EPA-650/1-74-013
Final Report
USE OF LbJCOCYTE METABOLISM
AS A HEALTH EFFECTS INDICATOR
Prepared for:
ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK
NORTH CAROLINA
CONTRACT 88-02-0713
STANFORD RESEARCH INSTITUTE
Menlo Park, California 94025 • U.S.A.
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Final Report
April 1974
USE OF LEUCOCYTE METABOLISM
AS A HEALTH EFFECTS INDICATOR
By. KENNETH D. LUNAN
Prepared for:
ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK
NORTH CAROLINA
CONTRACT 68-02-0713
SRI Project LSU-2430
Approved by:
W. A. SKINNER, Executive Director
Life Sciences Division
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ABSTRACT
The objective of this study was to evaluate the use of leucocytes as
a responsive bioindicator of lead, cadmium, and platinum intoxication in
rabbits. Adult rabbits were injected intraperitoneally with cadmium
chloride, lead acetate, and saline daily for one or three weeks. Toxicity
studies established the maximum permissible dosages for the metal treat-
ments. Leucocytes were isolated by density gradient centrifugation and
examined for their ability to synthesize deoxyribonucleic acid, ribonucleic
acid, protein, and phospholipid and to catabolize protein and phospholipid.
Rabbit leucocytes were also treated in vitro with sodium hexachloro-
platinate, and the same metabolic capabilities were assessed. Lead and
cadmium treatments produced a mild anemia, but the white cells were only
slightly affected. The one-week cadmium treatment and the three-week lead
and cadmium treatments depressed the synthesis of both nucleic acids. The
synthesis and degradation of protein and phospholipid were unaffected by
the metal treatments. Leucocytes from three-week control rabbits synthe-
sized all four classes of biomolecules at a faster rate than leucocytes
from the one-week control rabbits. In leucocytes treated with the platinum
salt in vitro, nucleic acid and protein synthesis were depressed, but
phospholipid synthesis was unaffected.
These results demonstrate that leucocytes may serve as a responsive
bioindicator of trace metal contamination.
This report was submitted in fulfillment of Contract 68-02-0713,
under the sponsorship of the Bioenvironmental Laboratory Branch, Environ-
mental Protection Agency.
iii
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ACKNOWLEDGMENTS
The successful completion of this research is due largely to the
technical skill and personal vigor and determination provided by Miss
Christine Feliton. I am very grateful for her participation.
Special thanks are due Mr. Theodore Jorgenson and Drs. Gordon
Newell (Director) and Daniel Sasmore of the Toxicology Department for
their cooperation and assistance in helping us overcome the difficult
problems associated with the preparation of leucocytes.
I would like to thank Drs. J. M. Mansfield and J. H. Wallace of the
University of Louisville School of Medicine for assisting us in success-
fully applying their method of preparing leucocytes.
Finally, I thank Drs. George Goldstein, Edward Faeder, and Anthony
Colucci of the Bioenvironmental Laboratory Branch, Environment Protection
Agency, Research Triangle Park, North Carolina, for their vital assistance
in obtaining funds to support this research and for many helpful dis-
cussions.
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CONTENTS
ABSTRACT ill
ACKNOWLEDGMENTS v
LIST OF ILLUSTRATIONS ix
LIST OF TABLES xi
INTRODUCTION .' 1
METHODS AND RESULTS 3
Preliminary Experiments 3
Biochemical Assays 3
Toxicity Studies 3
Intraperitoneal Toxicity (LD5Q) 3
Repeated Intraperitoneal Injections 8
Preparation of Leucocytes . 8
Isolation Experiments Using Ficoll-Hypaque ... 8
Isolation Experiments Using Gelatin 18
Isolation Experiments Using Dextran-Hypaque ... 20
Isolation Experiments Using Methycellulose-
Hypaque 22
The Procedure of Mansfield and Wallace 28
Phytohemagglutinin Standardization 29
Preliminary Lead and Cadmium Experiment 30
Control Experiment 36
One-Week and Three-Week Lead.and Cadmium Experiments ... 38
Animal Data 38
Blood Data 42
Biosynthetic Data 48
In Vitro Platinum Exposure Experiments 66
DISCUSSION 77
SUMMARY 87
GLOSSARY 89
REFERENCES 91
vii
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ILLUSTRATIONS
1 PHA Standardization . . . . 31
2 Leucine Depletion in Preliminary Lead and Cadmium
Experiment 34
3 Choline Depletion in Preliminary Lead and Cadmium
Experiment 35
ix
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TABLES
1 Variation in RNA and DNA from Replicate Samples of Rat
Lung Extracted with Alkali for One and Four Hours ..... 4
2 Replicate Analysis of Rat Lung Tissue ........... 5
3 Preliminary Analysis of Rat Leucocytes .......... 6
4 I. P. LD5Q Data for Rabbits Treated with Lead Acetate ... 7
5 I. P. LD5Q Data for Rabbits Treated with Cadmium Chloride . 7
6 Summary Data for Repeated Intraperitoneal Toxicity
of Lead Acetate and Cadmium Chloride ........... 9
7 Biosynthesis and Degradation of Lipids, Nucleic Acids,
and Protein in Control Rat Leucocytes ........... 11
8 Viability of Cultured Leucocytes ............. 13
9 Recoveries of Leucocytes from Rat and Rabbit Blood
in Plastic Centrifuge Tubes ................ 13
10 Recovery of Leucocytes from Rabbit Blood ........ . 14
11 Recovery of Rabbit Leucocytes in Various
Density Gradients ..................... 15
12 Recovery of Rabbit Leucocytes Following Hypotonic Lysis . . 16
13 Leucocyte Recovery from Rat Blood Following
Hypotonic Lysis ............ , ......... 17
14 Recovery of Rat and Rabbit Leucocytes
After Sedimentation in Gelatin .............. 19
15 Incorporation of Label in Preliminary
Lead and Cadmium Experiment ................ 33
16 Incorporation of %-Thymidine and
in Leucocytes from Three Control Animals ......... 37
17 Incorporation of 3H-Leucine«and l^C-Choline
in Leucocytes from Three Control Animals ......... 37
xi
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18 Degradation Rate of Protein and Phospholipid
in Three Control Animals 39
19 Rabbit Body Weights During the One-Week Exposure
Experiment
20 Rabbit Body Weights During the Three-Week Exposure
Experiment 41
21 Whole Blood Data - One-Week Experiment 43
22 Whole Blood Data - Three-Week Experiment 44
23 Differential Counts for One-Week Experiment 46
24 Differential Counts for Three-Week Experiment 47
25 Leucocyte Viability for One-Week Experiment 49
26 Leucocyte Viability for Three-Week Experiment 50
27 A Ranking of the Relative Ability of Individual
Rabbits to Incorporate Four Radioactive Substrates .... 51
28 Carbon 14 to Tritium Ratios in One-Week Experiment .... 53
29 Carbon 14 to Tritium Ratios in Three-Week Experiment ... 53
30 One-Week Experiment—Thymidine and Uridine Incorporation . 54
31 One-Week Experiment—Ratios of Uridine-to-Thymidine
Incorporation 55
32 One-Week Experiment—Leucine Incorporation and Depletion . 56
33 One-Week Experiment—Rate of Leucine Depletion 57
34 One-Week Experiment—Choline Incorporation and Depletion . 58
35 One-Week Experiment—Rate of Choline Depletion 59
36 Three-Week Experiment—Thymidine and Uridine
Incorporation 60
37 Three-Week Experiment—Ratios of Uridine-to-Thymidine
Incorporation 61
38 Three-Week Experiment—Leucine Incorporation
and Depletion 62
xii
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39 Three-Week Experiment—Rate of Leucine Depletion 63
40 Three-Week Experiment—Choline Incorporation
and Depletion 64
41 Three-Week Experiment—Rate of Choline Depletion 65
42 Summary of Effects of Lead and Cadmium Treatment 67
43 Incorporation of Labeled Substrates into In Vitro
Platinum-Treated Leucocytes—First Experiment -
Incorporation and Degradation Data 69
44 Incorporation of Labeled Substrates into In Vitro
Platinum-Treated Leucocytes—First Experiment -
Percent Incorporation and Degradation 70
45 Incorporation of Labeled Substrates into In Vitro
Platinum-Treated Leucocytes—Second Experiment -
Incorporation Data 72
46 Incorporation of Labeled Substrates into In Vitro
Platinum-Treated Leucocytes—Second Experiment -
Percent Incorporation 73
47 Incorporation of Labeled Substrates into In Vitro
Platinum-Treated Leucocytes—Third Experiment -
Incorporation and Degradation Data 74
48 Incorporation of Labeled Substrates into In Vitro
Platinum-Treated Leucocytes—Third Experiment -
Percent Incorporation and Degradation 75
xiii
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INTRODUCTION
Of increasing concern are the public health hazards associated with
the insidious accumulation of heavy metals in the bodies of man and ani-
mals as a result of contamination of the environment with an increasing
amount and variety of trace metals derived from industrial sources. Any
excess of environmentally derived material beyond that needed for optimun
growth and development may be termed a "pollutant burden." It can be
assumed that any biological response resulting from a pollutant burden
is undesirable. In a practical sense, it may be sufficient to limit a
pollutant burden to that level that produces a biological response with
uncertain physiological significance. Higher levels that may herald in-
cipient disease would be regarded as intolerable.
The problem of recognizing the level of a pollutant burden at a pre-
clinical stage in an individual is formidable. A preclinical biological
response induced by the pollutant burden may manifest itself as a meta-
bolic or physiologic alteration. The use of leucocytes as a bioindicator
offers considerable advantages not available in other readily accessible
biological materials. The most important feature is that the leucocyte
is a complete cell that functions in all major areas of metabolism. By
examining the response of the leucocyte to a test reagent, it may be
possible to assess the metabolic status of the individual. Numerous
examples exist in which the leucocytes from individuals suffering from
inborn errors of metabolism exhibit the same metabolic characteristics
as their host. Also, because it is derived from the reticuloendothelial
system and therefore possesses immunological characteristics not found
in other cells and because of its broad range of body functions, the leu-
cocyte undoubtedly will find wider use as a bioindicator in many areas
of environmental research.
The research described in this report was undertaken to assess the
use of leucocytes as a bioindicator of trace metal contamination using
lead and cadmium. In the event that one or more measurable metabolic
processes in leucocytes are effected by heavy-metal treatment of mammals,
then these results may be applicable to the determination of damage in
man due to chronic exposure to heavy metals.
A large portion of the work on this contract was devoted to develop-
ing appropriate methods for the isolation and culture of leucocytes in
the rabbit. Although rats were originally proposed as the experimental
animal, rabbits were finally chosen by mutual agreement between EPA and
SRI to facilitate the isolation of larger quantities of leucocytes than
would be possible from the rat. The methods found in the literature for
leucocyte preparation and culture are largely devoted to human cells, and
it soon became apparent that these methods were not adequate for animal
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cells. Late in the contract year, Mansfield and Wallace described solu-
tions for many of the difficulties encountered in this research. This
led to modifications resulting in a successful method for the preparation
and culture of rabbit leucocytes. The principal difference in methodol-
ogy is the inclusion in the culture medium of autologous rabbit plasma
as the blood protein supplement.
The main body of results reported herein concerns the experiments
in which animals were injected daily for one or three weeks with physio-
logical saline, lead acetate, or cadmium chloride solutions. Some pre-
liminary results with control animals and with those treated for two
weeks are also reported.
The final experiment deals with the in vitro effect of sodium hexa-
chloroplatinate on rabbit leucocytes. This form of platinum was chosen
by mutual agreement between EPA and SRI as the third metal to be studied
in this research.
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METHODS AND RESULTS
Preliminary Experiments
Biochemical Assays
Before leucocytes became available to us, lung tissue was used to
perfect various biochemical assays. Lipids were extracted with chloro-
fonn/methanol (2:1) and weighed [1]. Acid-soluble nucleotides were re-
moved in ice-cold 0.2% perchloric acid. RNA was removed by digesting
the residue for four hours at 37°C in 1 N KOH. DNA was subsequently re-
moved by digesting for one hour at 65°C in 10% perchloric acid. These
materials were determined spectrophotometrically [2], The residual pro-
tein was dissolved in base and determined by the Lowry method [3],
The data in Tables 1 and 2 represent typical analyses of lung tis-
sue. Table 1 shows that longer alkaline hydrolysis yields slightly more
RNA and less DNA, with the total nucleic acids remaining about the same.
These data attest to the validity of the observations of Fleck and Munro
[4] that the efficiencies of the alkaline hydrolysis for RNA and of the
acid hydrolysis for DNA depend on the tissue being analyzed. Further,
the separation of DNA and protein is a balance between maximizing DNA
extraction and minimizing protein degradation [4]. Thus, the hydrolysis
conditions employed here for lung tissue may not be optimal for leucocyte
analysis. Table 2 shows the reproducibility of these assays. The lack
of agreement between the two tables is due to the differences in age of
the lung tissue.
Table 3 presents data for one leucocyte preparation.
Toxicity Studies
Intraperitoneal Toxicity (LD5p)
Adult New Zealand white rabbits of mixed sex, each weighing
1.87 to 2.97 kg, were used. Lead and cadmium salts were administered
intraperitioneally in graded dosages. Tables 4 and 5 summarize the
intraperitoneal toxicity (LD,-0) of lead and cadmium, respectively, cal-
culated by the methods of Thompson and Weil [5] and Weil [6].
Lead—The source of lead was lead acetate (Baker, AR Grade),
expressed as Pb (CH3COO)2'3 H20. Water was the diluting vehicle used
in administering the compound, as a 50% (w/v) aqueous solution.
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Table 1
VARIATION IN RNA AND DNA FROM REPLICATE SAMPLES OF RAT LUNG
EXTRACTED WITH ALKALI FOR ONE AND FOUR HOURS3
Experiment
Conditions
Acid-Soluble
Nucleotides
RNA
DNA
Total
Nucleic Acids
1.0 N NaOH, 1 hr
1.0 N KOH, 4 hr
1.49
1.42
Avg 1.46
1.44
1.43
Avg 1.44
2.51
2.80
2.66
3.24
4.03
3.64
6.40
6.47
6.44
5.31
5.35
5.33
8.91
9.27
9.09
8.55
9.38
8.96
Units in mg constituent per gram wet tissue.
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Table 2
REPLICATE ANALYSIS OF RAT LUNG TISSUE
Acid-Soluble Total
Sample No. Lipids Nucleotides RNA DNA Nucleic Acids
Wet Tissue Weights3
1
2
3
4
5
6
Mean
Std.
Std.
Dev.
Error
88.
88.
89.
96.
99.
96.
93.
+ 4.
+ 1.
5
7
1
3
4
0
0
79
96
0.
0.
0.
0.
0.
0.
0.
+ 0.
+ 0.
433
406
388
352
326
385
382
0380
0166
3.
3.
3.
3.
3.
3.
3.
+ 0.
+ 0.
17
34
25
18
19
13
21
0753
0307
2
2
2
2
2
2
2
+ 0
+ 0
.63
.61
.40
.60
.64
.40
.55
.112
.0456
5.
5.
5.
5.
5.
5.
5.
+ 0.
+ 0.
80
95
65
78
83
53
76
147
0599
Dry Tissue Weightsb
1
2
3
4
5
6
Mean
Std.
Std.
Dev.
Error
532
527
539
570
588
570
554
+ 24.9
+ 10.2
1.
1.
1.
1.
1.
1.
1.
+ 0.
+ 0.
70
58
52
33
21
46
47
175
0714
12.
13.
12.
12.
11.
11.
12.
+ 0.
+ 0.
4
1
8
0
9
8
3
515
210
10.
10.
9.
9.
9.
9.
9.
+ 0.
+ 0.
3
1
43
80
81
10
77
448
183
22.
23.
22.
21.
21.
20.
22.
+ 0.
+ 0.
7
2
2
8
7
9
1
808
330
a
Units in mg constituent per gram wet tissue.
b
Units in mg constituent per gram dry tissue.
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Table 3
PRELIMINARY ANALYSIS OF RAT LEUCOCYTES3
Acid-Soluble Total
Sample Lipids Nucleotides RNA DNA Nucleic Acids
1 58.0 0.0703 0.762 5.83 6.59
aUnits in mg constituent per gram wet tissue.
Levels of 79, 178, 400, 1350 and 4560 mg/kg were used in the
rangefinding study. Based on these preliminary dosages, subsequent
treatment levels of 178, 267, 400, 600, 900 and 1350 mg/kg were used to
determine the single dose LD5Q for lead, as lead acetate. Restlessness
and rapid breathing, lasting from 10 minutes to three hours, occurred
as the dose increased from 600 to 1350 mg/kg. At time of sacrifice or
death, blood-tinged fluid was found in the peritoneal cavity at these
dosages. At lower doses of 178 to 400 mg/kg, the rabbits appeared rest-
less and exhibited rapid breathing immediately after injection and for
the next 15 to 40 minutes; necropsy of all animals revealed no gross
pathological changes.
The LD5Q of lead acetate is 470 mg/kg, with 95% confidence
limits of 283 to 780 mg/kg.
Cadmium—The source of cadmium was cadmium chloride (Mallin-
ckrodt, AR grade), expressed as CdCl2.2-l/2H20. The compound was ad-
ministered as an aqueous solution at varying concentrations in a con-
stant volume of 1 ml/kg.
Levels of 35.1, 52.7, 79, 118, 178, and 267 mg/kg were used
in the range-finding study. Based on these preliminary dosages, subse-
quent treatment levels of 3.08, 4.62, 6.93, 10.4, 15.6, and 23.4 mg/kg
were used to determine the single dose LD5Q for cadmium, as cadmium
chloride. Restlessness and rapid breathing were observed at all levels,
with increasing severity and duration of effects as the dosage increased.
Necropsy of all animals showed the presence of blood-tinged fluid in
the peritoneal cavity.
The ID50 of cadmium chloride is 8.5 mg/kg, with 95% confidence
limits of 2.46 to 29.3 mg/kg.
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Table 4
I.P. LD5Q DATA FOR RABBITS TREATED WITH LEAD ACETATE
Dose
(Mg/Kg)
178
267
400
600
900
1350
Number Dead/
Number Treated
0/4
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Repeated Intraperitoneal Injections
Adult New Zealand white rabbits, each weighing 2.35 to 2.97 kg,
were used. Three rabbits per treatment level received 21 repeated in-
jections 24 hours apart. Table 6 summarizes the toxicity data for both
lead and cadmium. Levels of 75 and 25 mg/kg for lead acetate and 1 mg/kg
for cadmium chloride were used.
The three rabbits that received 75 mg/kg lead acetate showed
diarrhea and emaciation after five days of treatment, with weight losses
in excess of 25%. Treatment was terminated after five days, but the
rabbits were held to observe post-treatment effects. Recovery from
treatment did not occur; the three rabbits died at two, three, and seven
days post-treatment.
At 25 mg/kg, one rabbit died after ten injections and another
after 15 injections. The third rabbit completed the 21 injection regi-
men, but died six days post-treatment. Body weight losses were 20, 37,
and 28%, respectively, for the three rabbits. All three rabbits treated
with 1 mg/kg cadmium chloride survived the 21-day exposure period, body
weight losses were 15% or less.
After evaluation of the data on repeated intraperitioneal in-
jections, 10 mg/kg for lead acetate and 1 mg/kg for cadmium chloride were
selected as the treatment levels for the one-week and three-weeks ex-
periments .
