a procedure
provided many technical advantages. Since no radioactive isotope exists
before activation, the samples of interest could be collected, and later
activated, when convenient. However, problems still remained. Biological
material contains high concentrations of such elements as Na, K and C1.

These elements are readily activated by neutron irradiation, interfering

with the identification of elements, especially those present in only

20

trace quantities, whose characteristic Y-rays are low energy. (See

Appendix for explanation of this phenomenon.) The radioactive platinum

species most efficiently formed, 199Pt, emits low energy‘Y—rays and,

moreover, has a relatively short half—life—--approximately 31 minutes.

199

Thus, with a biological matrix, even qualitative analysis of Pt
becomes a formidable task.
Fortunately 199Pt decays by 8--emission to the radioactive isotope

199Au. Although its characteristic y-rays are also of low energy (158

and 208 keV)26, its half—life is approximately 3.15 days. The inter—
ference from high concentrations of 24Na (half-life - 15 hours), however,
still presented a problem. A radiochemical separation, for the removal
of any and all interfering y-ray emitters, was thus sought.

Initial tests were done with normal mouse liver samples, spiked

with a cis-Pt(II)(NH solution containing 6ug of platinum. (Eventu-

3)2C12
ally, tests with spiked samples of all tissues of interest were used to
calibrate the entire system.) A control solution of the compound,
containing the same amount of platinum in doubly distilled water, was
used for comparison and calibration. These test samples, and all sub-
sequent samples, were irradiated in the Michigan State University Triga
Mark I research reactor (Gulf General Atomic, Inc.). Figures 2 and 3
show cut—away views of the reactor tank and its components and a close-
up of the sample holders, respectively. As seen, the sample holders

(40 in number) are equally spaced around a lazy-susan type apparatus,
which is kept rotating constantly during irradiation to insure even flux
distribution for all samples. For all tests described here, an 8 hour

irradiation at the maximum allowable sustained power level (ZSOkW and

approximately 2x1012 neutrons/cmz/sec.) was employed. The samples were

21

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TRIGA MARK I REACTOR

 

Figure 2: Cut-away view of the Triga Mark I reactor.

22

 

Figure 3: View of the lazy susan-type holder of the Triga Mark I.

23

heat—sealed in 2 dram polyethylene vials supplied by the reactor person-
nel and then placed within reactor sample containers and loaded, by means
of an electrically operated fishrod assembly, into the reactor sample
holders.

Approximately 60 hours were allowed to elapse between termination
of irradiation and initiation of radiochemical separation procedures,
both for personal safety (the samples were initially considerably radio-

active as a result of the high concentration of 24Na, 38C1, etc. formed)

199Pt.to 199

and for efficient conversion of Au (a lapse of at least 10
half-lives of the parent nuclide is recommended for efficient production
of the daughter isotope27). The biological samples were then removed
from the polyvials and digested in a minimum amount of a 1:1 solution of
HNO3 and H2804 acids, which was heated until digestion was complete and
all HNO3 fumes were driven off. (A small amount of H202 was also added
to clear colored solutions of both liver and intestine samples.) The
solution was then brought to volume (5 or 10 milliliters depending upon
the amount of tissue initially present) with 6N HCl, to convert the

heavy metal---the 199Au---to its hexachloro salt. When cool the solution
was passed over a Cl- balanced anion-exchange column (Dowex l-X8). (The
hexachloro salt of the 199Au remains tenaciously affixed to the top
portions of the resin and the interfering ions, principally cations, pass
through.) The column was then washed with 6N HCl to insure the complete
removal of interferences. Initial studies were undertaken to ascertain
the amount of wash for a given amount of a given tissue. The integrated
area, under the 158keV photopeak of the 199Au y-ray spectrum, of both

the washed column material and a simultaneously irradiated control

solution were compared, as a measure of radiochemical separation

24

efficiency. When the agreement between the two measurements reached a
reproducible 2-5%, the separation was considered suitable and the condi-
tions for that tissue then set. Five inch lengths of 5/8" inner
diameter polyvinylchloride tubing (Plastics Manufacturing and Supply Inc.
Lansing, Michigan) were usedas the ion—exchange columns and the top of
the washed resin merely pushed flush with the upper end of the tube, the
entire column suitably packaged in polyethylene bagging to prevent con-
tamination and the entire column placed within the well of the detection
crystal. This allowed for a consistent and readily disposable system.
(The column was prepared using a one—hole rubber stopper, from which a
short glass tube, equipped with a short length of tubing, extended. The
flow rate of the column, which was supported in a burette holder, was
controlled, with the use of a simple Hoffmann clamp, to approximately
3 or 4ml per minute.) Subsequent to irradiation, all work with samples
was carried out in a vented hood, behind a wall of lead bricks, and
rubber gloves and apron were constantly employed. A detection devise
was placed close to the work area to constantly monitor the radiation
levels and Michigan State university Public Safety officers performed
weekly tests for radioactive contamination. All contaminated solid and
liquid waste was suitably disposed of by the Department of Public Safety.
Each tissue of interest was individually calibrated with a control
solution, irradiated simultaneous with tissue being tested. Thus, the
conditions of the radiochemical separation were determined prior to
actual data collection, and it was assumed that approximately 100%

retention of the 199Au salt was constantly achieved. (The calibration

work, as well as other work with such an ion-exchange system,28’29

indicate this to be an accurate assumption.)

25

The 199Au activity of the anion-exchange columns was detected with

the use of a 3" by 3" well type Packard Instruments NaI(Tl) scintillation
crystal. (The well measured approximately 1 1/8" by 2".) This well-type
crystal was in a lead housing, lined with cadmium and copper to suppress
lead X-rays. The plastic tubing plus a "Lucite" disc served to suppress
bremsstrahlung. The anion-exchange column was placed in the well such

that the surface of the resin containing the 199Au salt was flush with

the bottom of the well. All samples and the individual control solutions
(one 6ug platinum control solution was included per reactor run, containing
approximately 11 test samples) were analyzed for a 20 minute "live time".
An EMI ten stage photomultiplier tube was directly connected to the scin—
tillation crystal, located within the lead shielding apparatus. The output
of the photomultiplier tube has fed directly into a Nuclear Data 512-
channel analyzer-computer (Series One-thirty), which, for test purposes,
was set to cover a 500keV range. The analyzer was connected to a Tetronix
RM503 Oscilloscope, a Moseley Autograf x-y recorder and an IBM electric
typewriter, model B, "input-output" type. The final Y—ray spectrun could
thus be obtained by any or all of these methods-—-visually, graphically
and/or digitally. The analyzer, moreover, was equipped with an integrator,
with which the integrated area under the photopeak of interest could be
automatically ascertained. This integration system was employed to provide
the integrated area under the 158keV 199Au photopeak. This integrated
area, as well as the counts in the channels immediately abutting the photo-
peak of interest, were then typed out. The integrated area could then be
corrected for background (by subtracting the average of the counts abutting
the photopeak, times the number of channels in the peak) and compared with
the corrected count so obtained for the control solution. A lapse of

199

approximately four hours, or about 5% of the Au lifetime, elapsed

26

between counting of the control and the last sample per run. This was
considered negligible and not taken into consideration at any time.

A pilot test, undertaken to obtain some idea of the time course of
the platinum within the body, was carried out as soon as a calibration
for each tissue was successfully obtained. Four cages, containing four
animals each, were prepared. Each animal received a tumor on day 0. On
the 8th day of tumor growth the animals received 8mg/kg of the
cis-Pt(II)(NH ) C1

3 2 2'

the injection of each of the 4 cages. At various intervals after injection

A 15 minute time interval was maintained between

---1, 6, 12 and 24 hours--one animal from each cage was sacrificed by
cervical dislocation. (The 15 minute spacing of the injections allowed
for autopsy and maintenance of accurate timing of sacrifices.) The liver
+ gall bladder; the complete intestinal tract (from duodenum to rectum)
with contents; the stomach, with contents, + the esophagus; the spleen;
the lungs; the kidney + urinary bladder; endocrines (adrenal glands,
pancreas and thymus); the heart; the brain; and the tumor were removed
and placed into separate, pre-weighed polyethylene vials. Three tissues
of each type were pooled per time (except for the intestinal tract, since
only two complete tracts could be placed in a polyvial) for neutron acti—
vation analysis. The tissues from the animals in the fourth cage were
placed in buffered formalin solutions for histopathological examination.
The activation analysis vials were weighed to ascertain the wet tissue
weight and then heat-sealed. They were kept refrigerated until irradiated.
The results of this pilot study indicated that longer, rather than
shorter time intervals following injection were still necessary. It also
indicated that measurement of samples of blood, obtained within the first

24 hours following treatment, would be of considerable interest. Conse-

27

quently, 0.6cc of blood, obtained by heart puncture, (0.2cc from each of

3 mice) was collected every hour for the first 6 hours following injection,
then 8, 10, 12, 16, 20 and 24 hours respectively. Separate samples were
simultaneously obtained from both tumored and non-tumored groups of
animals. No anti-coagulent solutions of any sort were added. The poly-
vials were heat—sealed and treated in the same manner as the other animal
tissues. (Calibration of spiked (6ug platinum) samples of blood was
carried out before the actual data was accumulated.)

For further tests on the other body tissues, 2 sets of animals, 4
cages apiece, were obtained, for the collection of all necessary samples.
Ten animals per cage in the one set received tumors, while the 14 animals
in each cage of the 2nd set remained non-tumored. IP injection with the
platinum compound was accomplished on the 8th day of tumor growth. Non—
tumored animals were sacrificed, and tissues for both activation analysis
and histopathology collected, at 1, 6, 12 and 24 hours after treatment.
Both tumored and non-tumored animals were subsequently sacrificed and
tissue so collected at 12 hour intervals, from 24 hours on, for 144 hours
or 6 days. All samples were weighed, heat-sealed and kept refrigerated
until irradiated.

The integrated areas obtained from these test samples were graphi-
cally converted into Hg of platinum. A calibration curve was prepared
for each separate irradiation run, by employing the value obtained for
the 6ug platinum control for the given run. A plot of counts versus
pg Pt was then developed. The corrected 6Ug Pt value obtained was
plotted and counts for multiples or fractions of this 6ug value calculated
and plotted. (Preliminary tests had shown such a calibration curve gave

results accurate to within 5% or less.)

28

Histopathology

 

The tissue samples for histological study were collected, as
described above, at the same time as samples were collected for activa—
tion analysis studies. Thus, tissue samples were examined at times
corresponding to those at which the platinum distribution within these
same tissues was ascertained. The entire spleen, two sections of the
main lobe of the liver, the section of the pancreas between the stomach
and the duodenum (same as for activation studies), the esophagus at its
gastric end, the glandular portion of the stomach, a section of duodenum
and of either jejenum or ilium, the adrenals, the kidneys, the urinary
bladder, the entire thymus, the heart, two sections of lung, and the
brain (halved) were removed and immediately placed in a buffered formalin
solution for fixation. (Tissues can remain indefinitely in buffered
formalin and its action is progressive. This fixative consists of 1
liter 10% formalin solution containing 4.0gm NaHzPO4 - H20 and 6.5gm
NazHP04.30) The entire tumor was excised in all cases and sectioned
according to the particular size. All sections cut included central
(possibly necrotic) and outer tissue portions. The animal marked for
sacrifice and histological use at 84 hours after injection lost the
majority of its tumor, by "fall out," by the time of sacrifice. However,
some tumor tissue did remain around the open wound and this was removed
and placed into fixative. After at least a month in fixative the various
sets of tissues were dehydrated, cleared, infiltrated and embedded
according to the paraffin method.30 (The brain sections were never

embedded, as no platinum was found in brain tissue at any time, and

other available data, with the same platinum compound, revealed no

29
change in brain tissue following treatment.31)

The tissues were first washed overnight under running water to
remove the formalin. They were then progressively dehydrated by a series
of increasing percentages of ethyl alcohol in distilled water---50%, 70%,
95% and absolute alcohol. The time in these particular alcohols was 1
hour, 1 hour, two 3/4 hour washes and two 3/4 hour washes, respectively.
The tissues were then cleared by two 3/4 hour washes in analytical
reagent grade toluene. (The toluene was supplied by Mallinckrodt, and
95% and absolute alcohol, used for the preparation of all other dilutions,
was purchased from Michigan State University Stores.) Two 3/4 hour
washes in liquid tissue mat, melting point 55.00C :_0.50, followed.

