xvEPA
                                United States
                                Environmental Protection
                                Agency
                                Health Effects Research
                                Laboratory
                                Research Triangle Park NC 2771 1
                                 Research and Development
                                EPA-600/S1-81-015  Aug  1981
Project  Summary
                                Sensitive  Biochemical  and
                                Behavioral  Indicators  of
                                Trace  Substance  Exposure:
                                Part  II.   Platinum
                                Edward J. Massaro, Bradley A  Lown, John B. Morganti, Carl H. Stineman,
                                and Rosemary B. D'Agostino
                                  The overall objective of this project
                                was to characterize Pt [Pt(SO4)2,
                                NA2PtCle] intoxication in a model
                                mammalian system (the mouse) em-
                                ploying acute (single) and serially
                                repeated (multiple) dose exposure
                                paradigms. Platinum lethality para-
                                meters were defined in both sexes of
                                the adult mouse and Pt tissue/organ
                                distribution was monitored as a func-
                                tion of time and dose. In coordination
                                with the tissue distribution study,
                                investigations of the effects of Pt on
                                selected parameters of open field,
                                exploratory and social behavior, pas-
                                sive avoidance learning and the acqui-
                                sition of two-way active avoidance
                                learning were undertaken. Correla-
                                tions among tissue levels of Pt and
                                open field behavioral parameters (am-
                                bulations and rearings)  were probed
                                employing statistical methodology.
                                  Since numerous xenobiotics are
                                known to cross the placenta and/or
                                accumulate in maternal milk, studies
                                of Pt tissue/organ distribution in the
                                gravid female and the embryo/fetus/
                                offspring were undertaken.  Platinum
                                levels were monitored as a function of
                                time. Coordinate studies examined
                                developmental and behavioral para-
                                meters of the offspring (as neonates,
                                pups and adults) of exposed mothers.
                                In addition, maternal behavior was
                                examined as a function of pup retrieval
                                and neonate and pup activity levels
                                were assessed. Ambulations and rear-
                                ings in the open field, passive avoid-
                                ance learning and rotarod performance
                                were assessed  in adult offspring of
                                exposed mothers.
                                 This Project Summary was devel-
                                oped by EPA's Health Effects Research
                                Laboratory, Research Triangle Park,
                                NC, to announce key findings of the
                                research project that is fully docu-
                                mented in a separate report of the
                                same title (see Project Report ordering
                                information at back).


                                Results
                                 Presently, we have no data to explain
                                the disproportionately high tissue con-
                                centrations of Pt in  animals receiving
                                Pt(S04)z intragastrically (IG) at the LD25
                                level as compared  to the LD5 level.
                                Conceivably, the higher Pt dose may
                                perturb the Gl tract in some manner,
                                resulting in facilitated Pt absorption.
                                The high Pt concentrations found in the
                                blood of such animals may damage the
                                kidneys (which normally eliminate Pt
                                quite efficiently), thereby decreasing Pt
                                excretion and increasing tissue uptake.
                                In addition, it is well known that some
                                metals, such as cadmium and lead, are
                                able to induce the synthesis of their own

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binding proteins. Platinum may have a
similar ability.

  The analysis of variance (ANOVA) of
the open field behavioral measures
revealed that the higher Pt dose de-
pressed ambulations significantly and
rearings marginally, relative to other
treatments, from 4 hours through 7
days post administration. A correlation
analysis revealed highly significant
negative correlations between behavioral
measures and tissue levels of Pt in all
tissues except brain  and muscle. Since
brain Pt levels were not related  to
behavior, it is not clear how non-CIMS
tissue Pt levels affect behavior.  In
earlier work with alveolar macrophages
in tissue culture, a 50% loss of viability
(ability to exclude trypan blue) after a 20
hour exposure to 300 mM PtCI* was
reported. Protein  and RNA synthesis
were inhibited by 50% by 60 mM Pt and
DNA synthesis was inhibited by 50% by
10  mM Pt. The maximum tissue  Pt
concentration  observed in our  study
was 50 ppm in the kidney, 4 hours post
administration. Fifty ppm is approx-
imately equivalent to 250 mM Pt. There-
fore, cellular processes may have been
disrupted to some extent in the LD25
animals which, in turn, may produce a
general  malaise and depression of the
activity component of certain behaviors.


Pt(SO4)2-Single (Acute) Dose
Effects on Exploratory
Behavior

  The high  rate of explorations of the 4
hour low pH control group, for which no
satisfactory explanation is presently
available, may have sufficiently accen-
tuated the  difference between  the 4
hour time point and all other times  of
observation to account for the finding of
a significant main effect of time as well
as the significant time by treatment
interaction.