Preparation of Leucocytes
In these and all subsequent experiments, rabbit blood was
obtained by cardiac puncture (see the section "The Procedure of
Mansfield and Wallace," pg. 28).
Isolation Experiments Using Ficoll-Hypaque
A suspension of purified, erythrocyte-free leucoytes was pre-
pared by a one-step, density gradient centrifugation, using Ficoll and
Hypaque* as the gradient material [7,8]. Heparinized rat blood diluted
1:1 with saline was layered over the gradient and centrifuged. The leu-
cocytes layered on top of the gradient, while the erythrocytes sedimented
to the bottom of the tube below the gradient and completely separated
*Hypaque is the trade name for sodium 3,5-diacetamide-2,4,6-triiodoben-
zoate, also called sodium diatriazoate (Winthrop Laboratories). Most
literature references refer to the use of Isopaque, also a product of
Winthrop, as a gradient material. However, Isopaque, the N-methyl de-
rivative of Hypaque, is no longer commercially available because of its
toxicity.
8
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Table 6
SUMMARY DATA FOR REPEATED INTRAPERITONEAL TOXICITY
OF LEAD ACETATE AND CADMIUM CHLORIDE
Dose Rabbit No. Body Wt (kg) % Body
(Mg/Kg) and Sex 0_ 1 Wk 2 Wk 3 Wk Wt. Loss Time to Death
Lead Acetate
75a 237 M 2.73 2.05 — — 25 7 days
238 M 2.66 1.78 — — 33 8 days
239 M 2.35 1.72 — — 27 12 days
25 243 F 2.29 1.70 1.44 — 37 15 days
244 F 2.74 2.44 2.42 1.96 28 27 days
245 F 2.97 2.37 — — 20 10 days
Cadmium Chloride
1 240 F 2.96 2.63 2.56 2.53 15
241 F 2.70 2.35 2.60 2.52 7
242 F 2.91 2.54 2.27 2.48 15
aTreatment terminated after 5 days—rabbits severely dehydrated, with
body weight loss.
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from the leucocytes. The bulk of the plasma layer above the leucocytes
was aspirated, and the leucocytes were removed with a Pasteur pipette,
washed with Hank's balanced salts solution (BBSS), and resuspended in
HBSS. Approximately 0.8 x 10^ leucocytes/ml of whole blood was recovered.
Although cell recovery was not determined in this experiment based on a
typical WBC of 5 x 10^ leucocytes/ml of whole blood, the recovery can be
estimated at approximately 16%. The Trypan blue dye exclusion test for
cell viability indicated over 95% viability.
Rat leucocytes were prepared and incubated overnight at a con-
centration of 2 x 10° cells/ml in Eagle's minimal medium (MEM) (contain-
ing 20% calf serum, 2 mM glutamine, and 250 units/ml each of penicillin
and streptomycin) at 37°C.* Difco phytohemagglutinin-M (PHA-M) (0.05
ml/2 ml of cell suspension) was added, and the cultures were incubated
for four days. At the end of four days, the cell count, initially
2 x 106/ml, had risen slightly and viability remained at about 97% of
total cells.
The cultures then received 1 //c/ml of each of the following
radioactive substrates: 5-3H-uridine, 2-l^C-thymidine, 3H-A-leucine (U),
and 1,2-^C-choline chloride. After incubation of the tubes for two
hours, the cells were harvested and washed with medium containing a 100-
fold excess of the above unlabeled compounds. Lipid, acid-solubles, RNA,
DNA, and protein were then extracted from one portion of the cells and
assayed for radioactivity in a liquid scintillation spectrometer. The
remaining cells were resuspended in more medium containing the unlabeled
compounds and were incubated an additional two hours. The cells were
then harvested, washed, and extracted in the same way as the first group
of cells.
The data in Table 7 follow the expected pattern, with the cells
from Tubes 2 and 4 (representing a two-hour degradation period) having a
significantly lower level of counts than the cells from Tubes 1 and 3.
Thus, at the end of two hours, lipid had declined to 3% of the zero-time
value. Similarly, RNA, DNA, and protein declined to 12%, 18%, and 22%,
respectively. Therefore, these data indicate a significant rate of deg-
radation of lipid, protein, and nucleic acid.
The major difficulty encountered in this experiment was the
small amount of cellular material and, consequently, the small amounts
of lipid, RNA, DNA, and protein extractable from the cells.
This initial success in both the preparation and culture of
leucocytes was found difficult to reproduce. One problem that became
readily apparent was due to the pH of the medium. When the gas phase
above the medium was replaced with 5% C02 in air at the time of inocula-
tion, the medium retained the proper pH throughout the culture period.
Nevertheless, the practice was adopted of renewing the gas phase daily.
The deleterious effect of not controlling the pH is indicated by the
*The culture conditions employed were a synthesis of the best features
described in References 9-12.
10
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Table 7
BIOSYNTHESIS AND DEGRADATION OF LIPIDS, NUCLEIC ACIDS,
AND PROTEIN IN CONTROL RAT LEUCOCYTES
Radioactive
Label:
Measurement
Tube
1
2
S
4
substrate:
•
•
Post-
Incubation
0
2 hr
0
2 hr
Lipid
Choline
c14
DPM-C14
Serum
Fetal
Fetal
Calf
Calf
Acid-
Solubles RNA
H3 or C
DPM-C14
Lipid
420b
14.1°
Uridine
14 H3
DPM-H3
Acid-
Solubles3 RNA
1074 95.6
200 22.4
1383 86.1
220 0
DNA
Thymidine
c14
DPM-C14
DNA
286
0
422
131
Protein
Leucine
H3
DPM-H3
Protein
52.8
51.3
45.3
0
aWhen counted for H3, Tube 3 gave a value of 49.72 disintegrations per minute (DPM);
the others were zero.
Combined counts for Tubes 1 and 3,
°Combined counts for Tubes 2 and 4.
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data in Table 8. Although sterile conditions were not strictly enforced
in preparing these preliminary cultures, bacterial contamination was not
evident in any of these culture experiments, thus attesting to the effi-
cacy of the antibiotics included in the culture medium.
Rat and rabbit leucocytes obtained by the density gradient cen-
trifugation method developed for human blood [7,8,13] were consistently
low in white cell count and had a high proportion of residual erythrocytes.
Considerable effort was devoted to improving the leucocyte yield
and lowering the residual erythrocyte count prior to the development by
Mansfield and Wallace [14] of their improved technique, which was adopted
in this research. Although it appears that the erythrocytes cannot be
completely eliminated, acceptable yields of white cells have been attained.
Initially separation experiments were performed in plastic dis-
posable centrifuge tubes until it was observed that white cells apparently
adhered to the walls of these tubes. Table 9 shows the low recovery of
white cells in these experiments. In all succeeding experiments, glass
tubes were used, and recoveries were generally improved. The leucocyte
count varied with the individual animals but averaged about 5 x 10"/ml of
whole rabbit blood.
Table 10 shows the improved recoveries of leucocytes from rab-
bit blood when glass tubes were used and when the centrifugation step was
eliminated. Higher recovery is favored by separation at 37°C, but longer
separation time, dilution of the blood, application of blood to the gra-
dient, and increase of the osmolarity of the gradient are all somewhat
deleterious to high white cell counts. These factors also raise the pro-
portion of red cells in the white cell suspension. Increased temperature
tended to reduce red cell count. Elimination of the centrifugation step
increased the proportion of red cells nearly 20-fold; at the same time,
the white cell count was doubled (Table 11).
Table 11 also shows the improved white cell yield obtained when
the density of the gradient was reduced. (The density is directly related
to the relative proportion of Hypaque; thus, when the proportion of Ficoll
is increased, the density decreases.) However, even though the red cell
count is diminished at lower densities, it is still too high.
Previously, hypotonic lysis has been employed to eliminate con-
taminating erythrocytes from human leucocyte suspensions, evidently without
adverse effects on the white cells, as judged by electron microscopy, via-
bility, and several biochemical parameters [15]. In one case, rabbit white
cells were subsequently cultured for 18 hours [16].
Hypotonic lysis [15] was employed to reduce the red cell count,
as illustrated in -Table 12. Although these data are not included in this
table, the red cell count averaged less than one red cell per white cell.
Thus, the lysis procedure was markedly effective in reducing the number
of red cells but not in eliminating them. However, comparison of the
data from Tables 11 and 12 reveals that the lysis also reduces the white
12
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Table 8
VIABILITY OF CULTURED LEUCOCYTES
Without C02
Day
0
1
2
3
4
Experiment 1
Cell Viability
Count
1.70
0.376
0.472
0.304
0.336
0.226
0.304
97
63
68
7
Experiment 2
Cell Viability
Count
1.12
1.15
0.08
0.048
5% CC-2
95
94
40 1.7
33
Experiment 1
Cell Viability
Count
2.2
96
97
Experiment 2
Viability
2.0
94
0.78
69
Table 9
RECOVERIES OF LEUCOCYTES FROM RAT AND
RABBIT BLOOD IN PLASTIC CENTRIFUGE TUBES3
Experiment
1
2
3
4
Blood
Sample
Rabbit
Rabbit
Rabbit
Rat
Viability
>95
0.66
73
whole blood was diluted with four volumes of saline
and layered onto five volumes of Ficoll/Hypaque
solution [9% (w/v) Ficoll in water, 24 volumes, and
34% (w/v) Hypaque in water, 10 volumes]. After
centrifuging-at 700-1700 x j» for 30'min, the inter-
facial band containing leucocytes was drawn off. The
leucocytes were washed in saline three times.
WBC recovered per ml of whole blood, x 10" .
13
-------
Table 10
RECOVERY OF LEUCOCYTES FROM RABBIT BLOODa
Temperature Time
Experiment Gradient (°C) (min)
1
2
3
4
5
6
7
Standard
Standard
Standard
Standard
Standard
Standard
Standard6
20
37
5
20
20
20
20
60
60
60
240
60
60
60
Blood
Whole
Whole
Whole
Whole
c
Wholed
Whole
WBCb
1.76
2.30
1.82
0.80
0.35
1.49
1.39
RBC
WBC
24.4
21.3
26.3
40.0
125
34.5
40.0
Reparation performed as in Table 9, except that whole blood was layered
onto gradient and, instead of centrifugation, the samples were allowed
to stand for the specified time.
WBC recovered per ml of whole blood, x 10~6.
One volume of blood/saline (1:1) was layered onto the gradient.
Two volumes of whole blood were layered onto the gradient.
^icoll/Hypaque made up in 1% saline.
14
-------
Table 11
RECOVERY OF RABBIT LEUCOCYTES
IN VARIOUS DENSITY GRADIENTS
Experiment la Experiment 2b
Ficoll: RBC RBC
Hypaquec WBCd WBC WBCd WBC
1.26 25.0
1.76 24.4
«
2.19 21.3
20:10
24:10e
26:10
28:10
30:10
0.93
1.08
1.94
1.04
Performed as in Table 9; centrifuged
at 700 x £.
Performed as in Table 10; no centri-
fugation.
°Gradient composition as in Table 9,
except proportions are varied.
WBC recovered per ml whole blood, x 10" -
eStandard gradient used in Tables 9 and 10.
cell count by a factor ranging from 5 to 20. Another disturbing obser-
vation is that vital staining revealed possible damage to the nuclear
membrane as a result of the lysis step. After lysis, nuclear material
was observed throughout the cell interior, whereas before lysis, a clear
nuclear structure could be observed.
Rat blood gave markedly higher white cell counts following
hypotonic lysis than did rabbit blood, as shown in Table 13. Increasing
the time of separation, the temperature of separation, or the time of
hypotonic lysis, as well as including low-speed centrifugation following
separation, significantly reduced the subsequent white cell count. In
all cases, the white cells appeared damaged, as judged by vital staining.
Other attempts to lower the red cell count were unsuccessful.
A second separation on Ficoll-Hypaque before hypotonic lysis and PHA-M
treatment prior to separation was not effective in either heparinized
"blood or in EDTA-treated blood. PHA-M is a red cell agglutinating agent
as well as a mitogen.
15
-------
Table 12
RECOVERY OF RABBIT LEUCOCYTES FOLLOWING HYPOTONIC LYSIS3
Time for: Centrifugation
Separation Hemolysis After ^
Experiment (min) (sec) Separation WBC
1 30 30 No 0.13
2 60 30 No 0.43
3 90 30 No 0.21
4 120 30 No 0.45
5 30 15 No 0.35
6 30 30 No 0.13
7 30 60 No 0.29
8 30 90 No 0.08
9 30 30 No 0.13
10 30 30 100 rpm 0.15
11 30 30 200 rpm 0.03, 0.19
12 30 30 400 rpm 0.93
13C 30 30 No 0.48
14d 30 30 No 0.48
a
The procedure is the same as that in Table 10, except that the
separation time was 30 min at 5°C. The plasma layer was removed
and centrifuged. The plasma was decanted, and the cells were re-
suspended in 2 ml of saline. Hypotonic lysis was effected by
adding 6 ml of water and mixing by inversion for 30 sec. Then
2 ml of 3.5% saline was added. The cells were sedimented and
washed three times in saline.
b 6
WBC recovered per ml of whole blood, x 10 .
°After removal of the plasma, the cells were resuspended in saline
containing 0.06% EDTA.
Whole blood was mixed with 0.2 volumes of saline and 0.02 volumes
of PHA-M.
16
-------
Table 13
LEUCOCYTE RECOVERY FROM RAT BLOOD FOLLOWING HYPOTONIC LYSIS3
Time for:
Separa- Hemo- Centrifuga- Separation
Experi- tion lysis tion After Temperature , RBC
ment (min) (sec) Separation (°C) WBC WBC
1 60 30 No 25 1.3
2 60 30 No 5 2.5
3.0
3 60 30 100 rpm 5 2.2
2.2
4 60 30 200 rpm 5 1.1
1.9
5 60 30 No 5 1.7 0.71
1.7 0.35
6 60 60 No 5 1.2 2.6
1.1 1.8
7 90 30 No 5 1.0 2.8
1.3 2.4
8° 60 30 No 5 1.7 1.6
1.3 0.65
The procedure is the same as that in Table 12, except that the
separation time was 60 min.
WBC recovered per ml of whole blood, x 10 .
cWhole blood was pretreated with PHA-M as in Table 12, footnote d.
17
-------
Isolation Experiments Using Gelatin
The second procedure tried was the older method of using gela-
tin solutions as a settling medium [17], The general procedure is as
follows. All glassware is sterile and silicon-treated. A fresh solution
of 3% gelatin in 0.85% saline is prepared and maintained at 45°C until
it is used; gelatin solutions over 2 hours old are not used. Blood is
collected in EDTA (or is defibrinated), mixed with gelatin (3:1), and
incubated at 37°C for 30 to 90 minutes. The clear supernatant containing
the leucocytes is centrifuged for 10 minutes at 1500 x £ and washed with
an equal volume of HBSS. The cells are resuspended in MEM-S culture med-
ium containing 25 mM HEPES and incubated at 37°C. The following experi-
ments were conducted using this procedure except where noted.
Experiment 1. In this initial experiment, rat or rabbit blood
was used with varying concentrations of gelatin and times of separation.
As shown in Table 14, this resulted in a high yield of white cells and
low red cell counts. Longer separation times in the gelatin, diluted
blood, and defibrinated blood diminish the recovery of white cells. The
latter two procedures also produce high red cell counts in the final sus-
pension. Gelatin at 3.5% appears to give the best yield of white cells.
Experiment 2. Rabbit or human blood in EDTA or heparin was
separated in silicon-treated und untreated glassware. As judged by the
relative clearness of the supernatant layer after separation, the rabbit
blood in EDTA separated in silicon-treated glassware appeared to give
the best removal of RBC. The rabbit blood seemed to be as well separated
as the human blood. White cells from untreated glassware were observed
to clump.
Experiment 3. White blood cells were isolated from defibrin-
ated or EDTA-treated rabbit blood and then cultured. After 24 hours, the
leucocytes had settled out of the medium. The cells isolated from EDTA
blood were difficult to resuspend and showed fibrin-like strands. The
cells isolated from defibrinated blood resuspended easily and did not
form strands. The cells were examined under a microscope but were not
counted. The RBC contamination appeared high (RBC/WBC « 5 to 10), and
WBC viability appeared low (below 20%), as judged by the Trypan blue test.
Experiment 4 (incubation time). We attempted to improve RBC
removal by varying the incubation time of the defibrinated blood-gelatin
mixture between 60 and 120 minutes. The best separation time was found
to be 90 minutes.
Experiment 5. WBC were isolated from 4 samples of defibrinated
rabbit blood at a separation time of 90 minutes. Counts were made immed-
iately following isolation. The following tabulation presents the results:
18
-------
Table 14
RECOVERY OF RAT AND RABBIT LEUCOCYTES
AFTER SEDIMENTATION IN GELATIN3
Experiment Blood
1 Rat
2 Rat
3 Rat
4 Rabbit
5 Rabbit
6 Rabbit
7 Rabbit
8 Rabbit
9 Rabbit
10 Rabbit
Gelatin
(%)
„
3.0
2.5
3.0
3.0
3.0
2.5
3.5
3.0d
3.0e
Time of
Separation
(min)
60C
90
90
60
90
v 120
90
90
90
90
V,
WBC
0.76
0.78
0.48
0.93
0.67
7.59
5.83
5.05
7.75
9.66
4.11
3.23
Percentage
of WBC
Recovery
29
29
18
35
25
61
47
41
62
78
33
47
RBC
WBC
0.19
0.19
4.3
2.3
2.3
2.03
1.43
1.60
1.12
1.35
9.05
10.2
One volume of EDTA-treated whole blood is mixed with 0.5 volume of
3% gelatin in saline and allowed to stand for 90 min at 37°C. The
clear upper layer is removed and the white cells are isolated by
centrifugation. The cells are washed twice in saline and resuspended
in culture medium at a concentration of 1 x 10 cells/ml.
WBC recovered per ml whole blood, x 10~6.
cWhite cells were isolated as described in Table 8, with a 60-min
separation followed by hypotonic lysis for 20 sec.
One volume of whole blood diluted 1:1 with saline mixed as in
footnote a.
eBefibrinated whole blood mixed as in footnote a.
19
-------
RBC
Sample WBCa % Viable WBCb WBC
1 1.36 73 4.3
2 1.09 74 3.4
3 1.40 70 3.9
4 1.40 63 4.3
aWBC (10~6) recovered/ml of blood.
^Using dye exclusion test (Trypan blue).
The gelatin method is improved by the use of fresh gelatin, a
90-minute separation time, silicon-treated glassware, and defibrinated
blood instead of an anticoagulant. The method appeared to give a good
yield of WBC, but the RBC counts were consistently high except in Exper-
iment 1.
Isolation Experiments Using Dextran-Hypaque [7]
Experiment 1. Rabbit blood collected in EDTA was layered over
gradients in the proportion of 1 part of blood to 1 part of gradient and
allowed to stand at room temperature for 90 to 120 minutes to achieve
separation. Three gradients were used: (1) 10 parts 33.9% Hp + 20 parts
6% Dextran; (2) 10 parts 33.9% Hp + 25 parts 6% Dextran; and (3) 10 parts
33.9% Hp + 22 parts 9% Dextran. After separation, the leucocyte layer
was removed and centrifuged for 10 minutes at 400 x j». The leucocyte
pellet was resuspended in HBSS, recentrifuged, and resuspended in MEM-S
medium. All three preparations had a very high RBC count (RBC/WBC =
10 to 25).