(The tissue mat was supplied by Fisher Scientific Company and the tempera-
ture of the liquid paraffin was maintained just slightly above its
melting point in a Stabil-therm Blue M Electric Oven, Blue Island,
Illinois.) The tissues were now ready for embedding. Bond boxes,
fashioned from 5" x 3" index cards, were used for the embedding. These
were filled 3/4 full with liquid paraffin and the tissues transferred

to them with the use of heated forceps, two or three tissues per box
depending upon tissue size. Each box flap was marked with the identi—
fication of the tissues placed therein. The paraffin was cooled
immediately in water, at a temperature of lO—lSOC. When a solidified
scum had formed on the surface, the potential block was dipped below the
surface of the water. These were then stored in a cool placed until
ready for trimming and slicing.

The embedded blocks were trimmed into rectangles or squares,
depending upon the shape of the tissues, attempting to keep opposite

sides parallel. These trimmed blocks were mounted, using a hot knife,

30
onto wooden blocks cut to fit the vise attachment of the microtome used
(Swift #650120 microtome). The paraffin blocks were sectioned until the
tissues were exposed, to the level of interest, and the tissues then
soaked in a Tide detergent solution to soften. The soften tissues were
then cooled in the freezer to allow for easier sectioning of the paraffin
and 6p slices were cut. (A Lipshaw, stainless steel microtome blade was
used for all sectioning.) Ribbons of tissue were removed to a Fisher
Tissuemat water bath (550C : 1.50C) where the sections were flattened.
Several consecutive sections of the given tissue were then mounted on
pre-cleaned, non-corrosive microsc0pe slides (Sargent) with the use of
Mayer's Albumen fixative, which had been personally prepared, according
to standard formulations.30 The slides were then allowed to dry, until
excess water had evaporated, and were then heated over an alcohol burner
to just melt the paraffin and thus allow the tissue sections to flatten
out on the slide surface. The slides were individually coded with a
diamond-point marker and stored in wooden slide boxes until ready for
staining.

A simple hematoxylin-eosin staining procedure was used for all
sections. Harris' hematoxylin was the specific hematoxylin employed,
and an adequate amount for all staining was graciously supplied by Mrs.
Mae Sunderlin of the Michigan State University Department of Pathology.
Eosin Y was obtained from the National Biological Stains Department of
Allied Chemical and an alcohol solution prepared according to standard
procedures.30 The staining series employed is diagrammatically shown
in Figure 4. The timing in all solutions was standard. After staining
was complete, 2 to 3 drOps of undiluted Permount (Fisher Scientific

Company) were placed over the tissue sections and a #1 24" x 60" Sargent

31

 

 

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32

cover glass affixed. After the mounting solution was sufficiently dry
(several weeks) the slides were cleaned with xylene and labeled.
Histopathological investigation of these tissues was then undertaken
with the aid of Dr. Richard Kociba. All pathological changes, as well
as their time course, were noted, and representative photomicrographs
taken. (The photomicrographs were taken with the use of an American
Optical Microstar 10 microscope and camera ensemble---the camera back
being produced by Kodak. Kodak Ektachrome-X, EX 135-20 film was used

for all photographs.)

RESULTS

LDSO Test

The results of the LDSO study are shown in Figure 5, as a plot of
percent mortality versus the dose level (the dosages being plotted on a
logarithmic scale). A straight line is tentatively drawn "through" the
data points, as is typically done for such data.32 It is obvious, how-
ever, that this data does not fit a straight line when so plotted. The
data point for l6mg/kg deviates most drastically from such a simple
curve, and appears to represent a dose level at which some change in
physiological treatment of the compound occurs. This test was repeated
by another member of the laboratory, and the results showed the same

general trend.33 From the present data, the LD appears to lie between

50

14 and 15mg/kg for a one injection dose schedule.
66.6% of the deaths occurring during this study occurred on the 4th

day after treatment, with 18% of the deaths on the 5th day and only 3,

2 and 2 deaths respectively on the 3rd, 6th and 7th days following

tr ea tment 0

Gross Changes Resulting from Treatment

Figure 6 graphically presents the data obtained from the weight
studies on the various sets of animals, ranging from complete controls

to animals receiving both a tumor and platinum. Each data point

33

80

4O

PERC E N 'l’ MORTALITY

Figure 5:

Results of LD

50

34

 

LD 20

cos: [mg/k9]

study.

30

[9m]

WEIGHT

Figure 6:

35

x YUMOIID
o NON-VUMOIID

— CONTIOI.
- - YIIAYID

 

DAYS

Average daily weights of various groups of mice.

36

represents the average of 12 animals, individually weighed (except the
last points for both tumored animal plots, as one tumored control
animal died on day 12 and one treated tumored animal died on day 13).
The arrows indicate the respective average weights on the day on which
8mg/kg of the platinum compound was administered. It is obvious that
the tumored animals are ill prior to platinum injection, as revealed by
the stability or even loss in their average weight during the first 8
days of tumor growth, as compared to the obvious weight gain for non—
tumored animals during this same time period. It is also obvious that
the treated, tumored animals lose more weight following platinum injec-
tion than those animals receiving only platinum--—an average weight loss
of 2.5 grams for the non-tumored animals and 3.6 grams for the tumored
animals. Both groups of treated animals, however, are capable of
recovering from the drug effects, as evidenced by the reversal in their
average weight plot at 12 days, or 4 days following treatment.

Autopsies were performed during the course of the LD50 studies and
at the completion of the above-mentioned weight studies. Moreover,
observations concerning organ differences were noted when samples were
collected for both the activation analysis and histopathology studies.
The spleen was an organ in which differences were consistently observed.
Following tumor implantation, the spleen increased in over-all size and
thickness as the tumor developed, so that for an animal with an 8 day
tumor growth, the spleen weighed approximately twice as much as that of
a non—tumored animal. Treatment with even a therapeutic dose of
cis-Pt(II)(NH3)2Cl2 caused a general shrinkage in spleen size, for both
the tumored and non-tumored animals, with a gradual return to normal as

the animal recovered from the platinum effects. (See Table 2.)

37

 

 

 

 

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38

The thymus appeared similar in size in the non-treated tumored and
non—tumored animals, and, in both sets of animals, it responded quite
dramatically to platinum treatment, shrinking quite markedly, until it
was almost non-existant. (The greatest amount of thymus atrophy was
evident in tumored as compared to non-tumored animals.) As with the
spleen, the thymus appeared to recover as the animal appeared to grossly
rebound from the drug effects.

In animals which received toxic doses of platinum---the LD test

50
---post-mortem autopsies revealed distended and hemmorhaged upper intes—
tinal tracts, with food accumulation in a grossly over~stretched stomach.
Tumored animals, sacrificed at various times following a therapeutic
dose, showed empty stomachs and intestinal tracts, evidencing at least

an anorexia even at therapeutic doses.

At toxic doses, moreover, the adrenals appeared shrunken and darker
in color than normal and the gall bladder was grossly enlarged. Adrenal
effects were never evident at therapeutic doses and gall bladder enlarge—
ment rare.

Although not grossly evident, the tissue weights measured for
activation analysis purposes revealed that the liver in the tumored
animal shrinks somewhat following treatment at therapeutic levels. This
can be seen from the values in Table 2. These values were measured for
activation analysis studies and represent the weight of three pooled
organs per time. The livers of animals receiving toxic doses of platinum
were occasionally observed to be grossly shrunken, in post-mortem autopsies.

Most 8 day tumor growths have extensive blood supplies, evident as
a complex system of capillaries. The inner portions of the tumor are

bloody and necrotic in appearance; the outer portions appearing pink and

39

"healthy". Following treatment with 8mg/kg cis-Pt(II)NH C12, it can

3)2
be observed that a large number of tumors progressively lose their
extensive blood Supply and the remaining tumor mass appears to be walled
off from the Surrounding, normal tissue. (A normally-growing tumor, in

the forward axillary region, is quite firmly anchored to both the wall of

the rib cage and to sub-cutaneous tissue.)

Neutron Activation Analysis

 

A typical y-ray spectrum for 199Au is Shown in Figure 7. It was

plotted after a 20 minute count of a typical 6ug platinum control sample.
(Note the ordinate is logarithmic.)

The data obtained from both the tumored and non-tumored animal
tissue samples can be expressed both numerically and graphically. Since
each data point was obtained from a single pooled sample of tissue, the
absolute values may not be statistically representative, although the
general trend of values should be typical. Although, in general, all
tissues were measured but once at a given time period, three sample
values for tumored Spleens, obtained 24 hours after injection (three
spleens per sample), are available-——1.58, 1.49 and 1.59 ug Pt/gm tissue.
These values point out the reproducibility of the technique as well as
the relative consistency of the platinum distribution.

Tables 3 and 4 present the data, as pg Pt/gm tissue for the tumored
and non-tumored samples, reSpectively, as well as total percent
recoveries for the various times. (Since only two complete intestinal
tracts were measured at each time, the percent recovery from the three

pooled animal tissues would be Slightly, though not significantly higher,

40

if the third intestinal tract had been included at each time.) Figure
8 graphically presents this percent recovery data for both sets of
animals. Figures 9 through 15 present the distribution data for the
various body organs, directly comparing the values for tumored and non—
tumored animals in all cases. Figure 16 presents the distribution data
for the tumor.

The peculiar distribution pattern over the first 24 hours following
injection---the fall and subsequent rise in the amount of platinum
recoverable in the major body organs---suggested the possible retention
of the platinum in the blood, combined with some blood element or chemical.
Thus, the distribution of platinum in the circulating blood was examined
and the results, for both sets of animals, are presented in Table 5 and

Figure 17.

41

IO
74
I SI

 

 

COUNTS

ENERGY [keV]

Figure 7: ‘Y—ray Spectrum of 199Au.

42

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43

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53

Table 5: Amount of platinum (pg Pt/cc blood) measured in the blood
samples from both tumored and non-tumored animals

 

 

Hours After Injection Tumored Non—Tumored
1 2.50 3.23
2 1.30 0.60
3 0.83 1.37
4 1.53 2.47
5 1.22 1.67
6 0.90 1.20
8 0.57 0.63

10 0.82 0.57
12 1.00 1.47
16 0.50 0.17
20 1.67 3.53

24 1.63 2.67

54

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55

Histopathology

 

As with most anti-cancer drugs,(cis—Pt(II)NH affects tissues

3)2C12
with relatively high turn-over rates most dramatically. In the present
study, normal body tissues in this class include intestines, stomach,

and the lymphoid elements of spleen and thymus. Tissue samples, from
both tumored and non-tumored animals, of pancreas, lung, heart, and
urinary bladder Showed no histological alterations of any type. Esophagus
samples from tumored animals revealed more extensive keratinization than
normally appears, at least for the first few days following treatment.
However, as mentioned above, anorexia was common in tumored animals
following treatment and lack of food passage through the esophageal

lumen would easily explain such keratinization.

