Biomedical subjects

S G Chaney

Publications and source records attributed to S G Chaney.

At least 55 records · Page 3Linked to original sources

PubMed1991

Treatment of DNA with ammonium bicarbonate or thiourea can lead to underestimation of platinum-DNA monoadducts.

Thiourea and NH4HCO3 are widely used to block the conversion of Pt-DNA monoadducts to diadducts prior to the enzymatic digestion of DNA and subsequent analysis of the relative proportion of the different types of Pt-DNA adducts. Our data show that NH4HCO3 (100 mM, 18 h, 25 degrees C) is much less effective than thiourea (10 mM, 10 min, 25 degrees C) at blocking monoadducts, apparently because considerable monoadduct-to-diadduct conversion occurs during the incubation of platinated DNA with NH4HCO3. Under these incubation conditions, neither NH4HCO3 nor thiourea treatment causes significant diadduct-to-monoadduct conversion. At 25 degrees C, thiourea causes no significant removal of either ethylenediamine(en)- or diaminocyclohexane(dach)-Pt monoadducts. However, at 37 degrees C, both en-Pt and dach-Pt monoadducts are selectively removed. Pt-DNA diadducts are stable to 10 mM thiourea at either temperature. These data suggest that previous experiments using NH4HCO3-blocked DNA are likely to have underestimated Pt-DNA monoadducts and to have overestimated diadducts. As a consequence, such studies are likely to produce inaccurate estimates for the repair of individual adducts. The data also show that although thiourea treatment is suitable for blocking Pt-DNA monoadducts under the conditions generally used (10 mM, 10 min, 25 degrees C), it can selectively remove Pt-DNA monoadducts at higher temperatures.

Bicarbonates↗

PubMed1991

Comparisons of tetrachloro(d,l-trans)1,2-diaminocyclohexane-platinum(IV) biotransformations in the plasma of Fischer 344 rats at therapeutic and toxic doses.

Plasma biotransformations of tetrachloro(d,l-trans)1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) were determined in vivo at both therapeutic (3 mg/kg) and toxic (12 mg/kg) doses in Fischer 344 rats. Tetraplatin was rapidly converted to dichloro(d,l-trans)1,2-diaminocyclohexaneplatinum(II) [PtCl2(dach)]. This conversion was complete at the earliest time measured (7.5 min) at the therapeutic dose, but some unreacted tetraplatin was detectable in the circulation at the toxic dose. Three other major biotransformation products were observed in plasma: (d,l-trans)1,2-diaminocyclohexaneaquachloroplatinum(II) [Pt(H2O)(Cl)(dach)]+, the Pt-methionine complex, and another biotransformation product tentatively identified as either the Pt-cysteine or Pt-ornithine complex. Several other minor plasma biotransformation products were detected. Two of these were most likely formed intracellulary from tetraplatin. Two or more other platinum complexes appeared to lack the diaminocyclohexane carrier ligand and were most likely formed intracellulary by trans-labilization of the carrier ligand. Tetraplatin, PtCl2(dach), and [Pt(H2O)(Cl)(dach)]+ all rapidly disappeared from the circulation. The other biotransformation products were persistent through at least 3 h and could be responsible for the delayed toxicity of tetraplatin. Although some minor differences were observed between tetraplatin biotransformations at the toxic vs therapeutic doses, most biotransformation products were simply present at much greater concentrations at the toxic dose than at the therapeutic dose. Thus, our data suggest that dose-dependent differences in tetraplatin toxicity are probably attributable to the amount, rather than the type, of biotransformation products present in the plasma.

Animals↗

PubMed1991

Role of DNA replication in carrier-ligand-specific resistance to platinum compounds in L1210 cells.

