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 ------- 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 ------- 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 ------- United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 Postage and Fees Paid Environmental Protection Agency EPA 335 Official Business Penalty for Private Use $300 • HS uOun3?s U S tMvTK FPUTc.r ITU'l MJ^C Hfc'.GJU'J S M^K^Y -"- " nF^PuOH*! SfPt^T if. ------- |