The adrenal showed no pathological changes for either set of animals.
However, a hyperplasia of medulla and/or cortex was evident at various
times. This may merely reflect the "stress” conditions platinum treat-
ment produces within the animal. Liver samples, likewise, revealed no
damage as a result of treatment. A non-consistent picture of fat
accumulation, known as fatty metamorphosis, was seen in various sections
of both tumored and non-tumored animal tissue. Its significance is
unknown and it is not considered a pathological alteration.

The pathological alterations (If the intestinal mucosa were quite
marked. Although no changes were evident 1 hour following injection,
by 6 hours inhibition of division in the crypt cell regions was complete.
Both pyknosis and karyorrhexis of the crypt cell nuclei were evident
and the villi were edematous in appearance. By 12 hours after treatment,

Vacuolization of the crypt region was evident. At this time, the villi

56

of non—tumored tissue sections were squared in appearance, most probably
as a result of a general shrinkage of the villi, resulting from lack of
generation of replacement cells in the inhibited crypt regions. In
tumored tissue sections, the villi tips appeared to be Sloughing into
the lumen and their lamina propria areas were edematous. Although the
tiSSue appearance was similar at 24 hours, by 36 hours several mitotic
figures were evident in sections of non-tumored tissue. The onset of
regeneration in the intestines of the tumored animals was not apparent
until 48 hours, however. Regeneration progressed rapidly in both sets
of tissue, with a regenerative increase evident. The intestines of the
non-tumored animals appeared completely normal, with no remnants of
inhibition remaining, by 72 hours after treatment. Debris resulting
from earlier inhibition remained for a longer period of time in tumored
animals, however, normal appearing sections not found until 108 hours
after treatment. Figures 18 through 21 Show photomicrographs of normal
crypt regions and villi as well as the appearance of these regions
following platinum treatment.

Inhibition in the stomach appeared quite similar to that explained
above for the intestines. A11 mitotic activity was inhibited by 6 hours
and the rugae and gastric pits became shrunken in appearance. However,
by 24 hours for the non-tumored animal and 36 hours for the tumored
animal, regeneration was evident and a normal appearing section was
again evident by 48 and 60 hours, respectively. (See Figure 22 for a
comparison of a normal and inhibited sections of stomach.)

Figure 23 shows a normal section of thymus tissue. Note the thick
outer section (cortex) which appears quite heavily stained. This region

contains large numbers of closely packed lymphoid elements. Following

57

 

 

 

Figure 18a: Villi of control small intestine section. Magnification
= 60x.

 

 

 

Figure 18b: Crypt region of control small intestine section. Magnifi-
cation = 240x.

58

 

Figure 19: Crypt region of treated small intestines. Tumored animal,
48 hours after treatment. Magnification - 240x.

59

.._4._'l'1', .., -. ,._. .~ ..

 

Figure 20: Squared villi of treated small intestines. Non—tumored
animal, 12 hours after treatment. Magnification - 60x.

 

 

Figure 21: Sloughing villi of treated small intestines. Tumored
animal, 12 hours after treatment. Magnification - 60x.

60

 

    

A

Figure 223: Control section of stomach. Magnification - 60x.

‘N- ‘V“‘£I241‘&Jié!:;

 

 

 

Figure 22b: Section of treated stomach. Non-tumored animal, 12 hours
after treatment. Magnification a 60x.

61

 

Magnification - 24x.

Control section of thymus.

Figure 23

 

 

Non—tumored animal, 36 hours

Magnification = 24x.

Section of treated thymus.

after treatment.

Figure 24

l

62

 

 

Figure 2

I _ . . ll__

 

5: Apparent reversal of thymus regions. Tumored animal, 108
hours after treatment. Magnification - 24x.

63

treatment with platinum, these elements are greatly depleted. In the
non-tumored animal this depletion never progresses beyond that illus—
trated in Figure 24 (maximum at 36 and 48 hours), and regeneration is
evident by 60 hours, with the normal appearance again obvious at 108
hours. In the tumored animal, however, depletion progresses considerably
beyond this point, until the section shows an apparent reversal of
regions. This apparent reversal results from such a dramatic depletion
of lymphoid elements in the cortical region, that the scantily populated
medullary region becomes the area of greatest cellularity. (See Figure
25.) This apparent reversal was first evident 36 hours following treat-
ment. Regeneration appeared obvious from 48 to 72 hours, but an apparent
reversal was again evident from 84 to 120 hours. Regeneration did again
occur, following this second depletion, but no normal appearing Sections
were yet obvious at the longest time interval examined (144 hours).

The white pulp of the Spleen, as the cortical aspect of the thymus,
is largely lymphoid in composition. It exists as relatively distinct
regions, separated by the splenic red pulp---the region of extramedullary
hematOpoiesis, particularly in young animals. (Figure 26 is a photo-
micrograph of a section of normal spleen.) Although descriptions of
the progressive development of Sarcoma 180 in mice, indicate no metastatic
invasion and only rare local invasion into the lungs35’36, sections of
spleen from animals with 8 day or older tumor growths revealed the pres-
ence of neoplastic invasion. In one animal, the metastatic tissue had
completely obliterated the normal splenic architecture. However, most.
spleens merely contained metastatic nodules admixed with normal Splenic
tissue---as shown in Figure 27.

Treatment with a therapeutic dose of cis-Pt(II)(NH3)2C12 resulted

64

 

 

Magn

Control spleen.

Figure 26

ification - 60x.

I

 

Control spleen of tumored animal.

 

Magnification = 240x.

Figure 27:

(Arrows indicate neoplastic cells.)

65

 

 

Section of treated Spleen.

after treatment.

Figure 28a

Non-tumored animal, 12 hours

Magnification - 60x.

 

 

Tumored animal, 84 hours after

Section of treated spleen.
treatment. Magnification - 120x.

Figure 28b

66

in a depletion of lymphoid elements and a destruction and inhibition of
the hematopoietic elements, in both tumored and non-tumored animals. In
the tumored animal tissue, any and all neoplastic tissue was likewise
inhibited. AS with all other inhibited tissue, splenic regeneration
rapidly occurred (evident by 24 hours in non-tumored samples and 36

hours in tumored samples). However, unlike the other tissues, the

normal appearance of the spleen was not recovered in the time interval
examined. Instead, a hyperplasia existed, of both the lymphoid elements
and those of the extramedullary hematopoietic system, as long as 144 hours
following treatment, with no leveling off of these elements evident.

Although not an organ whose tissue normally Shows rapid turn—over,
the kidney also showed some indications of pathological damage. Unlike
the above—mentioned tissues, however, no damage was evident until 24 hours
following treatment. A capillary congestion, in both cortical and
medullary regions (mainly medullary), became obvious at that time. This
persisted and several instances of pyknosis and vacuolization of the
cells of the tubular epithelium were observed. Tubular epithelium, so
damaged, eventually sloughed into the tubular lumen and was replaced.
Such tubular damage, however, was minimal. See Figures 29 through 31
for control and treated sections of kidney.

The changes in the tumor tissue, subsequent to platinum treatment,
were similar to those obvious for all tissues with rapid turn-over rates.
Mitotic activity was inhibited and nuclear degeneration was evidenced by
pyknosis and karyhorrexis. Unlike the other tissues, however, abnormal
mitotic figures could be seen in treated tumor sections (Figures 33, 34
and 35b). Moreover, the general appearance and staining prOperties of

the entire cell changed, as evident from a comparison of the control

67

and treated sections. (Figure 35a shows a capsule forming around the
outer aspects of the tumor mass.)

Tissue sections, prepared from tumors excised 132 and 144 hours
following treatment, appeared almost identical to control tumor sections.
Thus, as with all other affected tissue, the tumor is capable of regen-

eration, under the proper conditions.

68

 

 

 

Magnification = 60x.

Control section of kidney.

Figure 29:

 

 

 

Non-

Capillary congestion in medullary region of kidney.

Figure 30:

Magnification -

24 hours after treatment.

tumored animal

60x.

69

 

 

 

Figure 31: Tubular Sloughing in treated section of kidney. Non—tumored
animal 48 hours after treatment. Magnification = 120x.
CArrows indicate Sloughing tubular epithelium.)

71

 

 

 

Figure 33: Section of tumor, 48 hours after treatment. Magnification -
120x.

 

 

 

Figure 34: Section of tumor, 72 hours after treatment. Magnification =
120x.

 

 

 

Figure 35a: Section of tumor, 120 hours after treatment, showing forma-
tion of a capsule. Magnification - 60x.

 

 

 

Figure 35b: Section of tumor, 120 hours after treatment. Magnification
= 120x.

.HII \ILTI. ['1 III

DISCUSSION

Platinum Distribution

 

It is readily obvious from the distribution data that there is no
Specific uptake into tumor tissue. In fact the greatest portion of the
platinum was found in the filtering (liver and spleen) and excretory
(kidney mainly) organs. This raises a problem as to how the platinum
compound, generally inhibitory to all rapidly proliferating tissue, can
successfully regress both small and large established tumor growths
without permanent damage to such vital tissues as bone marrow, intestinal
and gastric mucosa and lymphoid tissue. A readily obvious solution to
this dilemma is the suggestion that, for some as yet unknown reason,
tumor tissue is more sensitive to the action of the platinum compound
than is normal body tissue. Although no conclusive evidence for this
postulate exists, the finding that primary tissue culture lines appear
less sensitive to platinum inhibition than transformed lines, and are
apparently reversibly inhibited while transformed lines appear to respond
irreversibly,23 gives such a suggestion at least some probability.

The data also reveal .a low total percent recovery of platinum.
(This figure was calculated using the total amount of platinum injected
into the three animals, sacrificed at the given time interval, as the
base value.) As mentioned above, if the platinum content of the third
intestinal tract, for the given set of tissues measured, was included,

the percent recovery would be somewhat, though not Significantly, higher.

73

74

Unfortunately, not all parts of the test animals were assayed for plati—
num content. Data with rats indicate £1 marked affect of the platinum
on bone marrow.37 Thus, some platinum would most probably be found in
the bone marrow, at least for the first day or so following treatment.
The amount, however, may not be significant, as present data shows
little if any relationship between tissue damage and platinum content.
Moreover, the animals' skeletal muscle; bone, nails and teeth; and Skin
and fur may also obtain various quantities of platinum. Despite such
unmeasured possibilities, even the highest percent recovery actually
attained appears considerably low. This and the high, early levels of
platinum in the liver and kidneys suggest a high, rapid excretion rate
via the urine. Such a suggestion would also satisfactorily explain the
delayed minimal renal damage, which is more suggestive of a constant
level of irritation than of a direct renal toxicity. Whole body tests
with a radioactive platinum isotope, to measure the total amount of
platinum remaining within the animal at any given time following injec-
tion, are soon to be accomplished and should provide valuable information
along this line.