Experiment 2. Rabbit blood collected in heparin was mixed with
6% Dextran in MEM in the proportion of 3 parts blood to 2 parts Dextran.
The mixture was allowed to stand in an ice bath for 60 minutes. The
white cell layer was rembved and was counted for white and red cells and
for viability of the white cells. The results are tabulated below:
Sample WBCa % Recovery WBC % Viable
1 2.44 57 100 0.89
2 1.51 35 100 0.97
aWBC x 10~6 recovered/ml whole blood.
20
-------
Varying quantities of PHA-P were added to aliquots of 3 x 10°
WBC in 5ml of MEM containing 5% fetal calf serum, 2 mM glutamine and 250
units/ml each of penicillin and streptomycin contained in Falcon plastic
culture flasks. The cultures were incubated at 37°C for 46 hours; 0.5
ftc of 2-l^c-thymidine was then added to each sample, and the cultures
were incubated for an additional 2 hours. The cells were harvested by
centrifugation, washed twice with HBSS containing a 100-fold excess of
unlabeled thymidine, dissolved in 1 ml of NCS Solubilizer, and counted
for radioactivity. The results shown in the table below clearly indicate
that the cells were no longer viable at the time the labeled thymidine
was added.
Sample PHAa CPMb
1 0 143
2 0.02 115
3 0.02 98
4 0.10 96
5 0.10 106
6 0.20 121
7 0.20 102
vial of Difco PHA-P was
reconstituted in 5 ml of water
as directed, and the indicated
number of milliliters were added
to each culture tube.
"Counts per minute were corrected
for background.
Experiment 3. Dextran separations were carried out as described
previously in three kinds of containers: Falcon plastic disposable tubes,
nonsiliconized glass tubes, and siliconized glass tubes. After the sepa-
ration, all tubes were examined for red cells adhering to the walls of
the tubes and interspersed in the upper layer (plasma layer) containing
the white cell suspension. All tubes had red cells present in the plasma
layer. The red cells adhering to the walls of fhe tubes frequently re-
mained when the white cell suspension was removed, but when red cells
were present within the suspension, the resultant white cell preparation
always contained a high red cell count.
Further experiments with Dextran were not done because of the
high red cell contamination and because the Ficoll-Hypaque method was
recommended by Mansfield [14],
21
-------
Isolation Experiments Using Methylcellulose Hypaque [7]
The general procedure for this method is as follows. All glass-
ware are sterile and silicon-treated. Rabbit blood collected in EDTA is
layered over the gradient (10 parts 33.9% Hp + 16 parts 2% MC) in a ratio
of 1 part blood to 1 part gradient. The preparation is incubated at room
temperature until the plasma layer separates from the RBC layer. The
white cells are harvested from the plasma layer and washed with 4 volumes
of HBSS for each volume of plasma. Cells are resuspended in MEM-S medium
and incubated at 37°C. Viability counts are made using Eosin Y instead
of Trypan blue. A variety of experiments were conducted in an attempt to
find optimum conditions for this procedure.
Experiment 1. The general procedure was followed except that
the wash step was omitted, resulting in heavy platelet contamination.
Viability counts were made immediately after isolation. The results are
tabulated below.
RBC
Sample WBCa % Recovery WBC % Viable WBC
1 4.68 24 100 0.66
2 6.10 31 100 0.70
3 6.95 35 100 0.69
4 2.91 22 100 0.55
aWBC x 10~6 recovered/ml whole blood.
Experiment 2 (effect of varying ratio of MC to Hp). The usual
procedure was followed except that the MCtHp ratio was varied. Viability
counts were made after 24 hours in culture. The results were as follows.
RBC
Sample MC;Hp WBCa % Recovery WBC % Viable WBC
1 12:10 0.82 4.3 47 1.02
2 12:10 0.65 3.4 51 0.82
3 14:10 0.57 2.9 50 1.38
4 14:10 0.33 1.7 38 0.97
5 16:10 0.53 2.8 38 0.38
6 16:10 0.63 3.3 37 0.42
7 18.10 0.63 3.3 28 0.63
8 18:10 0.48 2.5 30 0.87
aWBC x 10~6 recovered/ml whole blood.
22
-------
The ratio 16:10 was selected for use in future experiments.
Experiment 3 (effect of Varying MC concentration) . The usual
procedure was followed except that various concentrations of MC were
used. The gradient consisted of 10 parts 33.9% Hp + 16 parts MC. Via-
bility counts were made immediately after isolation. The results are
tabulated below.
MC Concen- RBC
Sample tration (%) WBCa % Recovery % Viable WBC
1 1.25 1.25 9.4 93 2.76
2 1.25 1.38 10.5 89 3.58
3 1.50 0.83 6.25 86 0.89
4 1.50 0.93 7.0 90 0.96
5 1.75 1.49 11.3 93 1.71
6 1.75 1.47 11.1 87 1.53
7 2.0 0.60 4.5 95 1.05
8 2.0 1.06 8.2 87 1.26
aWBC x 10~6 recovered/ml whole blood.
The WBC yield using 2% MC was typically severalfold higher
than shown above. Thus, 2% MC was chosen rather than 1.75% MC. Also,
the latter gave higher RBC/WBC ratios than the former.
Experiment 4 (effect of temperature on MC-Hp system). The
usual procedure was followed except that two different temperatures were
used for separation. Viability counts were made immediately after isola-
tion. The results are tabulated below.
Sample Temperature WBCa % Recovery % Viable
1 25°C 2.63 26.3 98 1.01
2 37°C 1.57 * 15.7 93 0.69
3 37°C 1.41 16.9 86 0.89
aWBC x 10~6 recovered/ml whole blood.
23
-------
A temperature of 25°C was found to provide greater WBC re-
covery .
Experiment 5 (effect of diluting blood on the MC-Hp system)
The usual procedure was followed except that a 10:1 dilution of whole
blood with 0.9% saline was used. Viability counts were made after 24
hours. The following tabulation presents the results:
Dilution RBC
Sample (Blood; 9% Saline) WBCa % Recovery % Viable WBC
1 Whole 1.70 18.3 38 0.20
2 Whole 0.72 7.8 53 1.67
3 10:1 1.64 17.7 24 1.98
4 10:1 1.08 11.7 30 4.41
aWBC x 10 ° recovered/ml whole blood.
Whole blood will be used in future experiments.
Experiment 6 (effect of osmolar concentration of the gradient).
Two solutions of Hp were used, 33.9% and 29.75% Hp + 4% NaCl. Both solu-
tions (10 parts) were mixed with MC (16 parts) and used in the standard
procedure. Viability counts were made after 24 hours. The results are
tabulated as follows.
Sample Hp Solution WBCa % Recovery % Viable
1 Hp 0.57 14.8 34 1.92
2 Hp 0.44 11.5 40 1.08
3 Hp 0.41 10.7 37 1.55
4 Hp/salt 0.67 17.4 48 4.20
5 Hp/salt 0.67 17.4 55 1.65
6 Hp/salt 0.51 13.3 43 2.85
aWBC x 10 recovered/ml whole blood.
The MC-Hp system provides good RBC removal and separation of
large amounts of blood. The results of these experiments indicate that
if a large amount of blood is separated in one tube, the recovery of WBC
24
-------
increases. However, this requires an increased separation time, which
may be a cause of the WBC clumping observed after a 2-hour separation
time on the gradient.
Experiment 7 (effect of defibrination). Rabbit blood was de-
fibrinated with wooden sticks [18]. Blood was also collected in EDTA.
The usual separation procedure was then followed. The majority of red
cells from the defibrinated blood settled out of suspension as expected,
but a clearly defined plasma layer was not evident due to extensive
hemolysis. No further work was done with this preparation.
White cells recovered from the EDTA blood were washed with MEM
(instead of HBSS), resuspended in the usual medium, and cultured for 48
hours. The white cells in the plasma layer from Sample 3 were collected
in two parts (the upper half and the lower half) in an effort to demon-
strate a red cell gradient in the plasma layer. The data in the table
below clearly show that the ratio of red cells to white cells in the
lower half of the plasma layer is nearly double that in the upper half.
This is partially attributable to the higher white cell count in the
upper half. The marked decline in viability is also seen in these data.
% Viable
Sample
1
2
3(upper)
3(lower)
3(total)
WBC*
0.68
0.69
0.56
0.37
0.93
% Recovery 0 hr 24 hr
10.1
10.3
8.3
5.5
13.8
94
99
98
96
52
57
55
61
48 hr
13
28
44
37
RBC
WBC
2.19
2.13
1.42
2.77
1.92
aWBC x 10~6 recovered/ml whole blood.
Experiment 8 (effect of duration of centrifugation during
wash). Rabbit blood was collected in EDTA and separated in the usual way
except that the white cells were harvested from the plasma layer by cen-
trifugation at 400 x £ for 10 minutes, and washed by resuspending in MEM
and centrifuging at 400 x £ for either 5 or 10 minutes. The results are
tabulated below.
25
-------
Time of »
Sample Centrifugation WBCa % Recovery % Viable
1 5 minutes 0.68 10 72 2.38
2 5 minutes 0.78 12 73 2.36
3 10 minutes 0.88 13 77 2.11
4 10 minutes 0.55 8 69 2.29
aWBC x 10~° recovered/ml whole blood.
These data show that the duration of the wash centrifugation
has no effect on the white cell yield or on the red cell contamination.
%
One possible explanation for the generally low yield of white
cells in these and previous experiments, as well as for the rapidly de-
clining viability when various white cell preparations were cultured, is
that in comparison to human leucocytes, rabbit leucocytes are inherently
more fragile or are made so due to the conditions of isolation and cul-
ture This experiment was undertaken to learn the minimum time of cen-
trifugation required to recover the maximum number of white cells during
the wash step, based on the hypothesis that minimum exposure to stress
would maximize the yield of viable cells. The data obtained from this
experiment do not support this hypothesis.
Experiment 9 (effect of sodium and ammonium heparin). Rabbit
blood was collected in both ammonium and sodium heparin at 10 U/ml of whole
blood. The separations were carried out in the usual way. In addition, a
longer separation was included for the blood collected in sodium heparin.
The blood collected in ammonium heparin gave a poor separation
due to hemolysis and was discarded. The table below shows the results
of the separation of blood collected in sodium heparin.
Separation % Viable RBC
Sample Time (min) WBCa % Recovery (24 hr) WBC
1 70 0.24 3.3 61 6.83
2 70 0.26 3.6 85 8.27
3 120 1.72 24.0 63 2.72
4 120 1.56 22.0 56 2.03
aWBC x 10~° recovered/ml whole blood.
26
-------
These data show that a longer separation time improves both
the white cell yield and the red cell contamination. The higher viabil-
ity after 24 hours of culture may indicate that heparin is less harmful
than EDTA.
Summary of Viability Data from Methylcellulose-Hypaque Experi-
ments. Cell viability is almost always between 90 and 100% immediately
after separation (Experiments 1, 3, 4, and 7, but it decreases steadily
over a period of 3 days after separation (see following tabulation).
The cells appear to be enriched in polymorphonucleocytes, which are known
not to survive in culture. The following tabulation summarizes survival
data from Experiments 2, 5, 6, 7, and 9. Counts were made at 24, 48,
and 72 hours. Only samples separated from whole blood using 2% MC and
33.9% Hp (16:10) are included.
Experi- % Viable RBC/WBC
ment Sample 24 hr 48 hr 72 hr 24 hr 48 hr 72 hr
2 5 38 0.38
6 37 0.42
5 1 38 12 9.5 0.20 0.36 0.13
2 53 14 2.8 1.67 0.17 0.23
6 1 34 21 10 1.92 0.30 0.07
2 40 26 26 1.08 0.13 1.05
3 37 7.5 3 1.55 0.23 0.03
7 1 52 13
2 57 28
3U 55 44
3L 61 37
9 1 61
2 85
3 63
4 56
This method was abandoned due to the poor longevity of the
white cells and because of the research of Mansfield and Wallace [14]
who indicated that white cells can be maintained for at least 72 hours
by using RPMI-1640 medium supplemented with 5 to 10% fresh, heat-
inactivated autologous rabbit plasma.
27
-------
The Procedure of Mansfield and Wallace
The procedure described below is essentially the method of Mansfield
and Wallace, [14] as clarified by personal conversation with Dr. Mans-
field by telephone and by a visit to his laboratory at the University of
Louisville School of Medicine, Louisville, Kentucky. Other minor modi-
fications have also been made as a result of numerous experiments in our
laboratory using their method.
Rabbit blood was drawn by cardiac puncture using a 19-gauge needle
attached to a 50-ml syringe containing 1,000 U of heparin or 20 U of
heparin/ml of whole blood based on a 50-ml blood volume. However, the
amount of blood drawn varied from rabbit to rabbit, with the result
that the heparin concentration varied from 20 to 38 U/ml. Aliquots of
blood were reserved for analysis of whole blood and for making heat-
inactivated plasma as a medium supplement.
Leucocytes were separated from 40 to 60 ml of blood. The blood
was centrifuged in siliconized tubes at 160 x £ for 15 minutes. The
platelet-rich plasma was removed, and the volume was replaced with HBSS.
Aliquots (10 ml) of this blood were then transferred to sterile
screw-cap culture tubes (25 x 150 mm) and diluted with 25 ml of HBSS.
A 10-ml gradient (1 part 34% Hypaque +2.4 parts 9% Ficoll) was then
layered beneath the diluted blood with a 10-gauge needle. The tubes
were then centrifuged at 400 x j> for 25 to 30 minutes.
After centrifugation, the clear upper layer consisting of HBSS and
platelets was removed by aspiration. The white cell layer formed at
the density boundary was transferred to 50-ml screw-cap culture tubes
using a sterile disposable pipette, and the cells were resuspended in a
fivefold excess of HBSS. The cells were centrifuged at 400 x £ for 5
minutes, the supernatant was poured off, and the cells were resuspended
by aspiration in RPMI-1640 medium containing per ml 100 U of penicillin,
100 //g of streptomycin, 60 fig of tylosin, and 2 /moles of glutamine.
The cells were then incubated for 2 hours at 37°C.
Viability counts were then done on the isolated cells; 0.1 ml of
1% eosin Y in ethanol was placed in a test tube and then dried. A few
drops of cell suspension were added, and the nonstaining viable cells
were counted in a hemocytometer. Nonviable cells stained bright pink.
The concentration of viable leucocytes was brought to 1.0 x 10°/ml
by dilution with medium. Also at this time, heat-inactivated autologous
plasma was added to the medium to a concentration of 10%. The plasma
supplement was prepared by centrifuging whole blood for 1 hour at 400
x £, removing the plasma layer, and heating it for 30 minutes in a 56 °C
water bath.
The cell suspensions were then aliquoted to screw-cap culture tubes
(16 x 125 mm) in 2-ml volumes (i.e., 2 x 10^ viable leucocytes/culture.
28
-------
To each culture was added 25 ^g of PHA in 0.1 ml of HBSS. The cells
were gassed with 5% C02 in air, capped tightly, and incubated at 37°C
for 22 hours.
After 22 hours, the various radioactive substrates were added to
12 replicate cultures in the following order:
Tubes Labels Added (in 0.1 ml HBSS)
1,2,3 2 pC± 3H-thymidine + 1 yUCi 14C-uridine
4,5,6 1.5 //Ci 3H-leucine + 1 /UC± 14-choline
7, 8, 9
10, 11, 12 " "
The cultures were then gassed with 5% C02 in air, recapped, and incubated
for an additional 22 hours.
After 22 hours, samples 1 to 6 were harvested. Samples 7 to 12
were washed twice with HBSS containing a 100-fold excess of unlabeled
leucine and choline by centrifuging at 400 x j> for 5 minutes for each
wash step, and then 2 ml of fresh medium was added. The cells were then
gassed and incubated for an additional five hours (Tubes 7 to 9) or 24
hours (Tubes 10 to 12). At these times, the indicated cells were
harvested.
Cells were harvested in the following manner. The cultures were
diluted with 5 ml of cold 0.15 M NaCl and centrifuged at 400 x £ for 5
minutes. The supernatant was decanted, and the cells were washed again
in the same manner. The pellet was precipitated with 5 ml of cold 5%
TCA and allowed to stand at 4°C for at least 20 minutes. After centri-
fuging at 400 x £ for 10 minutes and decanting the supernatant, the
TCA precipitate was dissolved in 1.0 ml of NCS Solubilizer, transferred
to scintillation vials in 10 ml of counting fluid (6 g PPO + 75 mg POPOP
per liter toluene), and counted in a Searle Analytic Mark III Liquid
Scintillation Counter with external standard for dual-label counting.
In addition to the teletypewriter printout, the data were also recorded
on punched paper tape for computer processing by the Institute's CDC-
6400. All counting data are recorded as disintegrations per minute (DPM)
as corrected for counting efficiency in the dual-label mode.
Phytohemagglutinin Standardization
Newly purchased Difco PHA-P was used. The contents of six vials
were pooled and dissolved in 30 ml of saline. Aliquots of 5 ml were
dispensed into serum vials and stored frozen at 20°C until needed.
29
-------
This PHA-P preparation served for all the remaining experiments in this
research.
The PHA-P was first standardized using normal rabbit leucocytes pre-
pared and cultured using the procedure just described except that varying
amounts of PHA-P were added and only ^H-thymidine uptake was measured.
The mitogenic effect of PHA-P is evident from the data in the accompany-
ing table and is illustrated in Figure 1.
PHA-P 3H-Thymidine- Uptake3
0/g/ml) (PPM ± S.D.)
0 8284 + 1524
2.5 8534 + 1305
5.0 25487 + 2958
12.5 108064 + 10249
50.0 155256 + 95111
aAverage of three values.
The PHA-P concentration of 12.5 ^g/ml of culture medium was selected
as the standard amount of PHA-P to be used in all subsequent experiments
in this research. These data closely approximate those obtained by Mans-
field and Wallace, particularly in regard to the low uptake when PHA-P
is absent. When this same experiment was carried out earlier using cul-
ture conditions shown by others to be suitable for culturing human leu-
cocytes, we obtained higher thymidine uptake in the absence of PHA-P and
less stimulation with PHA-P than was found in the present experiment.
Mansfield and Wallace [14] state that this effect is primarily due to
the use of RPMI 1640 medium containing heat-inactivated autologous rabbit
plasma. Thus, the results of our work are consistent with this interpre-
tation.
Preliminary Lead and Cadmium Experiment
This preliminary experiment was undertaken to test all phases of
the separation and culture procedures as detailed previously, to mimic
the subsequent experiments on exposure of rabbits to lead and cadmium
for one and three weeks, and to determine if three samples could be han-
dled simultaneously. We felt it would also be desirable to gain an im-
pression of the metabolic effects of lead and cadmium prior to initiat-
ing the main experiments. The procedure differed in several ways from
the methods finally adopted. The lead and cadmium animals were treated
daily for two weeks; the total culture period was 48 hours instead of
44 hours; the medium contained 5% heat-inactivated autologous rabbit
plasma rather than 10%; during the degradation period the plasma supple-
ment was not included; an extra degradation period of 2-1/2 hours was
30
-------
DPM OF 3H-THYMIDINE x 10"3
U>
O
C
30
m
TJ
3
• z
o
>
3J
g
N
-------
added and the 5-hour period was increased to 5-1/2 hours; the various
labels were not combined in ^C-% pairs but each was added separately to
its own set of triplicate culture tubes; and ^H-uridine was used instead
of 14C-uridine.
