EPA-600/1-76-010b
January 1976
Environmental Health Effects Research Series
ASSESSMENT OF TOXICITY OF
AUTOMOTIVE METALLIC EMISSIONS
Volume II
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development,
U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:
. 1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS
RESEARCH series. This series describes projects and studies relating
to the tolerances of man for unhealthful substances or conditions.
This work is generally assessed from a medical viewpoint, including
physiological or psychological studies. In addition to toxicology
and other medical specialities, study areas include biomedical in-
strumentation and health research techniques utilizing animals -
but always with intended application to human health measures.
This document is available to the public through the National
Technical Information Service, Springfield, Virginia 22161.
-------
EPA-600/1-76-01 Ob
January 1976
ASSESSMENT OF TOXICITY OF AUTOMOTIVE METALLIC EMISSIONS. VOLUME II;
Relative Toxicities of Automotive Metallic Emissions Against
Lead Compounds Using Biochemical Parameters
By
David J. Holbrook, Jr., Ph.D.
Department of Biochemistry
School of Medicine
University of North Carolina
Chapel Hill, North Carolina 27514
Contract No. 68-02-1701
Project Officer
Ms. Frances P. Duffield
Catalyst Research Program Office
Health Effects Research Laboratory
Research Triangle Park, North Carolina 27711
U. S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
-------
DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U. S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the
views and policies of the U. S. Environmental Protection Agency, nor
does mention of trade names or commercial products constitute endorsement
or recommendation for use.
-ii-
-------
ABSTRACT
SECTION I
The intraperitoneal administration of PtCl, or Pd(NO ) at levels
of 28 or 56 ymoles/kg body weight decreased the thymidine incorporation
into DNA of spleen, liver, kidney and testis. Spleen was most sensitive
to both the platinum and the palladium salt. In liver, DNA syntheses in
parenchymal cells and stromal cells were about equally sensitive to
3
PtCl,. In control rats, only 20-30% of the H in the acid-soluble
fraction of liver or spleen was in the form of thymidine and its phosphate
3
esters 2 hr after the intraperitoneal injection of H-thymidine; prior
injection of PtCl, (56 ymoles/kg body weight) did not change the pattern.
SECTION II
The effects of various salts of platinum or palladium were determined
on the parameters of the microsomal mixed-function oxidase system from
rat liver. The intraperitoneal injection of PtCl, or Pd(NO ) at 56
ymoles/kg, increased the hexobarbital-induced sleeping time in vivo and
generally decreased the aminopyrine demethylase in vitro and the microsomal
content of cytochrome P-450. The dietary administration of various salts
of Pt or Pd for one week generally decreased or had no effect on the para-
meters of drug metabolism by isolated microsomes and after 4 or more weeks
generally had no effect on, or increased, the parameters. The addition
of 0.15-0.2 mM PtCl or 0.2-0.3 mM Pd(NO ) to the incubation medium
(containing 5 mM MgCl2) inhibited the aminopyrine demethylase of isolated
hepatic microsomes by approximately 50%.
-iii-
-------
SECTION III
Lethal-dose studies are reported following the intraperitoneal or
oral administration of salts of lead,, manganese, platinum and palladium
to young male rats. Studies have been conducted on the effect of the
dietary administration of salts of lead, manganese, platinum and palladium
on the following: the growth rate of male rats, the organ weight of five
tissues (liver, kidney, spleen, heart and testis), and the tissue content
of DNA, RNA and protein.
In general, dietary levels of PbCl2, PdCl^, PdO and PdSO, greater than
10 mmoles/kg feed were necessary to restrict the weight gain of the rats.
4+
Soluble salts of Pt , namely PtCl4 and Pt(S04)2.4H20, at levels of
approximately 2 mmoles/liter drinking fluid, were sufficient to restrict
the weight gain of treated rats.
Dietary PbCl_ markedly increased the size of kidneys in treated rats.
In almost all other studies, however, the dietary administration of salts
of lead, manganese, platinum or palladium did not markedly or consisently
alter the organ weights of the metal-treated rats. The dietary administration
of PbCl-, PtCl, or Pt(SO,)?.4H 0 for 4 weeks did not alter the content of
DNA, RNA or protein in liver, kidney or spleen (when the content is
expressed per gram of wet tissue).
-iv-
-------
TABLE OF CONTENTS
Page
SECTION I. EFFECTS OF PLATINUM AND PALLADIUM SALTS ON THYMIDINE
INCORPORATION INTO DNA OF RAT TISSUES 1 -
INTRODUCTION 1
MATERIALS AND METHODS 2
RESULTS . 4
DISCUSSION 9
REFERENCES 10
SECTION II. EFFECTS OF PLATINUM AND PALLADIUM SALTS ON PARAMETERS
OF DRUG METABOLISM IN THE RAT 18
INTRODUCTION 18
MATERIALS AND METHODS 19
RESULTS 22
DISCUSSION 28
REFERENCES 30
SECTION III.TOXICITY OF PLATINUM AND PALLADIUM SALTS IN THE RAT 40
INTRODUCTION 40
MATERIALS AND METHODS 41
RESULTS 43
DISCUSSION 48
REFERENCES 49
-v-
-------
LIST OF FIGURES
4+ 2+
Figure 1. Effect of Pt and Pd to the Incubation Medium on
the Amino-Pyrine Demethylase Activity in Vitro 38
Figure 2. Double Reciprocal Plots of the Inhibition of Aminopyrine
Demethylase by the Addition of PtCl, or Pd(NO.) to the
Incubation Medium 39
-vi-
-------
LIST OF TABLES
I§j
SECTION I
Table 1. Effect of PtCl, on Thymidine Incorporation into
DNA of Rat Tissues 12
Table 2. Effect of Pd(NO ) on Thymidine Incorporation into DNA
of Rat Tissues 14
Table 3. Effect of PtCl, on Thymidine Incorporation into Nuclei
of Parenchymal (N-l) and Stromal (N-2) Cells of Liver 15
Table 4. Effect of PtCl, on Thymidine Incorporation into Tissues
of CCl.-Treated Rats 16
3
Table 5. Distribution of H-Labeled Compounds in the Acid-
Soluble Fraction 17
SECTION II
Table 1. Increase in Hexobarbital-Induced Sleeping Times in Rats
Treated with PtCl4 or Pd(N03>2 31
Table 2. Effect of Intraperitoneal Injections of PtCl, or Pd(NO )
for Two Consecutive Days on Parameters of Drug Metabolism
by Isolated Hepatic Microsomes (MCS) 32
Table 3. Effects of Administration of Metal-Containing Diets on
the Parameters of Drug Metabolism by Isolated Hepatic
Microsomes (MCS) 34
SECTION III
Table 1. Lethal Doses of Various Metallic Compounds After
Intraperitoneal or Oral Administration in the Rat 50
Table 2. Effect of Dietary Metallic Salts on Weight Gain 51
Table 3. Effect of Dietary Metallic Salts on Tissue Weights 53
Table 4. Effect of Dietary Metallic Salts on the Concentration of
DNA, RNA and Protein in Various Tissues 57
-vii-
-------
SECTION I. EFFECTS OF PLATINUM AND PALLADIUM SALTS ON THYMTDTNF.
INCORPORATION INTO DNA OF RAT TISSUES
INTRODUCTION
Platinum and palladium compounds currently are being used as the
active components in the catalytic converters of air pollution control
devices on many motor vehicles. It is of interest, therefore, to determine
the biological effects of the compounds of these metals. The present
experiments were conducted to determine the acute effects of the soluble
salts of platinum and palladium on the incorporation of radioactive
thymidine into DNA.
-1-
-------
MATERIALS AND METHODS
All experimental studies were conducted with male Sprague-Dawley
rats (weighing 160-200 g) obtained from Zivic-Miller Laboratories. In
each of these experiments, the metallic salt and the %-labeled thymidine
were injected intraperitoneally 4 hr and 2 hr, respectively, before the
n
removal of the tissues. The JH-thymidine was injected at a level of 99
uCi/kg body weight, except that a level of 990 yd/kg body weight was
3
used when the distribution of H in the acid soluble fraction was under
study (i.e., Table 5). [ H-methyl]thymidine was purchased from New
England Nuclear, Pd(NC>3)2 aqueous solution from Research Organic/Inorganic
Chemical, and PtCl from the latter firm and from B.F Goldsmith Chemical
and Metal.
The administered doses of PtCl^ and Pd(N03>2 were 14, 28 and 56 ymoles/
kg body weight. For PtCl^, these doses corresponded to 2.8, 5.5 and 11
mg Pt^ /kg. For Pd(N03)2» the doses by weight were equal to 1.5, 3 and
2+
6 mg Pd /kg body weight. The highest dose of Pt, namely 56 ymoles/kg,
was equal to one-half of the intraperitoneal LD^Q. However, because of
the steep slope of the lethal dose curve, the dose of 56 ymoles PtCl^/kg
was appreciably less than the LD,..
Thymidine incorporation was measured in spleen, liver, kidney and
testis. Tissue macromolecules were precipitated with cold 0.5 M HCIO^
and, after centrifugation, the supernatant was collected as the acid-soluble
fraction. The RNA in the pellet was hydrolyzed with 0.3 M NaOH (37°; 1
hr) and the intact macromolecules were precipitated with cold HCIO^ (final
concentration of un-neutralized HC104, 0.5 M). The DNA was hydrolyzed
in hot HCIO^ (0.5 M; 90°; 20 min). After centrifugation, the supernatant
(hydrolyzed DNA) was collected. The acid-soluble fraction and the hydro-
lyzed DNA were analyzed for nucleotide concentration by measuring the
-2-
-------
absorbance at 260 run. Tritium was measured by scintillation counting
using a mixture containing toluene and Triton X-100 (1).
The radioactivity in the DNA is expressed as counts-per-minute/
ymole DNA-nucleotide. The radioactivity in the acid-soluble fraction is
expressed as counts-per-minute/ymole total acid-soluble nucleotide. The
radioactivity is also expressed as a ratio of the two values, counts-
per-minute/ymole DNA-nucleotide divided by counts-per-minute/ymole
total acid-soluble nucleotide. This ratio takes into consideration two
factors: (a) the total amount of radioactivity available for incorporation
into tissue DNA, and (b) any variation in isotope concentration in the
same tissue of different rats within an experimental group.
N-l nuclei from hepatic parenchymal cells and N-2 nuclei from hepatic
stromal cells were isolated according to the method of Potter and co-
workers (2,3). The method is similar in principle to the method used
previously in this laboratory for the fractionation of nuclei from control '
and regenerating liver (4).
The adsorption of nucleotides and related compounds by charcoal from
acidic solution was conducted according to the method of Tsuboi and Price
(5). The charcoal-adsorbed compounds were eluted from the charcoal by
two treatments with 1% ammonia in 60% ethanol (5). The eluted charcoal-
adsorbable compounds were chromatographed on thin layer sheets of polyethyleneimine-
cellulose with 0.02 M ammonium acetate-95% ethanol (1:1, v/v). Thymidine and
thymine migrated to the solvent front; thymidine phosphates remained at or
near the origin.