The distribution pattern for the first 24 hours after treatment-~—
the fall and Subsequent rise in total platinum recovered———presents some
interesting possibilities. Earlier suggestions of retention of the
platinum in the circulating blood can be ruled out from the distribution
pattern measured from the blood samples. It is possible, however, that
the platinum combines with some element of the serum and is shunted to
the periphery as a result of an initial, temporary edema. Most of the
animals are somewhat sluggish following injection, so that a temporary,

mild edema cannot be completely ruled out from presently available data.

75

It is also possible that there is a temporary peripheral Shunt of the
p1atinum--to skeletal muscle, skin, etc.--~and that redistribution
throughout the major body organs is rapidly accomplished. Any and all

such suggestions are, however, tentative and further information concerning
the in vivo reactivity of the platinum compound and its total metabolism
Scheme is necessary before anything further can be ascertained in this
regard.

Complete absence of platinum in brain tissue suggests that, by as
early as 1 hour after injection, the initially neutral platinum compound
has been converted into a charged molecule, incapable of passing the
blood-brain barrier. (Early entrance into the brain tissue, with subse-
quent exist, of minute quantities of neutral compound, cannot be completely

ruled out, however.) The ease of reaction of cis—Pt(II)(NH C12, as a

3)2
result of the extreme lability of its chloride ions, argues for rapid
and complete reactivity of the initial compound with reactive biological
molecules present in the blood or even the intraperitoneal fluid. The
actual rate of reactivity and the compound or compounds formed in vivo
are presently unknown, however, although various workers are presently
investigating such problems.

Another interesting feature of the distribution data is the obvious
cyclic nature of the platinum distribution in the livers of both tumored
and non-tumored animals. The fact that a rhythmic response should be
revealed for data concerning rodent liver, in general, is not unusual
or surprising. Various mouse liver activities and properties show
characteristic circadian rhythms--—phospholipid, RNA and DNA metabolism;

mitotic activity; glycogen content.38 The Stimuli which govern the

expression of such rhythms arise within the animal rather than from any

76

cyclic aspect of the environment---daily cycles of light and dark of
high and low temperatures merely regulating these rhythms so that they
coincide with the 24-hour cycle of the natural environment. (For instance,
a nocturnal animal subjected to constant light does not alter the perio—
dicity of its daily activity period but merely progressively Shifts its
phase-—-i.e. shows a daily delay in onset of activity of about 30 minutes.
Such a phase Shift is continued only as long as the animal is maintained
on a constant light regimen.)39

The real point of interest concerning the cyclic nature of the
hepatic data is the fact that while the established rhythm for the non—
tumored animals appears to have a 24—hour cycle, that of the tumored
animals appears to have a 36-hour cycle. Is the shift in periodicity a
result of the tumor or a combination of the platinum and the tumor? In
this regard it is interesting to note that various workers in the field
suggest that maintenance of circadian behavior in mammals may depend upon
feedback to the central nervous system from periodic sequences of periph-
eral metabolic events, that are loosely locked in phase with each other
via specialized hormonal (and autonomic nervous) controls. Thus, a
given rhythm in an animal results from mixing and weighing of psychogenic,
neurogenic, hormonal and metabolic factors as well as physicochemical
and socioecological signals from the environment.40 Thus, it seems
possible that the 12-hour periodicity shift observed in liver samples
from tumored animals could well reflect various metabolic, hormonal,
neural changes resulting from the growth of the tumor and/or in combina—
tion with the platinum treatment. Since the cycle obvious in the livers
of non-tumored animals is the common circadian rhythm, it would appear

that tumor effects, or alterations in such effects by platinum treatment,

77

are most likely responsible for this shift in periodicity.

With regard to drug adminiStration in general, it is interesting to
note that the phase of the 24-hour changes in physiologic State can
critically determine survival from noxious agents--—i.e. periodic changes
in susceptibility to a given agent occur. Moreover, such periodic
changes in susceptibility are not the same from one agent to another.40
It would be pharmacologically important if certain therapeutic-toxic
ratios also change periodically.

Returning to the distribution data, it is obvious that measurable
amounts of platinum are still present 144 hours or 6 days following
treatment. That the animal has recovered from the initial inhibitory

effects of cis-Pt(II)(NH C12 by this time, suggests that either the

3)2
remaining platinum is no longer in a biologically active form or that

its intracellular level is below threshold value for effectiveness. If
the platinum is biologically inactive and cannot cumulatively act as a
typical heavy-metal poison, no major problem exists. If, however, the
level is merely sub-threshold or if the inactive platinum species can
cumulatively act as a heavy-metal poison, dose schedules requiring
several injections of the platinum compound may result in enhanced
physiological damage. Further Studies in this regard, are, therefore,
necessary.

It would seem to be of major interest to compare the present distri-
bution data with that for various other potent anti-cancer agents, as
well as metallic elements normally present in the body. Such comparison
is, however, difficult since the individual workers do not follow any

general procedure for the presentation of their distribution data.

Therefore, in order to be able to present a few pertinent comparisons,

78
the author has taken the liberty of converting all data presented to a
<:onsistent numerical system. For tumored animal samples, the value
(Dbtained for the tumor sample was arbitrarily given the value 1.00 and
‘the values for all other tissues presented relative to the tumor value.
.I?or non-tumored animal systems, the value for the liver, the largest of
t:he body organs, was arbitrarily given the value 1.00. Table 6 shows
t:he pertinent portion of the platinum data evaluated according to such
as system. It is readily obvious that the filtering and excretory organs
(:ontain the greatest percentage of platinum meaSured. Although the
‘Jalues for the tumored data are considerably different from the non-
‘tumored, since the comparatively low tumor value was here used for a
basis, the ratio of liver values to the other tissue values can be easily
seen to lie within the same area for both sets, for most of the tissues.
Table 7 presents similar data, 24 hours after injection, for

triethylenephosphoramide-BZP (TEPA), a potent alkylating agent. (All
injections were given intraperitoneally.) The values for non~tumored
animals Show a more general distribution of this drug as compared to the
platinum compound. Although the filtering and excretory organs do
contain a considerable portion of the drug still present in the body,
other organs measured contain relatively high portions also. The dis-
tribution for the solid tumors---the Sarcoma 37 and Lymphosarcoma l—-—,
however, are quite similar to the values for the platinum compound in
Sarcoma l80—bearing animals. The different pattern for the leukemic
animals—--animals with L—1210—-may merely result from the more diffuse
'nature of the neoplasm. For all these systems, 60 to 80% of the 32P was
excreted within 24 hours. The major pathway for excretion was via the

kidneys, with very little radioactivity found in the feces.41 (Thus, a

79

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Table 7: Distribution of triethylenephosphoramide-BZP (TEPA), a potent
alkylating agent, in non-tumored and several tumored animal
systems.

A 24 Hours After Injection

“Tissue Non-tumored L-1210 Sarcoma-37 Lymphosarcoma 1

liver 1.00 1.67 1.31 1.05

0.1. tract 0.75 4.22 0.63 0.81

spleen A 1.29 1.33 1.74 1.00

lungs 1.17 1.33 1.05 1.24

kidneys 0.88 1.11 0589 0.86

brain 0.17 0.22 0.11 0.19 57

tumor 1.00 1.00 1.00

muscle 0.21 0.55 0.37 0.29

skin 0.33 0.78 0.58 0.67

bone 0.79 1.45 0.74 0.43

 

81
relatively high portion of drug in the digestive system need not indicate
that excretion is considerable via this route.)
5—F1uorouracil-2-14C, an antimetabolite, was administered, via the
jLntraperitoneal route, to mice containing 10 day growths of Sarcoma 180.
TIThe distribution data collected is presented in Table 8. An obvious
cilifference exists for this system---a specific uptake of the drug into
‘tzumor tissue, even as early as 1 hour after injection. The bone marrow
<::ontained a large portion of the drug, with filtering and excretory
<:>rgans containing less but yet significant amounts. 88% of the adminis-
t:ered dose was excreted within the first 24 hours. The majority of the
4C was found in the urine, with small quantities of 14CO2 collected in
the early hours.42
Unfortunately, available distribution data for the potent folic
acid inhibitor--amethopterin---are relatively sparse. The studies pre-
sented in Table 9 were performed following I.V. injection of the drug
into Akm mice. A disc method of assay (analogous to antibiotic disc
tests) was performed on various tissue homogenates, at various times
after injection. Serum levels were initially high (on the order of 300-
400 mY/ml) but the concentration either reached or approximated zero
within 4 hours after treatment. Amethopterin, not unexpectedly, was
found to accumulate in tissues containing high concentrations of folic
acid (particularly liver and kidney). In mice with advanced leukemia,
results were suggestive of a more widespread distribution, with higher
concentrations found within the tissues. This may be an indication of
the affinity of leukemic cells for amethopterin. High serum levels over
a considerable time period, however, suggest that a possible renal

<dysfunction, a secondary leukemic effect, may be responsible for the

82

Table 8: Distribution data following an intraperitoneal injection of
5-f1uorouracil-2—14C into mice containing 10 day growths of
Sarcoma 180.

 

Hours After Injection

. —_

 

 

 

 

 

 

 

 

 

 

 

 

Tissue 1 hour 4 hours 12 hours 24 hours 48 hours
liver 0.40 0.21 0.13 0.16
intestine 0.54 0.30 0.38 0.03
spleen 0.41 0.27 0.21 0.23 0.11
lungs 0.11 0.07 0.05 0.07
kidney 5 H 0.55 0.15 0.13 0.11 0.22 A
heart 0.07 0.01 0.01 0.05
800105". 5- 0.10 0.03 0.03 0.05
tumor 1.00 1.00 1.00 1.00 1.00
bone marrow 0.86 0.55 0.33 0.73
muscle 0.05 0.01 0.01 0.00
ovaries 0.06 0.05 0.02 0.04

 

fat 0.18 0.09 0.03 0.08

-0 -‘ ”fin-- 0*»- -~. - v .‘ - ' -._.. '~C‘ .h— .0»..__-—-— ~-“

83

Table 9: Distribution of I.V.—injected amethopterin in non—tumored Akm

 

 

 

 

 

 

 

 

 

mice.
Hours After Injection
'Tissue 1 hour 2 hours 4 hours 24 hours
liver 1.00 1.00 1.00 1.00
spleen 0.19 0.37 0.34 0.51
lung 0.08 0.10 0.14 ----
kidneys 0.59 0.77 0.69 0.94
‘muscle 0.02 ---— 0.06 ----

84

higher drug levels in this instance.43 Since the data are so sparse, it
.is difficult to make any significant comparison between cis-Pt(II)(NH3)2C12
23nd amethopterin. It is interesting to note, however, the difference in
the time course of serum levels between the two drugs.

Table 10 presents distribution data for the tritiated methyl ester
<3f streptonigrin, an antibiotic isolated from the broth of Streptomyces
jflocculus and found to show anti—tumor activity. This drug was adminis—
‘tered I.P. to mice containing 5 to 10 day growths of Sarcoma 180.
AAlthough for this drug the greater portion can be found in the filtering
23nd excretory organs, the kidney appears to be less important than the
intestines. However, excretory measurements~-—indicating approximately
65% excretion by 12 hours and 90% by 24 hours--—could not be easily
partitioned into urinary and fecal portions, as such results varied
widely from animal to animal and fecal samples were contaminated with
urine. (Metabolic cages were used for excretion collection.)44

Radiomanganese, injected I.V. into the tail vein, was followed with
time in the major mouse organs as well as in the excreta. Within the
first 2 hours, approximately 83% of the administered dose was excreted,
mainly in the urine. Small amounts of the radioactive manganese were
then found in the fecal matter for several days, until approximately
88% was eliminated after 7 days. Table 11 shows the distribution of the
manganese, 24 hours after injection. As with cis—Pt(II)(NH3)2012 the
greater portion of the element at this time was found in liver and kid-
ney. (The pancreas also contained a considerable portion, and such is
not true for the platinum compound at this time.) However, the blood
level is relatively insignificant in the manganese data, while 24 hours

represents a time just past the second major blood level for the platinum

85

Table 10: Distribution of the tritiated methyl ester of streptonigrin,
an antibiotic, in mice bearing Sarcoma 180.