The results demonstrated that the entire procedure was workable and
that three blood samples could be handled simultaneously.
As shown in Table 15, the level of incorporation of thymidine was
approximately the same as in the PHA standardization experiment.
-*
The incorporation of thymidine into leucocytes from the lead-treated
animal was 24% higher than in the control, but because there were only
two samples in each group, the difference is not statistically signifi-
cant. Thus, the effect of lead treatment on DNA synthesis is unclear
from this experiment. In contrast, the incorporation of uridine was
over twice that in the control, suggesting a stimulation of RNA synthe-
sis. In the white cells from the cadmium-treated animal, the incorporation
of both thymidine and uridine was very low, indicating a severe depres-
sion of DNA and RNA synthesis. These differences can also be seen by
calculating the ratio of uridine to thymidine incorporation in the three
animals:
Control Lead Cadmium
Uridine/thymidine 0.360 0.593 1.254
Choline/leucine 0.835 1.122 17.87
Protein biosynthesis in the lead-treated animal is very similar to
that in the control animal, as judged by leucine incorporation. The in-
corporation of leucine in the cadmium-treated rabbit, however, is sig-
nificantly lower than in the control and is consistent with the diminished
biosynthesis of DNA and RNA. The biosynthesis of phospholipid in both
treated animals is significantly higher than in the control. These dif-
ferences are reflected in the ratio of choline to leucine incorporation
in the three animals, as shown above.
The degradation of protein and phospholipid is illustrated in Fig-
ures 2 and 3, in which the data are normalized to 100% at zero time.
Here it can be seen that although the average retention of leucine in the
lead-treated animal was higher than in the control, the differences are
not statistically significant, indicating that lead treatment has a neg-
ligible effect on the turnover of protein in leucocytes. On the other
hand, cadmium treatment markedly accelerated the rate of protein turnover
during the first five hours of post-incubation, but had no effect during
the remainder of the incubation period.
Phospholipid degradation in both the control and lead-treated ani-
mals was negligible during the first 5-1/2 hours. Thereafter, the rat6
32
-------
Table 15
INCORPORATION OF LABEL IN
PRELIMINARY LEAD AND CADMIUM EXPERIMENT
Label
Control
Lead
Cadmium
u>
LO
3H-Thymidine
3H-Uridine
3H-Leucine 0 hr
97041a + 15048
34948 + 2598
22210 + 1890
148953 + 21649
88383 + 10956
24573 + 2345
1366 + 188
1713 + 771
1883 + 383
14
2.5 hr
5.5 hr
24 hr
C-Choline 0 hr
2.5 hr
5.5 hr
24 hr
19247 + 1360
16718 + 4738
10895 + 1890
18535 + 1335
17521 + 848
17665 + 748
14092 + 4318
21951 + 4209
19678 + 3271
13765 + 781
27573 + 2095
28211 + 3314
29783 + 2765
21802 + 1638
1037 + 211
775 + 235
786 + 170
33649 + 1777
28551 + 5341
21002 + 1281
12750 + 1595
aEntries are DPM + Standard Deviation.
-------
HOURS
FIGURE 2 LEUCINE DEPLETION IN PRELIMINARY LEAD AND CADMIUM
EXPERIMENT
34
-------
HOURS
FIGURE 3 CHOLINE DEPLETION IN PRELIMINARY LEAD AND CADMIUM
EXPERIMENT
35
-------
of degradation was very similar in both animals. Thus, the rate of phos-
pholipid turnover in lead-treated animals is probably very similar to
that in the control. Throughout the incubation period, the lead-treated
animal had significantly more labeled phospholipid than the control.
In contrast, phospholipid degraded rapidly in the cadmium-treated
animal during the first 5-1/2 hours and then slowed during the remainder
of the incubation period. Thus, cadmium treatment resulted in an in-
creased rate of phospholipid turnover throughout the 24-hour incubation
period.
Control Experiment
A second full-scale trial of the entire procedure was undertaken to
gain further experience prior to conducting the main experiments and to
determine the reproducibility of the experimental data derived from sev-
eral rabbits.
The results (Tables 16 and 17) show a two- to three-fold variation
in the level of incorporation in various samples. These data parallel
our earlier impression of the variability of the leucocyte preparations
from seemingly identical rabbits. This variability was observed both
in the efficacy of the separation of white cells from blood and in the
subsequent culturing, even though the procedures were carried out as
identically as possible.
Although the incorporation of %-thymidine (Table 16) is somewhat
higher than in the two previous experiments, the rate is comparable and
demonstrates again the mitogenic effect of PHA in stimulating DNA syn-
thesis. Similarly, the uptake of ^C-uridine (Table 16) indicates the
concomitant synthesis of RNA. Uridine incorporation in this and the
previous experiment cannot be directly compared because different uri-
dine preparations were used. Although 1 jUc was added to each tube in
both experiments, this amount represents 35.7 /jmol in the previous ex-
periment and 17.5 yumol in this one. However, by calculating the uridine/
thymidine ratio (shown below), some of the variability among rabbits is
removed. This procedure is discussed and documented fully in the next
section. The ratios presented here can be compared later with the one-
week and three-week exposure experiments.
The levels of incorporation of %-leucine and -^C-choline (Table 17)
demonstrate the synthesis of protein and phospholipid, respectively. The
ratio of choline to leucine in the three animals (shown below) is some-
what higher than in the previous experiment.
Animal Uridine/Thymidine Choline/Leucine
1 0.246 1.338
2 0.518 1.113
3 0.627 1.609
Mean 0.464 1.353
SD +0.196 +0.248
36
-------
Table 16
3 14
INCORPORATION OF H-THYMIPINE AND C-URIDINE
IN LEUCOCYTES FROM THREE CONTROL ANIMALS
Animal
3H-Thymidine
(PPM)
14
C-Uridine
(PPM)
1 173778 42776
2 129296 66954
3 98649 61822
Mean 134484 57184
SP +38457 +11904
Table 17
INCORPORATION OF 3H-LEUCINE ANP C-CHOLINE
IN LEUCOCYTES FROM THREE CONTROL ANIMALS
H-Leucine
(DPM)
Animal 0 hr
1 19001
2 21926
3 61442
Mean 34123
SP +20804
5 hr
13301
15917
50127
26448
+17982
24 hr
9026
8385
23376
13595
+8253
0 hr
25442
24401
98859
52705
+38266
14
C-Choline
(PPM)
5 hr
15745
20069
71530
35781
+27460
24 hr
9444
7825
24367
13879
+8498
Student t-Test
H— Leucine
5 hr
24 hr
5 hr
24 hr
vs 0 hr
vs 0 hr
loline
vs 0 hr
vs 0 hr
PF
16
16
15
15
t
0.837
2.752
1.057
2.974
P
58.5
98.6
<90
>99
37
-------
As shown in Table 18, if the incorporation of label at zero hours
is normalized to 100%, then the average degradation rate of protein is
nearly the same as that of phospholipid. When the Student's t-test is
applied to the individual normalized figures, the differences between
the 5-hour and the 0-hour values and between the 24-hour and the 0-hour
values become more statistically significant than shown in Table 17.
This is particularly true for the 5-hour -%-leucine data, which in
Table 17 is not statistically different from the 0-hour control but
which becomes 95% using the normalized values as shown in Table 18.
These data are summarized in the table below.
Raw Data Normalized Data
Table 17 Table 18
%-Leucine t P t
5 hr vs 0 hr 0.837 58.5 6.328 100.0
24 hr vs 0 hr 2.752 98.6 11.667 100.0
14C-Choline
5 hr vs 0 hr 1.057 <90 5.684 99.9
24 hr vs 0 hr 2.974 >99 19.438 99.9
Although the use of C/ ratios and normalization of the degra-
dation data remove much of the variability encountered in this experi-
ment, the one- and three-week experiments were carried out with six
rabbits in each group, with the expectation of improving the statistical
evaluation of the biosynthetic data.
One-Week and Three-Week Lead and Cadmium Experiments
Animal Data
x.
The animals used in the one-week and three-week exposure exper-
iments were adult New Zealand white rabbits of mixed sex (Tables 19 and
20), weighing 2.66 to 5.39 kg. They were given daily intraperitioneal
injections of saline (control animals) or aqueous solutions of lead
acetate or cadmium chloride. The metal dosages were determined from
intraperitoneal toxicity (LD^g) studies and from repeated i.p. injection
studies conducted earlier. The dosages were established as 10 mg/kg for
lead acetate and 1 mg/kg for cadmium chloride.
Lead Cadmium Units
Salt 10.0 1.0 mg/kg
Metal 5.46 0.492 mg/kg
Metal 26.4 4.38 j/mol/kg
38
-------
Table 18
DEGRADATION RATE OF PROTEIN AND PHOSPHOLIPID
IN THREE CONTROL ANIMALS
Animal
1
2
3
Mean
SD
H-Leucine
0 hr
100.0
100.0
100.0
100.0
+ 6.9
5 hr
70.0
72.6
81.6
74.7
+9.8
24 hr
47.5
38.2
38.0
41.3
+13.4
14
C-Choline
0 hr
100.0
100.0
100.0
100.0
+3.5
5 hr
61.9
82.2
72.4
72.2
+13.5
24 hr
37.1
32.1
24.6
31.3
+9.4
Student t-Test
H-Leucine
5 hr vs 0 hr
24 hr vs 0 hr
14C-Choline
5 hr vs 0 hr
24 hr vs 0 hr
DF
16
16
15
15
6.328
11.667
5.684
19.438
>99.9
>99.9
>99.9
>99.9
The lead was administered as a 50% (w/v) aqueous solution. The cadmium
was administered at varying concentrations in a. constant volume of 1 ml/kg.
During the first three days of treatment, all rabbits appeared
to be mildly depressed as indicated by a lack of alertness. At the same
time all animals were restless in that they could not seem to find a
comfortable position as though they were internally irritated by the in-
jections. The depression continued throughout the 7- and 21-day treat-
ment periods for the lead- and cadmium-treated animals. One animal died
after fifteen days of cadmium treatment. The others appeared to be in
good health throughout the treatment period.
All animals were weighed initially and at weekly intervals (Tables
19 and 20). All animals, including controls, showed a mild but statisti-
cally significant weight loss after one week of treatment.
39
-------
Table 19
RABBIT BODY WEIGHTS DURING THE ONE-WEEK EXPOSURE EXPERIMENT
Treatment Sex
Control
1 ?
2 a*
3 ?
4 ?
5 3
6 ?
Avg.
SD
Dose Body^
(mg/kg) 0
0 3.87
3.14
3.22
3.77
3.52
4.41
3.66
±0.47
Wt (kg)
1 wk
3.72
2.95
3.05
3.70
3.52
4.00
3.49
±0.41
Percentage
Wt . Loss
3.88
6.05
5.28
1.86
0.00
9.30
4.40
±3.27
Cadmium
1 95%
3.43
2.94
3.00
3.77
2.66
3.60
3.23
:t0.43
2.78
2.60
2.54
3.42
2.30
3.12
2.79
±0.41
18.95
11.56
15.33
9.28
13.53
13.33
13.66
±3.30
10
4.883
>99.9%
40
-------
Table 20
RABBIT BODY WEIGHTS DURING THE THREE-WEEK EXPOSURE EXPERIMENT
Dose
Treatment Sex (ing/kg)
Control
1
2
3
4
5
6
cT
0
Body Wt (kg)
0
3.00
4.59
4.36
4.71
4.58
4.07
1 wk
2.82
4.50
4.27
4.80
4.19
4.12
2 wk
3.03
4.22
4.73
4.66
4.21
4.08
3 wk
2.98
4.35
4.40
4.60
4.36
4.05
Percentage
Wt. Loss
1 wk
6.00
1.96
2.06
1.91
8.52
-1.23
3 wk
0.67
5.23
-0.92
2.34
4.80
0.49
Avg.
SD
4.22 4.12
±0.64 ±0.68
4.16 4.12
±0.61 ±0.59
2.57 2.10
±4.06 ±2.49
Cadmium
1
2
3
4
5
6
1.0
2.93
4.52
3.16
4.19
3.58
5.39
2.73
4.04
2.90
4.11
3.34
5.32
2.56
4.19
3.02
4.18
3.24
4.87
2.50
4.10
3.05
4.20
3.20
Died
6.83
10.62
8.23
1.91
6.70
1.30
14.68
9.29
3.48
-0.24
10.61
—
Avg.
SD
3.96 3.74 3.68 3.41 5.93 7.56
±0.92 ±0.96 ±0.87 ±0.72 ±3.64 ±5.93
v
t
P
Lead
1
2
3
4
5
6
Avg.
SD
v
t
P
10.0
O*
10
1.512
9
2.067
<90% <95%
3.46
4.42
3.51
3.57
3.08
3.98
3.67
±0.47
3.36
4.30
3.06
3.44
2.68
3.84
3.45
±0.57
3.31
4.24
2.86
3.38
2.80
3.77
3.39
±0.55
9
3.26
4.18
3.00
3.29
2.78
3.73
3.37
±0.51
2.89
2.71
12.82
3.64
12.99
3.52
6.43
±5.03
10
1.464
<90%
5.78
5.43
14.53
7.84
9.74
6.28
8.27
±3.46
10
3.546
99.5%
41
-------
In the one-week experiment, both, groups of raetal-treated arnnals
shoved a statistically significant weight loss compared to the control
group, with the lead—treated an-i^als shoving the greater loss (Table 19).
In the three-week experiment, the lead—treated an-i»alg again shoved a
greater weight loss after one week than did the radwiurn-treated aniaals,
but the differences from the control group were not significant (Table
20). However, at the end of one week the'animals in the three-week exper-
iment are equivalent to the animals in the one—week experiment so that
the weight loss data can be combined for statistical evaluation. The
table below shows that at one week, both metal-treated groups had a sig-
nificant weight loss compared to the control:
Control Lead Cadmium
Average 3.48Z 10.05Z 7.16Z
SD ±3.64 ±5.54 ±3.25
v 22 22
t 3.429 2.607
P >99.5Z >97.5Z
In the three-week experiment, the average weight loss was slightly
greater at three weeks than at one week, but the difference was not
statistically significant. After three weeks, the weight loss of the
lead-treated animals, but not of the cadmium-treated animals, was sig-
nificantly greater than that in the controls.
Blood Data
A sample of whole blood from each rabbit used in the one- and
three-week exposure experiments was analyzed for red and white cell
counts and hemoglobin and hematocrit determinations. These data, as
well as the mean corpuscular volume (MCV), mean corpuscular hemoglobin
(MCH), and mean corpuscular hemoglobin concentration (MCHC) are presented
in Tables 21 and 22.
In the one-week experiment, none of the red cell parameters shown
in Table 21 differed significantly from control values. Moreover, all
of the average values were within the normal limits (tabulated below)
for rabbit blood [19].
WBC (cells x 10~6/ml) 4-13
RBC (cells x 10-6/ml) 4500-7000
Hemoglobin (g%) 10.4-15.6
Hematocrit (%) 33-44
MCV fy/m3) 60-68
MCH (g x 1012) 19.4-22.6
MCHC (%) 31.3-34.7
42
-------
Table 21
WHOLE BLOOD DATA - ONE-WEEK EXPERIMENT
Hb Hematocrit MCV
Rabbit
WBCa
RBCa
(g%)
(%)
(tfm3)
MCH
(g x 1012)
MCHC
(%)
Control
1
2
3
4
5
6
Avg.
SD
1
2
3
4
5
6
Avg.
SD
V
t
P
5.0
9.0
5.0
4.7
8.1
4.7
6.1
± 1.9
11.1
7.4
6.3
4.8
9.6
10.6
8.3
± 2.5
10
1.706
<90%
5450 x
7350
6050
6280
4800
5500
5905
± 877
6750
5260
4250
5000
4580
5700
5257
± 890
10
1.271
<80%
13.91
16.79
9.63
10.27
10.32
12.05
12.16
±2.75
Lead
12.23
12.19
8.23
10.02
10.58
10.44
10.62
± 1.49
10
1.217
<80%
41.0
49.5
30.0
31.5
35.0
41.5
38.1
± 7.3
39.0
35.0
27.0
34.5
36.5
34.5
34.4
± 4.0
10
1.074
<80%
75.2
67.3
49.6
50.2
72.9
75.5
65.1
±12.1
57.8
66.5
63.5
69.0
79.7
60.5
66.2
± 7.8
10
0.150
<50%
25.5
22.8
15.9
16.4
21.5
21.9
20.7
± 3.8
18.1
23.2
19.4
20.0
23.1
18.3
20.4
± 2.3
10
0.145
<50%
29.5
29.5
31.2
30.6
33.9
34.5
31.5
± 2.2
31.9
28.7
32.7
34.5
34.5
33.1
32.6
± 2.2
10
0.823
<60%
Cadmium
1
2
3
4
5
6
Avg.
SD
V
t
P
13.4
10.1
10.4
8.5
9.4
16.9
11.4
± 3.1
10
3.561
99.5%
7080
4160
5250
5700
5550
6600
5723
±1030
10
0.329
<50%
16.23
-
10.45
10.02
10.87
11.62
11.83
± 2.53
9
0.201
<50%
51.0
—
33.0
34.5
37.5
40.0
39.2
± 7.1
9
0.255
<50%
72.0
_
62.9
60.5
67.6
60.6
64.7
± 5.0
9
0.051
<50%
22.9
—
19.9
17.6
19.6
17.6
19.5
± 2.2
9
0.477
<50%
31.4
_
31.6
34.4
34.5
34.4
33.3
± 1.6
9
1.244
<80%
aCells x 10"6/ml of whole blood.
43
-------
Rabbit
Table 22
WHOLE BLOOD DATA - THREE-WEEK EXPERIMENT
Hb
Hematocrit
WBC
RBC
Control
1
2
3
4
5 '
6
Avg.
SD
1
2
3
4
5
6
Avg.
SD
V
t
P
3.5
6.0
7.0
8.2
14.6
5.7
7.5
± 3.8
4.8
4.95
5.8
9.3
3.4
3.6
5.3
± 2.2
10
1.232
<80%
6900
5550
5900
7900
5600
4950
6133
±1076
4950
5250
4140
4400
3000
5050
4465
± 831
10
3.006
>97.5%
13.09
12.66
10.64
10.92
9.67
9.46
11.07
± 1.51
10.69
11.42
7.61
10.58
4.85
8.22
8.9
± 2.5
10
1.844
<95%
42.0
39.0
34.0
37.0
41.0
36.0
38.2
± 3.1
Lead
33.5
34.5
28.0
34.5
20.0
33.5
30.7
± 5.8
10
2.816
>97.5%
Cadmium
1
2
3
4
5
Died
Avg.
SD
V
t
P
5.6
4.35
4.7
5.3
6.4
-
5.3
± 0.8
9
1.277
<80%
4300
6100
6050
4650
5650
-
5350
± 827
9
1.329
<80%
6.25
9.41
9.17
8.60
8.76
-
8.44
± 1.27
9
3.105
>97.5%
20.0
31.0
31.0
28.0
34.0
-
28.8
± 5.36
9
3.654
>99%
MCV
MCH MCHC
(g x IP12)
60.9
70.3
57.6
46.8
73.2
72.7
63.6
±10.4
67.7
65.7
67.6
78.4
66.7
66.3
68.7
± 4.8
10
0.854
<60%
46.5
50.8
51.2
60.2
60.2
53.8
± 6.1
9
1.471
<90%
19.0
22.8
18.0
13.8
17.3
19.1
18.3
± 2.9
21.6
21.8
18.4
24.0
16.2
16.3
19.7
± 3.2
10
0.820
<60%
14.5
15.4
15.2
18.5
15.5
15.8
± 1.5
9
1.347
<80%
32.1
30.8
32.0
33.9
42.3
38.1
34.9
± 4.5
31.3
. 30.1
36.7
32.7
41.2
40.7
35.4
± 4.8
10
0.227
<50%
32.1
33.0
33.7
32.5
38.8
34.0
± 2.7
9
0.298
<50%
aCells x 10~6/ml of whole blood.
