Combination chemotherapy of L1210 leukemia with platinum compounds and cyclophosphamide plus other selected antineoplastic agents.

Six antitumor platinum compounds were used in combination with cyclophosphamide plut 1 of 7 other antitumor drugs for treatment of L1210 leukemia in B6D2F [C57BL/6 X DMA/2) F] mice. Data obtained from each three-agent regimen were compared with those obtained after administration of each compound alone and each appropriate two-agent combination. No cure (greater than 60-day survival) was obtained with any compound used alone. Combination of cyclophosphamide with a platinum compound (Pt+CY) yielded a collective cure rate of 193/420, and the addition of a third cure rate to 290/420 (P less than 0.001). Certain regimens produced 100% cure rates. The most effective drugs when used in combination with PT+CY were cytosine arabinoside, 5-fluorouracil, hydroxyurea, and Yoshi-864. Adriamycin, methotrexate, and vincristine were less effective at the doses used. Toxicity, as evidenced by maximum weight loss, was slightly greater with the three-agent combinations than with the Pt+CY regimens.

Antileukemic properties of organoplatinum complexes.

The antitumor activity of 46 cis-amineplatinum congeners was evaluated against L1210 leukemia in (C57BL/L X DBA/2)F1 mice. Several compounds in this series significantly prolonged the life-spans of mice with the leukemia. During the selection of the compound that yielded optimal activity [dichloro(1,2-diaminocyclohexane)platinum], the chlorides were substituted with various organic and inorganic anions. The aqueous solubility was greatly increased with retention of significant antileukemic activity. Most of the active compounds were synergistic with cyclophosphamide, and cure rates up to 80% were obtained with certain combinations.

Mapping the domains of the interaction of the vitamin D receptor and steroid receptor coactivator-1.

The vitamin D receptor (VDR) binds to the vitamin D response element (VDRE) and mediates the effects of the biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], on gene expression. The VDR binds to the VDRE as a heterodimeric complex with retinoid X receptor. In the present study, we have used a yeast two-hybrid system to clone complementary DNA that codes for VDR-interacting protein(s). We found that the human steroid receptor coactivator-1 (SRC-1) interacts with the VDR in a ligand-dependent manner, as demonstrated by beta-galactosidase production. The interaction of the VDR and the SRC-1 takes place at physiological concentrations of 1,25(OH)2D3. A 48.2-fold stimulation of beta-galactosidase activity was observed in the presence of 10(-10) M 1,25-(OH)2D3. In addition, a direct interaction between the ligand-activated glutathione-S-transferase-VDR and 35S-labeled SRC-1 was observed in vitro. Deletion-mutation analysis of the VDR established that the ligand-dependent activation domain (AF-2) of the VDR is required for the interaction with SRC-1. One deletion mutant, pGVDR-(1-418), bound the ligand but failed to interact with the SRC-1, whereas another deletion mutant, pGVDR-(1-423), bound the ligand and interacted with the SRC-1. We demonstrated that all the deletion mutants were expressed as analyzed by a Gal4 DNA-binding domain antibody. Deletion mutation analysis of the SRC-1 demonstrated that 27 amino acids (DPCNTNPTPMTKATPEEIKLEAQSQFT) of the SRC-1 are essential for interaction with the AF-2 motif of the VDR.

High-Quality Draft Genome Sequence of Francisella tularensis subsp. holarctica Strain OR96-0246.

The bacterial pathogen Francisella tularensis was recently renewed as a tier-one select agent. F. tularensis subsp. tularensis (type A) and holarctica (type B) are of clinical relevance. Here, we report the complete genome of a virulent F. tularensis type B strain and describe its usefulness in comparative genomics.

Relatedness of strains of methicillin-resistant coagulase-negative Staphylococcus colonizing hospital personnel and producing bacteremias in a neonatal intensive care unit.