 

Hours After Injection

 

—- F‘—

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tissue 24 hours 48 hours
liver 1.39 0.98'
small intestine 3.56 1.11
large intestine 1.10 0.72 $.-
stomacc;~-u-uw * 0 . 34 1. 35
£1.12... " ‘ W“ 1.39 -- ._._ 1.93
lung 01 1 3 - "*““””“'“”digé 0.59
kidney 0.48 1.43
heart 0.24 0.47
brain 0.08 0.05
tumor 1.00 1.00
muscle 0.54 0.51
skin 0.47 0.20
plasma 0.27 0.73
blood cells 0.56 1.34

 

—-—. ‘1-~.. . -m..---.—-—” 1....-- . .w ..

bile (1 mouse) 0.31

 

86

Table 11: Distribution of radiomanganese in normal mouse tissue 24 hours
after I.V. injection.

 

24 Hours After Injection

_. ..___.... -._._ . a . .—

 

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'I'issue

 

 

 

 

 

 

 

 

 

 

 

 

 

 

:l_iver ‘ W W '4 ~ WW_WW"~~."W “ '"“*—“—iT66»- -1 __
:i_ntestine 0.41
Espleen 0.33
.;;;;ncreas 1.90
L<idney 2.60 -
Ital.“ ~— 0.10 -..--..
blood 0.01
muscle - "“*““""“o.ll ‘—
skin 0.15
bone 0.22 ‘W
testis 0'22

 

eye 0.10

87
compound. Similar to the platinum, however, manganese can still be
measured as long as 7 days after injection, mainly in kidneys, liver and
muscle.

With the exception of 5-fluorouracil, probably the only anti-cancer
drug currently known which shows specific tumor uptake, the general dis—
tribution pattern for such drugs as here included is similar. The
filtering and excretory organs contain the greatest percentage of the
compound remaining in the body. Moreover, excretion begins almost
immediately, at a considerably high rate. Unlike most of these drugs,
however, cis-Pt(II)(NH3)2Cl2 does not appear in brain tissue at the
times studied. This may be more fortunate than unfortunate, as toxic
central nervous system effects can, thus, be largely avoided. Moreover,
for treatment of cancers of the brain, direct introduction of the drug
is always possible.

It is also interesting to note that, other than the relatively high
pancreatic content, manganese distribution and excretion properties
appear quite similar to those for platinum. The significance of such a

comparison is presently unknown, however.

Histopathology

 

The data shows little, if any, relationship between the platinum
content or its time course for a given tissue and the histological
picture of that tissue. The liver and kidney, which consistently show
the highest platinum levels, show little or no damage, while the stomach
and intestines, which show considerably lower levels of platinum over a

relatively short period of time, show considerable pathological altera-

88

tions. It would appear, therefore, that the platinum compound is
selectively attacking tissues which show considerable mitotic activity—--
have short turn-over times. Table 12 gives the relative mitotic activity
of the major organs. Even such a crude division of tissue type readily
explains why agents toxic to tissue with high mitotic activity would
affect such tissues as gastric and intestinal mucosa and lymphoid tissues
more dramatically than liver, kidney, pancreas, adrenal or brain. It
also predicts that the bone marrow—--a tissue not investigated in the
present study-—-would likewise be inhibited by platinum, as was found
following platinum treatment of rats.37 Such is, in fact, indicated by
the hyperplastic nature of both lymphoid and hematopoietic elements of
the recovered spleen. If the bone marrow was indeed inhibited, extra-
medullary hematopoietic sources would attempt to compensate whenever
possible. (Preliminary blood smears from treated mice do indicate a
general leukopenia, typical of bone marrow depression.47)

Moreover, the recovery rates of the inhibited tissue are representa—
tive of the turn-over abilities of the tissues. Although the generation
time for mouse intestinal crypt cells is only 11 hours, it takes from
1.5 to 2 days for total transit time——-i.e. for transit of new cells,
produced in the crypt regions, up to the tips of the villi.46 Thus, from
onset of regeneration, a lapse of approximately 2 days would not be
unreasonable before the mucosal regions appear normal and totally free
of the remnants of inhibition. This is, in fact, the approximate time
period observed.

Lymphocyte depletion, of both the spleen and thymus, is likewise
understandable in light of turn-over times. Moreover, a thymic recovery

time-—-from onset of regeneration to apparent normality-—-of approximately

Table 12:

89

46

Mitotic activity of body tissue (After Bond, et al. )

 

No mitosis and
no cell renewal

Low mitotic index and

little or no cell renewal

Frequent mitosis with
cell renewal

 

CNS

sense organs

adrenal medulla

liver

glands:
exocrine
pancreas
salivary
prostate

endocrine

thyroid

anterior pituitary
kidney

adrenal cortex

differentiated connective

tissue (derma, tendon,
cartilage, bone)

vascular endothelia

epidermis and appendages
respiratory system
digestive system

urinary system

genital system
blood-forming organs

pulmonary alveolar cells
(macropahages)

mesothelia

 

9O

2 days, for the non-tumored mouse, is within turn-over times for this
tissue. The replacement time for thymus small lymphocytes in the mouse
is 3 to 4 days, with mitotic indices 5 to 10 times higher for thymic
lymphoid cells than for those of lymph nodes or spleen.48 The greater
and more prolonged atrophy of the thymus in tumored animals is not
necessarily unusual either. It has been known for many years, for
instance, that thymic atrophy is common with progressive growth of the
Sarcoma 180 tumor itself.49 Moreover, it is known that the mouse thymus
may undergo involution following illness or severe stress, as well as
following food restriction, irradiation and the action of certain
"mitotic poisons" (such as nitrogen mustard and podophyllin).50 All such
factors, and many more common ones, are now believed to produce thymic
atrophy by stimulating an increased production of lymphocytolytic adrenal
corticosteriods. (Atrophy may likewise be induced via the action of sex
steroids, malnutrition and vitamin B deficiencies.)48 The hyperplastic
nature of the adrenal cortex as well as the general "stress“ conditions
of the treated mouse---complicated In! the presence of an advanced growth
of Sarcoma 180---can thus fairly well explain the prolonged and varying
involution picture of the thymus.

In general, the histopathological response of mouse tissue to

cis—Pt(II)(NH C12 is similar to that found for the rat.37 For the rat

3)2
studies, however, LD50 (12.2 mg/kg for the rat) rather than therapeutic
doses were administered to non-tumored animals only. Gastric and intes—
tinal mucosa inhibition and degeneration was similar, with greater damage
evident in the rat. (Regeneration of these elements was complete 9 days

after treatment, in those animals which survived.) Thymic atrophy, with

severe lymphocytic depletion and apparent reversal, was evident, regen—

II III! I 4 \[II

91
eration again complete by the 9th day after treatment. The spleen also
showed obvious lymphoid depletion with a hyperplastic regeneration, which
persisted as long as 20 days following injection (The maximum pathology
for all the above tissues was evident on the 4th day after treatment.)
The kidney showed tubular epithelial necrosis, with hyalin and granular
casts present in the lumen of Sloughing tubules. Regeneration occurred,
without evident persistent damage.

Unlike the mouse studies, rat experiments included tests of bone
marrow and various blood-serum levels. As with other tissues evidencing
high mitotic activity, bone marrow was greatly inhibited by platinum
treatment. The marrow elements appeared to be attacked according to
their level of maturity--—immature precursors were relatively absent
within 2 days after treatment. Typical nuclear degeneration was present
with pyknosis, karyorrhexis and vacuolization evident. However, regen—
eration was evident, in those animals which survived, from 9 to 20 days
following treatment.

Of the circulating blood elements, basically the more immature
forms and those with short life spans were affected. Leukocytes, in
general, were depressed to a low at day 3, with a subsequent, fluctuating
return to normal. Platelets and reticulocytes (immature red blood cells)
were likewise depressed, with the same time course. Mature red blood
cells—-—with a life span of 60 to 80 days———were untouched, with no
evidence of hemolysis. The various enzymes, and other protein constit-
uents, of the blood were either relatively untouched or slightly depressed,
indicating no damage to major organs and possibly a general enzyme-
depressive effect of the platinum compound.

It is of interest to compare the pathology of this system with that

92
of some of the other anti—cancer agents as well. The general toxic
effect to tissues of high mitotic activity is peculiar to cancer chemo-
therapeutic drugs as well as to X—rays. Although this is true, each
particular drug has some more or less characteristic effect which is not
necessarily shared by all.

The alkylating agents, in general, produce their most serious
effect on the bone marrow. However, as with the other drugs, they
attack rapidly dividing cells in general, causing marked lymphoid deple—
tion and atrophy of the thymus and spleen as well as inhibition and
degeneration of the intestinal mucosa. Nuclear swelling and loss of
polarity occur in the crypt cells of the intestine and such "giant-
appearing cells" can be seen along the villi on subsequent days. The
same type of mucosal inhibition is evident in the stomach. Most drugs
in this class-—-notably excluding the cyclic ethylenimmonium derivatives
--—also cause central nervous system stimulation with convulsions.
Alkylating agents in general are also mutagenic and teratogenic, and

’

suppress the immune mechanism. Moreover, two side effects are seen

with one agent each---alopecia with cyclophosphamide and skin pigmentation
with busulfan.51

Various anti-metabolites show peculiar combinations of effects. As
with most alkylating agents, the overt manifestations of 6~mercaptopurine
intoxication are delayed. The pathological effects in mice and rats
include hepatic necrosis, lesions of the intestinal epithelium and
depletion of bone marrow. The changes encountered in the small and large
intestine include epithelial atypia and edema, congestion and leukocytic

infiltration of the mucosa.52 Renal impairment may also occur.

The greater utilization of uracil by rat hepatomas, as compared to

93

normal rat liver, led to the design and synthesis of fluorouracil and
its deoxyribose derivative. The two major toxic effects of these
fluorinated pyrimidines, in man, are gastrointestinal damage and bone
marrow suppression. Diarrhea, stomatitis, nausea, vomiting, alopecia,
dermatitis, pharyngitis, esophagitis, epistaxis and leukopenia are also
common side effects.51

Use of the main folic acid antagonist, methotrexate (amethopterin),
results in damage primarily to normal rapidly proliferating tissues-—-
bone marrow, gastrointestinal mucosa, hair follicles, gonads and fetal
tissue. With bone marrow damage, anemia, granulocytopenia and thrombo—
cytopenia are the chief factors limiting the dosage of these agents.
Increased susceptibility to infection, bleeding, diarrhea, moderate to
complete alopecia, and ulceration of oral and intestinal tract mucosa
are also prominent. Depression of spermatogenesis and a variety of
skin rashes, usually in the form of a perifolliculitis, vesiculation,
and desquamation, are seen. Moreover, abortion or teratogenic effects
may prevail during the early stages of pregnancy.