-3-
-------
RESULTS
The effects of PtCl, on thymidine incorporation into DNA of rat
tissues are presented in Table 1. In the tissues studied, the incorpora-
tion of thymidine into spleen DNA was the most sensitive to platinum. In
spleen, thymidine incorporation was reduced by one-third at the
lowest dose of PtCl . The two higher doses of PtCl, decreased thymidine
incorporation by 50% or more. The ratios (DNA/acid-soluble fraction) gave
the same pattern of inhibition. The incorporation of thymidine into liver
DNA was not as sensitive to platinum as was the incorporation into spleen
DNA. Although no inhibition was observed in liver at a dose of 14 ymoles/
kg, thymidine incorporation was inhibited by 40% and the ratio approached
that observed in spleen at a dose of 56 ymoles/kg. At the latter dose,
the radioactivity in the acid-soluble fraction was increased by approximately
50%. In kidney, thymidine incorporation into DNA was inhibited 40-50% at
doses of 28 or 56 pmoles/kg. At the two highest doses, the ratios in kidney
approached those obtained in spleen. As in the case of liver, the
radioactivity in the acid-soluble fraction was increased by approximately
50% at the highest dose of PtCl/j. In testis, the thymidine incorporation
into DNA and the ratio were decreased by 25-35% by doses of PtCl^ of 28
or 56 ymoles/kg.
Thymidine incorporation into DNA of spleen was very sensitive to the
administration of Pd(N03)2, just as it was to PtCl^ (Table 2). Moderate
decreases were found in thymidine incorporation into DNA and in the ratio of
spleen at the lowest dose of PdCNO-K. At the two highest doses, 50-60%
inhibition was observed in the DNA and the ratio. PdCNOo)^ decreased
incorporation into DNA and the ratio of liver by 25-45%, depending on the
-4-
-------
dosage. The kidney was relatively resistant to treatment with
The maximum inhibition in incorporation in kidney was approximately 40%
with a corresponding decrease observed in the ratio. Thymidine incorporation
into DNA of testis was markedly inhibited at the two higher doses of
Pd (1103)2 and the inhibition ranged from 40% to 60%; the decrease in the
ratio was comparable at each dose.
The administration of Pt or Pd did not cause a decrease in radioactivity
in the acid-soluble fraction in any of the tissues studied (Tables 1 and
2). Thus, the administration of either PtCl, or Pd(N03)2 did not decrease
the circulation of the radioactive thymidine from its site of injection
and/or the entrance of the thymidine into the tissue. Increases in acid-
soluble radioactivity were noted in some experiments. The highest dose
of Pt caused this effect in liver, kidney and testis but not in spleen.
In contrast, the highest dose of Pd caused an increase only in testis.
Potter and coworkers (2,3) have developed a method for the fractionation
of liver nuclei into two classesnuclei derived from parenchymal cells
or hepatocytes (called N-l nuclei) and nuclei derived from stromal cells ,.
or non-hepatocytes (called N-2 nuclei). The effects of PtCl^ on the
incorporation of thymidine into DNA of N-l and N-2 nuclei are presented in
Table 3. In experiment A, no inhibition in thymidine incorporation occurred
in either class of nuclei cind only a small decrease in the ratio was ob-
served at a dose of PtCl, of 28 ymoles/kg. However, in experiment B,
a dose of 56 ymoles Pt/kg decreased thymidine incorporation into DNA to 60%
of control values. This dose of PtCl, also decreased the ratios to approximately
40% of control values. Thus, in experiment B, thymidine incorporation into
both N-l and N-2 nuclei was depressed equally when expressed either as DNA
specific activities or as the ratios. Therefore, thymidine incorporation
was inhibited to an equal extent in both parenchymal cells and stromal cells.
-5-
-------
In control rats, the specific activities of DNA are in the following
decreasing order: DNA of N-l nuclei > total cellular DNA > DNA of N-2
nuclei; the specific activities of DNA from N-2 nuclei are approximately
two-thirds those of DNA of N-l nuclei. In Table 3 (lower portion), the
specific activities of DNA from N-2 nuclei are expressed as a fraction
of the specific activities of DNA from N-l nuclei in the same experiment.
Although the higher dose of PtCl^ (Table 3) markedly inhibits thymidine
incorporation in both cell types, the ratio of the N-2 to N-l specific
activities is not significantly altered by treatment with PtCl^.
The administration of CCl^ to rats results in the death of some
hepatic cells and a rapid DNA synthesis and mitotic activity in
surviving cells in order to replace the lost tissue. In data not
shown, it was found that thymidine incorporation into DNA is approximately
8-10-fold greater in liver of the CCl^-treated rats than in the control
rats. Incorporation has been studied in four tissues of rats which
received CCl^ and PtCl^ (Table 4). The data for each tissue are
compared to the values of animals in group B, which received CC14 and a
low dose of PtCl^.
In liver, thymidine incorporation into DNA of group B rats was
10-fold greater than that of rats receiving no CC!A. Moreover, increasing
the dose of PtClA to 28 ymoles/kg did not inhibit thymidine incorporation
into liver DNA of CCl^-treated rats. For spleen, kidney and testis,
thymidine incorporation into DNA of each tissue of groups A and B was
essentially equal. In these three tissues, in contrast to the pattern
seen in liver, thymidine incorporation in rats of group C (CCl^ and 28
ymoles PtCl,/kg) was decreased approximately 30% in comparison with
group B animals. The same pattern of results was obtained for all
-6-
-------
three tissues if the ratios of DNA to acid-soluble fraction are examined.
The ratio in liver of group C animals was apparently (but not statistically)
greater than in liver of group B rats; in contrast, the ratios in spleen,
kidney and testis were approximately 40% less in group C animals. Thus,
PtCl^ (at 28 ymoles/kg) apparently did not inhibit the stimulated
synthesis of DNA of liver in CCl^-treated rats but did inhibit thymidine
incorporation into DNA of the other three tissues, similar to the pattern
seen above (Table 1).
In these experiments, the radioactivity in the acid-soluble fraction
has been used as a reference for the total availability of radioactive
precursor in individual tissue samples. It was of interest, therefore,
to examine the distribution of radioactivity in the acid-soluble fraction.
The acid-soluble fractions of liver and spleen were examined in control
animals and in rats treated with 56 ymoles PtClA/kg the highest dose
used in the prior incorporation studies.
Charcoal adsorption was used to separate intact pyrimidine compounds
from their open-ring metabolites. Only those compounds which had the
pyrimidine ring intact were adsorbed by charcoal from an acidic solution.
At the end of the 2-hour incorporation interval, the majority of the
radioactivity in the acid-soluble fraction was in the form of the open-
chain metabolites and other non-adsorbed metabolites in liver and spleen
of control rats (Table 5). In liver, 30% of the radioactivity in the
acid-soluble fraction was in the form of compounds having the pyrimidine
ring intact. Furthermore, the administration of the highest dose of
PtCl, used in these experiments did not alter this distribution. In
spleen the situation was similar. Approximately 22% of the radioactivity
in the acid-soluble fraction of spleen of control rats was in the form
-7-
-------
of intact pyrlmldine compounds and the administration of PtCl/ did not
alter this value.
The charcoal-radsorbable compounds were separated by thin layer
chromatography on polyethyleneimine-cellulose. Two classes of compounds
were separated: (a) the thymidine phosphates, and (b) a mixture of
thymidine and thymine. In liver and spleen of control animals, one-
half of the total charcoal-adsorbable radioactivity was in the form of
thymidine phosphates (Table 5). Administration of PtCl^ did not
appreciably alter the values in these two tissues.
-8-
-------
DISCUSSION
The present study indicates that PtCl^ and Pd(N03)2 inhibit the
synthesis of DNA as measured by the incorporation of radioactive
thymidine. Waters et al. (6) report that the incorporation of thymidine
into DNA is more sensitive to inhibition by PtCl, than the incorporation
of uridine into RNA or of leucine into protein in cultured cells. The
inhibition by PtCl* may be analogous to the effect of Pt-containing
antitumor compounds (7-9). The inhibition of thymidine incorporation
into DNA is consistent with an inhibition of DNA polymerase due to the
interaction of the metallic cations with the template DNA. The
interaction in vitro of the Pt-containing antitumor compounds and of
Pd2+ ions with DNA have been demonstrated (10-12).
The structural features of the active antitumor, Pt-containing
compounds have a major role in the activities of these compounds (8).
It is unresolved what modifications in biological effects are made by
the selection of the salt PtCl^ for these studies. It is unknown
whether the rates of ionization or hydration, and resultant biological
effects, may be significantly different if an alternate soluble Pt1*"*"
salt such as Pt(804)2 ^ad been selected for these studies.
-9-
-------
REFERENCES
1. Patterson, M. S., and Greene, R. C. Measurement of low energy beta-
emitters in aqueous solution by liquid scintillation counting of
emulsions. Analyt. Chem. 37: 854 (1965).
2. Bushnell, D. E., Whittle, E. D., and Potter, V. R. Differential
utilization of pyrimidines for RNA synthesis in two classes of rat
liver nuclei. Biochim. Biophys. Acta 179: 497 (1969).
3. Sneider, T.W., Bushnell, D. E., and Potter, V. R. The distribution
and synthesis of DNA in two classes of rat liver nuclei during azo dye-
induced hepatocarcinogenesis. Cancer Res. 30: 1867 (1970).
4. Fisher, R. F., Holbrook, D. J., Jr., and Irvin, J.L. Density gradient
isolation of rat liver nuclei with high DNA content. J. Cell Biol.
17: 231 (1963).
5. Tsuboi, K. K. and Price, T. D. Isolation, detection and measure of
microgram quantities of labeled tissue nucleotides. Arch. Biochem.
Biophys. 81: 223 (1959).
6. Waters, M. D., Vaughan, T. 0., Abernethy, D. R., Garland, H. R., and
Coffin, D. L. Toxicity of platinum for cells of pulmonary origin.
Environ. Health Perspect., this issue, preceding paper (1975).
7. LeRoy, A. F. Interactions of Platinum Metals and Their Complexes in
Biological Systems. Environ. Health Perspect. 10: 73 (1975).
8. Harder, H. C., and Rosenberg, B. Inhibitory effects of anti-tumor
platinum compounds on DNA, RNA and protein synthesis in mammalian
cells in vitro. Intern. J. Cancer 6: 207 (1970).
9. Howie, J. A., and Gale, G. R. Cis-dichlorodiammine platinum (II).
Persistant and selective inhibition of deoxyribonucleic acid synthesis
in vivo. Biochem. Pharmacol. 19: 2757 (1970).
-10-
-------
10. Shishniashvili, D. M., et al. Investigation of the interaction of
DNA with palladium ions. Biophysics 16: 1003 (1972); translation
of Biofizika 16: 965 (1971).
11. Howie, J. A., Gale, G. R., and Smith, A. B. A proposed mode of action
of antitumor platinum compounds based upon studies with
3
cis-dichloro-((G- H)dipyridine)platinum(II). Biochem. Pharmacol. 21:
1465 (1972).
12. Hovacek, P., and Drobnik, J. Interaction of
c is-d ichloroammineplat inum (II) with DNA. Biochim. Biophys. Acta
254: 341 (1971).
-11-
-------
SECTION I: Table 1.
Effect of PtCl^ on thymidine incorporation into DNA of rat tissues.