The emergence of methicillin-resistant coagulase-negative Staphylococcus as a major bacterial pathogen in neonatal intensive care units has stimulated interest in the epidemiology of spread of the organism. During a 12-month "epidemic" of bacteremias with methicillin-resistant coagulase-negative Staphylococcus we compared the characteristics of bacteremic and personnel nasally-carried strains by traditional and biomolecular methods. Sixty-two percent of neonatal intensive care unit nurses were colonized with methicillin-resistant coagulase-negative Staphylococcus with similar speciation to bacteremic strains. Inspection of plasmid profiles revealed a moderate degree of similarity between bacteremic and colonizing strains although genomic DNA restriction patterns showed diversity. Ribotype patterns were highly conserved (90%) in personnel strains. A 2.6-kilobase plasmid DNA probe hybridized to similarly sized plasmids and larger plasmids in one-half of the strains. We hypothesize that related methicillin-resistant strains may be transferred among personnel and neonates in the neonatal intensive care unit. Epidemiologic studies of coagulase-negative staphylococci should consider multiple molecular techniques to relate strains.

Increased frequency of large R-plasmids in Klebsiella pneumoniae colonizing patients with spinal cord injury.

From 1978 to 1988 strains of gentamicin-susceptible (Gms) and gentamicin-resistant (Gmr) Klebsiella pneumoniae were saved from annual surveillance cultures of the perineal region of patients with spinal cord injury (SCI). Of 38 strains selected for further study (24 Gms and 14 Gmr), there were 23 different serotypes (two nontypable). Fourteen Gms as well as 14 Gmr strains displayed no common plasmid patterns, but all contained a large plasmid of 168-208 kb. Among the 14 Gmr strains, nine had large conjugative plasmids of approximately the same size (166-193 kb), which conferred to a susceptible Escherichia coli host an identical resistance pattern: ampicillin, chloramphenicol, gentamicin, piperacillin, trimethoprim-sulfamethoxazole, tetracycline, and tobramycin. Of the nine transconjugants, eight contained a single plasmid. One transconjugant contained a 168- and 80-kb plasmid. Restriction endonuclease digestion patterns of the R-plasmids revealed minimal similarity. We conclude that, during a 10-year period, different large R-plasmids have spread among multiple serotypes of K. pneumoniae in spinal cord injury (SCI) patients in one rehabilitation hospital. We hypothesize that other genes located on large, R-, and non-R-plasmids may confer an additional advantage for colonization by K. pneumoniae in SCI patients.

Effect of chelate treatments on kidney, bone and brain lead levels of lead-intoxicated mice.

The effects of chelating agent treatment with meso-2,3-dimercaptosuccinic acid (DMSA), Na2CaEDTA, Na2ZnEDTA, and Na3ZnDTPA on the organ lead levels of lead-loaded mice have been determined. At 1 mmol/kg/day i.p., all caused reductions in the lead levels of the kidney after four injections, but only Na2CaEDTA produced a significant reduction in brain lead. All chelating agents caused significant reductions in kidney and brain lead levels when administered at a daily dose of 1 mmol/kg/day for eight days, but only DMSA reduced the bone lead level. In animals given 50 mg Pb/kg or 100 mg Pb/kg, the administration of Na2CaEDTA or DMSA at 1 mmol/kg/day x 8 produced significant reductions in kidney, bone and brain lead levels, but DMSA produced greater reductions of bone lead in both groups and of kidney lead in the group given 100 mg Pb/kg. An examination of published data describing the effect of chelating agent treatment on brain lead levels indicates that DMSA produces a reduction in brain lead levels under all conditions examined to date.

Mobilization of lead in mice by administration of monoalkyl esters of meso-2,3-dimercaptosuccinic acid.

The following six monoalkyl esters of meso-2,3-dimercaptosuccinic acid (DMSA) were synthesized and evaluated for relative activities in mobilizing lead from kidneys and brains of lead-bearing mice: n-propyl (Mn-PDMS), i-propyl (Mi-PDMS), n-butyl (Mn-BDMS), i-butyl (Mi-BDMS), n-amyl (Mn-ADMS) and i-amyl meso-2,3-dimercaptosuccinate (Mi-ADMS). DMSA was used as a positive control. When each was administered intraperitoneally (i.p.) as a single dose of 2.0 mmol/kg, DMSA lowered the kidney lead concentration 52%, while the monoesters effected reductions of 54-75%. Mn-ADMS was toxic at this dose. DMSA lowered the brain lead level 20% when given as a single dose, while the monoesters conferred reductions of 64-87%. When given as 5 daily i.p. injections at 0.5 mmol/kg, DMSA reduced the kidney lead concentration 45%, while the monoesters caused reductions of 56-73%. DMSA lowered the brain lead concentration 35% on the 5-day treatment regimen, while the monoesters evoked reductions of 59-75%. Mi-ADMS was equally effective when given orally or i.p. The i.p. LD50 value of this analog in mice is 3.0 mmol/kg, a value which lies between the reported LD50 doses of DMSA (16.0 mmol/kg) and dimercaprol (1.1 mmol/kg). It is suggested that the ability of these monoesters to cross cell membranes may account for their superiority to DMSA in mobilizing brain lead in this animal model.

