Effect of platinum and palladium salts on thymidine incorporation into DNA of rat tissues.

The intraperitoneal administration of PtCl4 or Pd(NO3)2 at levels of 28 or 56 mumole/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 PtCl4. In control rats, only 20-30% of the 3H in the acid-soluble fraction of liver or spleen was in the form of thymidine and its phosphate esters 2 hr after the intraperitoneal injection of 3H-thymidine; prior injection of PtCl4 (56 mumol/kg body weight) did not change the pattern.


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 soluble salts of platinum and palladium on the incorporation of radioactive thymidine into DNA.

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 3Hlabeled thymidine were injected intraperitoneally 4 hr and 2 hr, respectively, before the removal of the tissues. The 3H-thymidine was injected at a level of 99 ,uCi/kg body weight, except that a level of 990 ,uCi/kg body weight was used when the distribution of 3H in the acid-soluble fraction was under study (i.e., Table 5). (3H-methyl) thymidine was purchased from New England Nuclear, Pd(NO3)2 aqueous solution from Research Organic/Inorganic Chemical, and PtCl4 from the latter firm and from B.F. Goldsmith Chemical and Metal.
The administered doses of PtCl4 and Pd (NO3)2 were 14, 28, and 56 ,tmole/kg body weight. For PtCl4, these doses corresponded to 2.8, 5.5, and 11 mg Pt4+/kg. For Pd(NO3)2, the doses by weight were equal to 1.5, 3, and 6 mg Pd2+/kg body weight. The highest dose of Pt, namely 56 ,mole/kg, was equal to one-half of the intraperitoneal LD50. However, because of the steep slope of the lethal dose curve, the dose of 56 ,umole PtCl4/kg was appreciably less than the LD5. Thymidine incorporation was measured in spleen, liver, kidney, and testis. Tissue macromolecules were precipitated with cold 0.5M HCl04 and, after centrifugation, the supernatant was collected as the acid-soluble fraction. The RNA in the pellet was hydrolyzed with 0.3M NaOH (37°C, 1 hr) and the intact macromolecules were precipitated with cold HCl04 (final concentration of HCl04 not neutralized, 0.5M). The DNA was hydrolyzed in hot HCl04 (0.5M, 90°C, 20 min). After centrifugation, the supernatant (hydrolyzed DNA) was collected. The acid-soluble fraction and the hydrolyzed DNA were analyzed for nucleotide concentration by measuring the absorbance at 260 nm. Tritium was measured by scintillation counting by using a mixture containing toluene and Triton X-100 (1).
The radioactivity in the DNA is expressed as counts per minute (cpm)/,mole DNA-nucleotide. The radioactivity in the acid-soluble fraction is expressed as cpm/,umole total acid-soluble nucleotide. The radioactivity is also expressed as a ratio of the two values, i.e., as cpm/Aumole DNAnucleotide divided by cpm/,umole 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-1 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 absorption of nucleotides and related compounds by charcoal from acidic solution was conducted according to the method of Tsuboi and Price (5). The charcoal-absorbed compounds were eluted from the charcoal by two treatments with 1% ammonia in 60% ethanol (5). The eluted charcoal-absorbable compounds were chromato-graphed on thin layer sheets of polyethyleneimine-cellulose with 0.02M ammonium acetate-95% ethanol (1:1, v/v). Thymidine and thymine migrated to the solvent front; thymidine phosphates remained at or near the origin.