Thymidine incorporation
Dose of PtCl/ (ymoles/kg body weight)
0 14 28 56
Tissue Sample
Spleen No. of samples
DNA
Acid- soluble
fraction
Ratio
Liver No. of samples
DNA
Acid-soluble
fraction
Ratio
Kidney No. of samples
DNA
Acid-soluble
fraction
Ratio
Testis No. of samples
DNA
Acid-soluble
fraction
Ratio
cpm/ymole,
or ratio
6
1560
±160
4950
±140
0.319
+0.039
14
824
+106
2050
+180
0.428
+0.064
8
382
+58
5110
+170
0.075
+0.011
8
402
+74
6880
+320
0.057
±0.009
% of control + S
4
67*
+6
105
+4
63*
+7
4
100
+24
118
+19
100
+47
4
99
+19
99
+10
107
+31
4
127
±8
113t
+4
116
+11
4
48**
±2
not
+4
43**
+2
10
87
+15
113
+10
78
+14
4
58*
+5
118t
+8
50*
+7
4
75
±10
114*
±3
67
+9
.E.
6
42**
+3
105
+5
40**
±5
8
60*
+11
147*
+17
45
+14
8
50**
+4
151**
+15
35**
+4
8
74
±9
118**
±3
64 1
+7
-12-
-------
Table 1 (continued)
Statistical analysis (t-test): **, P < 0.01; *, P < 0.05; t, 0.05 < P < 0.10.
PtCl^ and 3H-thymidine were injected intraperitoneally at 4 hr and 2 hr,
respectively, before removal of the tissues.
-13-
-------
SECTION I: Table 2.
Effect of Pd(NOo)2 on thymidine incorporation into DNA of rat tissues.
Thymidine incorporation
Tissue Sample
No. of samples
Spleen DNA
Acid-soluble
fraction
Ratio
Liver DNA
Acid-soluble
fraction
Ratio
Kidney DNA
Acid-soluble
fraction
Ratio
Test is DNA
Acid-soluble
fraction
Ratio
Dose of
0
cpm/ymole,
or ratio
6
1550
±200
5030
±300
0.316
'±0.052
700
±129
2230
±520
0.342
±0.047
263
±57
5590
±100
0.048
±0.011
380
±53
6310
±250
0.062
±0.012
Pd(N03)2 (pinoles /kg body weight)
14 28 56
(% of controls ±.
4
78
±12
119t
±6
65
±11
73
±16
115
±27
60*
±6
88
±14
108
±7
84
±20
107
±7
105
±10
102
±14
6
52*
±12
111
±7
49*
±13
70
±19
138
±22
55t
±21
93
±31
104
±3
88
±29
59t
±17
108
±5
56
±18
S.E.)
7
44**
+11
107
±4
41*
±11
59
±15
105
±19
55*
±10
59
±14
105
±6
56
±13
40**
±9
118*
±4
33**
±7
Statistical analysis (t-test): **, P < 0.01; * P < 0.05: t, 0.05 < P < 0.10.
o
and H-thymidine were injected intraperitoneally at 4 hr and 2 hr, respec-
tively, before removal of the tissues.
-14-
-------
SECTION I: Table 3.
Effect of PtCl^ on thymidine incorporation into nuclei of
of parenchymal (N-l) and stromal (N-2) cells of liver.
Experiment
Dose of
(ymoles/kg)
No. of samples
28
56
DNA
cpm/pmole + S.E
Total cellular
N-l nuclei
N-2 nuclei
Ratio: DNA/
Acid-soluble
fraction + S.E.
Total cellular
N-l nuclei
N-2 nuclei
DNA
N-2 nuclei/N-1
nuclei of
same sample
± S-E-
810
+220
1590
+380
700
+140
0.51
+0.15
0.99
+0. 28
0.43
+0.09
0.51
+0.15
910 (112%)a
+260
1670 (105%)
+190
710 (101%)
+130
0.45 (89%)
+0.12
0.83 (84%)
+0.14
0.34 (79%)
+0.06
0.42
+0.05
660
+230
730
+240
450
+60
0.28
+0.04
0.31
+0.05
0.21
+0.04
0.71
+0.10
410 (62%)a
+70
400 (55%)
+160
300+ (65%)
+40
0.11* (41%)
+0.01
0.11** (36%)
+0.02
0.09* (41%)
+0.02
0.82
+0.18
a Expressed as % of the control values in the same experiment.
Statistical analysis (t-test): **, P < 0.01; *, P < 0.05; +, 0.05 < P < 0.10.
PtCl/ and ^H-thymidine were injected intraperitoneally at 4 hr and 2 hr,
respectively, before removal of tissues.
-15-
-------
SECTION I:' Table 4.
Effect of PtCl^ on thymidine incorporation into tissues of CCl^-treated rats.
Group
Dose of CC1,
(ml/kg)
ABC
0 1.0 1.0
ABC
0 1.0 1.0
ABC
0 1.0 1.0
Dose of PtCl^
(ymoles/kg)
No. of samples a
Tissue
Spleen
Liver
Kidney
Testis
0 14 28
454
DNA
cpm/ymole nucleotide
+S.E. (% of group B)
1800 1990 1410
+200 ±530 ±210
(91) (71)
580 6070 7610
+130 +1540 +1940
(10) (125)
300 300 200
+60 +50 +40
(100) (67)
540 470 310*
+80 +50 +40
(114) (65)
0 14 28
454
Acid-soluble fraction
cpm/ymole nucleotide
+S.E. (% group B)
3790 3720 4420
+1120 ±850 ±490
(102) (119)
1850 3180 2710
+90 +690 +1030
(58) (85)
6240 5790 7090
+1150 +290 +1530
(108) (122)
7450 7050 6860
+120 +290 +360
(106) (97)
0 14 28
454
Ratio: DNA/Acid-
soluble fraction
+S.E. (% of group B)
0.57 0.70 0.35
±0.16 +0.37 +0.11
(82) (50)
0.32 2.00 3.15
+0.09 ±0.50 +0.46
(16) (157)
0.052 0.052 0.032
+0.012 +0.009 +0.009
(100) (61)
0.073 0.067 0.039+
+0.012 +0.005 +0.011
(108) (58)
Statistical analysis (t-test): *, P < 0.05; +, 0.05 < P< 0.10.
CC1/, diluted in corn oil, was injected intraperitoneally at
42 hr, PtCl^ at 4 hr, and 3H-thymidine at 2 hr before removal
of the tissues.
a Except 3 rats each in spleen samples of groups A and B.
-16-
-------
SECTION I: Table 5.
Distribution of H-labeled compounds in the acid-soluble fraction.
Tissue Liver Spleen
Dose of PtCl^, ymolesAg 0 56 0 56
Charcoal adsorption,
% of 3H in
acid-soluble
fraction
intact pyrimidine 30 28
compounds
(adsorbed) (28; 31) (22; 34)
pyrimidine 70 72
catabolites
(not adsorbed) (69; 72) (66; 78)
Thin layer chromatography,
% of 3H of charcoal-
adsorbable fraction
thymidine phosphates 48 44
(47; 48) (37; 51)
thymidine and thymine 43 47
mixture
(43; 44) (42; 52)
22 23
(20; 24) (19; 26)
78 77
(76; -80) (74; 81)
54 59
(54; 55) (54; 64)
41 36
(41; 42) (33; 40)
Means of values of two rats; percentage values of each rat are given in
parentheses.
PtCl and H-labeled thymidine were injected intraperitoneally 4 hr and
2 hr, respectively, before removal of the tissues.
-------
SECTION II. EFFECTS OF PLATINUM AND PALLADIUM SALTS ON PARAMETERS OF
DRUG METABOLISM IN THE RAT
INTRODUCTION
Platinum and palladium compounds are used as the active components
in the catalytic converters of air pollution control devices of various
motor vehicles. It is of interest, therefore, to determine the biological
effects of the salts of these metals.
The acute exposure of rats to various metallic cations such as Cd^+
2+
and Pb markedly decreases the parameters of the hepatic microsomal mixed-
13
function oxidases. However, longer-term administration of these
metallic salts to rats typically does not result in impaired microsomal
4-6
enzymatic activities. Likewise, in the current study, the injection
of Pt or Pd salts (or in some cases, 1-week dietary administration) results
in decreased activities of hepatic microsomal "drug metabolizing" enzymes
whereas dietary administration of 4 weeks or longer did not decrease these
activities.
-18-
-------
MATERIALS AND METHODS
Materials. Male Sprague-Dawley rats, obtained from Zivic-Miller
Laboratories, were used in all experiments. The Pt and Pd salts were
purchased from the following sources: B.F. Goldsmith Chemical and Metal,
Pt(S04)2 . 4H20, Pt02, PtCl4, PdO, PdCl2, PdSO^; Research Organic/Inorganic
Chemical, PtCl2, PdCl2.2H20, PtCl4, Pd(N03)2 aqueous solution, Pt02; Var-
Lac-Oid Chemical, Pt(S04>2-AH20; Ventron/Alfa Products, PdO, PtCl^; ICN/K
and K Laboratories, Pt(SO/4)2.4H20, PdSO^; and Apache Chemicals, PtCl2.
Glucose-6-phosphate, NADP, and glucose-6-phosphate dehydrogenase (type XV)
were obtained from Sigma Chemical.
Diet treatments. The dietary administration of metallic salts was
conducted for one week (7.5-8.5 days), four weeks (28.5-32.5 days), or 13
weeks (87-93 days). The rats were housed four rats/cage. When initially
placed on metal-containing diets, the mean body weights were 100-110 g
(age, 4-5 weeks). Body weights of individual rats and consumption of feed
and fluid (per cage) were measured every seventh day. The metallic salts
were administered either in the drinking fluid or by mixing in the dry feed
(Purina laboratory chow). The volume of fluid consumed (in ml) was approxi-
mately 1.6 times the weight of feed consumed (in grams).
Isolation of microsomes and assay methods. Rats were fasted for 14 hr
before isolation of microsomes. Liver was homogenized in 0.15 M KC1-50
mM Tris-HCl (pH 7.7 at 5 ). The homogenate was centrifuged at 9000 g for
20 min; the resulting supernatant was then centrifuged at 159,000 g-av. for
30 min. The microsomal pellet was resuspended in 0.15 M KC1-50 mM Tris-
HCl and recentrifuged at 159,000 g-av. for 30 min. The washed microsomes
were resuspended in 0.1 M Tris-HCl for transfer to incubation mixtures (final
pH, 7.6-7.7 at 37°).. Glucose-6-phosphate, glucose-6-phosphate dehydrogenase
-19-
-------
and NADP were used as the NADPH-generating system. Aniline hydroxylase
was measured (at 37° and 1.5-2.0 mg microsomal protein/ml) by the method
7 8
of Imai et al., modified by the addition of HgC^ during the assay for
o
p-aminophenol. Aminopyrine demethylase was measured (at 37 and 1.5-2.0
9
mg microsomal protein/ml) by the formation of formaldehyde (Nash reaction).
Protein was measured by the method of Lowry et al. Microsomal cytochrome
P-450 and cytochrome b^ were analyzed essentially by the methods of Omura
and Sato.
The isolated hepatic microsomes were routinely assayed for the following
parameters of drug metabolism: yield of microsomal protein (mg/g liver),
aniline hydroxylase activity (nmoles p-aminophenol produced/min/mg micro-
somal protein and nmoles p-aminophehol produced/min/nmole cytochrome P-450),
aminopyrine demethylase activity (nmoles formaldehyde produced/min/mg
microsomal protein and nmoles formaldehyde produced/min/nmole cytochrome
P-450), and microsomal content of cytochrome P-450 and of cytochrome b^
(each expressed as nmoles/mg microsomal protein). Aminopyrine and aniline
were selected as representatives of substrates which give type I and type
II binding spectra, respectively. The data in the tables are expressed
as the percentage of the mean control values and the control values are given
the-legends.