  At 1, 3 and 7 days post IG administra-
tion, the mean number of explorations
for the 2 Pt groups (LDS, LD25) and the
low pH  control group was lower than
that of the saline control group, but not
significantly. Possibly, this trend was
due to some nonspecific effect of the
low pH or sulfate content of the solutions.
In any case, it is apparently not a specific
Pt effect. Thus, in this study, Pt exposure
per se, does not appear to have effected
changes in  exploratory behavior.
Pt(SO4)2-Single (Acute) Dose
Effects on Activity Wheel Per-
formance
  Platinum at either the LD5 or LD25
levels appeared not to  affect  wheel
running scores. All groups exhibitedthe
lowest scores during the  first 24 hours
post administration. This may have
resulted from trauma induced by han-
dling the dose administration.  The
relative depression of scores  observed
on the last 2 days of testing may have
been due to the effect of time (habitua-
tion). Trend analysis demonstrated that
the performance curves of all treatment
groups followed the same trend, inde-
pendent of treatment. Thus, there were
no overt  effects of Pt  exposure on
activity wheel performance.

PtfSOth-Single (Acute) Dose
Effects on Passive Avoidance
Learning
  Intragastric administration of PtfSCMg
at the LDs or LD2s levels had no observ-
able effect on passive avoidance learning
by the adult mouse.

Pt(SO4)2-Single (Acute) Dose
Effects on Active Avoidance
Learning
  Platinum sulfate at the LD5 or  LD25
levels had no effect either on the acqui-
sitions  of two-way  active avoidance
learning or activity in the apparatus.

Pt(SO4)2-Single (Acute) Dose
Effects on Social Behavior
  At the IG LD5 or LD25 levels, Pt (SO4)2
had no effect on the measure of murine
social behavior investigated.

NazPtCk-Single (Acute) Dose
Tissue Distribution and Open
Field and Exploratory
Behavioral Studies
  Relative to the dose administered, the
fairly low tissue/organ Pt levels observed
suggest that  Na2PtCI6 is only  poorly
absorbed from  the gut. With the excep-
tion of the spleen, the biological half-life
of Pt administered as Na2PtCI6 appears
to be short.
  Na2PtCle depressed ambulations and
rearings in the  open field. However, the
effect was transitory and clear-cut
effects were found only at 4 hours post
administration. At 1 and 3 days post
administration, the LD5 exposed groups
had depressed rearings relative to the
saline controls groups but not the low
pH control groups.
  Na2PtCle had no observable effect or
exploratory behavior at 1, 3 and 7 day;
post administration. Unfortunately
exploratory behavior was not examinee
4 hours post administration when th<
strongest depression of open fielc
behavior had been observed.

Pt(SO4)2-Multiple (Serially
Repeated) Dose Effects on
Tissue Distribution and
Open Field Behavior
  This study was undertaken to gair
insight into  the effects of subchronn
exposure to  Pt on certain  behaviors o
the mouse.  Subchronic exposure wa:
simulated via the  use of  a limitei
repeated dose experimental design (1 ti
10 doses)  Adult  male mice were sub
jected  to repeated IG administratioi
(every 72 hours) of relatively high dose
of Pt(S04)2 (each dose equaled the 7da
LDi, which is approximately 40% of th'
7 day LD5o). This design resulted only ii
marginal adverse effects on the genere
activity and exploratory behavior of th.
adult animal. Tissue/organ Pt level^
while highly variable, generally  in
creased with the number of dose
except for the brain, in which no Pt wa
detected in any animal. The absence c
Pt in the brain is not surprising since th
highly charged  Pt cation should b
excluded by  the blood brain barrier. 0
the three behavioral measures studiec
only rearings in the open field showed
significant correlation with tissue  F
levels. Since this correlation involved
number of tissues and not the brain,
general systemic effect, rather  than
neurotoxic effect, seems to be mdicatec
Inorganic Pt compounds have bee
shown to inhibit DNA, RNA and protei
synthesis in tissue culture. Thus, it ma
be that Pt has a depressive effect o
cellular metabolic processes and e>
posure to Pt above a threshold lev(
results in general malaise which i
reflected in  the  slight depression  i
behavior observed in this study.

Pt(SO4J2 Developmental
Studies
  The predominant effect of  matern;
administration of Pt(S04)2 on days 7 ar
12 of gestation was in offspring weigh
The in utero  Pt exposed offspring wei
lighter than  low pH exposed offsprin'
This effect was powerful, beginning c
day 0 and continuing through  day i