Results
The effects of PtCl4 on thymidine incorporation into DNA of rat tissues are presented in Table 1. In the tissues studied, the incorporation 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 PtCl4. The two higher doses of PtCl4 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 ,umole/kg, thymidine incorporation was inhibited by 40% and the ratio approached that observed in spleen at a dose of 56 ,mole/kg. At the latter dose, the radioactivity in the acid-soluble fraction (ASF) was increased by approximately 50%. In kidney, thymidine incorporation into DNA was inhibited 40-50% at doses of 28 or 56 ,umole/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 PtCl4. In testis, the thymidine incorporation into  'All values ± S.E. Statistical analysis (t-test): dagger (t) denotesp < 0.01; asterisk (*), p < 0.05; double dagger (t), 0.05 <p < 0.10. PtCl4 and :3H-thymidine injected intraperitoneally at 4 hr and 2 hr, respectively, before removal of the tissues.
DNA and the ratio were decreased by 25-35% by doses of PtCl4 of 28 or 56 ,Amole/kg. Thymidine incorporation into DNA of spleen was very sensitive to the administration of Pd(NO3)2, just as it was to PtCl4 (Table 2). Moderate decreases were found in thymidine incorporation into DNA and in the ratio of spleen at the lowest dose of Pd(NO3)2. At the two highest doses, 50-60% inhibition was observed in the DNA and the ratio. Pd(NO3)2 decreased incorporation into DNA and the ratio of liver by 25-45%, depending on the dosage. The kidney was relatively resistant to treatment with Pd(NO3)2. 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(NO3)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 PtCl4 or Pd(NO3)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 et al. (2, 3) developed a method for the fractionation of liver nuclei into two classes: nuclei derived from parenchymal cells or hepatocytes (called N-1 nuclei) and nuclei derived from stromal  ,umole/kg decreased thymidine incorporation into DNA to 60% of control values. This dose of PtCl4 also decreased the ratios to approximately 40% of control values. Thus, in experiment B, thymidine incorporation into both N-1 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. In control rats, the specific activities of DNA are in the following decreasing order: DNA of N-1 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-1 nuclei. In Table 3 the specific activities of DNA from N-2 nuclei are expressed as a fraction of the specific activities of DNA from N-1 nuclei in the same experiment. Although the higher dose of PtCl4 (Table 3) markedly inhibits thymidine incorporation in both cell types, the ratio of the N-2 to N-1 specific activities is not significantly altered by treatment with PtCl4.
The administratioh of CC14 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 to 10-fold greater in liver of the CCl4-treated rats than in the control rats. Incorporation has been studied in four tissues of rats which received CC14 and PtCl4 ( 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 PtCl4.
In liver, thymidine incorporation into DNA of group B rats was 10-fold greater than that of rats receiving no CC14. Moreover, increasing the dose of PtCl4 to 28 ,mole/kg did not inhibit thymidine incorporation into liver DNA of CCl4-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 (CC14 and 28 ,umole PtCl4/kg) was decreased approximately 30% in comparison with group B animals. The same pattern of results was obtained for all three tissues if the ratios of DNA to acid-soluble fraction were 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, PtCl4 (at 28 Amole/kg) apparently did not inhibit the stimulated synthesis of DNA of liver in CCl4-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 Environmental Health Perspectives fractions of liver and spleen were examined in control animals and in rats treated with 56 ,umole PtCl4/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-hr incorporation interval, the majority of the radioactivity in the acid-soluble fraction was in the form of the open-chain metabolites and other nonadsorbed metabolites in. liverand spleen..of -control rats ( Table 5). In liver, 30% of the radioactivity in the acid-soluble fraction was in the form of compounds aMeans of values of two rats; percentage values of each rat are given in parentheses. PtCl4 and 3H-labeled thymidine were injected intraperitoneally 4 hr and 2 hr, respectively, before removal of the tissues.
having the pyrimidine ring intact. Furthermore, the administration of the highest dose of PtCl4 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 of intact pyrimidine compounds, and the administration of PtCl4 did not alter this value.
The charcoal-adsorbable 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 PtCl4 did not appreciably alter the values in these two tissues.

Discussion
The present study indicates that PtCl4 and Pd(NO3)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 PtCl4 than the incorporation of uridine into RNA or of leucine into protein in cultured cells. The inhibition by PtCl4 may be analogous to the effect of Pt-containing antitumor compounds (7)(8)(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 has been demonstrated (10)(11)(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 PtCl4 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 Pt4+ salt such as Pt(SO4)2 had been selected for these studies.