Effects of addition of metallic salts to the incubation medium. In
all cases the incubation medium contained 5 mM MgCl«. The addition of PtCl^,
Pd(N03)2 and PbCl^ to the incubation medium at the concentrations used
caused negligible changes (< 0.1) in the pH of the medium; the addition of
MnC^^I^O did cause some decrease in the pH of the medium. The addition
of the salts to the incubation medium did not result in the formation of a
microsomal sediment or in an observable turbidity. The addition of the
metallic salts in vitro, at the concentrations used, did not inhibit the
-20-
-------
activity of the NADPH-generating system (glucose-6-phosphate, glucose-6-
phosphate dehydrogenase and NADP).
-21-
-------
RESULTS
Hexobarbital-induced sleeping time. The effects of intraperiotoneal
Injections of PtCl^ or PdCNOOo on tne hexobarbital-induced sleeping time
in rats are presented in Table 1. The metallic salts were administered
for two consecutive days, each day at the designated dose, and sleeping
times were measured on the third day. PtCl^, at doses of 28 umoles (5.5
4+ /.+
mg Pt ) and 56 ymoles (10.9 mg Pt^/kg body weight, increased hexobarbital-
induced sleeping times by approximately 25 and 50%, .respectively. The
higher dose of PtCl^ is equal to one-half of the intraperitoneal LDcn dose
of 113 ymoles/kg body weight (administered as a single dose with a 14-day
observation interval) but is less than the intraperitoneal LDc- The two-
r\ |
day intraperitoneal administration of Pd(N03)2 at 56 ymoles (6.0 mg Pd )
or 113 ymoles (12 mg Pd2+)/kg body weight also increased the hexobarbital-
induced sleeping time by approximately 60% (Table 1). Thus, the administra-
tion of PtCl^ or Pd(N03>2 apparently decreased the ability of the treated
animals to metabolize hexobarbital in vivo.
Acute effects on microsomal enzymatic activities. Various parameters
of drug metabolism were measured in hepatic microsomes isolated from rats
injected with PtCl, or Pd(N03>2 at 18 and 42 hr before isolation of the
microsomes. The intraperitoneal injection of PtCl^ for two consecutive days
prior to the isolation of the microsomes generally produced only small
changes in the measured parameters of drug metabolism (Table 2). Decreases
of approximately 15-25% were observed in the microsomal content of cyto-
chrome P-450 (nmoles/mg microsomal protein) and in the aminopyrine demethylase
activity (per mg microsomal protein), respectively. Small changes (<15%)
also were observed in the microsomal content of cytochrome b (nmoles/mg
microsomal protein) and in the yield of hepatic microsomal protein (mg protein/
g liver). Thus, at doses relatively high in comparison to the LD5Q, injection
-22-
-------
of PtCl/ had only moderate effect on the measured parameters of drug metabolism
Injection of PdCNO^^ decreased the amiinopyrine demethylase activity (per
mg microsomal protein) by one-third at doses of - 56 or 113 umoles/kg body weight
(Table 2). The microsomal content of cytochromes P-450 and br were also
decreased but by only one-fifth and about one-fourth, respectively.
In an additional series of experiments.(data not shown), each Pt-
treated rat received a single intraperitoneal injection of PtCl^ (100 ymoles
(19.5 mg Pt)/kg body weight) and hepatic microsomes were isolated 45 hr
later. The following parameters of drug metabolism were reduced in micro-
somes of the Pt-treated rats: the yield of microsomal protein (mg/g liver),
by 24% (P<0.001); aniline hydroxylase (nmoles p-aminophenol produced/min/
mg protein), by 28% (P<0.01); aminopyrine demethylase (nmoles formaldehyde
produced/min/mg protein), by 42% (P<0.01); and microsomal content of cyto-
chrome P-450 (nmoles/mg microsomal protein), by 33% (P<0.01). The microsomal
content of cytochrome b5 was reduced by only 13% (P>0.1).
-23-
-------
Effect of dietary administration on microsomal activities. One of the
objectives in this study was to determine the effects of " long-term, low-
level" (dietary) administration of the metallic salts on the ability of iso-
lated microsomes to function in drug metabolism. With one exception, the
dietary administration (via drinking fluid or solid feed) of Pt or Pd^+
salts resulted in the following pattern of changes (Table 3). (a) If a
1-week metal-containing diet resulted in any changes, there was a decrease
in the parameters of drug metabolism (e.g., activities of aniline hydroxylase
and aminopyrine demethylase), consistent with the acute effects of the intra^
peritoneally injected metallic salts. (b) If a 4- or 13-week metal-containing
diet resulted in any changes, there was an increase in the parameters of
drug metabolism
The dietary administration of PtCl^ had relative little effect on any of
the measured parameters of drug metabolism (Table 3). The only changes
which appear to occur were increases of 20-30% in aminopyrine demethylase,
aniline hydroxylase and/or cytochrome b5 after dietary administration of PtCl^
for 4 weeks at 13.2 mmoles (2.58 g Pt +)/kg solid feed or for 13 weeks at
0.54 mmoles (106 mg Pt^+)/liter drinking fluid; each rat consumed a mean of
1.58 g and 1.4 g, respectively, of Pt^+ during the diet treatments. A one-
week dietary treatment with Pt (804)2 -^2° s^owec* a decreased activity of
aniline hydroxylase whereas a four-week treatment showed little or no change
in the enzymatic activities. Pt02» an insoluble salt, had marginal effects
on the measured parameters even when the concentration in the feed was 29.8
mmoles (5.81 g Pt )/kg feed; each rat consumed a mean of 4.9 g of Pt
during the four weeks on the diet.
In each of two experiments, the administration of PdCl2.2H20 (a partially
soluble salt) as a saturated solution in the drinking fluid for one week,
decreased the activities of aniline hydroxylase and aminopyrine demethylase
in isolated microsomes (Table 3). In contrast, the 4-week dietary administration
-24-
-------
of PdCl2 (an "insoluble" salt), at 13.2 mmoles (1.40 g Pd2+)/kg feed,
resulted in an increase in aniline hydroxylase and aminopyrine demethylase
(per mg microsomal protein) with an equivalent increase in the microsomal
content of cytochrome P-450. The increase in enzymatic activities did not
occur when the dietary concentration of PdC^ was increased to 29.8 mmoles
2+
(3.17 g Pd )/kg feed but the Pd-treated rats in the latter experiment showed
a 25% reduction in weight gain.
The dietary administration of PdSO^ did not produce any (statistically
significant) changes in the parameters of drug metabolism even when administered
2+
at a concentration of 29.8 mmoles (3.17 Pd )/kg feed. Likewise, PdO caused
no changes in the measured parameters except for a decrease in the yield of
2+
microsomal protein at a PdO level of 29.8 mmoles (3.17 g Pd )/kg feed.
2+
Dietary PbCl2» 4 weeks at 29.8 mmoles (6.17 g Pb )/kg feed, increased
the yield of microsomal protein and decreased the activity of aniline hydroxyl-
ase; other diet schedules produced only minor changes (Table 3). MnCl-^H^O,
2+
administered in the drinking fluid for 13 weeks at 8.3 mmoles (0.46 g Mn )/
2+
liter or 18.6 mmoles (1.02 g Mn ) /liter, did not alter any of the measured
parameters of drug metabolism.
4+
Effect of prior administration of Pt on survival following an LDcQ
dose of PtCl^. Decreases in the ability of isolated microsomes to metabolize
drugs in vitro are observed only following the acute administration (i.e.,
18 and 42 hr prior to isolation of microsomes) of PtCl, or Pd(NOo)2 or following
a short diet period (i.e., one week). After 4- or 12-week diet periods,
the drug-metabolizing activities of isolated microsomes from Pt -treated !
2+
(or Pd -treated) rats generally were equal to or greater than those from
94- 2+
controls rats. Such a pattern has been observed for Cd and Pb salts. In
2+ 9+
the case of Cd , the toxicity is reduced by the induced synthesis of a Cd -
74- 2+
binding protein in rats treated with Cd^T or Zn salts. In such cases, the.
prior administration of Cd (or Zn ) protects rats against a subsequent
-25-
-------
2+ . 12,13
administration of a normally lethal dose of Cd salts. A similar
type of study was conducted with PtCl/ . In rats weighing approximately 270
g, one group of rats received an intraperitoneal injection of PtCl/
(56 pmoles/kg body weight) and control rats received saline injections.
After 48 hr, each rat received an intraperitoneal injection of PtCl, (113
ymoles/kg). After one week following the higher dose of PtCl^, 33% and 100%
4+
of the saline-pretreated and of the Pt -pretreated rats, respectively,
survived; there were a total of 12 rats in each of the groups. Although an
induced synthesis of a Pt -binding protein in the Pt -pretreated rats
has not been demonstrated, the observed protection against PtCl^-lethality
by Pt -pretreatment is consistent with the production of such a protein
2+
and might be analogous to findings with Cd where the production of such a
14
protein has been experimentally demonstrated.
Effect of addition of cations to the incubation medium on activity of
aminopyrine demethylase. The alterations in the activity of aminopyrine
demethylase of isolated hepatic microsomes from control rats were measured
after the addition of various metallic cations to the incubation medium.
The Pt and Pd2+ ions were appreciably more inhibitory than Pb2+ or Mn
ions. At aminopyrine concentrations of 0*25 or 1 mM, 50% inhibition was ob-
tained at' 0.15-0.2 mM Pt4+ (Fig..' 1A)
However, the extent of inhibition
4+
of aminopyrine demethylase by Pt is markedly decreased as the concentration
of the substrate (aminopyrine) is increased. At an aminopyrine concentration
of 2 mM, 50% inhibition of the demethylase occurred at Q.5 mM PtClA. At
aminopyrine concentrations of 1 or 4 mM, 50% inhibition occurred at 0.2-0.3
mM Pd2+ (Fig. IB). In contrast to the pattern with Pt^+, however, the inhibi-
2+
tion in vitro of aminopyrine demethylase by Pd is not affected by changes
in the aminopyrine concentration. A 4-fold increase in aminopyrine concen-
2+
tration (i.e., 1 and 4 mM ) altered the- percentage inhibition by Pd by <5%.
-26-
-------
2+
Pb , at concentrations of 0.1-1.0 mM (and at 1-4 mM aminopyrine),
produced only 15-25% inhibition of the aminopyrine demethylase activity of
94-
isolated microsomes. Mn , at either 1 or 4 mM aminopyrine, inhibited the
2+
aminopyrine demethylase approximately 20% and 40% at 6.4 and 12.8 mM Mn ,
respectively. However, a MnCl2~induced change in the pH of the incubation
2+
medium may contribute to the lower activity seen at the higher Mn con-
centration.