Although present toxicologic data is limited to mice and rats, it
appears that cis-Pt(II)(NH3)2C12, while attacking rapidly proliferating
tissue as most such drugs, is not as generally toxic as most cancer
chemotherapeutic agents. Diarrhea does not occur until lethal doses are
reached and therapeutic doses do not appear to cause any mutagenic or
teratogenic effects. Moreover, no central nervous system involvement has

been witnessed, even at LD doses or higher in mice and rats.

50
The general pathological effects of the platinum compound are some-

what similar to those resulting from acute radiation illness in mice.

Table 13 summarizes the early lesions found in various mouse organs

94

 

 

 

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95

following radiation.53 Although lymph nodes were not specifically
investigated for the mouse in the present studies, several lymph nodes
were included in treated sections of intestine, and a depletion of
lymphocytes, similar to that for spleen and thymus tissue at the same
time interval, was evident. Although the gonads were not directly
investigated, tests with mating of "cured" animals20 tend to deny at
least permanent destructive effects of the platinum compound on
spermatogenesis.

Although the effect and time course of irradiation on intestinal
mucosa is similar to that for platinum, several specifics vary. Large
nuclei, with prominent, rounded nucleoli and abundant nuclear sap were
formed among the crypt cells which survived immediate cellular destruction
by irradiation. These cells are commonly referred to as possessing
"owl's eye” nuclei and are present in the deeper portions of the mucous
glands of the large intestines and in the crypts of the jejenum and
ileum, beginning as early as 12 hours after irradiation. Cells so
affected did not appear to die but gradually moved up the sides of the
glands and villi to the tips, whence, presumably, they were sloughed in
the usual process of attrition.53 Such cells were not evident in either
mouse or rat intestinal tissues following platinum treatment.

Although it appears that soluble platinum compounds do not, as a
rule, act as the soluble salts of heavy metals usually do (e.g. the
extremely soluble (Pt(II)(NH3)4)++ salts are tolerated as high as 400
mg/kg on a daily schedule), it is still of interest to compare toxic
effects of platinum compounds with those of soluble heavy metal salts.
Some heavy metals are known to exert their biological effects through

combination with sulfhydryl groups. However, they are all known to

96

react with other essential biological ligands as well (~OH, -COO-, PO4H2_
9

NH and imidazole).54 As with cis—Pt(II)(NH C12, there is often no

2 3)2
correlation between the organs and tissues affected by a metal and the
tissues of greateSt concentration.55

Soluble arsenic salts lead to mild vasodilation or even injury to
capillary beds; edema resulting from transudation of plasma; disruption
and inhibition of gastrointestinal epithelia; renal capillary, tubular
and glomerular damage; bone marrow depression and anemia; and hepatic
toxicity evidenced as fatty infiltration, central necrosis and cirrhosis.
Such salts are slowly excreted in both urine and feces, starting 2 to 8
hours after administration. Their slow excretion is the basis for their
cumulative toxic action. Chemically and biologically, antimony salts
resemble those of arsenic, but are more caustic locally.55

Most silver salts are insoluble but the few that are can cause
corrosion of the mucosa of the digestive tract, resulting in local
trauma and hemmorhagic gastroenteritis, which often proceeds to shock
and death. They have an initial stimulatory effect on the brain stem,
followed by depressive effects, with respiratory depression leading to
death. Such salts are cumulative in the body tissues.55

The most common toxic effects of soluble gold compounds involve skin
and mucous membranes, usually of the mouth. Lesions of the mucous mem-
branes, including stomatitis, pharyngitis, tracheitis, glossitis,
gastritis, colitic and vaginitis, are comon. Gold salts are also usually
toxic to the hematopoietic organs, with thrombocytopenia, leukopenia,
agranulocytosis and aplastic anemia not uncommon. Toxicity to the kidneys
is usual, with renal insufficiency often resulting in death. Encephalitis,
peripheral neuritis, hepatitis and nitroid crisis are also possible toxic

effects.55

97

Soluble mercury compounds cause disruption and inhibition of gastric
and intestinal mucosa; kidney damage; central nervous system involvement;
and electrolyte imbalance. Like other heavy metal salts they are cumu-
lative, as a result of their slow excretion rate. Lead salts also
result in severe gastrointestinal distress; neuromuscular problems and
paralysis; and severe central nervous system disorders.5

It is obvious that even the fairly toxic platinum compounds, such

as the cis-Pt(II)(NH C12 used for the present studies, do not cause

3)2
as general damage to major body organs as do typical soluble heavy metal

compounds. The actual differences between platinum compounds and those

of most heavy metals is presently unknown, however.

General Considerations

 

Available data concerning cis-Pt(II)(NH suggest that this

3)2C12
drug is a potent cancer chemotherapeutic agent. It successfully
regresses and even cures a wide variety of transplantable tumor systems
commonly employed for the screening of potential anti-cancer drugs.
Although it shows effects in common with known classes of anti—tumor
agents, it also evidences significant differences. Its distribution
pattern, as presented here, is similar, in general, to that for various
other agents, as well as for the naturally occurring metal element,
manganese. However, the various differences obvious in this data—--the
peculiar distribution pattern for the first 24 hours; the return of
measurable amounts of platinum into the circulation after as long as 20

hours; the long term retention of measurable quantities---suggest a

possible basic difference in physiological treatment of this drug.

98

Moreover, the pathological effects induced appear minimal when compared
to the numerous toxic side effects of the various other drugs. It
appears to attack rapidly proliferating tissue (with the possible excep—
tion of the reproductive organs) almost exclusively, with little if any
direct effect on other major body organs.

The present studies, by themselves, establish little, if anything,
positive concerning the biological activity of the compound. What is,
however, presented is evidence contrary to several previous suggestions
---such as the belief that the potent tumor activity resulted from
specific uptake into neoplastic tissue--~as well as guidelines, or even
limits, to further experimentation and speculation. For instance, it
suggests that a biologically active form of the compound (or a threshold
level of the same) is relatively short-lived---only 1 or 2 days—--
although measurable amounts of platinum are present for considerably
longer periods of time. This, combined with the probability of rapid
reactivity and possible temporary peripheral shunting, provides a
starting point for investigation of the active chemical compound. Under-

standing of the fate and mode of action of cis—Pt(II)(NH C12 is obviously

3)2
in its infancy, and no present theory can be satisfactorily supported

or denied.

LI ST OF REFERENCES

10.

11.

LIST OF REFERENCES

Roe, F.J.C. "Cancer as a Disease of the Whole Organism," The
Biology g£_Cancer, edited by E.J. Ambrose and F.J.C. Roe.
New York: D. Van Nostrand Company Ltd., 1966. Pp. 1-32.

Koller, P.C. "Chromosomes: Genetic Component of Tumour Cell,"
The Biology 9f_Cancer, edited by E.J. Ambrose and F.J.C. Roe.
New York: D. Van Nostrand Company Ltd., 1966. Pp. 33-51.

 

Stock, J.A. "The Chemotherapy of Cancer," The Biology 9£_Cancer,
edited by E.J. Ambrose and F.J.C. Roe, New York: D. Van
Nostrand Company Ltd., 1966. Pp. 176—222.

 

Rutman, R.J. "Molecular Basis of Chemotherapy," Current Perspectives

 

ig_Cancer Therapy, edited by W.S. Blakemore and 1.8. Rovdin.
New York: Harper and Row, Publishers, 1966. Pp. 24-34.

 

Double, J.A. "Extracellular Factors Affecting the Reponse of
Tumors to Chemotherapeutic Agents," Scientific Basis 9£_

Cancer Chemotherapy, Recent Results in Cancer Research,

 

 

 

Connors, T.A. "Anti-Cancer Agents. Their Detection by Screening
Tests and their Mechanism of Action," Scientific Basis of
Cancer Chemotherapy, Recent Results in_Cancer Research,
Volume 21, 1-17, 1969.

 

 

 

Knock, F.E. Anticancer Agents. Springfield: Charles C. Thomas,
Publishers, 1967.

 

Greenwald, E.S. Cancer Chemotherapy. New York: Medical Examination
Publishing Company, Inc., 1967.

 

Sternberg, S.S., F.S. Phillips and J. Scholler. "Pharmacological
and Pathological Effects of Alkylating Agents," Ann. N.Y.
Acad. Sci., 68, 811-825, 1958.

 

Baserga, R. "Biochemistry of the Cell Cycle: A Review," Cell
Tissue Kinet., 1, 167-191, 1968.

 

Rosenberg, B., L. Van Camp and T. Krigas. "Inhibition of Cell
Division in Escherichia coli by Electrolysis Products from
a Platinum Electrode," Nature, 205, 698-699, 1965.

 

99

12.

l3.

14.

15.

l6.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

100

Rosenberg, B., L. Van Camp, E.B. Grimley and A.J. Thomson. ”The
Inhibition of Growth or Cell Division in Escherichia coli by
Different Ionic Species of Platinum (IV) Complexes," J. Biol.
Chem., 232, 1347-1352, 1967.

Rosenberg, B., E. Renshaw, L. Van Camp, J. Hartwick and J. Drobnik.
"Platinum-Induced Filamentous Growth in Escherichia coli."
J. Bacteriol., 2;, 716-721, 1967.

L. Van Camp. (Personal Communication.)

Renshaw, E., and A.J. Thomson. "Tracer Studies to Locate the Site
of Platinum Ions within Filamentous and Inhibited Cells of
Escherichia coli,” J, Bacteriol., 23, 1915—1918, 1967.

Rosenberg, B., L. Van Camp, J.E. Trosko and V.H. Mansour. "Platinum
Compounds: A New Class of Potent Antitumor Agents," Nature,
222, 385-386, 1969.

L. Van Camp. (Personal communication.)
Rosenberg, B., and L. Van Camp. "The Successful Regression of Large

Solid Sarcoma 180 Tumors by Platinum Compounds," Cancer Res.,
in press.

 

L. Van Camp. (Personal communication.)

L. Van Camp. (Personal communication.)

G. Valentine. (Personal communication.)

Harder, H.C. and B. Rosenberg. "Inhibitory Effects of Anti-Tumor
Platinum Compounds on DNA, RNA and Protein Synthesis in
Mammalian Cells In Vitro," Intern. J, Cancer, in press.

H.C. Harder. (Personal communication.)

Kleinberg, J. (editor), Inorganic Synthesis, Volume VII. New York:
McGraw-Hill Book Company, Inc., 1963.

 

"An Outline of Procedures for Preliminary Toxologic and Pharmacologic
Evaluation of Experimental Cancer Chemotherapeutic Agents,"
Cancer Chemotherapy Rpts., g1, 1-34, 1964.

 

Dams, R. and F. Adams. "Gamma-Ray Energies of Radionuclides Formed
by Neutron Capture Determined by Ge(Li) Spectrometry,“
Radiochimica Acta, 19, l-ll, 1968.

 

Lyon, W.S., Jr. Guide £9 Activation Analysis. New York: D. Van
Nostrand Company, Inc., 1964.

 

 

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

101

Jervis, R.B. and K.Y. Wong. "Chromatographic Group Separation
Scheme Used with Gamma Spectrometry for Multi—Element Activa—
tion Analysis Surveys," Nuclear Activation Techniques ig_£hg.
Life Sciences, Proceedings of the Symposium, Amsterdam. IAEA,
1967. Pp. 137-158

 

 

Van Den Winkel, P., A. Speecke and J.J. Hoste. "Separation Scheme
for the Determination of Nine Elements in Biological Material."
Nuclear Activation Techniques Jn_the Life Sciences, Proceedings
of the Symposium, Amsterdam. IAEA, 1967. Pp. 159-172.