Effects of sodium N-methyl-N-dithiocarboxyglucamine on cadmium distribution and excretion.

Sodium N-methyl-N-dithiocarboxyglucamine (MDCG) was evaluated for its efficacy in mobilizing and promoting excretion of metallothionein-bound 109Cd using mice which had received 0.03 mg of CdCl2 . 2 . 5H2O along with 1.0 muCi of 109CdCl2 three weeks earlier. The MDCG-induced change in the fecal excretion of Cd ranged from a 15-fold increase over the control rate at the lowest dose level used (2.2 mmol/kg; 684 mg/kg) up to a 72-fold increase at the highest dose (8.8 mmol/kg; 2736 mg/kg) following three daily injections. The latter treatment regimen resulted in a fecal excretion of almost 30% of the administered Cd over a 3-day period of observation. Urinary Cd excretion was insignificant in both the control and treated groups. The whole body burden of Cd was reduced by over 50% following seven thrice-weekly i.p. injections of MDCG at 8.8 mmol/kg. There was a 60-65% reduction in both the liver and kidney Cd levels following the same treatment regimen. Radioassay of ten other organs and tissues revealed only modest changes. Testicular Cd was decreased slightly at the highest dose level, and heart tissue from each treated group contained slightly more Cd than controls. Results indicated a rather marked specificity of MDCG in lowering the Cd content of two organs most susceptible to Cd-induced toxicity.

Selective removal of cadmium from aged hepatic and renal deposits: N-substituted talooctamine dithiocarbamates as cadmium mobilizing agents.

The preparation and examination of three dithiocarbamates derived from N-substituted D-gluco-L-talooctamine reveals that the 4-methoxybenzyl derivative (MeOBGD) is superior to any previously prepared dithiocarbamates as an agent for the mobilization of aged intracellular hepatic cadmium deposits from mice. All of these compounds are also quite effective in reducing whole body burdens of cadmium. The use of these compounds does not result in any increase in the cadmium content of the brain. The selection of these chelating agents for synthesis was suggested by an analysis of the log dose-response curves for the mobilization of renal cadmium by previously studied dithiocarbamates. This revealed that the slope of the percentage renal cadmium mobilized vs the log dosage curve is determined to a considerable extent by the sum of the Hansch pi parameters for the substituents, while the intercept is largely determined by the molecular weight of the compound. The implication of such a correlation is that the ability of a chelating agent to remove cadmium from its aged deposits is determined to some extent by its molecular weight, provided the polarity of the overall molecule is appropriate.

Dithiocarbamate treatment of chronic cadmium intoxication in mice.

The dithiocarbamate analogs, N-benzyl-N-dithiocarboxy-D-glucamine (BDCG) and N-cyclohexyl-N-(2-hydroxy-3-sulfonatopropyl)dithiocarbamate (CAPSO-DTC), were evaluated as cadmium (Cd) antagonists in mice which had received repetitive injections of Cd to effect accumulation of substantial levels of metallothionein-bound Cd in kidneys and livers. BDCG was highly effective in lowering whole body Cd stores and renal Cd concentrations. While the percent of renal Cd mobilized decreased with increasing Cd concentrations, the total amount of Cd mobilized increased. CAPSO-DTC was also effective in reducing whole body Cd levels, but appeared to have less affinity for renal Cd than did BDCG. Treatment of Cd-laden mice with BDCG provoked only a modest elevation of serum creatinine levels, suggesting that the complex of Cd with BDCG may be less nephrotoxic than the complex of Cd with EDTA or dimercaprol. The log of the percent reduction of renal Cd by BDCG was found to be a linear function of the pretreatment renal Cd concentration, and reductions of whole body Cd burdens correlated closely with reductions of liver and kidney Cd concentrations. It was suggested that a Cd complexing agent of the dithiocarbamate class may have ultimate application in a provocative methodology to estimate body or organ Cd stores based upon the amount of Cd excreted following a standard dose of the chelator.

Schedule dependency of a cadmium-complexing dithiocarbamate analog in mice.