L1210 cell lines have been described that are sensitive to most platinum compounds (L1210/0), resistant to ethylenediamine (en)-Pt but sensitive to diaminocyclohexane (dach)-Pt (L1210/DDP), and resistant to dach-Pt but sensitive to en-Pt (L1210/DACH). We have examined the effect of the dach and en carrier ligands on the ability of Pt-DNA adducts to inhibit DNA replication. Alkaline sucrose gradient sedimentation was used to determine the influence of both carrier ligands on the inhibition of replicon initiation and DNA chain elongation. Initiation of replicons was inhibited by Pt-DNA adducts to a greater extent than chain elongation in all three cell lines. Inhibition of replicon initiation was affected by the nature of the platinum carrier ligands only in the L1210/DACH cells in which 7.8-fold more dach-Pt adducts than en-Pt adducts were required to reach 63% inhibition. However, a strong carrier ligand effect was observed on the inhibition of DNA chain elongation in both the L1210/DDP and L1210/DACH cell lines. The L1210/DDP cell line required 4-fold more en-Pt adducts than dach-Pt adducts to inhibit DNA chain elongation by 63%. In the L1210/DACH cell line, 2.7-fold more dach-Pt adducts than en-Pt adducts were required for 63% inhibition. The L1210/0 cell line demonstrated no carrier ligand specificity for inhibition of chain elongation. Significant replicative bypass of Pt-DNA adducts was observed even in the L1210/0 cell line in that greater than 50 Pt-DNA adducts per 100 kb were required for 63% inhibition. The same level of inhibition was reached with 1.25 adducts of benzo[a]pyrene diolepoxide I per 100 kb. These data suggest that L1210 cells are capable of substantial replicative bypass of Pt-DNA adducts. Furthermore, the bypass of Pt-DNA adducts is increased in resistant L1210 cells and is markedly dependent on the nature of the platinum carrier ligand.

Animals↗

PubMed1990

Role of carrier ligand in platinum resistance in L1210 cells.

We have examined the effect of carrier ligands on platinum accumulation, incorporation of platinum into DNA, cytotoxicity of Pt-DNA adducts, and repair of Pt-DNA adducts in three L1210 cell lines: L1210/0, which is sensitive to most types of platinum compounds; L1210/DDP, which is resistant to platinum compounds with the ethylenediamine (en) carrier ligand but sensitive to those with the diaminocyclohexane (dach) ligand; and L1210/DACH, which is resistant to dach-Pt compounds but sensitive to en-Pt compounds. There was a selective decrease in accumulation of dach-Pt in the L1210/DACH line and of en-Pt in the L1210/DDP line. Intracellular dach-Pt was incorporated into DNA to a lesser extent than en-Pt in both resistant cell lines. Cytotoxicity of en-Pt adducts was less than that of dach-Pt adducts in the L1210/DDP line, while the reverse was true in the L1210/DACH line. Increased repair was seen in both resistant cell lines; a carrier ligand effect was seen only in the L1210/DDP line, which showed a greater initial rate of repair for en-Pt than dach-Pt adducts. These data suggest that carrier ligand effects seen in resistant cell lines may be due, in part, to differences in accumulation of platinum, repair of Pt-DNA adducts, and tolerance of Pt-DNA adducts.

Animals↗

PubMed1990

In vitro biotransformations of tetrachloro(d,l-trans)-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) in rat plasma.

The in vitro biotransformation of tetrachloro(d,l-trans)-1,2,-diaminocyclohexaneplantinum(IV) (tetraplatin) in the plasma of Fischer 344 rats were studied by the two-column high-performance liquid chromatography technique described previously (Mauldin et al., Cancer Res., 48: 5136-5144, 1988). The reduction of tetraplatin to dichloro(d,l-trans)-1,2-diaminocyclohexaneplatinum(II) [PtCl2(dach)] was extremely rapid. From experiments with diluted plasma, it was possible to estimate a t1/2 for tetraplatin of approximately 3 s at 37 degrees C in undiluted plasma. By titrating with N-ethylmaleimide, it was possible to show that sulfhydryl groups were responsible for 70-80% of the total reducing potential of plasma. The rapid reduction of tetraplatin to PtCl2(dach) was followed by slower substitution reactions involving the chloro ligands of PtCl2(dach). The t1/2 for PtCl2(dach) in plasma at 37 degrees C was 1.5 h. The monoaquamonochloro complex was an important biotransformation product at early times, reaching 10 to 12% of the total platinum present from 15 min to 2 h, when it was gradually replaced with more stable biotransformation products. Three major stable biotransformation products accumulated in the plasma. One of these biotransformation products was identified as the Pt(methionine)(dach) complex. The other two were tentatively identified as the Pt(cysteine)(dach) or Pt(ornithine)(dach) complex and the Pt(urea)(dach) or Pt(citrato)(dach) complex on the basis of coelution in two different high-performance liquid chromatography separation systems. These biotransformation products could play a role in tetraplatin effectiveness and/or toxicity.