44
-------
After three weeks of lead treatment (Table 22), the red cell count,
hematocrit, and hemoglobin content were all low, and the red cell size had
increased, as shown by the elevated MCV value. These data are consistent
with a mild hemolytic anemia characteristic of chronic lead poisoning.
In the cadmium-treated animals, on the other hand, the hemoglobin content
and the hematocrit were depressed even thouugh the red cell count was not
significantly lower than that of the controls. This implies that the red
cells were smaller in size and had less hemoglobin than the larger control
red cells. The hemoglobin concentration, however, was normal as shown by
the MCHC value (Table 22).
In the one-week experiment (Table 21), the white cell count from the
cadmium-treated animals was significantly elevated compared with the
controls. The white cell count from the lead-treated animals was higher
than that of the controls, but the difference was not significant and was
within the normal range. At the end of three weeks (Table 22), the white
cell count from the lead-and cadmium-treated animals did not differ from
that of the controls.
Differential counts were done on the white cells from each rabbit.
Whole blood smears were prepared and stained by standard methodology and
submitted to Veterinary Reference Laboratory, San Jose, California for
analysis. The normal white cell distribution is as follows [19]:
Neutrophils (%) 43 (30-50)
Lymphocytes (%) 42 (30-50)
Monocytes (%) 9 (2-16)
Eosinophils (%) 2 (0.5-5)
Basophils (%) 4 (2-8)
In the one-week experiment (Table 23), the distribution in the
lead-treated animals was not significantly different from that of controls.
However, the cadmium-treated animals had a significantly elevated neutro-
phil count and a correspondingly low lymphocyte count. None of the aver-
age values were significantly outside normal ranges. The lead-treated
animals were characterized by several cellular abnormalities, the most
predominant of which were polychromasia and anisocytosis. These charac-
teristics were also observed in the control group but to a lesser extent.
Polychromasia was seen in only two of the cadmium-treated animals.
At the end of three weeks (Table 24), the differential counts
in both metal-treated groups were not significantly different from that
in controls, although the average lymphocyte counts are higher and the
average monocyte counts are lower than normal values in both treated
groups. The average neutrophil count for the cadmium-treated group is
low because of one extraordinarily low value. The frequency of cellular
abnormalities had diminished markedly. However, anisocytosis was ob-
served in three of the five lead-treated animals. None of the control
rabbits exhibited aberrant white cell forms.
45
-------
Table 23
DIFFERENTIAL COUNTS FOR ONE-WEEK EXPERIMENT
Rabbit
1
2
3
4
5
6
Avg.
SD
1
2
3
4
5
6
Avg.
SD
V
t
P
1
2
3
4
5
6
Avg.
SD
V
t
P
Neut. '
26
39
36
40
34
46
37
± 6.7
25
36
17
49
45
46
36
±13.8
10
0.150
<50%
42
54
39
64
51
54
51
± 9.1
10
3.004
>97.5%
Lymph.
63
54
57
58
63
51
58
± 4.8
67
60
80
47
52
50
59
±12.5
10
0.305
<50%
56
35
50
26
47
45
43
±10.9
10
2.996
>97.5%
Mono. Eos.
/ w\ fV\
\/e) \nj
Control
10
7
6 1
2
3
3
5
± 3
Lead
5
7
6
3 1
3
4
4
± 1.2
10
0.748
<60%
Cadmium
2
11
11
9 1
2 1
1
6
± 5
10
0.364
<50%
Baso
(%)
1
—
1
-
-
—
3
—
—
—
-
—
.
-
—
—
—
—
NRBCa Comments
b
b,c
b,c
b,c,d,e
b,c
b,d
b,c
b,c
b,f
b(rare)
Nucleated red blood cells/100 WBC, bPolychromasia, cAnisocytosis,
dBasophilic stippling, eSeveral macrocytes, ^Few Howell-Jolley bodies.
46
-------
Table 24
DIFFERENTIAL COUNTS FOR THREE-WEEK EXPERIMENT
Rabbit
1
2
3
4
5
6
Avg.
SD
1
2
3
4
5
6
Avg.
SD
V
t
P
1
2
3
4
5
6
Avg.
SD
V
t
P
Neut.
(%)
_
33
40
70
13
55
42
±21.7
61
36
31
29
Too few
38
39
±12.8
8
0.284
<50%
59
55
31
13
-
21.6
±18.6
7
0.186
<50%
Lymph.
(%)
63
58
20
85
44
54
±24
38
60
68
71
to count
62
60
±13
8
0.475
<50%
40
45
67
87
—
60
±22
7
0.372
<50%
Mono . Eos . Baso .
(%) (%) (%)
Control
_
4 - -
2 - -
55-
2 - -
6 - -
3
± 1.6
Lead
1 - -
4 - -
1 - -
_ _ _
_ _ _
_ _ _
1
± 1.6
8
1.547
<90%
Cadmium
1 - -
_ _ _
2 - -
_ _ -
_ _ _
0.75
± 0.96
7
0.961
<80%
NRBCa Comments
7
1
b,c
c
c
1
1
b,c(severe)
c
d
Nucleated red blood cells/100 WBC, bPolychromasia, cAnisocytosis,
done.
47
-------
Thus, in general, the lead and cadmium treatments have only marginal
influence on the blood values although it may well he that small changes
may represent profound alterations in the physiology of hlood cell pro-
duction.
After separation of the leucocytes by the method detailed previously,
the white cell yield and the proportion of red and white cells were mea-
sured and the viability of the white cells was determined. These data
are presented in Tables 25 and 26 for the one- and three-week experi-
ments, respectively.
The predominant features of these data are the generally very
high proportion of viable white blood cells obtained and the low yield
of white cells. The poor yields contrast with the experience of Mansfield
and Wallace [14]. In spite of intense effort and consultation with Drs.
Mansfield and Wallace, who generously donated their time, better yields
could not be obtained and the cause of the low yields has not been dis-
covered .
Occasionally a very high proportion of red cells are obtained
in the white cell preparations. The data show that there is a strong
inverse correlation between white cell yield and red cell contamination.
Our present view is that blood platelets play an important role in both
parameters.
Biosynthetic Data
These experiments were carried out exactly as described pre-
viously with the exception that an extra step was included in an attempt
to reduce the platelet population during the preparation of the leucocyte
suspension. Whole blood was centrifuged at 160 x £ for 15 minutes. The
platelet-rich plasma was removed by aspiration and an equal volume of
HBSS was added. The blood was then worked up as described previously.
This procedure typically resulted in the removal of about 20% of the whole
blood volume although there was considerable variation, ranging from 10%
to 50%. The one-week data are presented in Tables 30 to 35 and the three-
week data are in Tables 36 to 41. One feature common to all the data is
the variability in the amount of the incorporated label among the differ-
ent animals. This is consistent with the previous experiment and rein-
forces the conclusion that it is due to normal variations in the metabolic
activities of leucocytes from the individual rabbits. Further support
for this conclusion can be drawn from Table 27, in which each animal is
ranked according to its level of incorporation of the four radioactive sub-
strates into its leucocytes. It is seen that in most groupings the white
cells from one particular rabbit are most active in taking up all four
labels, the cells from another animal are least active with respect to the
four labels, and cells from the other animals are ranked in between.
A quantitative measure of this correlation is provided by Ken-
dall's coefficient of concordance;[20], which indicates that in five of
the six groupings this correlation is highly significant.
48
-------
Table 25
LEUCOCYTE VIABILITY FOR ONE-WEEK EXPERIMENT
WBC
Viable RBC Yield
Rabbit WBCa (%) WBC (%)
Control
1
2
3
4
5
6C
0.55
1.13
0.55
0.09
0.83
1.50
98
98
95
97
88
99
3.2
2.2
4.3
18. 5b
1.9
1.2
11
12.5
11
1.9
10
32
Lead
1
2
3
4
5
6
0.67
0.48
1.13
0.20
0.55
1.73
95
98
99
95
97
99
8.2
4.0
5.5
29. 4b
5.4
1.2
6.0
6.5
18
4.2
5.7
16.3
Cadmium
1
2
3
4
5
6
0.53
0.76
2.00
1.00
0.55
0.90
96
96
95
95
94
96
3.5
2.6
0.64
0.84
6.7,
2.6
4.0
7.5
19
12
5.9
5.3
aCells x 10~6 isolated/ml whole blood.
^Because of the high red cell contamination,
the leucocyte preparation was not used.
cLeucocytes from this rabbit were used for
the first in vitro platinum exposure ex-
periment (see next section).
49
-------
Table 26
LEUCOCYTE VIABILITY FOR THREE-WEEK EXPERIMENT
Viable RBC
Rabbit WBCa (%) WBC
Control
1
2
3
4
5
6
0.65
0.41
0.83
0.06
1.53
0.65
95
98
97
98
98
0.48
1.1
0.67
24. lb
0.30
2.24
18.6
6.7
11.9
0.7
10.5
11.4
Lead
1
2
3
4
5
0.52
1.12
0.33
0.53
-
88
97
95
100
-
4.3
3.1
40. Ob
3.6
high>
10.8
22.6
5.7
5.7
-
0.40 100 37.5 11.1
Cadmium
1
2
3
4
5
6C
0.37
0.65
0.50
0.41
0.50
—
87
95
90
100
95
_
1.7
1.34
2.0
3.94
1.56
—
6.6
14.9
10.6
7.7
7.8
—
aCells x 10~6 isolated/ml whole blood.
^Because of the high red cell contamination,
the leucocyte preparation was not used.
cAnimal died on 15th day of treatment.
50
-------
Table 27
A RANKING OF THE RELATIVE ABILITY OF INDIVIDUAL RABBITS
TO INCORPORATE FOUR RADIOACTIVE SUBSTRATES3
Animal
No.
1
2
3
4
5
6
s
W
P
1
2
3
4
5
6
s
W
P
1
2
3
4
5
6
s
W
P
One Week
4
1
2
5
3
U L C
Control
444
112
221
533
355
Three Weeks
T U L C
122
0.76
>99%
2
3
4
5
1
Lead
124
345
453
531
212
74
0.46
<95%
Cadmium
3
4
5
1
6
2
208
0.74
>99%
314
541
455
222
666
133
1
4
5
3
2
223
554
445
111
332
128
0.80
>99%
2
1
3
4
50
0.625
>95%
111
234
323
442
1
3
4
2
5
111
222
443
335
554
132
0.82
>99%
aRadioactive substrates thymidine, uridine,
leucine and choline are symbolized by T,
U, L, and C, respectively. Entries are the
ranking, e.g., in the 1-week experiment con-
trol animal #1 ranks 4th for all substrates.
51
-------
Thus, it is valid to normalize the one- and three-week incor-
poration data by forming the l^C/^H ratio for uridine/thymidine and for
choline/leucine and comparing the averages for the lead and cadmium groups
with the control group. These correlations are presented in Tables 28
and 29.
The most noteworthy feature of Tables 28 and 29 is the eleva-
tion in the uridine/thymidine ratio for the one- and three-week cadmium-
treated animals. In both experiments, the cadmium ratios are nearly 4.5
times the control ratios. This effect of cadmium treatment is primarily
due to the marked diminution in the %-thymidine incorporation, as seen
in Tables 30 and 36, although -"-^C-uridine incorporation is also depressed.
The decrease in the ratio at three weeks is due primarily to an increased
thymidine incorporation relative to the control while the uridine uptake
remained nearly the same. .Thus, in the one-week cadmium group the thy-
midine uptake was 6.5 times less than in the controls, but by three weeks
the difference was reduced to a factor of 3.3. Cadmium treatment dimin-
ishes uridine incorporation by a factor of 1.7 after one week and of 2.5
after three weeks.
The ratios for the one-week lead-treated group exhibit wide
variation and reflect the lack of correlation in ranking the rabbits in
the incorporating ability, as seen in Table 27. In both the one- and
three-week experiments, the uridine/thymidine ratios for the lead-treated
animals are not significantly higher than for the controls. At one week,
the absolute incorporation of thymidine and uridine in the lead-treated
animals are identical to the controls, but at three weeks the incorpora-
tion was depressed 41% and 35%, respectively (Tables 30 and 36). The
difference between these two figures is not statistically significant,
however, and the resultant uridine/thymidine ratio is the same as that
of the controls at three weeks.
Leucine and choline incorporation is not affected by either one-
or three-week treatment with lead or cadmium. Also, the choline/leucine
ratios for both metal-treated animal groups do not differ significantly
from that of the controls. These conclusions are apparent from Tables
32, 34, 38, and 40, in which the incorporation data are presented, as well
as from Tables 28 and 29, which show the choline/leucine ratios. It is
noteworthy that the incorporation of both leucine and choline at three
weeks is nearly double the level at one week.
The depletion rate of labeled leucine is unaffected by the lead
or cadmium treatments or by the length of treatment. Approximately
55% of labeled leucine remains after 24 hours post-incubation in all
cases (Tables 33 and 39).
Choline depletion in the one-week lead- and cadmium-treated ani-
mals is somewhat more rapid than in the controls, but the Student's t-test
indicates that the differences are not statistically significant (Table
35). The levels of labeled choline remaining after 24 hours in the three-
week lead-treated animals and after 5 hours in the three-week cadmium-
treated animals are significantly lower than in the controls (Table 41).
52
-------
Table 28
CARBON 14 TO TRITIUM RATIOS IN ONE-WEEK EXPERIMENT
14c-Uridine/3H-Thymidine ^C-Choline/3H-Leucine
Animal
1
2
3
4
5
6
Avg.
SD
V
t
P
Control
0.784
0.954
0.511
_
0.644
0.805
0.740
±0.169
Lead
1.206
0.863
1.104
_
0.483
0.442
0.820
±0.349
8
0.461
<90%
Cadmium
3.905
2.200
4.941
2.528
3.371
3.010
3.326
±0.994
9
10.822
>99.9%
Control
2.579
1.416
2.603
_
2.157
1.506
2.052
±0.569
Lead
1.217
1.952
3.016
_
6.005a
1.367
2.711
±1.972
8
0.718
<90%
Cadmium
0.915
4.207a
2.185
1.459
2.361
1.937
2.177
±1.124
9
0.225
<90%
when these values are deleted, the
ically not significantly different
average values remain statis-
from the average control value.
Table 29
CARBON 14 TO TRITIUM RATIOS IN THREE-WEEK EXPERIMENT
l^C-Uridine/3H-Thymid ine l^C-Choline/3H-Leucine
Animal
1
2
3
4
5
6
Avg.
SD
V
t
P
Control
0.290
0.236
0.370
-
0.595
0.218
0.342
±0.153
Lead
0.478
0.252
—
0.455
_
(4.739)a
0.395
±0.124
6
0.504
<90%-
Cadmium
(0.275)a
2.360
1.397
1.207
1.042
—
1.502
±0.590
7
4.284
>99%
Control
1.596
4.535
2.041
-
1.300
1.832
2.261
±1.301
v
Lead
1.390
1.524
-
1.611
_
3.364
1.972
±0.932
7
0.372
<90%
Cadmium
1.394
1.892
3.276
1.536
5.638
-
2.745
±1.775
8
0.492
<90%
^Values in parentheses not used in computing averages.
53
-------
Table 30
ONE-WEEK EXPERIMENT—THYMIDINE AND URIDINE INCORPORATION
Ol
ANIMAL
1
2
3
4
5
6
MEAN
STD DPV
STUDENT T-TEST
PB
CD
H3-THYMIDINE
CONTROL PB
20050. 43358.
95356. 32041.
74603. 24590.
0. 0.
11801. 7984.
25277. 115969.
41850. 45699.
33003. 40436.
OF* T
VS CONTROL 26. .276
VS CONTROL 30. 4.548
CD
5825.
4860.
7958.
10021.
1384.
8533.
6430.
3158.
P
21.517
99.992
C14-URIDINE
CONTROL PB
15724.
91024.
38118.
0.
7600.
20357.
30532.
27857.
OF
PB VS CONTROL ?6.
CO VS CONTROL 30.
52268.
27655.
27142.
0.
3856.
51221.
32770.
19358.
T
.247
1.773
CO
22747.
10691.
20471.
25337.
4666.
25686.
18266.
8481.
P
19.305
91.356
*Same as "v" in text on Tables 30 through 41.
-------
Table 31
ONE-WEEK EXPERIMENT—RATIOS OF URIDINE-TO-THYMIDINE INCORPORATION
Ol
Ol
ANIMAL
1
«?TD
STUOfcNT T-TEST
PR
nF
VS CONTROL 26.
C14-URIDINE
CONTROL
.855
.779
.728
.930
0.000
.979
.495
.511
.533
0.000
0.000
0.000
.692
.604
.648
1.029
.732
.700
.730
.170
T P
•676 61.106
/ H3-THVMIOINE
PB CO
1.188
1.234
1.197
0.000
.843
.885
1.224
.988
1.123
0.000
0.000
0.000
.485
.498
,468
.429
.439
.461
.819
.340
CD
3.707
4.144
3.913
2.351
1.416
3.216
2.344
2.710
2.702
2.698
2.354
2.532
3.275
1.892
5.110
2.520
3.804
3.090
2. 988
.894
VS CONTW
OF T P
30. 9.266 100.000
-------
Table 32
ONE-WEEK EXPERIMENT--LEUCINE INCORPORATION AND DEPLETION
-------
Table 33
ONE-WEEK EXPERIMENT—RATE OF LEUCINE DEPLETION
Ol
ANIMAL
1
2
3
4
b
•b
MEAN
*TD DFV
STUDENT T-TEST
5 HP
?4 HR
5 HR
?4 HR
5 HR
74 HR
<• HR
«; HR
?4 HR
o HR
? HH
?4 HR
0
100.000
100.000
100.000
0.000
100.000
100.000
100.000
12.627
CONTROL
CONTROL
PR
PB
CD
CD
PB
PB
PB
CD
CD
CD
CONTROL
5
94.761
90.799
87.514
VS
VS
VS
vs
VS
VS
VS
VS
VS
VS
VS
vs
0.000
69.278
54.538
79.378
16.866
(i
0
0
0
0
r
(i
5
?4
li
5
?4
HR
HR
HR
HP
HR
HR
HR
HR
HR
HP
HR
HR
24
65.912
68.329
57.328
0.000
54.642
3P.726
55.089
16.919
CONTROL
CONTROL
PB
PB
CD
CD
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
H3-LEUCINE
PB
0 5
100.000 90.204
100.000 88.447
100.000 80.351
0.000 0.000
100.000 0.000
100.000 68.782
100.000 82.612
7.291 9.330
DF
27.
25.
23.
28.
31.
34.
27.
23.
26.
30.
28.
29.
24
43.187
74.329
61.339
0.000
44.894
53.726
55.495
12.726
T
3.706
7.856
5.2?6
11.752
3.333
9.303
.000
.550
.072
.000
.46n
.214
0
100.000
100.000
100.000
100.000
100.000
100.000
100.000
15.326
F
99.