A number of dosing regimens was assessed to determine the optimum schedule of administration of N-(4-methoxybenzyl)-N-dithiocarboxy-D-glucamine (MeOBDCG) in depleting whole-body, renal and hepatic levels of metallothionein-bound cadmium (Cd) in mice. A comparison of 4.0 mmol/kg given as a single injection versus 0.5 mmol/kg given as 8 hourly injections revealed the latter regimen to be superior in reducing renal Cd levels, but less effective than a bolus dose in lowering hepatic Cd concentrations. Administration of 1.33 mmol/kg for 3 consecutive days or 0.8 mmol/kg for 5 days effected a more extensive depletion of renal Cd concentrations than did a single injection of 4.0 mmol/kg. Three injections of 1.0 mmol/kg given at 4- to 7-day intervals were generally more effective in reducing renal Cd concentrations than were 3 consecutive daily injections in mice which had low or moderately high total Cd burdens. The lowest effective dose of MeOBDCG in lowering whole-body, liver and kidney Cd levels when given repetitively was about 0.2 mmol/kg. While schedule variations did not alter appreciably the whole-body Cd reductions at any given total dose of MeOBDCG, repetitive dosing schedules in which injections were given at intervals of several days rather than daily were typically more effective in reducing renal Cd levels. Based upon consideration of pharmacological response as influenced by body surface area, it was calculated that doses of MeOBDCG of the order of 2.0 g/d may be effective in reducing renal Cd levels in individuals with chronic renal dysfunction secondary to chronic Cd intoxication.

Structural influences on intracellular cadmium mobilization by dithiols.

An examination of 5 dithiols, N-(2,3-dimercaptopropyl)phthalamidic acid (DMPA), benzene-1,2-dithiol (BDT), toluene-3,4-dithiol (TDT), alpha, alpha'-dimercapto-o-xylene (DOX), and 4,5-dimethyl-alpha,alpha'-dimercapto-o-xylene (DDOX), reveals that these compounds are all inferior to previously reported compounds as agents for the in vivo mobilization of cadmium from its intracellular sites, although all possess sulfhydryl groups capable of reacting with cadmium. The results demonstrate the considerable importance of those parts of the molecule which do not participate directly in the formation of chelate rings in the determination of the ultimate behavior of such compounds in vivo.

N,N-disubstituted dithiocarbamates as cadmium antagonists: N-(4-methoxybenzyl)-N-dithiocarboxy-D-glucamine.

The newly synthesized dithiocarbamate analog, N-(4-methoxybenzyl)-N-dithiocarboxy-D-glucamine (MeOBDCG) reduced whole-body cadmium levels 66% in cadmium-laden mice when given as 3 injections at 1.0 mmol/kg. Renal and hepatic Cd concentrations were reduced 78 and 85%, respectively. After 6 injections, the whole-body cadmium burden was reduced 71%, while renal and hepatic levels were lowered 84% and 91%, respectively. Mobilized cadmium was excreted almost exclusively by the fecal route. There was no evident toxicity consequent to treatment as judged by mouse body weights and by gross appearance of organs upon dissection. On a molar dose basis, MeOBDCG was more effective than N-benzyl-N-dithiocarboxy-D-glucamine (BDCG) in removing cadmium from both renal and hepatic deposits. An in-vitro assessment of the interaction of MeOBDCG, BDCG and N-methyl-N-dithiocarboxy-D-glucamine with murine cadmium-metallothionein (Cd-MT) revealed a direct relationship between the extent of cadmium depletion from Cd-MT and the relative in-vivo efficacies of the 3 analogs.

Effects of diethyldithiocarbamate on organ distribution and excretion of cadmium.

Sodium diethyldithiocarbamate (DDTC) was evaluated for its efficacy in promoting organ mobilization and excretion of metallothionein-bound cadmium (Cd) using mice which received a single injection of CdCl2 X 2.5 H2O, 0.03 mg per mouse, along with 109CdCl2 three to six weeks earlier. After seven or 13 i.p. injections of DDTC, 500 mg per kg, over a two or four week interval, DDTC was highly effective in mobilizing Cd from kidney (Ki) and spleen (Sp), but less effective in removing it from liver (Li). Treatment with DDTC moderately enhanced Cd levels in lung (Lu), heart (He) and testes (Te), and increased brain (Br) levels to over 500 percent of control values. Relative accumulation of Cd in organs of control mice were in the order Li greater than Ki greater than Sp greater than Lu greater than He greater than Te greater than Br. The extreme values were Li = 57 percent and Br = 0.07 percent of the Cd administered. Even though a major portion of Cd mobilized was from the kidneys, excretion was apparently exclusively by the fecal route.