Amino Acids↗

PubMed1990

Effect of the diaminocyclohexane carrier ligand on platinum adduct formation, repair, and lethality.

Platinum compounds with the diaminocyclohexane (dach) carrier ligand are of particular interest because cell lines that have developed resistance to platinum compounds in general often retain sensitivity to dach-platinum compounds, suggesting that the dach carrier ligand affects the formation, repair, or lethality of platinum-DNA adducts. The effect of the dach ligand on platinum adduct formation was assessed by using the (HaeIII-HindIII)146 fragment of pBR322 treated to give equal amounts of dach- or ethylene-diamine-platinum adducts. The sites of adduct formation were mapped by digestion with Escherichia coli ABC excinuclease. There were no significant effects of the dach carrier ligand on the types or sites of platinum adduct formation. The effect of the dach ligand on platinum adduct repair was determined by using synthetic oligomers designed to have single, specific platinum adducts (G monoadduct; GG, AG, or GNG diadduct) with either the dach or ethylenediamine (en) carrier ligand. These adducts differed significantly in their ability to serve as substrates for ABC excinuclease with GNG greater than or equal to G greater than AG greater than GG. The dach carrier ligand had little effect on the recognition of AG and GG adducts by ABC excinuclease, but significantly improved the ability of ABC excinuclease to excise G monoadducts and GNG diadducts. These data suggest that if the carrier ligand has any effect on the repair of platinum adducts, it is more likely to exert that effect on the repair of platinum monoadducts or GNG diadducts rather than on the more abundant AG or GG diadducts. [14C]Thiourea incorporation was used to quantitate the rate of monoadduct to diadduct conversion.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1990

Inhibition of DNA synthesis in P388 lymphocytic leukemia cells of BDF1 mice by cis-diamminedichloroplatinum(II) and its derivatives.

cis-Diamminedichloroplatinum(II) (cisplatin; cDDP) derivatives were found to afford T/C% values greater than 200 against the growth of P388 lymphocytic leukemia cells in vivo. The parent compound, cDDP, preferentially inhibited DNA synthesis. The RNA synthesis was elevated, whereas protein synthesis was unaffected after two or three daily ip doses. Radiolabeled drug studies demonstrated cellular uptake and binding of cDDP derivatives to the DNA molecule. cis-Diamminedichloroplatinum(II) (cDDP) treatment resulted in DNA strand scission after a single dose, but caused cross-linking of DNA strands after two or three ip doses. There was an accumulation of deoxynucleoside triphosphates [d(NTP)s] on day 2 and 3, indicating that incorporation of nucleotides into the DNA strand had been blocked. Thymidine kinase, thymidine monophosphate kinase, carbamoyl phosphate synthetase, and aspartate transcarbamoylase activities were inhibited in vivo after three doses of cDDP at 1.5 mg/kg/day. However, only the inhibition of a cytoplasmic preparation of DNA polymerase alpha by cDDP appeared to be directly related to the inhibition of DNA synthesis and the accumulation of d(NTP) pool levels. Thus, the primary target for cDDP appears to be DNA itself, although direct inhibition of DNA polymerase alpha may play a minor role in the inhibition of DNA replication by cDDP.

Animals↗

PubMed1990

Reduction of tetrachloro(dl-trans)1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) toxicity by the administration of diethyldithiocarbamate (DDTC), S-2(3-aminopropylamino)ethylphosphorothioic acid (WR-2721), or sodium selenite in the Fischer 344 rat.