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post partum. As indicated by the signifi-
cant  multivanate  effect for  postnatal
exposure, type of foster mother exposure
also has a significant effect on offspring
weight. Thus, on day 45 post partum,
pups (exposed to Pt or low pH in utero)
reared by Pt exposed foster mothers
weighed less than Pt or low pH gesta-
tionally exposed pups reared by low pH
exposed mothers. The  reason for the
weight differences  is unknown. How-
ever,  it  may be that reduced  weight
could have important consequences,
such as reduced viability.
   Administration of PtfSCUfe to lactating
mothers resulted in symptoms of toxicity
in  offspring.   Offspring  assigned  to
mothers administered Pt  during lactation
exhibited depressed neonatal activity
(p< 0.006) and lower adult weights (p<
0.015) than controls.  Pt may have
affected these offspring directly through
the  maternal  milk  supply and/or in-
directly  by affecting some aspect of
maternal behavior.
     PtCle Developmental
 Tissue Distribution and
 Behavioral Studies
   Maternal blood, kidney, and whole
 embryo Pt levels were highest 1 day
 post administration of NajPtCle on day 7
 of gestation. No Pt was detected in the
 maternal brain. By day 5 post adminis-
 tration,  Pt was undetectable in fetal
 tissues, but the placental level was 2.5
 fold greater than that of maternal blood.
   Except for the maternal spleen, the Pt
 levels of maternal, fetal, and placental
 tissues  peaked on day  1  post adminis-
 tration of NaaPtCle on day 12 of gesta-
 tion. A low level of Pt (0.43 ± 0.01 ppm)
 was detected m maternal brain tissue.
 Placental Pt levels were greater than
 blood levels throughout the course of
 the experiment, reaching concentration
 factors,  compared to blood, of 1.3 and
 4 2 on days 1 and 5 post administration,
 respectively.
  Whole body Pt levels in the suckling
 offspring of dams receiving Pt on day 2
post partum were highest on day 1 post
 administration. By day 1 2 post adminis-
 tration, except for the digestive tract and
 its contents, no Pt was detectable in the
tissues  of the sucklings.
  Effects of maternally administered
 NaaPtde on neonatal and adult off-
spring behavior were limited to day 12
 gestational exposure. The neonatal
effects were expressed as lower day 8
post partum activity scores for in utero
Pt exposure compared to PBS exposure.
For the adult offspring,  there  was a
prenatal x postnatal exposure interaction
indicating that Pt exposed mothers
reduced the performance of offspring on
those tasks with a large activity compo-
nent (i.e., ambulations, passive  avoid-
ance trial  1 latency and passive avoid-
ance  difference scores).  In this case,
gestational exposure to Pt appears to
have  persistent effects on the mother
that are transmitted postnatally to
offspring. This may occur directly
through residual  Pt in the milk or
indirectly through maternal behavioral
alteration resulting in neglect  of the
offspring.
Conclusions
  In summary, PtfSCMz administered
via  the  IG  route appears to be poorly
absorbed. However, with the exception
of brain tissue, the absorbed Pt achieves
general systematic distribution. With
repeated exposure, tissue concentra-
tions tend to increase with dose; again,
with the exception of brain which  did
not accumulate Pt. While Pt did affect
behavior under conditions of single and
multiple dose exposure, these effects
were weak except when very high [LDzs)
individual doses were employed In the
latter case, the behavioral effects were
more pronounced and lasted for up to 7
days post administration (end of observa-
tion). The consistent pattern  of rela-
tionship between tissue levels of Pt and
behavior is suggestive of interference
with cellular processes induced by Pt at
concentrations  greater than some
threshold value.  This interference is
manifested as behavioral  malaise.
  It is difficult to predict  the potential
danger of  increased anthropogenic
redistribution of Pt in the  environment.
Toxicity has been observed  in humans
exposed to high  Pt levels in the work-
place or during antitumor chemotherapy.
However, such dose  levels are many
times those that would ever by expected
to occur in the general environment.
Nevertheless, since exposure  to  Pt
appears to alter certain behavior of the
mouse, the possibility gf subtle adverse
biological and behavioral effects result-
ing from long-term low level (environ-
mental) exposure of humans cannot be
disregarded especially in the light of the
fact that Pt can  be  methylated in a
manner analagous to that of mercury.
By altering  its physicochemical  proper-
ties, methylation  may, as in  the case of
mercury, greatly enhance the toxicity of
Pt and its bioaccumulatability
  Edward J. Massarois with the Center for Air Environment Studies, Pennsylvania
    State University, University Park, PA 16802; Bradley A. Lown and John B.
    Morganti are with the Department of Psychology, State University College at
    Buffalo, Buffalo, NY 14222; Carl H Stineman and Rosemary B. D'Agostino are
    with the School of Medicine, State University of New York at Buffalo, Buffalo,
    NY 14214
  George M. Goldstein is the EPA Project Officer (see below).
  The complete report, entitled "Sensitive Biochemical and Behavioral Indicators
    of Trace Substance Exposure.  Part II. Platinum," (Order No. PB 81-160 897;
    Cost' $8 00, subject to change) will be available only from-
          National Technical Information Service
          5285 Port Royal Road
          Springfield,  VA 22161
          Telephone 703-487-4650
  The EPA Project Officer can be contacted at
          Health Effects Research Laboratory
          U S Environmental Protection Agency
          Research Triangle Park,  NC 27711
  us GOVERNMENT PRINTING OFFICE 1981 -757-012/7283

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