100.
99.
100.
99.
100.
.
41.
5.
0.
35.
16.
CD
5
87.200
76.902
87.471
67.318
0.000
91.580
82.094
15.419
904
000
997
000
776
000
000
263
711
000
120
790
24
50.951
66.362
71.790
42.820
32.055
59.31B
53.883
14.402
-------
Table 34
ONE-WEEK EXPERIMENT—CHOLINE INCORPORATION AND DEPLETION
Vl
00
CONTROL
0 5
ANIMAL
1 27863. 28630.
2 43063. 30172.
3 46861. 43633.
4 0. 0.
5 30204. 17769.
6 12106. 5524.
MEAN 3123B. 25150.
*TD OEV 13029. 13319.
STUPENT T-TEST
5 HR CONTROL VS 0 HR
24 HR CONTROL VS 0 HR
5 HR PB VS 0 HR
?4 HR PB VS 0 HR
5 HR CD VS 0 HR
24 HR CD VS 0 HR
f HP PB VS 0 HR
5 HP PB VS 5 HR
24 HR PB VS 24 HR
r HR CD VS 0 HR
5 H» CD VS 5 HR
74 HP CD VS ?4 HR
24
18590.
18212.
29074.
0.
11542.
2597.
13992.
8109.
CONTROL
CONTROL
PB
PB
CD
CD
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
C14-CHOLINE
PB
0 5 24
29061. 24854. 9961.
20663. 14619. 9441.
29924. 18222. 13696.
0* 0. 0.
65248. 0. 20265.
35125. 21032. 11560.
36044. 20053. 13024.
16024. 4171. 4164.
OF T
27. 1.243
25. 4.090
23. 3.067
28. 5.385
31. 2.729
34. 5.979
27. .862
23. 1.165
26. .405
30. .060
28. 1.075
29. 1.360
CO
0 5
25960. 21239.
55695. 23749.
25745. 17423.
33657. 18021.
15062. 0.
32644. 25711.
31527. 21228.
13937. 4704.
P
77.541
99.961
99.454
99.999
98.963
100.000
61.465
74.418
31.142
4.746
70.855
81.556
24
12629.
17626.
11785.
8440.
3562.
11402.
10907.
4457.
-------
Table 35
ONE-WEEK EXPERIMENT—RATE OF CHOLINE DEPLETION
cn
CONTROL
0 5
ANIMAL
1 100.000 102.755
2 100.000 70.034
3 100.000 93.073
4 0.000 0.000
b 100.000 58.897
6 100.000 45.631
MFAN 100.000 74.n78
STD DFV 12.854 22.868
STUDENT T-TEST
5 HR CONTROL VS 0 HR
?4 HR CONTROL VS 0 HR
5 HR PB VS 0 HR
24 HR PB VS 0 HR
5 HR CD VS 0 HR
74 HR CD VS 0 HR
r HP PB VS (1 HP
5 HR PB VS b HR
?4 HR PB VS 74 HR
«• HR CD VS 0 HR
5 HP CD VS 5 HR
?4 HR CD VS ?4 HR
24
66.719
4?. 272
62.018
0.000
38.212
21.450
43.691
18.319
CONTROL
CONTROL
PB
PB
CD
cn
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTPOL
C14-CHOLINE
PB
0 5
100.000 85.521
100.000 70.069
100.000 60.693
0.000 0.000
100.000 0.000
100.000 59.679
100.000 69.914
7.562 12.080
OF
27.
25.
23.
28.
31.
34.
27.
23.
26.
30.
28.
29.
24
34.274
45.252
46.437
0.000
31.058
32.912
37.987
7.411
T
3.725
9.302
7.687
22.684
5.743
14.3C4
.000
.526
1.108
0.000
1.215
1.710
CO
0 5
100.000 81.814
100.000 42.488
100.000 67.673
100.000 53.276
100.000 0.000
100.000 78.761
100.000 64.792
16.436 18.786
P
99.909
100.000
100.000
100.000
100.000
100.000
.000
39.638
72.215
0.000
76.555
90.209
24
48.646
31.533
45.776
24.92V
23.646
34.929
34.910
10.129
-------
Table 36
THREE-WEEK EXPERIMENT--THYMIDINE AND URIDINE INCORPORATION
CONTROL
ANIMAL
1 308093.
«• 125*72.
3 99850.
* 0.
5 235633.
0 6 281430.
MEAN 210095.
CJTD DEV 88063.
STUDENT T-TEST
PB VS CONTROL
CD VS CONTROL
H3-THYMIDINE
PB
183953.
245082.
0.
84936.
0.
2660.
124182.
104307.
OF T
24. 2.274
28. 3.966
CD
278547.
14675.
5006.
19157.
2235.
0.
63924.
111349.
P
96.781
99.957
Cl
CONTROL
89497.
29562.
36935.
0.
140133.
61173.
71460.
42221.
OF
PB VS CONTROL 24.
CD VS CONTROL 28.
4-URIUINt
PB
87840.
61685.
0.
38606.
0.
12701.
46787.
29805.
T
1.655
3.232
CD
76574.
34630.
6994.
23121.
2329.
0.
28729.
28959.
P
88.912
99.686
-------
ANIMAL
1
MFAN
STO DEV
Table 37
THREE-WEEK EXPERIMENT—RATIOS OF URIDINE-TO-THYMIDINE INCORPORATION
C14-URIDINE / H3-THYHIDINE
CONTROL PB CD
.310
.?74
,?86
,?55
.214
.?35
.362
.402
.324
P. 000
0.000
0.000
.580
.59*
.611
.229
.223
.195
.341
.14*
.500
.4*9
0.000
.248
.232
.283
0.000
0.000
0.000
.310
.472
.568
0.000
0.000
0.000
4.959
5.566
4.125
1.61P
2.130
.339
.225
.265
1.8*1
1.901
3.109
1.239
1.350
1.722
1.434
.909
1.207
1.305
.978
.707
0.000
0.000
0.000
1.235
.748
STUDENT T-TFST
PB
DF T
VS CONTROL 24. 2.318
97.072
CD
VS CONTROL
T
4.548
P
99.990
-------
Table 38
THREE-WEEK EXPERIMENT—LEUCINE INCORPORATION AND DEPLETION
I
to
CONTROL
0 5
ANIMAL
1 33351. 31652.
2 10*77. 75*9.
3 21509. 17722.
* 0. 0.
5 46374. 36931.
6 31779. 2*887.
MEAN ?8698. 237*8.
STD DEV 12937. 11573.
STUDENT T-TEST
5 HR CONTROL VS 0 HR
2* HR CONTROL VS 0 HR
5 HR PB VS 0 HR
?* HR PB VS 0 HR
S HR CD VS 0 HR
24 HR CD VS 0 HR
r HP PB VS 0 HR
"5 wR PB VS 5 HR
2* HR PB VS ?* HR
r HP CD VS 0 HR
S HR CD VS S HR
2* HR CD VS ?* HR
2*
J7928.
5580.
13640.
0.
32217.
17178.
16444.
88*2.
CONTROI
CONTROL
PB
PR
CD
CD
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
PB
052*
7*468. 0. 39076.
21252. 17478. 12*75.
0. 0. 0.
21818. 16278. 11*07.
0. 0. 0.
15997. 10789. 7569.
33384. 14520. 18547.
25472. 3351. 14008.
OF T
28. 1.104
26. ?.879
18. 2.064
21. 1.708
21. 1.775
23. 2.453
25. .621
21. 2.185
22. .447
28. .290
21. 1.712
21. 1.290
CD
0 5
669*8. 0.
39939. 32263.
1*7*8. 115**.
2*317. 0.
7*?5. 7635.
0. 0.
30675. 15258.
23057. 10820.
P
72.120
99.211
9*. 629
89.755
90.966
97.783
45.996
95.968
34.073
22.576
89.838
78.888
2*
0.
18636.
6601.
135*2.
7211.
0.
12355.
5323.
-------
Table 39
THREE-WEEK EXPERIMENT—RATE OF LEUCINE DEPLETION
ANIMAL
i
2
3
4
b
6
MFAN
STO DFV
100
100
100
0
100
100
100
14
0
.000
.000
.000
.000
.000
.000
.000
.173
CONTROL
5
94.904
72.054
B2.393
0.000
79.638
76.313
ftl.460
15.208
24
53.757
53.261
63.415
o.noo
69.472
54.055
57.615
9.663
H3-LEUCINE
PB
0 5
100.000
100.000
0.000
100.000
0.000
100.000
100.000
20.317
0.000
62.245
0.000
74.610
0.000
67.443
73.831
9.417
24
52.474
56.700
0.000
52.285
0.000
47.314
53.182
8.410
100
100
100
100
100
0
100
17
0
.000
.000
.000
.000
.000
.000
.000
.032
CO
5
0.000
80.781
78.279
0.000
102.636
0.000
86.113
23.684
24
0.000
46.661
44.761
55.691
97.125
0.000
53.846
16.165
GO
STUDENT T-TFST
5
24
5
?4
5
?4
o
5
?4
-
"
76
HR
HR
HR
HR
MR
HP
HR
HP
HP
HR
HR
HP
CONTROL
CONTROL
PB
PB
CO
CD
PB
PB
PB
CD
CD
CD
VS
VS
VS
VS
VS
VS
VS
VS
VS
VS
VS
VS
0
r.
0
"
c
0
f)
5
?4
0
5
?4
HR
HR
HR
HR
HR
HR
HR
HR
HR
HR
HP
HM
CONTROL
CONTROL
PB
PB
CD
CD
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
Ci^TPOL
DF
28.
26.
18.
21.
21.
23.
25.
21.
22.
2B.
21.
21.
3
9
3
7
1
6
1
1
T
.454
.095
.386
.095
.392
.771
.000
.286
.187
.000
.623
.697
99
100
99
100
62
100
78
75
0
56
so
p
.82?
.000
.671
.000
.156
.000
.000
.74?
.213
.000
.010
.639
-------
Table 40
THREE-WEEK EXPERIMENT—CHOLINE INCORPORATION AND DEPLETION
o
CONTROL
0 5
ANIMAL
1 53231. 42753.
2 47508. 31199.
3 43905. 26619.
4 0. 0.
5 60308. 43055.
6 58209. 39009.
MEAN 52632. 36527.
STD DfV 7808. 8560.
STUDENT T-TEST
5 HR CONTROL VS 0 HR
24 HR CONTROL VS 0 HR
5 HR PB VS 0 HR
24 HR PB VS 0 HR
5 HR CD % VS 0 HR
?4 HR CD VS 0 HP
r HR PB VS 0 HR
5 HR PB VS 5 HR
74 HR PB VS 24 HR
r HR CD VS 0 HR
5 HR CD VS 5 HR
24 HR CD VS 74 HR
24
25224.
2564P.
17702.
0.
25551.
26866.
24592.
3914.
CONTROL
CONTROL
PB
PB
CO
CD
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
C14-CHOLINE
PB
0 5 24
103540. 0. 38169.
32390. 24531. 14074.
0. 0. 0.
35151. 21179. 15234.
0. 0. 0.
53809. 24861. 14405.
56222. 23398. 21022.
30423. 2965. 12676.
OF T
28. 5.384
26. 11.715
18. 3.015
21. 3.559
21. 3.054
?3. 4.576
25. .441
21. 4.168
22. .966
28. .993
21. 1.339
21. .963
CO
0 5
93332. 0.
75582. 48614.
48314. 28943.
37339. 0.
41784. 20597.
0. 0.
59270. 30731.
24695. 12113.
F
99.999
100.000
99.256
99.815
99.398
99.987
33.717
99.956
65.544
67.06?
80.511
65,339
24
0.
30129.
20793.
19005.
17508.
0.
22729.
5381.
-------
Table 41
THREE-WEEK EXPERIMENT—RATE OF CHOLINE DEPLETION
05
Ol
CONTROL
0 5
ANIMAl
1 100.000 00. 316
i. 100.000 65.672
3 100.000 60.626
4 0.000 0.000
5 100.000 71.391
6 100.000 67.015
MFAN 100.000 69.005
«TO OEV 7.884 11.260
STlintNT T-TEST
5 HR CONTROL VS 0 HR
24 HR CONTROL VS 0 HR
5 HR PB VS 0 HR
24 HR PB VS 0 HR
•5 HR CD VS 0 HR
?4 HR CD VS 0 HR
i HP PB VS 0 HR
* HR PB VS 5 HR
24 HR PB VS ?4 HR
P HR CD VS 0 HR
5 HR CD VS b HH
?4 HR CD VS ?4 HP
24
47.386
53.970
40.319
0*000
42.368
46.154
46.762
6.503
CONTROL
CONTROL
PB
PR
CD
CD
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
C14-CHOLINE
PB
0 5 24
100.000 0.000 36.664
100.000 75.737 43.451
0.000 0.000 0.000
100.000 60.253 43.339
0.000 0.000 0.000
100.000 46.203 26.771
100.000 58.655 38.591
9.540 13.074 9.326
DF T
28. U.722
26. 19.300
18. 8.156
21. 15.566
21. 6.942
23. 10.664
25. 0.000
21. 1.947
22. 2.521
?8. .000
21. 2.521
2). .969
CO
0 5
100.000 0.000
100.000 64.320
100.000 59.906
100.000 0.000
100. QUO 49.293
0.000 0.000
100.000 57.030
15.626 9.936
P
100.000
100. QUO
100.000
100.000
100.000
100.000
0.000
93.495
98.054
0.000
96. nU
65.625
24
0.000
39.863
43.036
50.899
41.900
0.000
44.330
5.173
-------
One final feature of Tables 28 and 29 is the constancy of the
effect of lead and cadmium treatment on the l^C/^H ratios compared to
the controls. This is illustrated in the table below.
14C-Uridine/3H-Thymidine 14C-Choline/3H-Leucine
1 wk 3 wk 1 wk 3 wk
Control 1.00 1.00 1.00 1.00
Lead 1.11 1.15 0.92 0.87
Cadmium 4.49 4.39 0.86 0.90
Thus, while the relative incorporation of thymidine and uridine in
the lead- and cadmium-treated animals does not change compared to the
controls in the two experiments, individual incorporation of the two sub-
strates changes markedly from one week to three weeks. Thus, in the
control animals, thymidine uptake increases five-fold, compared to a
2.5-fold increase in uridine uptake (Table 30 versus Table 36). In the
lead-treated animals, the corresponding values are 2.6 and 1.2, while in
the cadmium group the increases are 1.6 and 0.9, respectively (values
for the first cadmium-treated animal are not included in the three-week
calculations). In each case, the uridine ratio at three weeks is about
half that at one week.
In marked contrast, the choline/leucine ratios are virtually con-
stant for all three groups in both the one- and three-week experiments,
although the absolute incorporation of labeled choline and leucine at
three weeks is approximately double the values at one week (Table 32 versus
Table 38 and Table 34 versus Table 40). The incorporation data for the
one- and three-week experiments are summarized in Table 42.
In Vitro Platinum Exposure Experiments
Leucocytes from the sixth control rabbit of the one-week exposure
experiment were used for the first experiment. Twelve cultures contain-
ing 2 x 10*> viable WBC were prepared in 2 ml of medium. To each was
added 1 mg of sodium hexachloroplatinate (IV) (^PtClg•6H20) in a 0.1-
ml volume of HBSS. The final concentration of the platinum salt was 0.476
mg/ml of culture medium or 0.848 |/M. This is equivalent to 165 //g/ml as
metallic platinum. The cultures were then gassed with 5% C02 in air and
incubated for 2 hours at 37°C.
At the end of the 2-hour incubation, each culture was mixed with 2
ml of HBSS and centrifuged at 400 x £ for 5 minutes; the supernatant
was decanted. The cells from each culture were then washed with 3 ml of
HBSS and resuspended in 2 ml of medium and 0.1 ml of PHA in HBSS. At
this point, the platinum-treated cells were firmly attached to the wall
66
-------
Table 42
SUMMARY OF EFFECTS OF LEAD AND CADMIUM TREATMENT3
A A B
One Week Three Weeks Three Weeks
Lead Cadmium Lead Cadmium Control Lead Cadmium
3H-Thymidine NC 85l 4li 951 402? 172? 60t
14C-Uridine NC 40 1 35 1 771 134T 43T 57T
14C-Uridine/3H-Thymidine NC 309T NC 339T 54i 52 1 551
3H-Leucine NC NC NC NC 88 T 106 T 84?
14C-Choline NC NC NC NC 68 T 56? 88?
14C-Choline/3H-Leucine NC NC NC NC NC NC 14?
3H-Leucine 5 hr NC NC NC NC NC NC NC
3H-Leucine 24 hr NC NC NC NC NC NC NC
14C-Choline 5 hr NC NC NC 17? NC 161 NC
14C-Choline 24 hr " NC NC 17? NC NC NC 27?
aEntries show percentage by which the tabulated group differs (? = increase,
1 = decrease) from the control group (A) or from the corresponding one-week
group (B), or by which the tabulated 5-hr or 24-hr group differs from the
zero-hour group. Differences less than 10% or those not statistically sig-
nificant are indicated by NC (no change).
-------
of the culture tubes. They did not come off until they were harvested.
The untreated control cells, which did not stick as much or as tightly
to the culture tubes, were not washed as were the treated cells.
After the addition of PHA, the cultures were incubated, exposed to
radioactive substrates, and harvested exactly as described previously,
except that no 5-hour pulse was done.
The second in vitro platinum experiment was carried out exactly
like the first except that an extra set of triplicate cultures were in-
cluded and treated with 0.238 mg/ml of platinum in addition to treatment
at 0.476 mg/ml. Tfie cells for this experiment were from the third con-
trol rabbit of the three-week exposure experiment. The platinum-treated
cells adhered to the culture tube walls and the controls did not, as in
the first experiment.
The third and final in vitro platinum experiment was carried out
exactly like the first except that each culture contained only 0.5 x 106
viable WBC/ml of medium (one-half of normal). Nine cultures received no
platinum salt (controls), nine received 95.2 //g/ml, and nine received
0.476 mg/ml of platinum salt. There was no 5-hour pulse. The cells used
in this experiment were from an untreated rabbit. The blood analysis
and viability data are presented below.
WBCa 2.15
RBCa 6450
Hb (g%) 11.79
•Rematocrit (%) 43.0
Differential count:
Neutrophils (%) 14
Lymphocytes (%) 85
Monocytes (%) 1
Eosinophils (%) 0
Basophils (%) 0
Nucleated RBCb 1
WBC isolated 0.52
Viable WBC (%) 100
RBC/WBC 2.2
WBC yield (%) 24.2
acells x 10~6/ml of whole blood.
bcells/100 WBC.