Diethyldithiocarbamate (DDTC), S-2(3-aminopropylamino)ethylphosphorothioic acid (WR-2721), and sodium selenite have all been shown to effectively reduce cisplatin toxicity. As a result, we have investigated the efficacy of these compounds to reduce the toxicity associated with tetrachloro(dl-trans)1,2-diaminocyclohexaneplatinum(IV) (tetraplatin), a second-generation platinum compound recently approved for phase I/II clinical trials. The dose-limiting toxicities associated with tetraplatin (16.5 mg/kg) in the Fischer 344 male rat were nephrotoxicity, myelosuppression, and gastrointestinal toxicity. The nephrotoxicity in Fischer 344 rats was effectively reduced by treatment with either DDTC (750 mg/kg ip) 0.5 hr after tetraplatin or WR-2721 (200 mg/kg ip) 0.5 hr before tetraplatin as determined by blood urea nitrogen and creatinine values. Diarrhea was evident in 95% of the rats treated with tetraplatin alone while it was not evident in any of the DDTC- or WR-2721-protected rats. Only DDTC was moderately effective in preventing tetraplatin-induced decreases in platelet and lymphocyte counts. Histopathology confirmed DDTC protection of renal, intestinal, and lymphoid tissues and WR-2721 protection of renal and intestinal tissues. Sodium selenite was ineffective in reducing tetraplatin-induced damage when administered 4 hr before tetraplatin at doses of 0.5, 1.0, or 2.0 mg/kg. The results suggest that DDTC may allow for increased dosages of tetraplatin by ameliorating the toxic side effects of the drug. WR-2721 may also have some usefulness in tetraplatin therapy, but it does not reduce as wide a variety of toxic side effects as DDTC.

Amifostine↗

PubMed1990

Renal, hepatic, cardiac and thymic acute toxicity afforded by bis(helenalinyl)malonate in BDF1 mice.

Bis(helenalinyl)malonate [BHM], a pharmacologically active sesquiterpene lactone potentially useful as an antineoplastic agent, proved to be less toxic than its parent compound, helenalin. Its LD50 in BDF1 mice, i.p. was more than twice that of helenalin. Its lower toxicity allowed a higher therapeutic dose (15 mg/kg/day vs. 8 mg/kg/day for helenalin) which, in turn, afforded a greater T/C% (261 vs. 161). When BHM was employed at its therapeutic dose of 15 mg/kg/day, no marked toxicity was evident after three daily doses. However, continuation of treatment at this level led to both kidney and liver toxicity as measured by clinical chemistry parameters. Histological lesions in the thymus and kidney were demonstrated within 48 h at 25 mg/kg as a single dose. Apparently the toxicity was delayed with BHM but accumulated over time. Transient cardiotoxicity occurred with the drug and the agent was suspected of causing intestinal blockage.

Animals↗

PubMed1990

Inhibition of nucleic acid synthesis in P388 lymphocytic leukemia cells in culture by cis-platinum derivatives.

Cis-diaminedichloroplatinum(II) [cDDP] and three related derivatives Pt(mal)(NH3)2, PtCl2(dach) and Pt(mal) (dach) have been observed to possess cytotoxicity against the growth of P388 lymphocytic leukemia cells. DNA synthesis in P388 cells was inhibited by the agents in a manner which was consistent with their ED50 values for cytotoxicity. When P388 cells were treated with these platinum complexes in vitro at doses which caused more than 80% inhibition of DNA synthesis, no significant inhibition was observed for thymidine, kinase, thymidine monophosphate kinase, carbamoyl phosphate synthetase, or aspartate transcarbamoylase activities. Thus, there was no evidence that these agents inhibited de novo purine, pyrmidine, or deoxynucleotide synthesis. All of the agents did inhibit the nuclear DNA polymerase activity, but the extent of inhibition was 20% or less at doses which caused greater than 70% inhibition of DNA synthesis. Thus, the inhibition of DNA synthesis appeared to be due to cisplatinum(II) drug binding to the DNA bases. This was estimated to be 1 atom of platinum per 1500-3000 DNA base pairs which is consistent with other studies. The platinum complexes with chloro leaving ligands caused considerable DNA strand scission by 24 h at 10 times the ED50 dose, most likely a measure of impending cell death. In contrast, the platinum complexes with malonato leaving ligands did not cause significant strand scission by 24 h at similar doses. They also exhibited a significant delay in the inhibition of DNA synthesis. These data were interpreted as resulting from slower monoadduct to diadduct conversion, but it is not possible to eliminate the possibility of a different mode of interaction with DNA or a different mechanism of cytotoxicity for the malonato compounds.