 

 

Humason, G.L. Animal Tissue Techniques. San Francisco: W.H.
Freeman and Company, 1962.

 

R. Kociba. (Personal communication.)

Skipper, H.E. and L.H. Schmidt. "A Manual on Quantitative Drug
Evaluation in Experimental Tumor Systems, Part 1," Cancer
Chemotherapy Rpts., 17, 1-144, 1962.

 

G. Valentine. (Personal communication.)

Smith, H.A. and T.C. Jones. Veterinary Pathology. Philadelphia:
Lea and Febiger, 1961.

 

Dunham, L.J. and H.L. Stewart. "A Survey of Transplantable and
Transmissible Animal Tumors," J, Nat. Cancer Inst., 12(2),
1299-1377, 1953.

 

 

Stewart, H.L., K.C. Snell, L.J. Dunham and S.M. Schlyer. Trans-
plantable and Transmissible Tumors 9f_Animals. Washington,
D.C.: Armed Forces Institute of Pathology, 1959.

 

Kociba, R. "Cancer Chemotherapeutic Properties and Toxologic
Effects of Cis-Platinum(II)diamminodichloride," Doctoral
Thesis, Department of Pathology, Michigan State University.

Barnum, C.P., C.D. Jardetzky and F. Halberg. "Time Relations Among
Metabolic and Morphologic 24-Hour Changes in Mouse Liver,"
Amer. J, Physiol., 195, 301—310, 1958.

Rawson, K.S. "Experimental Modification of Mammalian Endogenous
Activity Rhythms," Photoperiodism and Related Phenomena ig_
Plants and Animals, edited by R.B. Withrow. Washington, D.C.:
American Association for the Advancement of Science, 1959.

Pp. 791—800.

 

 

Halberg, F., E. Halberg, C.P. Barnum and J.J. Bittner. "Physiologic
24-Hour Periodicity in Human Beings and Mice, the Lighting
Regimen and Daily Routine," Photoperiodism and Related Phenomena
Jn_Plants and Animals, edited by R.B. Withrow. Washington,
D.C.: American Association for the Advancement of Science,
1959. Pp. 803-878.

 

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

102

Smith, P.K., M.V. Nadkarni, E.G. Trams and G. Davison. "Distribu-
tion and Fate of Alkylating Agents," Ann. N.Y. Acad. Sci., 68,
834-852, 1958.

Chaudhuri, N.K., B.J. Mbntag and C. Heidelberger. "Studies on
Fluorinated Pyrimidines. III. The Metabolism of
S—Fluorouracil-Z—C14 and S—Fluoroorotic-Z—C14 Acid in Vivo,"
Cancer Res., J§(l), 318-328, 1958.

 

Fountain, J.R., G.B. Waring, D.J. Hutchinson and J.H. Burchenal.
"Distribution of Amethopterin in Normal Mouse Tissue Following
Intravenous Injection," Proc. Soc. Exper. Biol. Med.,_§L,
193*196, 1958.

Mizuno, N.S. "Distribution of Tritiated Methyl Ester of Streptonigrin
in Mice Bearing Sarcoma 180," Biochem. Pharmacol., J2, 394-395,
1966. .

Kato, M. "Distribution and Excretion of Radiomanganese Administered
to the Mouse," Quart. J, Exper. Physiol., 48, 355-369, 1963.

 

Bond, V.P., T.M. Fleidner and J.0. Archambeau. Mammalian Radiation
Lethality. A_Disturbance Jn_Ce11ular Kinetics. New York:
Academic Press, 1965.

 

L. Van Camp. (Personal communication.)

Metcalf, D. The Thymus, Recent Results Jn_Cancer Research, Volume 5.
New York: Springer-Verlag, Inc., 1966.

 

 

Savard, K. and F. Homburger. "Thymic Atrophy and Lymphoid Hyperplasia
in Mice Bearing Sarcoma 180," Proc. Soc. Exper. Biol. Med., 19,

Dunn, T.B. "Normal and Pathological Anatomy of the Reticular Tissue
in Laboratory Mice, with Classification and Discussion of
Neoplasms," J, Nat. Cancer. Inst., J4(2), 1281-1430, 1954.

 

Oliverio, V.T. and C.G. Zubrod. "Clinical Pharmacology of the
Effective Antitumor Drugs," Ann. Rev. Pharmacol., 5, 335-356,
1965.

 

Philips, F.S., S.S. Sternberg, L. Hamilton and D.A. Clarke. "The
Toxic Effects of 6+Mercaptopurine and Related Compounds,"
Ann. N.Y. Acad. Sci., 69, 283-296, 1954.

 

Barrow, J. and J.L. Tullis. "Sequence of Cellular Responses to
Injury in Mice Exposed to 1,000R Total-Body R-Radiation,"
Arch. Path., 3;, 391—407, 1952.

 

Levine, W.G. "Heavy-Metal Antagonists," The Pharmacological Basis
9f_Therapeutics, edited by L.S. Goodman and A. Gilman. New
York: Macmillan Company, 1967. Pp. 929—942.

 

 

55.

56.

57.

58.

103

Harvey, S.C. "Heavy Metals," The PharmacologicalBasis gf_Thera-
peutics, edited by L.S. Goodman and A. Gilman. New York:
Macmillan Company, 1967. Pp. 943-975.

 

Bowen, H.J.M. and D. Gibbons. Radioactivation Analysis. New York:
Oxford University Press, 1963.

 

Lenihan, J.M.A. and S.J. Thomson. Activation Analysis. Principles

 

and Applications. New York: Academic Press, 1965.

 

Taylor, D. Neutron Irradiation and Activation Analysis. New York:
D. Van Nostrand Coupany, Inc., 1964.

 

‘

APPENDIX

APPENDIX

Neutron Activation Analysis

 

Activation analysis is a method of chemical analysis primarily based
on the principle that a stable isotope can be induced, by irradiation
with nuclear particles or photons of sufficient energy, to undergo a
nuclear transformation to a radioisotope, which can then be detected and
analyzed—--both qualitatively and quantitatively. Although activation
with charged particles and high-energy photons is technically possible,
these types of activation analysis have not been widely developed. For
charged particles there is always a threshold energy which must be
exceeded before activation can be achieved. Moreover, although light
elements have reasonable capture cross-sections for charged particles,
these particles have very little penetration power in solid materials
and exposed targets are thus raised to high temperatures. For photon
activation the threshold energies are even higher and the cross-sections
of most nuclides much lower.56

Activation with neutrons and, in particular with thermal neutrons,
has received the most attention and greatest degree of development.
Neutrons as neutral particles face no energy threshold for nuclear inter-
action and most nuclei have high capture cross—sections for thermal

neutrons. Moreover, high neutron fluxes are readily available from

modern nuclear reactors.

104

105

Various reactions are possible following bombardment with neutrons,
depending on the kinetic energy of the incident particles. For thermal
neutrons,(kinetic energy at 200C approximately 0.025eV) the predominant
reaction is the (n,y) process. With the capture of a neutron, the
nuclide is raised in atomic weight but not atomic number, thus retaining
its chemical identity. The new—formed nuclide is in an excited state
and promptly drops to its ground state with the release of characteristic
y—ray(s) called prompt y's.27 (Although possible, it is not ordinarily
feasible to perform analyses by detection of these prompt y—rays.) If
the new-formed nuclide is radioactive, as is the usual case, it will
decay with time as any radioisotope. For thermal neutron reactions, the
usual forms of decay are internal conversion or B-—particle emission, or
a combination of the two. These reactions are usually accompanied by the
release of one or more characteristic Y-rays.

Most neutron-rich nuclides decay by B-—emission, a transformation
which tends toward nuclear stability by conversion of a nuclear neutron
to a nuclear proton, with the emission of a B——particle and a
neutrino. The new, usually stable nuclide is one atomic number higher
than the original nuclide (e.g. 2(4Na--:E:D24Mg). Most artifically pro-
duced radionuclides are only slightly away from the stability diagonal
and thus decay to stable nuclides in a single decay event as opposed to
the long chains of consecutive decays found for the naturally radioactive
species.

Bombardment with fast neutrons (kinetic energies in the MeV range)
can produce several types of reactions———(n,n'), (n,p), (n,o) and (n,2n).
Such processes seldom have large capture cross-sections. Moreover, the

(n,p) and (n,a) reactions face Coulombic barriers against the escape of

106

the proton and alpha particle respectively. In the case of a few of the
heaviest elements, bombardment with fast neutrons with kinetic energies
in the lOOMeV range, causes spallation or breakdown of the target nuclei
into a mixture of lighter nuclides.

The activities produced by irradiation with neutrons can be

expressed as follows:

At

A = N00 (1 - e" i); A = 0.693/T,

u\

P

where A = activity induced, disintegration/second
N = number of target atoms
0 = capture cross-section, cm
0 = neutron flux, neutrons/cm /second
ti= irradiation time
TJ= half-life of product isotope

The term (1 - e—Ati) is called the saturation factor. As ti becomes
large compared to T1 this factor approaches unity or A = N00.
/2
As with any radioactive sample, the radioactivity induced decreases

with time according to

A = A (e-O.693/T%
t O

)

where A = initial activity
At = activity after time t
t elapsed time

Therefore, at a given time the activity of a given radionuclide is
dependent on several factors---some intrinsic to the nuclide; others
variables of the irradiation process. In cases of long irradiation,
radionuclides with relatively short half—lives may decay considerably
before irradiation is complete. Therefore, irradiation variables must
be suitably adjusted for the radionuclide(s) of interest. (For quali—

tative analysis of all elements present in a given sample, therefore,

107

several irradiations of different lengths may be required.)

Various instruments (Geiger-Mueller counters, ion chambers, scin-
tillation counters, gas counters, spectrometers, and semiconductor detec-
tors) are available for detection of the radioactive emissions induced---
charged particles and/or Y-rays. In termal neutron activation analysis,
B--partic1es and‘Y—rays are the types of emission usually investigated.
Beta particles radiated from a given nuclide are not monoenergetic but
exhibit a continuous energy distribution from zero to some maximum energy
characteristic of the emitting nuclide.37 Since most irradiated samples
will contain more than one 8--emitter, each with continuous energy
spectra, a chemical separation of the nuclides is necessary before
measurements can be made. After isolation of the various radionuclides,
a simple counter is adequate for quantitative measurement of the individual
nuclides---once the absence of extraneous y-ray producers has been
ascertained and the B-—spectrum has proved that of the separated B-~emitter
only.

Since nearly all activated nuclides emit y-rays of characteristic
energy, the kind and amounts of elements present in an activated sample
can be ascertained from the total y-ray spectrum. When y-rays pass
through matter they can ionize atoms and molecules, losing all or a
fraction of their kinetic energy in so doing. There are three basic
modes of interaction: photoelectric interaction, Compton interaction
and pair production. For the photoelectric effect the incoming y-ray
imparts all of its energy to an orbital electron, which is thus ejected
with a kinetic energy equal to that of the original y—ray minus the
binding energy of the electron. It is a major mode of interaction for

low—energy Y-rays (0.5 MeV or less) in elements of high atomic number.