The kinetics of the aminopyrine demethylase reaction in the presence
4+ 2+
of Pt or Pd are shown in Fig. 2. The differing response to inhibition
/ _L O I
of aminopyrine demethylase by Pt and Pd to increases in aminopyrine
concentration resulted in a different character in the inhibition. Double
4+
reciprocal plots (i.e., 1/v versus 1/S) in the Pt -inhibited demethylase
reaction gave a common intercept on the vertical axis (i.e., competitive
2+
inhibition) (Fig. 2A) whereas plots in the Pd -inhibited reaction had
a common intercept at, or very near, the horizontal axis (i.e., non-
competitive inhibition) (Fig. 2B). However, the patterns of the Lineweaver-
Burk plots shown in Fig. 2A and 2B occurred only where the concentrations of
Pt4+ and Pd2+ were relatively low (e.g., 0.1 mM PtCl^ and 0.2 mM Pd(N03>2).
Higher concentrations of either metal (e.g., 0.20-0.25 mM Pt^+ or 0.4 mM
Pd ) caused an inhibition in which the plots of 1/v versus 1/S of control
and metal-containing samples generally did not have common intercepts on either
axis.
-27-
-------
DISCUSSION
In the acute studies (Table 1), the PtCl, and Pd(N03)2 were injected in-
traperitoneally each day for two consecutive days before isolation of the
microsomes. With this schedule, decreases were observed in the aminopyrine
demethylase activity (per mg microsomal protein) and the microsomal content
1 2
of cytochrome P-450. However, the administration of methylmercury, '
2 3
PbCl2, or cadmium acetate, at (generally) appreciably lower molar doses,
caused appreciably greater decreases in the microsomal content of cyto-
chrome P-450 and in the enzymatic activities which utilize cytochrome P-450
when measured 1-2 days after single or 2-consecutive-day administration.
Thus, on a molar basis and at short intervals after administration, the
/ _i_ Q i
Pt and Pd salts produced appreciable lesser effects on the parameters of
drug metabolism than the effects reported for the salts of methylmercury,
Pb2+ or Cd2+.
The decreases in the parameters of microsomal drug metabolism in Pt -
2+
or Pd -treated rats were observed only after short-term exposure to the
metals (i.e., after intraperitoneal injection or 1-week diets). After longer
/ _I_ O-l-
exposure (4- or 12-week diets), Pt or Pd salts produced no effect or
small increases in the parameters of microsomal drug metabolism. Thus, the
Pt and Pd salts produce a pattern of effects similar to the pattern re-
2+ 2+ 2+ q 94-2
ported for Cd and Pb . Although Cd salts and Pb salts produce marked
decreases in parameters of drug metabolism at short intervals after administra-
tion, the long-term dietary administration of Cd salts or Pb salts '
do not alter or may increase various parameters of drug metabolism.
Depending on the aminopyrine concentration, 25% and 50% inhibition of
aminopyrine demethylase activity can be obtained in vitro at 0.1 and at
approximately 0.2 mM PtCl,, respectively. One of the lower dietary levels
of Pt used in the present experiments was 1.63 mmoles (318 mg Pt )/liter
-28-
-------
of drinking fluid. Rats on this diet consumed 60-80 mg Pt in an 8-9 day
interval and attained maximum Pt levels of 4.8 yg Pt/g kidney (nominal
concentration of 35 yM, assuming tissue water of 70%) and 0.8-2.2 yg
Pt/g liver (nominal concentration of 6-16 yM). Rats which survived
for 14-days after receiving an intraperitoneal dose of Pt(SO^)2-4H?0
(113 mg Pt/kg body weight) (equivalent to 90% of the LD5Q) attained liver
and kidney levels of approximately 35 yg Pt/g tissue (a nominal concentration
of 0.25 mM). Thus, although levels of Pt in tissues of experimental
animals can attain levels which are nominally sufficient to inhibit the
representative drug-metabolizing enzyme studied here, the amounts administered
to the animals far exceed an anticipated environmental exposure.
-29-
-------
REFERENCES
1. G.W. Lucier, H.B. Matthews, P.E. Brubaker, R. Klein and O.S. McDaniel,
Molec. Pharmac. _3, 237 (1973).
2. A.P. Alvares, S. Leigh, J. Cohn and A. Kappas, £. exp. Med. 135, 1406
(1972).
3. W.M. Hadley, T.S. Miya and W.F. Bousquet, Toxic, appl. Pharmac. 28,
284 (1974).
4. D.D. Wagstaff, Bull, envir. Contamin. Toxic. 10, 328 (1973).
5. W.E.J. Phillips, D.C. Villeneuve and G.C. Becking, Bull, envir. Contamin.
Toxic. £, 570 (1971).
6. W.E.J. Phillips, G. Hatina, D.C. Villenevue and G.C. Becking, Bull.
envir. Contamin. Toxic. ^, 28 (1973).
7. Y. Imai, A. Ito and R. Sato, J. Biochem. (Tokyo) 60, 417 (1966).
8. R.S. Chhabra, T.E. Gram and J.R. Fouts, Toxic, appl. Pharmac. 22,
50 (1972).
9. J.B. Schenkman, H. Remmer and R.W. Estabrook. Mol. Pharmac. ^, 113
(1967).
10. O.K. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. biol. Chem.
193, 265 (1951).
11. T. Omura and R. Sato, J. biol. Chem. 239, 2370 (1964).
12. C.J. Terhaar, E. Vis, R.L. Roudabush and D.W. Fassett, Toxic, appl.
Pharmac. ]_, 500 (1965).
13. G. Gabbiani, D. Baic and C. Deziel, Can. J. Physiol. Pharmac. 45,
443 (1967).
14. M. Webb, Biochem. Pharmac. 21, 2751 (1972).
15. D.J. Holbrook, Jr., M.E. Washington, H.B. Leake and P.E. Brubaker,
Envir. Hlth Perspect., in press (1975).
-30-
-------
SECTION II. Table 1.
Increase in hexobarbital-induced sleeping times in rats treated with
or
Dose of Hexobarbital-induced sleeping time
metallic
salt (% of mean, paired controls + S.E.)
(ymoles/
kg body ptclA Pd(N03)2
weight)
0 100 + 16 100 + 8
14 111 + 10
28 123 + 10
56 151 + 15* 159 + 20*
113 160 + 23*
Rats, initially weighing 162 g (+13, S.D.) were injected intraperitoneally
with PtCl4 or Pd(N03)2 ^2 and *& hr (each time at the designated dose) prior
to the intraperitoneal injection of hexobarbital (100 mg/kg body weight).
The mean hexobarbital-induced sleeping times of control rats were 43 min in
both the PtCl^ and Pd(N03)2 experiments. There were 6-7 and 8-11 values for
each dose in the PtCl4 and Pd(N03>2 experiments, respectively. Statistical
analysis (t-test): *, P < 0.05.
-31-
-------
SECTION II. Table 2.
Effect of intraperitoneal injections of PtCl^ or PcKNOg^ for two
consecutive days on parameters of drug metabolism by isolated hepatic
microsomes (MCS).
Treatment Dose Number Microsomal Aminopyrine
(ymoles values protein demethylase
salt/kg yield
body wt.) (mg/g
liver)
Control - 10 100
+2
PtCl4 3.5 3
14.1 6 91+
+2
28.2 5 94
+3
56 4 91+
+4
Control - 7 100
±2
Pd(N03)2 28 3 100
+4
56 5 100
+4
113 4 96
+5
(per mg
microsomal
protein
(% of mean
100
+3
92
+9
83*
+5
83**
+2
85
+13
100
+4
103
+15
69**
+6
62*
+14
(per nmoles
cytochrome
P-450)
Cytochrome
P-450
(nmoles/
mg MCS
protein)
Cytochrome
b5
(nmoles/
mg MCS
protein)
paired controls + S.E.)
100
+8
88
+6
93
+8
99
+7
115
+16
100
+11
92
+2
84
+7
74+
+5
100
+7
104
+13
86+
+4
81*
+5
74**
+3
100
+7
112
+18
81
+10
80
+15
100
+4
101
+4
97
+2
89+
+2
87*
+3
100
+6
111
+11
86
+5
72*
+7
Rats, initially weighing 163 g (+12, S.D.) were injected intraperitoneally with
or Pd(NO-j)2 42 and 18 hr (each time at the designated dose) prior to isolation of
hepatic microsomes. Values (mean + S.D.) for 14 control rats were: initial body
-32-
-------
Table 2 (Continued)
weight, 161 + 11 g; liver weight, 5.5 + 0.7 g; yield of microsomal protein, 25+2
mg/g liver; aminopyrine demethylase, 5.9 + 0.9 nmoles formaldehyde produced/min/mg
protein; microsomal cytochrome P-450 content, 0.58 + 0.16 nmoles/mg protein; and
microsomal cytochrome b5 content, 0.29 + 0.05 nmoles/mg protein. Statistical
analysis: **, P < 0.01; *, P < 0.05; +, 0.05 < P < 0.10; no designation
used where P > 0.10.
-33-
-------
SECTION II. Table 3.
Effects of administration of metal-containing diets on the parameters of drug metabolism by isolated hepatic microsomes
CMOS).
Metallic
salt
ptci4
Pt(S04)2
4H20
Pt02
PdCl,.
2H20
PdCl2
Diet
duration
(weeks)
1
1
4
4
4
13
1
4
4
1
4
4
Dietary
metal
concn.
(mmoles/
1 or kg)
1.63/1
2.45/1
1.63/1
5.9/kg
13.2/kg
0.54/1
1.63/1
5.9/kg
29.8/kg
(satd.soln)
13.2/kg
29.8/kg
Number
ratsa
12
4
12
4
4
4
8
4
4
8
8
4
Microsomal
protein
yield
(mg/g
liver)
101
84*
98
101
97
95
103
110+
103
102
95
104
Aniline
hydroxylase
(activity/ (activity/
mg MCS nmoles
protein) cytochrome
P-450)
(% of
100
110
97
88
92
123+
79*
88
104
72**
121**
96
Amlnopyrine
demethylase
(activity/
mg MCS
protein)
(activity/
nmoles
cytochrome
P-450)
Cytochrome
P-450
(nmoles/
mg MCS
protein)
Cytochrome
bs
(nmoles/
mg MCS
protein)
paired mean controls)
.
-
_.
-
-
128*
-
-
109
-
98
87
106
102
100
99
122+
114
-
102
110
78**
125**
100
-
-
-
-
117
120
-
-
120
-
101
92
nm
-
-
-
102
97
nfTl
XUD
93
nm
125**
109
nm
115
97
94
129*
107
t^m
Tim
94
nm
110*
103
-------
Table 3. Continued
Metallic
salt
Diet
duration
(weeks)
Dietary
metal
concn .
- (nsnoles/
1 or kg)
Number Mlcrosomal
ratsa protein
yield
(mg/g
liver)
Aniline
hydroxylase
(activity/
mg MCS
protein)
(activity/
nmoles
cytochrome
P-450)
Amlnopyrine
demethylase
(activity/ (activity/
mg MCS nmoles
protein) cytochrome
P-450)
Cytochrome
P-450
(nmoles/
mg MCS
protein)
Cytochrome
b5
(nmoles/
mg MCS
protein )
(% of paired mean controls)
PdS04
PdO
PbCl2
MnCl,.
4H20
aAn
1
4
4
4
4
4
4
13
13
13
(satd. soln.)
(satd. soln.)
29.8/kg
29.8/kg
3.7/1
8.3/1
29.8/kg
3.7/1
8.3/1
18.6/1
equal number of rats were
4 101
4 98
4 97
4 84*
8 104
4 103
8 110*
6 94
6 102
4 99
109
90
93
97
92
101
89+
86
92
107
102
98
82+
96
-
112
79*
-
112
88 81
96 106
102 90
93 89
91
nm :
97
-
95
102 108
108
92
111
106
-
90
110
nm
nm
95
99
92
100
99
93
99
107*
nm
nm
102
used for control animals.