Animals↗

PubMed1989

In vitro inhibition of mouse hepatic mixed-function oxidase enzymes by helenalin and alantolactone.

The sesquiterpene lactones (STL) helenalin and alantolactone were effective in vitro inhibitors of the mouse hepatic microsomal mixed-function oxidase (MFO) enzymes, aminopyrine demethylase (APD), aniline hydroxylase (ANH) and 7-ethoxyresorufin deethylase (ERD). Helenalin and alantolactone concentrations of 0.5 mM produced a 50-60% inhibition of APD and ERD, and a 20-30% inhibition of ANH. An increase in substrate (aminopyrine) concentration from 0.5 to 25 mM decreased STL inhibition of APD by 12-32%. APD was also inhibited at low aminopyrine concentrations (0.5 mM) by the helenalin derivative 2,3,11,13-tetrahydrohelenalin (tetrahydrohelenalin). The STL produced type I binding spectra with oxidized microsomes; Ks values for helenalin and alantolactone were 161 and 9 microM respectively. These results suggest that STL inhibition of the MFO system results, in part, from STL binding to the substrate-binding site of cytochrome P-450. It has been reported that the irreversible alkylation of protein cysteinyl residues is responsible for STL inhibition of several different enzymes, and second-order rate constants for the reaction of helenalin and alantolactone with glutathione were 25.1 and 1.80 mM-1.hr-1 respectively. Tetrahydrohelenalin did not react with glutathione. However, the subsequent addition of 3.0 mM thiols, i.e. L-cysteine, N-acetylcysteine or glutathione, to STL-treated (0.5 mM) microsomes reversed helenalin and alantolactone inhibition of APD and ERD by 50-80%. The ability of thiols to reverse STL inhibition of APD was decreased 20-43% by the coincubation of STL and microsomes with an NADPH-generating system. In addition, established effects of sulfhydryl-reactive compounds on the MFO system, i.e. inhibition of NADPH-cytochrome c reductase and conversion of cytochrome P-450 to cytochrome P-420, were not observed after addition of helenalin (1.0 mM) or alantolactone (0.5 mM) to mouse hepatic microsomes. These results suggest that STL inhibition of MFO enzymes may not be dependent upon the reactivity of the STL towards sulfhydryl groups. Instead, we suggest that STL binding to the substrate-binding site of cytochrome P-450 and subsequent metabolism of the STL may contribute to inhibition of the MFO system.

Aminopyrine N-Demethylase↗

PubMed1989

Rapid reduction of tetrachloro(D,L-trans)1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) in RPMI 1640 tissue culture medium.

Tetrachloro(D,L-trans)1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) is a new platinum analogue which is less nephrotoxic than cisplatin and is effective in some cell lines which have become resistant to cisplatin. Since platinum(IV) compounds are thought to require reduction to their platinum(II) analogues for activity, the biotransformations of tetraplatin and its platinum(II) analogue, dichloro(D,L-trans)1,2-diaminocyclohexaneplatinum(II) [PtCl2(trans-dach)], were studied. For L1210 cells cultured in RPMI 1640 medium, the time course for inhibition of DNA synthesis and cytotoxicity was virtually identical for both drugs. The time course for binding to fetal calf serum in the tissue culture medium was also the same for both drugs. In the complete tissue culture medium, tetraplatin was reduced to PtCl2(trans-dach) with a half-life of 5 to 15 min, depending on the level of protein sulfhydryl in the medium. The major reducing agent in the medium was the protein sulfhydryl, with glutathione and glucose making minor contributions. No other component of the medium reacted with tetraplatin. The rapid reduction of tetraplatin to PtCl2(trans-dach) was followed by much slower substitution reactions involving the chloro ligands of PtCl2(trans-dach). The major transformation products which accumulated in RPMI 1640 medium were identical for both drugs. These data suggest that tetraplatin should be considered a prodrug which is very rapidly converted to PtCl2(trans-dach) with subsequent biotransformations as expected for the platinum(II) analogue. These data also indicate that in tissue culture most of the tetraplatin is reduced extracellularly.