108

The Compton effect (or Compton scattering) occurs when the incoming
y-ray loses only a fraction of its energy in an interaction with an
orbital electron. Provided the y-energy is appreciably greater than the
electron binding energy, the Y—photon is deflected as though an elastic
collison had taken place.56 The photon moves off at some angle, dependent
on the particular collision event, with less energy than the incident
Y-ray. Likewise the Compton electron is scattered at an angle, with a
given kinetic energy. This type of interaction is important at y—energies
above 1.0 MeV.

In pair production the incident y-ray reacts with the nucleus of an
atom and is completely converted into an electron—positron pair, with
kinetic energy equal to that of the original photon less the energy of
creation. Since the rest mass energy of each particle is 0.51MeV, the
threshold for this event is 1.02 MeV.57 When the positron slows down
it reacts with any free electron present, annihilating both particles
and producing two 0.51 MeV photons. Pair production predominates in
the high photon-energy region, particularly in media of high atomic
number.

If the above reactions occur in a phosphor (highly fluorescent
solid or liquid substance such as doped NaI or anthracene) the de-
excitation and recombination processes convert the absorbed energy into
light pulses or scintillations, the brightness of which is proportional
to the total energy absorbed.56 Such is the basis of detection with
scintillation detectors. The most commonly used detector of this type
which is employed in activation analysis is the single-crystal NaI(Tl)
scintillation counter~——employed either as a flat or well-type crystal.

The short—range photo-electrons generated by the photoelectric reactions

109

are stopped in the crystal, imparting all their energy to the crystal.
This results in a total absorption or photoelectric peak with energy
characteristic of the originally emitting substance. The original inter-
action takes place on an iodide atom leaving a vacancy in the atom. As
electrons from higher orbitals "drop" to fill the vacancy created, a
characteristic iodine X-ray of 28 keV is emitted. If this reaction
occurs close to the crystal surface, the X-ray can escape the crystal
without detection. Thus the photoelectric event would not possess the
full energy of the original photon and a peak will instead appear at an
energy corresponding to the energy of the photoelectric peak minus 28
keV. This peak is called an "escape peak“.

Compton electrons formed have energy values from essentially zero
to a maximum cutoff point called the Compton edge, which represents the
maximum energy transfer between the photon and electron. Large angle
Compton processes (1800) cause a characteristic spectral feature called
the "back scatter peak" which looks quite like a photopeak at an energy
of about 200 keV. The ratio of photopeak height to Compton distribution
increases with the size of the detection crystal. (The actual sizes, of
course, are limited by numerous factors—-—a 3" by 3" NaI(Tl) crystal being
a commonly used size.)

When the emitting substances produce y-rays of sufficient energy to
cause pair-production in the scintillation crystal, this is evidenced in
the y-spectrum by the appearance of an "annihilation peak“ at 0.511 MeV.
The annihilation of a positron and electron produces two 0.511 MeV
photons, emitted in opposite directions. One or both of these photons
may escape the crystal and thus detection, but those that are detected

contribute to the height of the 0.511 MeV peak.

110

The "light flashes" or scintillations resulting from the above—
mentioned interactions in the crystal are converted to voltage pulses
and amplified by a multistage photomultiplier tube. For NaI(Tl) spec-
trometry, the crystal-photomultiplier ensemble is quite often connected
directly to an analyzer---single-channel or multi-channel depending upon
the requirements of the particular experiment. For most experiments a
multi-channel analyzer is most practical, usually with 512 to 1024
channels. When the sample has been counted for a preset "live time” and
the counts per channel suitably programmed in the memory, the resulting
y-ray spectrum can be read out in various ways, depending on the parti-
cular requirements and the equipment available. A visual display of the
spectrum can be produced by channeling the output onto an oscilloscope
screen or to a chart recorder. It can also be dumped out digitally as
actual counts per channel or punched onto tapes. By suitable calibration
the energy per channel, usually measured as keV, can be ascertained and
energies associated with the various photopeaks assigned.

A "live time" reading was stressed above as all analyzers have a
dead time which limits the counting rate which the equipment can handle.
(For a multi—channel analyzer the dead time is in the order of 80 usec.)58
Therefore, for samples with high count rates—--greater than the capa-
bility of the analyzer---counting times are in reality longer than the
preset "live time".

(Although the NaI(Tl) scintillation system has been used effectively
for y—ray analysis, it lacks the peak resolution often required for
analysis of multi-nuclide samples. To achieve greater resolution, semi-
conductor detectors can be employed——-e.g. the lithium—drifted germanium

detector, Ge(Li). Gamma-spectra measured with the use of such detectors

111

evidence excellent peak resolution but low counting efficiency.)

The information obtained either as 8 counts versus channel (or
energy) plot or as actual digital counts per channel must now be analyzed
according to the information desired. For a purely qualitative analysis
of the elements present this can, in many cases, by a straight—forward
identification of the nuclides present by the characteristic y-ray
energies found. From this information, identification of the elements
present in the original sample can be determined. In many instances
this analysis is complicated by various factors-—-several possible pre-
cursors forming the same radionuclide; y-energies of nuclides being too
close for complete resolution (at least with NaI(Tl) detectors); or a
general matrix interference in which the chief constituents of the
samples are activated very efficiently, causing high backgrounds and
poor counting efficiencies for detection of trace element y—rays. Except
for the first complication, radiochemical separation following irradiation
of the sample would be of benefit. The radionuclides formed are separated
into small groups or individually and then counted. In this way inter-
fering matrix constituents can be significantly removed and nuclides
producing y-rays of like energies can be separated from each other.

Quantitative measurements of the trace or matrix are possible.
Although absolute measurements are possible, they are seldom employed as
the variables of the detection system (counting efficiency, amplification
factors, etc.) as well as those peculiar to the nuclide itself and to
the irradiation process must be known with good accuracy. Comparative
methods are instead used in which the area Under the photopeak character-
istic of the radionuclide of interest (suitably corrected for background)

is compared with the corrected area resulting from a standard, containing

112

a known amount of the nuclide and irradiated and counted under conditions
identical to the sample being analyzed. For exact comparison, flux
variation at the various sample positions in the reactor or the other
neutron source employed must be controlled or known accurately and then
taken into consideration in the final calculations. Moreover, the
standard must have the same type of matrix as the samples being measured,
as far as feasible and necessary for the given system, as self-shielding
effects can differ significantly from medium to medium. Also, the
counting geometry of standard and sample should likewise be controlled.
For accurate and reproducible quantitative measurement, adequate
counting statistics must exist for the photopeak of interest. Suitable
adjustment of the "live time" for counting, considering sample activity
and the half—life of the nuclide, can produce satisfactory count sta-
tistics. (The radioactive decay process itself is statistical in nature—-—
all randomly occurring processes being subject to fluctuations around a
mean. Thus, even if all other variables are carefully controlled, the
counting rate for a given sample will vary from measurement to measure-
ment.) Numerous statistical techniques, usually computerized for con-
venience in handling large quantities of data, have been devised for
determining quantitatively amounts of trace elements and major matrix
constituents. Although such non—destructive methods of detection are
preferred, interferences often make it difficult if not impossible to
undertake accurate, reproducible quantitative measurements, even with
the best statistical analyses of the data. (For example the large
amount of 23Na, which is readily converted to 24Na, T111 = 15 hours, by
thermal neutron irradiation, makes it extremely difficult to measure

short—lived isotopes of trace elements generated in biological matrices-~—

113

particularly for those nuclides whose characteristic -rays have low
energies which would be hidden beneath the extremely high Compton scatter
produced by large quantities of 24Na.) In such cases radiochemical
separation of the radionuclide(s) of interest must precede analysis.
Separation of nuclides is usually performed after rather than before
irradiation to avoid possible contamination which might be introduced in
the separation procedure---either from the reagents used and/or from
mechanical manipulation of the sample.

Since activation analysis is often used for the determination of the
levels of trace elements present, a radiochemical separation can entail
recovery of microgram amounts or less of the desired nuclide. Unless it
has been previously ascertained that the particular procedure of
separation consistently yields close to 100% recovery, carriers are
usually added. A measured amount---usually several milligrams-—-of a
compounds containing a known amount of the element to be separated, in
the same valence state, is added to the sample before any separation
steps are undertaken. Measurement of the amount of carrier present at
the end of the separation procedure, by ordinary analytical techniques
(e.g. spectral analysis), will give the percent recovery for the procedure
in that particular instance. (No carrier was added in the present experi-
ment, as the hexachloro salts of heavy metals are known to be, for all

practical purposes, 100% retained on the resin.28’29

)

If all sources of error are considered and suitably controlled and/
or included in the calculations, neutron activation analysis, with Y—ray
analysis, can be a powerful qualitative and quantitative analytical tool.
Since each field of interest presents different matrices and conditions,

the applicability of this type of analysis will vary not only from field

to field but among various problems of a given field.

114

For the present investigations---detection of microgram amounts of
platinum in a biological matrix——-a relatively long irradiation time,
followed by a suitable radiochemical separation proved to be an amenable
system. Table 14 lists various elements, found in relatively high con—
centrations in biological media, which form radionuclides with neutron
bombardment. Many elements produce isotopes with half-lives comparable
to or longer than that of 199Pt (30.0 minutes) and with Y-ray photopeaks

of considerably higher energy. However, 199Au has a considerably longer

half-life—--3.15 days26 and is formed as follows:

198 n 199
‘--‘9185.9; 316.9; 542.8 keV Y—rays
B-
199Au
‘--‘9158.3 and 208.2 keV Y-rays
B-
199
“8

The extremely high concentration of 24Na and 42K in all tissue and 59Fe
in blood—--each with reasonably long half-1ives——-still represent inter-
ferences to accurate Y-ray analysis. Use of the anion—exchange resin
successfully eliminates these interferences.

Although actual limits of detectability were never accurately deter-
mined for this system, a sample containing 6Ug Pt consistently gave
approximately 3-4 x 103 counts above background at maximum peak height,
for the 158 keV photopeak, for a 20 minute "live time" count (a total
peak area of approximately 200,000, with corrected area approximately
90,000). Thus, for most samples (with the possible exception of the
intestines, as interferences from fecal debris contamination were present

here) 0.IUg Pt was easily detectable and smaller amounts were actually

115

detected, for several spleen samples, with reasonable counting sta-

tistics.

Table 14:

116

Elements of biological media activated by neutron bombard-

 

 

ment.26
Element Radionuclide formed T,E Main Y~ray photopeak (keV)
24
Na Na 15 h. 1368.4; 2753.6
K 42x 12.52 h. 1524.7
47
Ca Ca 4.7 d. 160.0; 1296.9
490a 8.8 min. 3083; 4071
Mg 27Mg 9.45 min. 844.0; 1014.1
c1 3801 37.29 min. 1642.0; 2166.8
Fe 59Fe 45.1 d. 192.5; 1098.6; 1291.5
Zn 652n 245 d. 1115.4
69mZn 13.8 h. 438.7
7lZn 2.2 min. 121.8; 511.6; 910.1
I 1281 25.4 min. 442.7; 526.3
Cu 640u 12.8 h. 1345.5
66Cu 5.1 min. 1039.0
80
Br Br 4.5 h. 640.4; 617.0; 665.7
82Br 35.87 h. 554.3; 619.0; 776.6
86
Rb Rb 18.66 d. 1076.6
86mRb 1.02 min. 555.8
88Rb 17.8 min. 898.0; 1836.1; 2677.6

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