Each experiment was conducted generally with 4 control and 4 metal-treated rats and several experiments were repeated; nm,
not measured. Values (mean ± S.D.) for 92 control rats (except 68 rats for cytochromes P-450 and be) in the 4-week diet
-------
experiments vere: liver weight, 10.5 ± 1.5g; yield of mlcrosomal protein, 26 ± 3 mg/g liver; amlnopyrine demethylase,
7.4 ± 1.4 nmoles formaldehyde produced/mln/ mg protein; aniline hydroxylase, 1.0 ± 0.2 nmoles p-amlnophenol produced/mln/mg
protein; microsomal cytochrome P-450 content, 0.70 ± 0.17 nmoles/mg mlcrosomal protein; and mlcrosomal cytochrome b_ content,
0.32 ± 0.04 nmoles/mg mlcrosomal protein. Statistical analysis (t-test): **, P < 0.01; *, P < 0.05;
t, 0.05 < P < 0.10.
-------
LEGENDS TO FIGURES
9+
Fig. 1. Effect of Pt + and Pd addition to the incubation medium on the amino-
pyrine demethylase activity in vitro. PtCl4 (1A) or Pd(NOo)2 (IB),
the concentrations expressed on a logarithmic scale, were added to
the standard incubation medium for aminopyrine demethylase. It should
be noted that the medium contains 5 mM MgCl2- There were 6-9 and
3-4 values for each Pt^+ point and Pd^+ point, respectively. The
vertical lines show the standard error. (A) Pt ; aminopyrine at
A, 0.25 mM; 0, 1 mM; , 2 mM; and Q, 4 mM. (B) Pd2+j aminopyrine
at 0, 1 mM; andD, 4 mM.
Fig. 2. Double reciprocal plots of the inhibition of aminopyrine demethylase
by the addition of PtCl, or Pd(1^)3)2 to the incubation medium.
The lines drawn are the calculated least-squares regression lines.
In the six experiments on the kinetic parameters, the Vmax of amino-
pyrine demethylase of control hepatic microsomes was 7.3 + 0.4 (mean +_
S.E.) nmoles formaldehyde/min/mg protein and the Km was 0.93 + 0.23 mM.
1/v, I/(nmoles formaldehyde produced/10 min/mg protein); 1/S, I/(amino-
pyrine, expressed in mM). The incubation medium contains 5 mM MgC^.
Each point is the mean of duplicate samples. (A) 0, control; A, 0.1
mM PtCl4. (B) 0, control;D, 0.2 mM Pd(N03)2.
-37-
-------
Figure 1
75
50
.o
2 25
c
V
o»
o
75
O»
Z 50
4)
QL
25
O.I 0.2 0.4 0.5 0.8
Cation Concentration (mM)
-38-
-------
Figure 2
0.15
I/V
0.05
-I
I/S
l/v
0.05
0.04
0.03
B
I 2
I/S
-39-
-------
SECTION III. TOXICITY OF PLATINUM AND PALLADIUM SALTS IN THE RAT
INTRODUCTION
The incorporation of platinum and palladium as components in the
catalytic converters of motor vehicles may be accompanied by the release
of various salts of these metals into the environment. Consequently, it
is of interest to determine the toxicity of these compounds in mammalian
systems. Most of the prior studies on the toxicity and biochemical actions
of platinum salts have been concerned with the properties of platinum-
containing antitumor compounds. A number of these compounds interact with
tissue macromolecules and such interactions may contribute to the biochemical
and toxic effects of these compounds.
Since it has been proposed that various manganese compounds be
substituted for the tetraethyl lead in fuels, various compounds of
manganese and lead are included in this study.
-40-
-------
MATERIALS AND METHODS
All studies were conducted with male Sprague-Dawley rats obtained
i
from Zivic-Miller Laboratories. The animals were received at 3-3.5 weeks
of age and were maintained for 1-1.5 weeks before use. The mean body
weights were usually 10(H110' g when the rats were used for lethal-dose
studies or started on the diets.
In the lethal dose experiments, a 14-day observation interval was
used and survivors were weighed 7 and 14 days after treatment. The LD50
values were calculated by the method of Litchfield and Wilcoxon (1949).
In diet experiments, four rats were maintained per cage and,at 7-day
intervals, the following measurements were made: body weight of
individual animals and consumption of feed and drinking fluid per cage.
The metallic salt under study was either dissolved in the drinking fluid
or mixed in the ground dry feed. Animals consumed feed and drinking
fluid ad libitum. In control animals, the volume of fluid consumed
(in ml) was approximately 1.6 times the weight of the feed consumed (in
grams). Analyses for metals were performed on samples from three lots
of feed (Purina Laboratory Chow); the feed contained (mean + S.D.):
56 + 5 mg Mn/kg feed and 0.99 + 0.07 mg Pb/kg feed. The analyses for
platinum in the three lots were 0.09, < 0.02 and < 0.02 mg/kg.
At the end of the diet intervals the rats were weighed, then fasted
for 14-15 hr, and tissues removed and weighed. The rats were fasted
because parameters of drug metabolism in vitro were measured on isolated
hepatic microsomes (to be reported elsewhere). Tissue samples were
frozen for the analyses of metals and for the measurement of the tissue
content of DNA, RNA and protein. For the latter analyses, tissue samples
-41-
-------
were homogenized in water, and tissue macromolecules were precipitated and
washed with cold 0.5 M HC104. The RNA was hydrolyzed in 0.5 M NaOH (1 hr,
37°C) and the DNA and RNA were precipitated and washed with cold 0.5 M
HC104. The DNA was then hydrolyzed in 0.5 M HC104 (20 min, 90°C). The
DNA and the RNA were determined from the absorbance at 260 nm (Gilford
model 2400 spectrophotometer). The protein in the final precipitate
was measured by the method of Lowry et al. (1951).
From a least-squares linear regression line of organ weight versus
body weight for all control animals, an "expected weight" was calculated.
Statistical comparisons were made on the organ weights of metal-treated
rats and their paired controls, each value being expressed as a percentage
of the "expected weight". This method corrects for the change in organ
weights (expressed as percentage of body weight) which occurs with changes
in body weight. The equations of the expected wet weight of organs of
control rats (fasted 14-15 hr before removal of the tissues) in the weight
range of 130-620 g; i.e., after 1-, 4- and 12-week diets, were: liver
weight (g) = 0.0240 (body weight, g) + 2.66; kidney weight (g) = 0.00624
(body weight, g) + 0.98; spleen weight (g) = 0.000705 (body weight, g)+
0.80; heart weight (g) = 0.00211 (body weight, g) + 0.27, and testis
weight (g) = 0.00559 (body weight, g) + 1.19. Statistical analyses of
organ weights and of growth rates were made by the t-test.
-42-
-------
RESULTS
Lethal dose studies. The LD5Q, LD1Q and LD of the various metal
salts, after intraperitoneal or oral (stomach tube) administration, are
given in Table 1. Various other salts were examined in lethal dose
experiments but lack of acute toxicity at the highest oral doses tested
did not permit determination of LDjQ values. Those salts which caused
lower levels of lethality after oral administration were the following
(percentage survival given, with number of rats tested in parentheses):
60% (10) and 82% (11) at Mn02 doses of 115 and 77 mmoles/kg, respectively;
90% (10) at a PbCl2 dose of 35 mmoles/kg; 64% (11) at a PbO dose of 45
mmoles/kg; 100% (6) at a PdO dose of 82 mmoles/kg; 71% (7) and 83% (6)
at PtO~ doses of 35 and 20 mmoles/kg, respectively.
Thus, following the intraperitoneal administration of the metallic
salts, the acute toxicities of the salts (expressed on a molar basis)
were in the following order: PtCl/ > Pt(SO^)2.4H20 = PdCl2.2H20 =
MnCl2.4H20 > PdSO^ > PtCl2 > PbCl2. Likewise, following the oral
administration of the metallic salts, the toxicities of the salts
(expressed on a molar basis) were in the following order: PtCl/ >
Pt(SO^)?.4H20 > PdCl2.2H20 > RuCl3 > MnCl2.4H20 > PbO = Pt02 > Mn02 >
PdO. Thus, PtClA was the most toxic salt tested by either route of
administration and when expressed on either a molar basis or as weight
of cation.
-43-
-------
Weight gains. The weight gain/rat was determined for the following
dietary intervals: each week during weeks 1-4 and, in the 12^week
experiments, for the fifth through eighth week and for the nineth through
twelveth week (expressed as 9/week/rat). The inclusion of MnCl?.4HxO in
the drinking fluid at levels of 8.3 or 18.6 mmoles/liter (1.64 and 3.69 g
salt/liter, respectively) did not alter the weight gain during any interval
of the 12 weeks on the diet. Likewise, PbC^ in the drinking fluid at a
level of 3.7 mmoles/liter (1.02 g salt/liter) did not affect weight gain
during any of the intervals through the 12 weeks on the diet. At a PbC^
concentration of 8.3 mmoles/liter (2.30 g PbCl2/liter) in the drinking
fluid, the weight gain/week/rat of the Pb-treated rats did not differ from
controls each week on a 4-week schedule (P > 0.1) but the total weight gain
during the entire 4 weeks was 79% (p > o.l) of that of controls. Increasing
the dietary intake by administration of PbCl2 in the feed at the levels 13.2
or 29.8 mmoles/kg feed (1.4 or 3.3 g salt/kg feed, respectively) resulted
in decreased weight gain during the first week at the lower level and for
each of the first two weeks at the higher level (Table 2). However, for
the last 2-3 weeks of the 4-week diets, weight gains by the PbC^-treated
rats were equal to that by controls (Table 2).
The use of a saturated solution of PdCLg^I^O (a "partially soluble"
salt) as the drinking fluid for one week did not alter the weight gain of
the Pd-treated rats. Likewise, the addition of PdCl£ (an insoluble salt)
to feed at a level of 13.2 mmoles (2.34 g salt)/kg feed did not decrease
the weight gain by Pd-treated rats during each of the 4 weeks on the diet
except for a slight decrease (80% of control; 0.05 < P < 0.1) during the
first week. An increase in the dietary PdCl2 concentration to 29.8
mmoles (5.38 g salt)/kg feed markedly decreased the weight gain of the
-44-
-------
Pd-treated rats for each of the first three weeks on the diet but not
during the fourth week (Table 2); the weight gain for the full 4-week
interval (172 + 12 g, S.D.) was 74% (P < 0.05) of the weight gain by
the paired control rats.
PdO, at a dietary level of 29.8 mmoles (3.64 g salt^kg feed, decreased
the weight gain in the Pd-treated rats only during the fourth week of the
four-week diet. However, the weight gain during the entire 4-week interval,
201 + 7 g (S.D.), was 81% (P < 0.01) of the gain by paired controls.
The use of a saturated solution of PdSO* as the drinking fluid for
4 weeks did not affect the weight gain/rat during any of the 4 weeks.
Likewise, a dietary level of 5.9 mmoles (1.19 g salt)/kg feed did not
affect the weight gain/week during any of the 4 weeks on the diet.