Antineoplastic Agents↗

PubMed1989

Disposition of cisplatin derivatives -3H-cis-1,2-diaminocyclohexanedichloroplatinum (II) and 3H-cis-1,2-diaminocyclohexanemalonatoplatinum (II) in BDF1 mice.

The disposition of 3H-cis-1,2-diaminocyclohexanedichloroplatinum (II) and 3H-cis-1,2-diaminocyclohexanemalonatoplatinum (II) was investigated in P388 tumor-bearing BDF1 mice. Fifteen minutes after IP administration of the drugs, the serum contained 12% of the 3H-chloride derivative and 20% of the malonate derivative. Both drugs distributed to all organs of the body but were not sequestered in any major internal organ. However, substantial amounts of the drugs were found in the carcass and skin. After 24 hr, approximately 43% of the radioactivity was excreted in the urine. Only 5 to 8% of the radioactivity was eliminated in the feces. The radioactivity half-life (t1/2 beta) for the 3H-chloride derivative was estimated from urinary excretion data to be 22.7 hr, and 30.0 hr for the 3H-malonate derivative.

Animals↗

PubMed1988

Intracellular biotransformation of platinum compounds with the 1,2-diaminocyclohexane carrier ligand in the L1210 cell line.

We have previously reported the development of a two-column high performance liquid chromatography system for separation of platinum(II) complexes with the 1,2-diaminocyclohexane (DACH) carrier ligand (Mauldin et al., Anal. Biochem., 157: 129, 1986). Here we report the application of this technique to the study of the intracellular biotransformations of (DL)-trans-1,2-diaminocyclohexanemalonatoplatinum(II) [PtCl2(trans-DACH)] and (DL)-trans-1,2-diaminocyclohexanemalonatoplatinum(II) [Pt(mal)(trans-DACH)] in the L1210 cell line. The two-column high performance liquid chromatography system allowed separation and identification of both parent drugs and intracellular biotransformation products containing glutathione, methionine, cysteine, arginine, lysine, aspartate or glutamate, and serine or threonine. With the exception of the platinum-glutathione complex, the relative abundance of each biotransformation product was independent of drug concentration. The relative abundance of the platinum-glutathione biotransformation product increased with increasing platinum concentration, suggesting that platinum drugs cause an increase in intracellular glutathione levels in a dose-dependent manner. This hypothesis was verified by direct measurement of intracellular glutathione levels. In continuous uptake experiments, the intracellular levels of the parent compounds peaked between 2 and 5 h and declined to negligible levels by 24 h. In pulse-chase experiments, the chemical t1/2 for PtCl2(trans-DACH) and Pt(mal)(trans-DACH) inside the cell at 37 degrees C was determined to be 12-15 and 21-28 min, respectively. This is far shorter than previously determined rates for the displacement of either ligand in vitro. The platinum-amino acid complexes accumulated gradually throughout the 24-h incubation. The free trans-DACH carrier ligand also accumulated to a level approaching 20% of filterable counts during the 24-h incubation, probably due to trans-labilization of the carrier ligand by sulfur-containing nucleophiles. A combination of reverse phase high performance liquid chromatography and a DNA binding assay was used to identify and quantitate the reactive biotransformation products. As expected from previous studies (Mauldin et al., Cancer Res., 46: 2876, 1986), the PtCl2(trans-DACH)-treated cells had approximately 3 times more reactive platinum biotransformation product at early times, but the levels of reactive biotransformation product fell much more rapidly than in Pt(mal)(trans-DACH)-treated cells. In the PtCl2(trans-DACH)-treated cells, the major reactive biotransformation product was the aquachloro species at all time points tested. In Pt(mal)(trans-DACH)-treated

Animals↗

PubMed1988

Displacement of the bidentate malonate ligand from (d,l-trans-1,2-diaminocyclohexane)malonatoplatinum(II) by physiologically important compounds in vitro.