The results of the first experiment (Tables 43 and 44) show a dra-
matic depression in the incorporation of thymidine in in vitro platinum-
treated leucocytes. Thymidine uptake in these cells was only 15% of that
68
-------
Table 43
INCORPORATION OF LABELED SUBSTRATES INTO DJ VITRO
PLATINUM-TREATED LEUCOCYTES
First Experiment - Incorporation and Degradation Data
Tritium
Carbon 14
Control
0 hr
5 hr
24 hr
Platinum
(0.5 mg/ml)
0 hr
24 hr
Thyroid ine
25439
± 3742
—
-
3818
± 393
-
Leucine
7923
±2354
4335
±1060
2531
± 635
5515
± 684
3269
± 489
Uridine
20479
± 2217
-
-
11153
± 1004
-
Choline
12265
± 3600
5690
± 2072
2628
± 650
11331
± 825
4207
± 1922
Student's t-Test
Substrate Comparison v t
Thymidine Pt vs control 4 9.952 >99.9%
Uridine Pt vs control 4 6.637 >99%
Leucine Pt vs control 4 1.702 <90%
Choline Pt vs control 4 0.438 <90%
69
-------
Table 44
INCORPORATION OF LABELED SUBSTRATES INTO IN VITRO
PLATINUM-TREATED LEUCOCYTES
First Experiment - Percent Incorporation and Degradation
Incorporation
Percent of Percent
Substrate Control Depressed
Thymidine
Uridine
Leucine
Choline
15.0
54.5
69.6
92.4
85.0
45.5
30.4
7.6
Degradation:
Percent .of Zero Hour
Control
Platinum
Substrate 5 hr 24 hr 5 hr 24 hr
54.7 31.9 - 59.3
37.1
Leucine
Choline
31.9
46.4 21.4
of the controls. Uridine uptake was reduced by one-half. These data in-
dicate that platinum treatment markedly interferes with DNA synthesis and
also diminishes RNA synthesis.
Choline incorporation, on the other hand, is only slightly affected,
with an 8% reduction compared to the controls. Leucine uptake is depressed
to 70% of normal, but the difference does not appear to be significant.
These data indicate that the in vitro platinum treatment has only a
slight effect of phospholipid biosynthesis, while the effect on protein
biosynthesis is uncertain. In contrast, the rate at which whole cell
protein and phospholipids are catabolized is clearly influenced by the
platinum treatment. In the control cells, both constituents show approx-
imately a 50% turnover after 5 hours, whereas in the treated cells a 50%
turnover for protein is not achieved until after 24 hours. Similarly,
the phospholipid turnover rate is also increased by nearly a factor of
two. Thus, while the biosynthesis of protein and phospholipid is virtu-
ally unaffected by platinum treatment, the rate of degradation is strongly
depressed.
70
-------
The results of the second experiment (Tables 45 and 46) do not appear
to corroborate the results of the first. Both thymidine and uridine uptake
are depressed by about 30% in the cells treated with 0.5 mg/ml of platinum,
the same dose level as in the first experiment. Similarly, both leucine
and choline incorporation are depressed to about the same extent, that is,
about 20%. At the lower platinum concentration of 0.25 mg/ml, incorpora-
tion of thymidine is lower but the difference is not statistically signif-
icant. Thus, the two platinum concentrations are equally effective in in-
hibiting thymidine and uridine uptake. Degradation studies were not car-
ried out in this experiment.
The results of* the third experiment, presented in Tables 47 and 48,
demonstrate the inhibition of thymidine and uridine incorporation at both
concentrations of platinum treatment. As expected, a greater inhibition
is observed with the higher platinum level of 0.5 mg/ml; this is in con-
trast to the second experiment, in which the two treatment levels did not
result in significantly different incorporation. As in the first experi-
ment, platinum inhibits the uptake of thymidine to a greater extent than
that of uridine, but the differences are not nearly so pronounced.
A surprising observation is that both platinum treatments appeared
to stimulate choline incorporation—by nearly 70% at the lower platinum
level and 40% at the higher level. However, these figures are in ref-
erence to a single control culture that remained out of three originally
prepared. Although it is possible that the values for the control choline
tube are abnormally low, it seems unlikely in view of the generally low
standard deviations found with the other triplicate cultures (Table 47).
Leucine incorporation is slightly depressed at the 0.1 mg treatment
level, but the difference is not significant. At the higher platinum
concentration, the incorporation is significantly less by more than 40%.
The degradation rates of protein and phospholipid in this experiment
appear to be only slightly retarded compared to the controls. Thus, 60%
of both leucine and choline remains in the 0.5 mg platinum-treated cells
after 24 hours, whereas about 55% remains in the control.
Choline retention is the same at both platinum concentrations after
24 hours, but at the lower level protein degradation is increased slightly,
with 48% of labeled leucine remaining.
71
-------
Table 45
INCORPORATION OF LABELED SUBSTRATES INTO IN VITRO
PLATINUM-TREATED LEUCOCYTES
Second Experiment - Incorporation Data
Tritium
Carbon 14
Control
Platinum
0.25 mg/ml
0.5 mg/ml
Thymidine
99501
±2950
61751
±19428
71903
±11841
Leucine
20267
± 802
-
15173
± 1151
Uridine
36744
± 4577
24436
± 4579
24503
± 2049
Choline
43310
± 2219
-
35210
± 2969
Student's t-Test
Substrate
Thymidine
Uridine
Leucine
Choline
Comparison
0.25 Pt vs control
0.5 Pt vs control
0.25 Pt vs 0.5 Pt
0.25 Pt vs control
0.5 Pt vs control
0.25 Pt vs 0.5 Pt
0.5 Pt vs control
0.5 Pt vs control
3 3.604 >95%
4 3.917 >98%
3 0.751 <50%
3 2.945 <95%
4 4.237 >98%
3 0.024 <50%
4 6.289 >99%
4 3.786 >98%
72
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Table 46
INCORPORATION OF LABELED SUBSTRATES INTO IN VITRO
PLATINUM-TREATED LEUCOCYTES
Second Experiment - Percent Incorporation
Incorporation
Percent of Percent
Control Depression
Substrate 0.25 0.50 0.25 Q.5Q
Thymidine 62.0 72.3 38.0 27.7
Uridine 66.5 67.7 33.5 33.3
Choline - 81.3 - 18.7
Leucine - 75.0 - 25.0
73
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Table 47
INCORPORATION OF LABELED SUBSTRATES INTO Jfl VITRO
PLATINUM-TREATED LEUCOCYTES
Third Experiment - Incorporation and Degradation Data
Tritium
Carbon 14
Thymidine Uridine Leucine Choline
Control
0 hours
24 hours
Platinum - 0.1 mg/ml
33055
± 2718
13367
±1118
8813
4874
±1218
Platinum - 0.5 mg/ml
0 hours
24 hours
8531
± 951
4068
± 318
5099
± 370
3076
± 65
15744
8413
± 1071
0 hours
24 hours
18200
± 3924
—
_
6352
±1005
—
_
7739
± 283
3725
409
26489
± 1491
15410
138
21757
± 858
12818
± 130
Student's t-Test
Substrate
Thymidine
Uridine
Leucine
Choline
Comparison
0.1 Pt vs control
1.5 Pt vs control
0.1 Pt vs 0.5 Pt
0.1 Pt vs control
0.5 Pt vs control
0.1 Pt vs 0.5 Pt
0.1 Pt vs control
0.5 Pt vs control
0.1 Pt vs 0.5 Pt
0.1 Pt vs control
0.5 Pt vs control
0.1 Pt vs 0.5 Pt
4 5.390 >99%
4 14.750 >99.9%
4 4.148 >98%
4 7.808 >99%
4 13.093 >99.9%
4 3.754 >98%
1 5.370 <90%
2 13.486 >99%
3 8.431 >99%
1 10.194 <95%
2 9.407 >98%
3 4.672 >98%
74
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Table 48
INCORPORATION OF LABELED SUBSTRATES INTO JEN VITRO
PLATINUM-TREATED LEUCOCYTES
Third Experiment - Percent Incorporation and Degradation
Incorporation
Percent of Percent
Control Depressed
Substrate 0.1 0.5 0.1 0.5
Thymidine 55.1 25.8 44.9 74.2
Uridine 47.5 30.4 52.5 69.6
Leucine 87.8 57.9 12.2 42.1
Choline 168.2 138.2 -68.2 -38.2
24-Hour Degradation:
Percent of Zero Hour
Platinum
Substrate Control 0.1 0.5
Leucine 55.3 48.1 60.3
Choline 53.4 58.2 58.9
75
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DISCUSSION
The animals were treated initially with varying doses of lead ace-
tate and cadmium chloride to determine the highest level at which the
animals would survive for the three-week experiment with daily i.p. in-
jections. These levels were found to be 10 mg/kg for lead acetate and
1 mg/kg for cadmium chloride.
The lead and cadmium treatments produced a mild depression that
lasted throughout the 7- and 21-day treatment periods. The weight losses
seen in the control and experimental animals after both lead and cadmium
treatments are thought to be in part a result of a lower food intake
caused by the depressive effect of the metal treatments. Similar results
were obtained with rats fed diets containing 300 ppm of lead in the form
of lead acetate. Both weight loss and reduced food intake were noted
[21].
Lead poisoning produces hemolytic, mild hypochromic, and sometimes
microcytic anemia. Features include reticulocytosis which may be tran-
sitory, and basophilic stippling in the peripheral blood cells [22].
Stippling appears within 24 hours of exposure and may persist for 10
days to three weeks or more in rabbits [23]. The origin of lead-induced
anemia is complex and not fully understood. Its course is profoundly
affected by the level of, and route of exposure to, lead. Currently, it
is believed that the anemia is primarily the result of shortened erythro-
cyte life span caused by mechanical fragility and reduced heme synthesis
in red cell precursors [24] resulting from direct interference with syn-
thesizing enzymes and possibly enzymes of glycolysis. Lead interacts
with erythrocytes causing swelling, shrinkage, and crenation, which are
typically seen in lead poisoning in man and animals, including rabbits.
Crenation later disappears, and the attendant fragility leads to the
shortened life span. The decrease in the red cell count is generally less
than that of hemoglobin which seldom falls below 60%. The red cell count
may diminish to 4 x 106/mm3. Severe anemia is often accompanied by aniso-
cytosis, poikilocytosis, and the appearance of Howell-Jolley bodies [23].
No consistent changes in white cell counts are observed, although
leucocytosis, up to 15,000/mm3, is more commonly observed than leuco-
penia, especially in animals. Various effects have been noted on the
differential count, including increases in the number of monocytes and
lymphocytes. Eosinophilia has been noted by some workers but not by
others. Granulocytosis that changes to a shift to the right as the lead
poisoning becomes chronic has been observed. An in vitro exposure of
leucocytes to lead chloride led to a loss of phagocytic ability [23],
77
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There is little in the literature concerning blood findings in
cadmium poisoning. In workers in a battery factory the hemoglobin con-
tents and red cell counts were normal or slightly low. The sedimenta-
tion rate was somewhat elevated. The white cell counts and differentials
were normal with possible eosinophilia [25]. Tests on rabbits exposed
to cadmium-iron dust or given daily subcutaneous injections of cadmium
sulfate yielded similar results. Hemoglobins were slightly low, and
the red cell counts were diminished from about 6.0 x lO^/mm^ to about
4.5 x l()6/mm3. White cell counts and differentials were normal with
possible eosinophilia [25],
The results obtained in this research show that the three-week, but
not the one-week, lead treatment produced a mild hemolytic anemia char-
acterized by a diminished red cell count with the cells being larger
than normal. The cadmium treatment produced an anemia characterized by
a reduced blood hemoglobin content. Because the red cell count was nor-
mal and the hematocrit was low, it appears that the red cells were re-
duced in size as shown by the low MCV. However, the cells had a normal
hemoglobin concentration as indicated by the MCHC value. These obser-
vations suggest an aberration in erythropoesis.
The effect of lead treatment on white cells was minor. White cell
counts at both treatment periods were not significantly different from
controls, and all values were within normal limits. The differential
counts after both one and three weeks of lead treatment were nearly iden-
tical to the controls. Polychromasia and anisocytosis were more preva-
lent in the treated animals but were also seen in the control animals.
It is interesting that basophilic stippling, a common characteristic of
plumbism, was seen in only two of the lead-treated animals after one
week of treatment and was not seen at all in the animals treated for
three weeks.
In the one-week, cadmium-treated animals, the white cell count was
elevated compared with that of the controls, but it was still within
normal limits. After three weeks of cadmium treatment, the white cell
count was again normal. The differentials showed a significant in-
crease in neutrophils and a decrease in lymphocytes after one week of
treatment. After three weeks, the neutrophil count became lower than
that of the controls, but the difference was not significant. The lym-
phocyte count was normal. At both treatment periods, the appearance of
aberrant cell forms was minor.
These observations for lead-treated animals are consistent with re-
ports in the literature, especially in view of the reported variability
of blood findings in plumbism, with the exception of anemia, stippling,
and crenation. The anemia observed here in the cadmium-treated animals
contrasts with Friberg's data [25] and may reflect differences in the
conduct of the two experiments. In both cases, the apparent insensitiv-
ity of leucocyte counts and differentials to cadmium poisoning suggests
that it is not a useful clinical parameter.
78
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The biochemical analyses anticipated for the isolated leucocytes
were abandoned when it was found that the dry weight associated with
2 x K)6 cells was 0.9 rag. Thus it was impractical to attempt the sepa-
ration of such a small mass into lipid, protein, RNA and DNA. It would
have been possible to add a known amount of a carrier, such as yeast
cells, to each sample to ensure the recovery of each fraction, but since
the purpose of isolating these fractions was to determine the specific
activity of the particular radioactive precursor in each fraction, the
addition of a carrier would preclude a mass determination.
For determining the radioactivity content of the cells, isotopically
labeled substrates were combined in ^-^C-% pairs for incubations. The
Searle Mark III LSC is capable of resolving the two types of radioactive
disintegrations. In all cases, the combination of washing labeled cells
with a 100-fold excess of unlabeled substrate and the subsequent TCA
precipitation ensures that unbound labeled precursors are eliminated in
the acid-soluble fraction from the cells.
During the 22-hour incubation in the presence of radioactive pre-
cursors, it is reasonable to expect some redistribution of label by
metabolism of the radioactive substrates. Thus, for example, the in-
ference that cellular tritium derived from ^H-leucine represents protein
is not entirely correct. However, the incorporation of radioactive sub-
strates in heavy metal-treated cells is always compared with that of
control cells, so that for the purpose of detecting altered rates of
metabolism due to heavy metal treatment, the procedure is adequate.
The incorporation of radioactivity observed in these experiments is
due to uptake by the leucocytes and not due to other potential sources
of metabolic activity. For example, bacterial contamination has never
been observed. Aliquots of blood samples were cultured on tryptic soy
agar plates and no bacterial growth was observed. Although this single
test does not ensure that more fastidious organisms are not present,
it is indicative of the absence of gross bacterial contamination. Mans-
field reports (private communication) that pleuropneumonia-like organ-
isms (PPLO) are a frequent accompaniment of rabbit blood cell cultures,
but that their growth is effectively suppressed by the inclusion of
tylosin in the culture medium. This precaution was routinely observed.
Blood platelets are metabolically active [26], but these cells are
largely removed during two steps in the preparation of the leucocyte
suspension: removal of the platelet-rich plasma and washing of the
final leucocyte suspension.
As indicated earlier, contamination of the leucocyte suspension
by red blood cells was unavoidable. The RBC/WBC ratio rarely fell below
1 and ranged up to 8 (Tables 25 and 26). Leucocyte preparations with a
greater red cell contamination were not used except that from the #6
lead-treated animal in the three-week experiment, which had a ratio of
37.5. The KBC control experiment clearly demonstrates that, in general,
the contribution of rad.ioactivity incorporation due to RBC is very much
79
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less than 10%. However, in two of the animals the WBC incorporations
were small enough that the potential contribution due to RBC invalidates
some of the data. The table below shows the percent of the WBC incor-
porations that may be attributable to the RBC content for several of the
WBC preparations having the lowest cell count combined with the highest
RBC contamination.
Experiment (weeks)
Treatment: Lead Lead Cadmium Cadmium Lead
Animal:
RBC/WBC:
Thymidine (%)
Uridine (%)
Leucine (%)
Choline (%)
#1
8.2
1.8
0.3
3.3
1.7
#5
5.4
6.6
2.9
—
—
#5
6.7
47
3.0
10
2.7
#5
1.6
6.9
1.4
2.1
0.2
#6
37.5
100
6.2
47
16
The presence of red cells in the leucocyte preparations seems un-
desirable but, at the present time, inescapable. As indicated earlier,
the one procedure attempted for the hypotonic lysis of RBC irreparably
damaged the leucocytes as well. However, this procedure was designed
for human cells. Mansfield (personal communication) reports that .an-
other procedure [27] for hypotonic lysis worked well with rabbit leuco-
cyte preparations. We have found that this method completely eliminates
red blood cells from the leucocute suspensions, and we are currently
evaluating the integrity of the resultant white cells in preparation
for future research.
The low incorporation of radioactive substrates into the red blood
cells is consistent with the known metabolism of these highly special-
ized cells [28,29], For example, since erythrocytes have no nucleus,
they cannot biosynthesize DNA or RNA. Thus, the incorporation of labeled
thymidine and uridine into a mixed population of red and white cells
should be due primarily to the white cells, particularly in the circum-
stance when DNA synthesis and also, therefore, RNA and protein synthesis
have been stimulated by the mitogen, phytohema,gglutinin. Some incorpor-
ation may be due to the small proportion of reticulocytes as well. Also,
mature red cells do not synthesize lipids or protein. There is, however,
an energy-independent exchange of cholesterol and, to a lesser extent,
phospholipid between plasma and the red cell membrane.
Similarly, in dogs and humans, the rate of glycolysis in erythro-
cytes was 0.014 to 0.028 fl% glucose/hr/10^ cells, whereas in leucocytes
the rate was 4.7 to 13.2 //g glucose/hr/106 cells [29]. Thus, the glyo-
lytic rate in erythrocytes was, at most, 0.6% of the rate found in leu-
cocytes. Only a few of the tricarboxylic acid enzymes persist in the
adult erythrocyte. It seems likely that the situation would be similar
in rat and rabbit cells.
80
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The greatest difficulty encountered in this research was associated
with the separation of white cells from rabbit blood and maintaining
their viability during the subsequent culturing. The major problems
were low white cell yields, white cell clumping or aggregation, high red
cell contamination, and short viability of the white cells. Repeated
efforts were made to improve these aspects of the procedure, using many
techniques that the literature indicated as being satisfactory for human
cells; however, all of them failed to produce acceptable rabbit white
cell preparations. Successful preparations were not obtained until the
paper of Mansfield and Wallace [14] appeared and we adopted their proce-
dures. Several important factors were indicated in their paper and in
direct communication with them: the use of heparin (and not EDTA) as
an anticoagulant; the use of up to 100 U/ml of heparin to prevent plate-
let aggregation and RBC agglutination; the use of RPMI-1640 culture med-
ium; the inclusion in the medium of heat-inactivated autologous plasma
as the protein supplement; the inclusion in the medium of tylosin (at a
level of 10%) as an anti-PPLO agent; the use of 1 x 106 white blood cells
per milliliter of culture medium; the use of low levels of PHA to stimu-
late mitogenesis; and extension of the PHA treatment time to 22 hours
prior to addition of the radioactive substrates. Other factors that we
found to be important, and in which Mansfield concurred (private communi-
cation), are the dilution of whole blood 1:2.5 with HBSS, and centrifu-
gation on the gradient for at least 30 minutes and at 400 x j* or more.
Because of the repeated inability to obtain a good leucocyte pre-
paration, we investigated numerous other variables, none of which had
a significant enough effect on the procedure to warrant a change. Among
these are: obtaining the blood by opening the chest cavity before card-
iac puncture; pretreating the rabbit with heparin prior to obtaining
blood by cardiac puncture; the heparin concentration within the limits
of 10-100 U/ml; the age of the blood up to about six hours after drawing;
filtering the blood through sterile cheesecloth or glass wool to remove
small clots of red cells; the freshness of the Ficoll and Hypaque solu-
tions (they are mixed just prior to use); autoclaving Ficoll but not the
Ficoll-Hypaque mixture; layering the gradient beneath the blood as opposed
to layering the blood on top of the gradient; and increasing the time or
speed of centrifugation by up to 50%.