However, PdS04 at a level of 29.8 mmoles (6.03 g salt)/kg feed decreased
the weight gain of the Pd-treated rats to 54% (P < 0.01) of control for
the entire 4-week interval of the diet.
The addition of PtCl, to the drinking fluid (1.63 mmoles (550 rag salt)/
liter) decreased weight gain/rat by 15% (P < 0.01) during the first week
but did not affect the weight gain during each of the remaining three weeks
on the diet (Table 2). When the PtCl4 is added to the drinking fluid at
a concentration of 0.54 mM (183 mg salt/liter)., the Pt-treated rats showed
normal weight gain throughout the 12 weeks on the diet. Likewise, when
PtCl, was added to the feed at the level of 5.9 mmoles (1.98 g salt)/kg
feed, normal weight gains were observed through each week of a 4-week
schedule. However, at a concentration of PtCl, of 13.2 mmoles (4.46 g salt)/
kg feed, the weight gains by Pt-treated rats were decreased to 27% and 76%
of the paired controls during the first week and during a total 4-week interval
respectively (.Table 2) , but the weight gains by Pt-treatedJ~rats was normal
during the second, third and fourth weeks on the diet.
-45- .
-------
, an "insoluble" salt, had no effect on weight gain during each
of 4 weeks when present in the feed at a level of 29.8 mmoles (6.76 g
salt)/kg feed. Pt(S04)2.4H20, at a concentration of 1.63 mmoles (750
mg salt)/liter in the drinking fluid, decreased the weight gain by Pt-
treated rats during a one-week diet (Table 2). When Pt(304)2.41^0 was
included in the feed at a level of 5.9 mmoles (2.70 g salt)/kg feed, the
weight gain of Pt-treated rats was decreased by 15-18% during the first,
second (P < 0.01), and third (P < 0.05) week of the treatment (Table 2).
Organ weights. The weight of five tissues.(liver, kidney, spleen,
heart and testis) are presented in Table 3. The data are expressed as
the percentage of the expected weight (based on the body weight of rats
on control and metal-containing diets). The administration of MnCl2.4H20
for 13-weeks, at the doses used, did not affect the weights of any of the
five tissues.
Dietary PbCl2 did not consistently affect the weights of any of the
tissues except kidney (Table 3). In all five dietary schedules used,
PbCl2 increased the weight of kidney (expressed as a percentage of the
kidney weight expected of rats of equal body weight) and the increases
often exceeded 25% above the expected weight. As discussed later, the
kidney enlargement in Pb-treated rats has been reported previously in
work by others.
In each of two experiments in which anhydrous PdCl2 was added to the
feed at the level of 13.2 mmoles/kg feed, there was a reduction in the
weights of the liver, kidney and spleen but not in the heart or testis.
The combined data of the two experiments (8 rats) are presented in Table 2.
Upon feeding other Pd-containing salts, however, no consistent pattern of
changes in organ weights was observed. In a single experiment (4 control
and 4 Pd-treated rats), PdO caused a decrease in the heart weight.
-46-
-------
The dietary administration of Pt4+ salts did not markedly change
the weights of any organs. The administration of Pt(S04)2.4H20 (1 week;
1.6 mmoles/liter drinking fluid) did decrease the liver weight and the
administration of PtCl^ (4 weeks; 1.6 mmoles/liter drinking fluid)
increased the kidney weight. Under most of the schedules of the dietary
administration of Pt4+ salts, small increases were noted in the size of
the kidney but the increase was statistically significant in only one
series (Table 3).
Tissue content of DNA, RNA, and protein. Following the dietary
administration of metallic salts, various tissues were analyzed for the
content of DNA, RNA, and protein. Rats which had received PbCl2 at a
level of 3.7 mmoles/liter drinking fluid for 4 weeks did not show altered
DNA, RNA or protein content in liver, kidney or spleen (Table 4). Although
in one series of rats there was an apparent decrease in the DNA content
of testis of Pb-treated rats, analyses on testis from a second series of
animals showed no such decrease.
The administration of Pt4+ salts, either as PtCl^ at 13.2 mmoles/kg
feed for 4 weeks or as Pt(S04)2.4H20 at 5.9 mmoles/kg feed for 4 weeks,
did not alter the tissue content of DNA, RNA or protein in liver, kidney
or spleen (Table 4).
-47-
-------
DISCUSSION
On a molar basis, the soluble salts of platinum and palladium are
among some of the more toxic metallic salts. It is anticipated that the
exposure of populations to these metallic salts will be low dde to the
relative rarity of the compounds.
The inclusion of the metallic salts in the diets of rats at the
doses used in this study commonly resulted in decreased weight gain by
the metal-treated rats. In most cases, however, the decreased weight
gain was reflected in the decreased feed consumption by these rats.
In a manner similar to the results reported by Hirsch (1973) and
by others, the dietary administration of lead salts resulted in enlargement
of the kidney. Hirsch showed that the increased weight in the kidney was
not due to an increased percentage of water content. The dietary
administration of platinum and palladium salts did not bring about major
and/or consistent changes in the weights of the five tissues examined.
Likewise, dietary treatment with salts of platinum or palladium did not
alter the content of DNA, RNA or protein in liver, kidney or spleen (when
the content is expressed per gram of wet tissue).
-48-
-------
REFERENCES
Hirsch, G. H. (1973). Effect of chronic lead treatment on renal function.
Toxicol. Appl. Pharmacol. 25, 84.
Litchfield, J. T. and Wilcoxon, F. A. (1949). A simplified method of
evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96,
99.
Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951).
Protein measurement with the Folin phenol reagent. J. Biol. Chem.
193, 265-275.
-49-
-------
SECTION III. Table 1.
Lethal Doses of Various Metallic Compounds After
Intraperitoneal or Oral Administration in the Rat
Compound
Route
Lethal Doseb
(mmoles/kg)
(mg cation/kg)
PbCl,
PdCl2.2H20
PdSO, ip
PtCl2 ip
PtCl^ ip
oral
Pt(S04)2.4H20 ipc
RuCl
ip 0.70 (0.61-0.80); 0.56; 0.87
oral 7.5 (7.0-8.1); 6.3; 9.0
ip 8.5 (5.0-14.4); 1.6; 16.8
ip 0.57 (0.45-0.72); 0.39; 0.82
oral 2.7 (2.2-3.4); 1.56; 4.8
ip 1.42 (1.11-1.81); ; 1.8
ip 2.5 (1.58-4.0);
ip 0.11 (0.09-0.15);
0.70 (0.51-0.96); 0.31; 1.57
0.68 (0.60-0.76); 0.56; 0.82
ipQ 0.3-0.4; 0.2-0.3; 0.4-0.6
oral 2.2 (1.57-3.1); 1.37; 3.5
oral 3.2 (2.4-4.0); 1.78; 5.4
38 (33-44); 31; 48
410 (380-450); 350; 490
1760 (1050-3000); 330; 3500
60 (48-77); 42, 87
290 (240-360); 166; 520
151 (118-193); ; 195
490 (310-770);
22 (17-29);
136 (99-188); 60; 310
132 (117-149); 110; 160
59-78; 39-59; 78-117
430 (310-600); 270; 690
310 (240-400); 180; 550
aMale Sprague-Dawley rats; initial body weight 100-llOg. A
14-day observation period was used in lethal dose studies.
Data are given in the following sequence:
in parentheses); LD; LD^Q
(and its 95% confidence limits,
cCompound obtained from ICN/K and K Laboratories
dCompound obtained from D. F. Goldsmith Chemical and Metal Corporation
-50-
-------
SECTION III. Table 2.
Effect Of Dietary Metallic Salts On Weight Gain
i
Ul
Metallic Salt . Group Dietary Level3
PbCl2
PdCl2
PdO
PdS04
ptci4
Control
Pb 13.2/kg
Control
Pb 29.8/kg
Control
Pd 29.8/kg
Control
Pd 29.8/kg
Control
Pd 29.8/kg
Control
Pt 1.63/A
Control
Pt 2.45/A
Control
Pt 13.2/kg
Parameter Measured
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
(4)
(4)
(12)
(8)
(4)
(4)
(4)
(4)
(4)
(4)
(12)c
(8)c
(4)
(4)
(8)
(8)
Week Number
1
110
72**
106
73**
106
52**
120
109ns
121
103t
99
84**
103
79**
103
21**
2
120
106ns
103
84*
105
75*
105
101ns
109
103ns
89
64*
3
89
85
99
89ns
105
71**
95
90ns
97
103ns
79
77ns
4
97
82
104
93ns
101
115ns
127
59**
100
98ns
96
70ns
1-4
104
8lt
103
84*
104
77*
112
91**
100
54**
100
94ns
91
57**
-------
Table 2 (Continued)
Metallic Salt Group
Pt(S04)2.4H20 Control
Pt
Control
Pt
}2 aDietary levels of metallic
i
Dietary Level3 Parameter Measured"
W (16)
1.63/£ W (8)
W (4)
5.9/kg W (4)
salts are expressed as mmoles/kg feed or mmoles/£iter
Week Number
1234
103
74** __
94 115 105 118
80ns 98** 87* 115ns
drinking fluid .
1-4
108
95*
^Parameters measured include weight gains (W). Weight gains are expressed
as percentage of the weight gains during the corresponding week by all control rats maintained
on diets for 4 or more weeks. Weight gains (g/rat/week) (mean + S.D.) by 120-124 control
rats were: week 1, 58 + 10; 2, 57 + 12; 3, 56 + 13; 4, 51 + 14. Weight gains by controls
during weeks 1-4 were 222 +_ 34.
cMinimum number of values for each time interval.
Statistical analysis (t-test): **, P < 0.01; *, P < 0.05; f, 0.05 < P < 0.1; ns, P > 0.1.
-------
SECTION III. Table 3.
EFFECT OF DIETARY METALLIC SALTS ON TISSUE WEIGHTS
Metallic Salt Group, No.
of Rats
MnCl2-4H20 Control,
8
Mn,
8
Control,
4
Mn,
4
PbCl2 Control,
8
Pb,
S 8
Control,
4
Pb,
4
Control,
Pb,
8
Control,
Pb,
Control,
8
Pb,
8
Dietary Level Duration
(weeks)
13
8.3M 13
13
18.6/£ 13
4
3.7/X, 4
4
8.3/A 4
13
3.7/£ 13
4
13.2/kg 4
4
29.8/kg 4
Body
Weightb
(g)
517
520
578
511
309
309
344
296
511
482
318
286
297
258*
Tissue Weight
Liver
92
91
93
88
104
107
105
102
89
98*
103
106
108
107
Kidney
(106)
(102)
(97)
(105)
(95)
(101)t
(96)
(112)t
(103)
(127)**
(107)
(131)*
(106)
(143)**
(% of Expected Weight)0
Spleen
92
105
(90)
(87)
(82)
(88)
96
104
(103)
(106)
(101)
(95)
(114)
(132)
Heart
(97)
(103)
(101)
(93)t
(104)
(108)
(107)
(104)
(99)
(99)
102
101
99
106
Testis
94
94
(103)
(95)
(102)
(100)
(101)
(96)
(100)
(97)
(98)
(99)
(96)
(100)*
-------
Table 3 (Continued)
Metallic Salt Group, No. Dietary Level3
of Rats
PdCl2.2H20 Control,
8
Pd, (satd. soln.)