Previous studies of platinum(II) compounds with bidentate leaving ligands have emphasized the contrast between the stability of the bidentate leaving ligand in vitro (T1/2 greater than 11 days in water) and the apparent reactivity of these bidentate platinum compounds in vivo. However, none of these studies actually measured the stability of these compounds in tissue culture medium (or in any other reaction mixture resembling in vivo conditions). The experiments described in this paper were designed to measure the stability and fate of (d,l-trans-1,2-diaminocyclohexane)malonatoplatinum(II) [Pt(mal)(trans-dach)] in RPMI-1640 tissue culture medium. The T1/2 for displacement of the malonate ligand in this medium was 9.5 hr at 37 degrees. Of the inorganic anions present in the medium, chloride accounted for the greatest displacement of the malonate ligand. However, at the concentrations with which it is found in tissue culture medium and in blood, bicarbonate was nearly as effective as chloride at displacing the malonate ligand. This observation is of particular significance because the bicarbonatoplatinum complex is unstable and the bicarbonate displacement reaction appears to represent a major non-enzymatic pathway for the formation of the biologically active aquated platinum complexes. At the concentrations with which they occur inside the cell, phosphates may play a similar role. Of the amino acids present in the medium, glutathione and the sulfur-containing amino acids were 50- to 400-fold more effective at displacing the malonate ligand than the other amino acids in RPMI-1640 medium. In the case of methionine, the reaction with Pt(mal)(trans-dach) was shown to be a direct displacement (SN2) reaction at physiological methionine concentrations. When Pt(mal)(trans-dach) was incubated at 37 degrees for 24 hr in RPMI-1640 medium, the major transformation products formed were (d,l-trans-1,2-diaminocyclohexane)methionineplatinum(II) (38%), other amino acid-platinum complexes (19%), and (d,l,-trans-1,2-diaminocyclohexane)dichloroplatinum(II) (14%). Eleven percent of the Pt(mal)(trans-dach) remained intact. Mass spectrometry and 1H-NMR indicated that the (d,l-trans-1,2-diaminocyclohexane)methionineplatinum(II) complexes that formed in RPMI-1640 medium consisted of approximately 60% of the bidentate mono-methionine complex coordinated to platinum at the sulfur and alpha-amino positions and 40% of the bis-methionine complex, presumably coordinated at the sulfurs.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗

PubMed1988

Inhibition of nucleic acid synthesis in P-388 lymphocytic leukemia tumor cells by helenalin and bis(helenalinyl)malonate in vivo.

Although the parent sesquiterpene lactone, helenalin, and its derivative, bis(helenalinyl)malonate, are structurally related chemically, they demonstrate differences in their antineoplastic activity, with bis(helenalinyl)malonate being much more active against P-388 lymphocytic leukemia cell growth (T/C% = 261) compared with helenalin (T/C% = 162). Previous studies have shown that both agents strongly inhibit protein synthesis in vivo by greater than 70% after 3 d of administration and in vitro by 50% at a 100 microM concentration of drug. This inhibition of protein synthesis of P-388 cells may be partially responsible for the cytotoxicity of the drug. These agents also inhibit nucleic acid synthesis in vivo, with DNA synthesis being suppressed by greater than 90% after 2 d of administration of drugs at the therapeutic dose. Of the sulfhydryl-bearing enzymes involved in nucleic acid synthesis that were assayed, only the activities of inosine-5'-monophosphate (IMP) dehydrogenase and the ribonucleotide reductase complex were inhibited by greater than 50% by these sulfhydryl-reactive drugs, which would account for the observed inhibition of nucleic acid synthesis in the P-388 cells. The inhibition of the activities of these enzymes lowered the deoxyribonucleotide levels in P-388 cells, which would explain the overall suppression of DNA synthesis by the sesquiterpene lactones.