The incorporation of labeled thymidine, uridine, leucine, and cho-
line by viable leucocytes implies the biosynthesis of DNA, RNA, protein,
and phospholipid, respectively. The problem of the redistribution of
label during the incubation period was discussed earlier.
The substrates employed are among the more metabolically stable
biochemicals found in living matter and are frequently used to monitor
the above biosynthetic processes.
The data clearly show that leucocytes demonstrated to be viable by
the eosin Y test are also very active metabolically in synthesizing the
major classes of biochemical constituents of nucleic acids, protein, and
81
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lipid. Furthermore, the leucocyte preparations exhibit the dynamic na-
ture of cellular metabolism characteristic of living cells in that the
labeled protein and phospholipid in the leucocytes undergo degradation,
thus implying a continual renewal of these materials. These observa-
tions serve to demonstrate that the leucocyte preparations are healthy—
or at least sufficiently so to serve as monitors for examining the meta-
bolic effects of heavy metal treatment in the host animals from which
the leucocytes are derived.
The data from the preliminary lead and cadmium experiment indicate
that lead and cadmium treatments of rabbits have important metabolic ef-
fects. The predominant effect noted is a severe depression in the bio-
synthesis of DNA in the cadmium-treated animal. This observation was
confirmed in the one-week and three-week experiments. The greatest de-
pression was seen after one week of cadmium treatment. By three weeks,
DNA synthesis was still reduced but less so than at one week.
RNA synthesis is also affected by cadmium, but the depression is
much less than with DNA. The depressive effect of cadmium on DNA syn-
thesis was about the same at one and three weeks.
In contrast to these results, lead treatment in the preliminary ex-
periment and in the one-week experiment had negligible effect on either
DNA or RNA synthesis. By three weeks, nucleic acid synthesis was sup-
pressed but less so than that due to cadmium exposure.
Protein and phospholipid biosyntheses were not affected by either
lead or cadmium treatment for one or three weeks. In the preliminary
experiment, protein biosynthesis was not affected by the lead treatment,
but was decreased by cadmium exposure. In contrast, phospholipid syn-
thesis appeared to be accelerated by both treatments. Since this result
was not repeated in the one- and three-week experiments in which more
animals were used, we assume that the phospholipid results from the pre-
liminary experiment are probably not representative.
In general, protein and phospholipid degradations were not affected
by lead or cadmium treatment in the one- or three-week experiments. Two
exceptions were noted in that phospholipid turnover was increased by lead
treatment at 24 hours and by cadmium treatment at 5 hours (Table 41).
The increases were small but statistically significant. The preliminary
experiment gave the same result for the lead treatment but indicated that
both protein and phospholipid turnover were increased by cadmium treat-
ment. Again, this result is not considered representative.
The effect of cadmium and lead treatment in vivo on the metabolism
of leucocytes does not appear to have been studied previously [30],
91-(1972), although extensive work has been carried out in other tissues
from many species. Lead and cadmium bind to a wide variety of biomole-
cules by virtue of their strong affinity for purine, pteridine, porphy-
rin, and phosphate moieties and for sulfhydryl, histidinyl, phenoxy, and
82
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side-chain amino acid carboxyl groups of proteins. Lead and cadmium in-
hibit many enzymes having functional sulfhydryl groups, particularly
those associated with oxidative phosphorylation and, hence, synthesis
[31] and with nucleic acids. On the other hand, they can either stimu-
late or inhibit several1 peptidases by substituting or interacting with
resident zinc atoms [30].
In this research, inhibition of the incorporation of thymidine into
nucleic acids by cadmium and lead treatment was the outstanding metabolic
feature. The incorporation of leucine into protein, of choline into
phospholipid, and of uridine into ribonucleic acid is energy-dependent
requiring the participation of ATP. Because these processes were not
affected by the lead and cadmium treatments, or only minimally so, it
may be deduced that energy metabolism and oxidative phosphorylation
were not appreciably affected.
Since the incorporation of thymidine into deoxynucleic acid is also
energy-dependent, it is reasonable to infer that the lead and cadmium
treatments interfered with DNA synthesis in a manner not involving energy
metabolism. Cadmium and other metals affect the physical properties of
DNA, but their biological significance has not been explored [32]. Lead
forms complexes of nucleotides and nucleic acids through the phosphate
group [33]. Lead concentrates in the cell nuclei of liver and kidney
[34,35] and probably also in leucocytes as judged by the appearance of
chromosomal abnormalities in human [36] and mouse white cells [37],
Thus, it is possible that the depressive effect of the cadmium and lead
treatments on DNA synthesis may be a result of a direct interaction of
these metals with DNA which subsequently disrupts the replication pro-
cess. Further research would be required to investigate this hypothesis.
Thymidine incorporation occurs at varying rates in leucocytes ac-
cording to cell type and to the biochemical and physiological status of
each cell type. This incorporation depends on such factors as the size
of the purine and pyrimidine pools, the number of mitotically active
cells at any given time, the extent to which labeled thymidine is di-
verted to non-DNA pathways, and the extent to which the labeled moieties
of DNA are reutilized [38]. The extent to which such factors influenced
the results of this research cannot be assessed, but it seems pertinent
to observe that the experiments were conducted as identically as possible,
with the exception of the treatment regimen, in order to minimize pro-
cedural variations. Thus, it seems reasonable to suggest that the dif-
ferences in incorporation of labeled thymidine is attributable to the
metal treatment which, in ways that are obscure at present, altered one
or more factors controlling the incorporation of labeled thymidine. Be-
cause the shifts in the white cell differentials caused by metal treat-
ment were small, it may be concluded that a shift in cell type is not
responsible for the variations in incorporation.
A final observation concerning the one- and three-week experiments
is that the level of incorporation of all four radioactive substrates
was significantly higher in the three-week control animals than in the
83
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one-week controls (Table 42, section B). The animals for the two exper-
iments were purchased together and randomly placed on the one- or three-
week treatment regimes; hence, variations due to age and sex can be
eliminated. There was no correlation between incorporation of radioac-
tive substrates and the sex of the rabbits. To obtain sufficient blood
for the leucocyte preparation, adult rabbits having a minimum weight of
3 kg were specified (three rabbits weighing less were used—see Tables
19 and 20). Because of differing growth rates, the ages of the animals
used also varied. Although data on ages were not presented, there was
no correlation between incorporation of radioactive substrates and the
age of the rabbits. Animal handling, care, and treatment were identical
for both groups of animals except for the duration of treatment. The
procedures for obtaining the blood and for white cell preparation and
use were conducted,in as identical a manner as possible to avoid any bio-
chemical effects that might be caused by procedural variations.
The primary difference between the one- and three-week controls was
that the latter incurred an additional two weeks of stress caused by
daily i.p. injections of a saline control. Biochemical alterations in
animals are well-known concomitants of stress and include release of
ACTH through activation of the adrenal-pituitary axis [39] and alterations
in microsomal drug-metabolizing enzymes [40]. In most studies, the stress
appeared to be more extreme than that caused by daily i.p. injections of
saline. However, subtle alterations of leucocyte metabolism may repre-
sent a sensitive response to stress not previously recognized.
Unexpectedly, the results of the three in vitro platinum experi-
ments lacked consistency. The third experiment was performed in the
most favorable manner in that both the control and platinum-treated cells
were washed twice in saline to maximize removal of platinum salt from
the treated cells. In the first two experiments, because the two wash
steps were omitted, it is possible that the leucocytes might have been
more numerous and more vigorous than those in the treated suspensions.
The results of the second experiment seem less satisfactory than the
other two in that (a) there is little variation in the percent depression
of the various labels, and (b) the depression of incorporation is higher
for the lower treatment level.
One point of constancy regarding the first and second experiments
is in the absolute incorporations of the four labeled substrates in the
control cells compared to the corresponding incorporations in the control
cells from the one- and three-week experiments. This is expected, of
course, since the same pool of leucocytes was used in both cases. While
the first and third experiments vary in the magnitude of the various
depressions of incorporation, there is a constancy in that platinum
treatment of 0.5 mg/ml depresses thymidine incorporation to the greatest
degree, uridine second, and leucine third. Choline uptake is the least
affected in both experiments, with only a slight depression in the first
experiment and an apparent stimulation in the third.
84
-------
Considering all three experiments together, it seems apparent that
platinum treatment at 0.5 mg/ml does interfere with cellular metabolism
and in particular with nucleic acid synthesis. Protein synthesis is also
probably affected, while the effect on phospholipid metabolism is small.
It also seems evident that platinum affects nucleic acid metabolism at
the lowest level tested, 0.1 mg/ml.
Although these three i.n vitro experiments show considerable varia-
tion in results, this type of experiment has a major advantage over the
type of experiments performed in the one- and three-week exposures. In
the in vitro experiments, the treated cells and the control cells are
derived from the same pool, whereas in the one- and three-week experi-
ments the control cells and the treated cells are derived from different
animals. This is,- of course, a necessary concomitant to the study of in
vivo effects of heavy metal treatment, but it does necessitate reliance
on a statistical evaluation of results from several animals. We believe
that such an analysis has been successfully accomplished, particularly
through using the tactic of comparing the uridine/thymidine and choline/
leucine ratios, which normalize the data and eliminate much of the in-
herent biological variation among a small population of animals.
Determining the extent of recent and past exposure to trace metals
is difficult. Blood and urine determinations are used as indices of
recent and current exposure to lead, but they are inadequate in assess-
ing previous exposure because of its storage in kidney, liver, bone, and
other tissues. Thus, the blood level may be normal or minimally elevated,
but the body burden may be very high. Determinations of lead in hair
gives information on previous exposures but not on present metabolic
status. One method currently used to estimate body burden involves
mobilization of tissue stores of lead using CaEDTA and measurement of
urinary excretion of the lead complex.
Cadmium is also stored in tissues, particularly in the liver and
kidney. Thus, levels of blood cadmium, like blood lead, may not reflect
the degree of intoxication. Also cadmium analysis presents such diffi-
culties as tissue preparation, interference from much larger amounts of
zinc, and sensitivity of the assay. More recent methods, such as flame-
less atomic absorption analysis using a carbon rod atomizer (Varian
Techtron), should facilitate such analyses. In both cases, tissue anal-
ysis would be desirable, but acquisition of tissue samples is generally
not feasible. Thus, other methods for the determination of body burden
of lead and cadmium would be useful.
The work described here demonstrates that the white blood cell is
a responsive bioindicator of lead and cadmium intoxication in the rab-
bit. Further work is required to relate both the body burden of these
metals to the degree of leucocyte response and the sensitivity of the
response to metal intake.
The leucocyte has several attributes that are ideal in a bioindicator
for trace metal contamination. It is a complete cell possessing a nucleus,
»
85
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mitochondria, and microsomes in contrast, for example, to the mature
red blood cell (erythrocyte) which lacks these organelles. Further,
normal leucocytes are important in many of the body's defense mecha-
nisms, and the appearance of abnormal forms is often a manifestation of
significant disease processes. Thus, the leucocyte may be expected to
possess a diversity of biochemical processes that equal or exceed those
of other nucleated and differentiated cells of the body. Finally, leu-
cocytes are a readily available biopsy tissue that can be separated from
other blood cells in relatively pure form for subsequent studies of mor-
phology, biochemistry, physiology, and immunochemistry. It is antici-
pated that the use of leucocytes as bioindicators will be expanded to
include a variety of compounds other than trace metals and extended in
relevancy by the use of human leucocytes.
86
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SUMMARY
1. Rabbit leucocytes were successfully separated from whole blood
and cultured, using the procedure of Mansfield and Wallace [14]. The
essential features of this procedure are separation of white cells from
heparinized whole blood by density gradient centrifugation on a Ficoll-
Hypaque solution and culture of white cells in RPMI-1640 medium contain-
ing 10% heat-inactivated autologous rabbit plasma.
2. The metabolism of the white cells was stimulated by a 22-hour
treatment with the plant mitogen, phytohemagglutinin (PHA).
3. Untreated, viable control cells were shown to synthesize DNA,
RNA, protein and phospholipid by the incorporation of %-thymidine, -^C-
uridine, ^H-leucine, and -^C-choline, respectively. In addition, protein
and phospholipid were shown to catabolize in previously labeled cells
following the removal of labeled leucine and choline, respectively, from
the culture medium.
4. The intraperitoneal injection of rabbits in vivo with lead
acetate or cadmium chloride solutions for one or three weeks resulted
in well-characterized alterations in the metabolism of leucocytes de-
rived from the treated rabbit.
5. The most outstanding effect noted was a severe depression in
nucleic acid synthesis, particularly DNA, caused by cadmium treatment
for one or three weeks or by lead treatment for three weeks. Lead
treatment for one week had no influence on nucleic acid synthesis.
6. Lead and cadmium treatment had little or no effect on the bio-
synthesis or degradation of protein or phospholipid after either one or
three weeks of treatment.
7. Leucocytes from three-week control rabbits, which received
daily i.p. injections of saline, incorporated 402% more thymidine, 134%
more uridine, 88% more leucine, and 68% more choline than did leucocytes
from the one-week control rabbits. It is suggested that the extra two
weeks of stress caused by the injections was responsible for the in-
creased incorporations.
8. The one- and three-week control and metal treatments produced
a mild weight loss in all animals. At the end of one week of treatment,
the weight loss in the metal-treated animals was significantly greater
than that in the controls, with the lead-treated animals exhibiting the
greatest loss. After three weeks, the weight loss of the lead-treated
animals, but not of the cadmium-treated animals, was significantly
greater than that of the controls. These losses are thought to result
from reduced food intake.
»
87
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9. The one-week lead and cadmium treatments had no effect on red
cell findings. The three-week lead treatment produced a mild hemolytic
anemia characterized by a diminished red cell count, hemoglobin content,
and hematocrit and an increased red cell size. Anemia occurred after
the three-week cadmium treatment as a result of a low hemoglobin con-
tent and small red cells with a normal hemoglobin concentration.
10. After one week of cadmium treatment, the white cell count was
significantly elevated, but by three weeks it had returned to normal.
Neither the one-week nor the three-week lead treatments had an effect
on the white cell count.
11. Lead treatment had no effect on the white cell differentials.
Polychromasia and anisocytosis were more prevalent after one week of
lead treatment and were normal by three weeks. Basophilic stippling, a
characteristic of chronic lead poisoning, was observed in only two ani-
mals and only after one week of treatment. Cadmium treatment produced
an elevated neutrophil count and reduced lymphocyte count at one week.
After three weeks, the neutrophil count was low, and the lymphocyte
count was normal. Aberrant cell forms were infrequently seen in the
cadmium-treated animals.
12. When control leucocytes were treated in vitro with sodium
hexachloroplatinate for two hours, synthesis of nucleic acid, partic-
ularly DNA, is depressed. This effect is noted at the lowest salt con-
centration used, 0.1 mg/ml of culture medium. Protein synthesis is also
depressed, but phospholipid synthesis is only slightly affected.
13. In one experiment, degradation of protein and phospholipid was
not affected by platinum treatment even at the lowest level used, whereas
in another experiment the degradation rate was slowed by a factor of two.
Thus, the effect of platinum treatment on the degradation of protein and
phospholipid is uncertain.
88
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GLOSSARY
Cd, CD
cpm, CPM
DNA
DF
dpm, DPM
EDTA
Hb
HBSS
Hp
LD50
MC
MCH
MCHC
MCV
MEM
MEM-S
fie
Solubilizer
NRBC
Cadmium
Counts per minute
Deoxyribonucleic acid
Degrees of freedom (see "v")
Disintegrations per minute
Ethylenediaminetetraacetic acid
Hemoglobin
Hank's balanced salts solution
Hypaque
Median lethal dose, one that is fatal to 50%
of the test animals
Methylcellulose
Mean corpuscular hemoglobin—the hemoglobin content
of the average red blood cell
Mean corpuscular hemoglobin concentration—the hemo-
globin concentration in the average red blood cell
Mean corpuscular volume—the volume of the average
red blood cell
Eagle's minimal medium
Eagle's minimal medium, spinner modification
Microcurie = 10~6 curie = 2.22 x 10^ dpm
A solution of a quaternary ammonium base in toluene
developed by Amersham/Searle for the solubilization
of biological materials
Nucleated red blood cells
89
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P The probability that the two samples examined are
statistically significantly different from each
other. P > 95% is considered significant.
Pb, PB Lead
PHA Phytohemagglutinin—this material is available from
Difco as either the M or the P form, the latter hav-
ing the highest purity
Pt Platinum
RBC Red blood cells
RNA - Ribonucleic acid
RPMI-1640 A nutrient medium for tissue culture devised at the
Roswell Park Memorial Institute, New York
s A statistical parameter relating to Kendall's
coefficient of concordance
SD Standard deviation
t, T The t-value from the Students' t-Test
U Units
v Degrees of freedom, a statistical parameter employed
in the Students' t-Test
W Kendall's coefficient of concordance
WBC White blood cells
90
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93
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA 650/1 74-013
4. TITLE AND SUBTITLE
Use of Leucocyte Metabolism as a Health Effects
Indicator
3. RECIPIENT'S ACCESSIOWNO.
5. REPORT DATE
April 1974
_Ap
>. PEf
RFORMING ORGANIZATION CODE
7. AUTHOR(S)
Kenneth D. Lunan
8. PERFORMING ORGANIZATION REPORT NO
LSU-2430
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Stanford Research Institute
Menlo Park, California 94025
10. PROGRAM ELEMENT NO.
1A1005
11. CONTRACT/GRANT NO.
68-02-0713
12. SPONSORING AGENCY NAME AND ADDRESS
Human Studies Laboratory
National Environmental Research Center
Environmental Protection Agency
Research Triangle Park, N. C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final -- 12 month^
14.' SPORSO RIN G"AG E NC Y CO I
CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The objective of this study was to evaluate the use of leucocytes as a responsive
bioindicator of lead, cadmium, and platinum intoxication in rabbits. Adult rabbits
were injected intraperitoneally with cadmium chloride, lead acetate, and saline daily
for one or three weeks. Toxicity studies established the maximum permissible dosages
for the metal treatments. Leucocytes were isolated by density gradient^ centrifugation
and examined for their ability to synthesize deoxyribonucleic acid, ribonucleic acid,
protein, and phospholipid and to catabolize protein and phospholipid.
Rabbit leucocytes were also treated in vitro with sodium hexachloroplatinate, and
the same metabolic capabilities were assessed. Lead and cadmium treatments produced
a mild anemia, but the white cells were only slightly affected. The one-week cadmium
treatment and the three-week lead and cadmium treatments depressed the synthesis of
both nucleic acids. The synthesis and degradation of protein and phospholipid were
unaffected by the metal treatments. Leucocytes from three-week control rabbits
synthesized all four classes of biomolecules at a faster rate than leucocytes from
the one-week control rabbits. In leucocytes treated with the platinum salt in vitro.
nucleic acid and protein synthesis were depressed, but phospholipid synthesis was
unaffected.
These results demonstrate that leucocytes may serve as a responsive bioindicator
of trace metal contamination.
17.
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b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
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Release Unlimited
19. SECURITY CLASS (This Report)
* Unclassified
20. SECURITY CLASS (Thispage)
Unclassified
21. NO. OF PAGES
im
22.PRICE
EPA Form 2220-1 (9-73)
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