8
PdCl, Control,
Z 8
Pd, 13.2/kg
8
Control,
4
Pd, 29.8/kg
4
PdO Control,
4
Pd, 29.8/kg
4
PdSOA Control,
4
Pd, 5.6/kg
4
Control,
4
Pd, 29.8/kg
4
PtCl, Control,
* 4
Pt, 0.54/S.
4
Duration
(weeks)
1
1
4
4
4
4
4
4
4
4
4
4
13
13
Body
Weight13
(g)
136
132
335
318
314
258*
324
287**
295
318
318
238*
547
538
Tissue Weight (1% of Expected Weight)0
Liver
85
85
113
101**
112
114
104
103
104
105
102
88
92
88
Kidney
(97)
(95)
(102)
(93)**
.f
(97)
(99)
(98)
(106) t
(104)
(108)
(128)
(124)
(107)
(114)
Spleen
(134)
(89)**
(100)
(82)
(79)
(96)
(93)
(101)
(103)
(86)
(92)
(94)
Heart
(95)
(96)
105
103
(104)
(95)*
(98)
(100)
102
103
(103)
(102)
Testis
(103)
(104)
(103)
(106)
(97)
(100)
(101)
(98)
(101)
(112) t
(96)
(95)
-------
Table 3 (Continued)
Metallic Salt Group, No. Dietary Levela
of Rats
PtCl, Control,
12
Pt, 1.63M
12
Control,
12
Pt, 1.63/d
12
Control,
4
, Pt, 5.9/kg
i
Control,
4
Pt, 13.2/kg
4
PtO? Control,
4
Pt, 29.8/kg
4
Pt(SO, )2.4H20 Control,
8
Pt, 1.63/X,
8
Control,
4
Pt, 5.9/kg
4
Duration
(weeks)
1
1
4
4
4
4
4
4
4
4
1
1
4
4
Body
Weight*5
(g)
152
145
306
296
287
279
288
252t
272
300
173
149*
328
285**
Tissue Weight (1% of Expected Weight)0
Liver
89
87
100
100
107
107
116
96
102
105
99
88*
109
113
Kidney
88
91
(89)
(95)**
(95)
(99)
(117)
(123)
(108)
(115)
(99)
(97)
(109)
(128)
Spleen
96
91
(78)
(81)
(124)
(112)
105
93
96
92
105
91
(104)
(120)
Heart
97
97
103
104
(101)
(104)
96
101
101
102
107
93
101
100
Testis
76
79
(99)
(106) t
(98)
(99)
(105)
(106)
(104)
(103)
89
87
(99)
(103)
-------
Table 3 (Continued)
Statistical analyses (t-test) : **, P < 0.01; *, P < 0.05; t, 0.05 < P < 0.01; no designation is used
were P > 0.1.
aDietary levels of metallic salts are expressed as mmoles/liter drinking fluid or mmoles/kg feed.
bfiody weight and tissue weights were measured after fasting for 14-15 hours.
cExpected tissue weights were calculated from the equations given in the MATERIALS AND METHODS,
except that values in parentheses were calculated from similar equations calculated only for control rats
on the same dietary duration (that is, 1-, 4- or 13-weeks).
^Except only 4 values for liver, heart and testis.
-------
SECTION III. Table 4.
EFFECT OF DIETARY METALLIC SALTS ON THE CONCENTRATION OF DNA, ENA AND PROTEIN IN VARIOUS TISSUES
i
Ul
Salt Group3 Dietary Level Duration Macromolecule*3
(weeks)
PbCl2 Control 4 DNA
RNA
Protein
Pb 3.7/£ 4 DNA
RNA
Protein
PbCl2 Control 4 DNA
RNA
i
Protein
Pb 3.7/A 4 DNA
RNA
Protein
PtCl, Control 4 DNA
4
RNA
Protein
-
Liver
5.19
+0.69
22.7
+0.9
298
+95
4.64
.+1.06
20.9
+2.1
279
+59
27.3
+0.7
27.1
1.2
6.82
+0.72
23.0
+1.6
188
+37
Kidney
6.86
+1.06
9.84
+1.01
92.2
+29.1
7.05
+0.47
11.90
+2.10
90.3
+18.2
8.42
+0.94
10.94
+1.59
137
+15
8.03
+0.66
11.20
+0.52
143
+4
6.51
+0.63
8.10
+0.51
113
+17
Tissue
Spleen
40.1
+1.7
18.6
,±2-7
122
+9
38.6
+3.8
22.2
+4.5-
129
+7
38.3
+6.0
23.6
+1.4
109
+12
35.8
+12.8
23.7
+6.3
117
+17
28.3
+3.1
17.7
+1.3
103
+45
Testis
6.06
+0.45
8.94
+0.51
117
+22
5.32*
+0.10
9.31
+1.00
129
+9
6.62
+0.52
9.90
+0.59
63.3
+10.0
6.67
+0.27
10.66
+1.42
64.6
+5.8
-------
Table 4 (Continued)
EFFECT OF DIETARY METALLIC SALTS ON THE CONCENTRATION OF DNA, RNA AND PROTEIN IN VARIOUS TISSUES
Salt Group3 Dietary Level
PtCl4 Pt 13.2/kg
Pt(S04)2
.4H20 Control
i< Pt 5.9/kg
00
Duration Macromolecule'-'
(weeks)
4 DNA
RNA
Protein
4 DNA
RNA
Protein
4 DNA
RNA
Protein
Liver
6.49
+0.38
21.2
+2.4
230
+36
7.64
+0.52
23.2
+1.5
164
+12
6.96
+1.44
21.lt
+1.1
170
+20
Kidney
6.50
+0.76
7.99
+1.02
93t
+8
6.66
+0.32
9.58
+0.50
111
+6
7.09
+0.71
9.40
+1.30
104
+12
Tissue
Spleen
27.4
+1.6
18.7
+2.0
102
+34
39.3
+7.9
19.2
+0.9
114
+5
36.4
+2.1
19.5
+2.3
116
+12
Testis
-.-
Statistical analysis (t-test): *, P < 0.05; t, 0.05 < P < 0.1; no designation is used where P > 0.1
a4 rats/group
DNA content is expressed as ymoles DNA-nucleotide/g wet tissue; RNA as ymoles RNA-nucleotide/g wet tissue; and
protein as mg protein/g wet tissue.
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/l-76-010b
3. RECIPIENT'S ACCESSIOI*NO.
4. TITLE AND SUBTITLE
ASSESSMENT OF TOXICITY OF AUTOMOTIVE METALLIC EMISSIONS
Volume II: Relative Toxiciti.es of Automotive Metallic
Emissions Against Lead Compounds Using Biochemical Parameters
5. REPORT DATE
January 1976
6. PERFORMING ORGANIZATION CODE
?.>AUTHOR(S)
-David J. Holbrook, Jr.
8. PERFORMING ORGANIZATION REPORT NO,
S.PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Biochemistry
School of Medicine
University of North Carolina
Chapel Hill, fl.C. 27514
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
68-02^1701
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, M.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
1. Results of \ntraperltoneal (IP 1 administration of PtCl4 or Pd(~N03J[2 are reported.
Administration at levels of 28 or 56 moles/kg body weight decreased the
thymidi.ne incorporation into DNA of spleen, liver, and testis,
2. Effects of various salts of platinum or palladium administered by intraperitoneal
injection or ingestion were determined on the parameters of the mlcrosomal
mixed function oxidase system from rat liver.
3. Lethal-dose studies are reported following the intraperitoneal or oral adminis-
tration of salts of lead, manganese, platinum, and palladium to young male rats.
Studi.es have been conducted on the effect of dietary administration of salts of
Pb, Mn, Pt, and Pd on the following: the growth rate of male rats, the organ
weight of five tissues (liver, kidney, spleen, heart, and testis), and the
tissue content of DNA, RMA, and protein.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COS AT I Field/Group
Exhaust Emissions
Lead (metal)
Manganese
Platinum
Palladium
Deoxyribonucleic acids
Ribonucleic acids
Toxicity
Thymidines
Metabolism
06" F,' T
21 D
8. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
63
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
59
-------
INSTRUCTIONS
1. REPORT NUMBER
Insert the EPA report number as it appears on the cover of the publication.
2. LEAVE BLANK
3. RECIPIENTS ACCESSION NUMBER
Reserved for use by each report recipient.
4. TITLE AND SUBTITLE
Title should indicate clearly and briefly the subject coverage of the report, and be displayed prominently. Set subtitle, if used, in smaller
type or otherwise subordinate it to main title. When a report is prepared in more than one volume, repeat the primary title, add volume
number and include subtitle for the specific title.
5. REPORT DATE
Each report shall carry a date indicating at least month and year. Indicate the basis on which it was selected (e.g., date of issue, date of
approval, date of preparation, etc.).
6. PERFORMING ORGANIZATION CODE
Leave blank.
7. AUTHOR(S)
Give name(s) in conventional order (John R. Doe, J. Robert Doe, etc.). List author's affiliation if it differs from the performing organi-
zation.
8. PERFORMING ORGANIZATION REPORT NUMBER
Insert if performing organization wishes to assign this number.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Give name, street, city, state, and ZIP code. List no more than two levels of an organizational hirearchy.
10. PROGRAM ELEMENT NUMBER
Use the program element number under which the report was prepared. Subordinate numbers may be included in parentheses.
11. CONTRACT/GRANT NUMBER
Insert contract or grant number under which report was prepared.
12. SPONSORING AGENCY NAME AND ADDRESS
Include ZIP code.
13. TYPE OF REPORT AND PERIOD COVERED
Indicate interim final, etc., and if applicable, dates covered.
14. SPONSORING AGENCY CODE
Leave blank.
15. SUPPLEMENTARY NOTES
Enter information not included elsewhere but useful, such as: Prepared in cooperation with, Translation of, Presented at conference of,
To be published in, Supersedes, Supplements, etc.
16. ABSTRACT
Include a brief ^200 words or less) factual summary of the most significant information contained in the report. If the report contains a
significant bibliography or literature survey, mention it here.
17. KEY WORDS AND DOCUMENT ANALYSIS
(a) DESCRIPTORS - Select from the Thesaurus of Engineering and Scientific Terms the proper authorized terms that identify the major
concept of the research and are sufficiently specific and precise to be used as index entries for cataloging.
(b) IDENTIFIERS AND OPEN-ENDED TERMS - Use identifiers for project names, code names, equipment designators, etc. Use open-
ended terms written in descriptor form for those subjects for which no descriptor exists.
(c) COSATI FIELD GROUP - Field and group assignments are to be taken from the 1965 COSATI Subject Category List. Since the ma-
jority of documents are multidisciplinary in nature, the Primary Field/Group assignment(s) will be specific discipline, area of human
endeavor, or type of physical object. The application(s) will be cross-referenced with secondary Field/Group assignments that will follow
the primary posting(s).
18. DISTRIBUTION STATEMENT
Denote releasability to the public or limitation for reasons other than security for example "Release Unlimited." Cite any availability to
the public, with address and price. /
19. &20. SECURITY CLASSIFICATION
DO NOT submit classified reports to the National Technical Information service.
21. NUMBER OF PAGES
Insert the total number of pages, including this one and unnumbered pages, but exclude distribution list, if any.
22. PRICE
Insert the price set by the National Technical Information Service or the Government Printing Office, if known.
EPA Form 2220-1 (9-73) (Reverse)
------- |