Animals↗

PubMed1988

Acute toxicity of helenalin in BDF1 mice.

The acute toxicity of helenalin, a sesquiterpene lactone isolated from Helenium microcephalum, was examined in male BDF1 mice. The 14-day LD50 for a single ip dose of helenalin in male mice was 43 mg/kg. A single ip injection of 25 mg helenalin/kg increased serum alanine aminotransferase (ALT), lactate dehydrogenase (LDH), urea nitrogen (BUN), and sorbitol dehydrogenase within 6 hr of treatment. Multiple helenalin exposures, ip injection of 25 mg helenalin/kg for 3 days, increased differential polymorphonuclear leukocyte counts and decreased lymphocyte counts. Serum ALT, BUN, and cholesterol levels were also increased by multiple helenalin exposures at 25 mg helenalin/kg/day. Helenalin significantly reduced liver, thymus, and spleen relative weights and histologic evaluation revealed substantial effects of multiple helenalin exposures on lymphocytes of the thymus, spleen, and mesenteric lymph nodes. No helenalin-induced histologic changes were observed in the liver or kidney. Multiple helenalin exposures (25 mg/kg/day) significantly inhibited hepatic microsomal enzyme activities (aminopyrine demethylase and aniline hydroxylase) and decreased microsomal cytochromes P-450 and b5 contents. Three concurrent days of diethyl maleate (DEM) pretreatment (3.7 mmol DEM/kg, 0.5 hr before helenalin treatment) significantly increased the toxicity of helenalin exposure. The present studies indicate that the hepatic microsomal drug metabolizing system and lymphoid organs are particularly vulnerable to the effects of helenalin. In addition, helenalin toxicity is increased by DEM pretreatments which have been shown to decrease glutathione concentrations.

Animals↗

PubMed1987

Inhibition of inosine monophosphate dehydrogenase by sesquiterpene lactones.

Inosine monophosphate (IMP) dehydrogenase had previously been determined to be a likely target enzyme for the sesquiterpene lactones, a class of potential anti-neoplastic drugs. IMP dehydrogenase was purified approx. 770-fold from the P-388 lymphocytic leukemia tumor cell line. The Km values for the substrates, IMP and NAD, were determined to be 12 microM and 25 microM, respectively. Xanthine monophosphate (XMP) was shown to be a competitive inhibitor with a Ki of 67 microM. Mycophenolic acid gave mixed-type inhibition with a Ki of 8 nM for the noncompetitive component and a Ki of 2 nM for the competitive component. Dissociation constants (Kd) and rate constants for inhibition of IMP dehydrogenase by nine different sesquiterpene lactones were determined. The highest Kd was seen with 2,3-dihydrohelenalin while the lowest Kd was observed with bis-helenalinyl malonate. Binding of the drugs by IMP dehydrogenase increased as the size of the drug increased. Also, changes in structure at position 6 had a relatively large effect on the Kd. There was no correlation with hydrophobicity, as determined by octanol/water partition. The first-order rate constants for the reaction of the sesquiterpene lactones with IMP dehydrogenase (k1) and the second-order rate constants for the reaction of the sesquiterpene lactones with glutathione (k2) were also determined. The rate constants for most of the sesquiterpene lactones with the alpha-methylene-gamma-lactone moiety were similar and were approximately twice as great as the rate constants for those sesquiterpene lactones with only the alpha, beta-unsaturated cyclopentenone ring. Microlenin had approximately 5-times the reactivity of the other sesquiterpene lactones towards IMP dehydrogenase, but had approximately the same reactivity towards glutathione, suggesting that it was bound to the enzyme in a way which facilitated its reaction with one or more essential sulfhydryls. The same procedure was used for a series of N-substituted maleimide compounds with the N-substituent ranging in size from a methyl group to a benzyl group. The binding of the maleimide compounds was generally tighter than for the sesquiterpene lactones and there was an increase in binding with size.

Animals↗