Comparative effects of diethyldithiocarbamate, dimercaptosuccinate, and diethylenetriaminepentaacetate on organ distribution and excretion of cadmium.

Diethyldithiocarbamate (DDTC), dimercaptosuccinate (DMSA), and diethylenetriaminepentaacetate (DTPA) were compared for their relative efficacies at equimolar doses in promoting mobilization of metallothionein-bound cadmium (Cd) from its sites of deposition several weeks after i.p. injection of a sublethal dose of Cd containing 109Cd. Routes and rates of excretion were also determined. The most rapid and extensive reduction of renal, hepatic, and splenic Cd was obtained with DDTC. Concurrently, however, DDTC caused moderate increases in lung, testicular, and heart Cd burdens, and increased the brain burden about ten-fold. Only renal and testicular levels of Cd were reduced by DTPA, and it was much less effective than DDTC in lowering the renal levels. No reduction of Cd levels in any organ was attained with DMSA treatment. Excretion of Cd following DDTC treatment was exclusively by the fecal route; DTPA promoted both urinary and fecal excretion, but the total amount excreted by both routes was considerably less than that observed following DDTC treatment. It was concluded that the effectiveness of a chelator in promoting mobilization and excretion of metallothionein-bound Cd cannot be predicted on the basis of its effectiveness in protecting mice against a lethal dose of Cd when given immediately after Cd and prior to induction of metallothionein synthesis. The possibility is discussed that the greater effectiveness of DDTC may be due to an interaction with nascent metallothionein rather than to chelation of Cd.

Mechanism of diethyldithiocarbamate, dihydroxyethyldithiocarbamate, and dicarboxymethyldithiocarbamate action of distribution and excretion of cadmium.

Diethyldithiocarbamate (DEDC), dihydroxyethyldithiocarbamate (DHDC), and dicarboxymethyldithiocarbamate (DCDC) were assessed for their relative efficacies in mobilizing metallothionein-bound cadmium (Cd) from selected organs and tissues of mice and in promoting Cd excretion. The DHDC was effective but less so than DEDC in mobilizing Cd from liver, kidney and spleen. However, while DEDC effected a redistribution of Cd which resulted in higher levels in lungs, testes, heart, and brain, DHDC after 13 injections reduced the levels of Cd in all four of these organs. The DCDC analog did not alter the Cd load of any organ. Bone Cd content was most effectively reduced by DEDC; DCDC was somewhat less active, and DCDC was without effect. Skin Cd burden was enhanced markedly after treatment with DEDC but was reduced by treatment with DHDC or DCDC. Treatment with DEDC increased the muscle Cd content about 250 percent over control values; treatment with DHDC reduced it significantly, while DCDC was without effect. Both DEDC and DHDC reduced the whole body Cd burden, but DCDC was ineffective. The initial rate of fecal excretion of Cd was much greater following DHDC treatment than after DEDC treatment, while DCDC did not promote excretion to any detectable extent. The pattern of mobilization, redistribution, and excretion of Cd following treatment with each chelator was related to the organic/aqueous partition coefficient of each dithiocarbamate-Cd complex.

Effects of combined treatment with diethyldithiocarbamate and diethylenetriaminepentaacetate on organ distribution and excretion of cadmium.

Diethyldithiocarbamate (DDTC) and diethylenetriaminepentaacetate (DTPA) were assessed to determine if combination treatment with these two chelators of different chemical classes would enhance mobilization and excretion of metallothionein-bound cadmium (Cd) from selected organs of mice which had earlier received 0.03 mg of CdCl2 . 2.5 H2O along with 1.0 microCi of 109Cd. In addition to measuring individual organ radioactivity after seven and after 13 injections of each compound individually as well as in combination, whole body Cd burden was measured, and the routes and rates of Cd excretion were determined. When used alone, DDTC was effective in mobilizing Cd from kidney, liver, intestine, and spleen. The DTPA when used alone was not consistently effective in reducing Cd burdens in any of the organs assessed. Coadministration of DDTC and DTPA promoted an enhancement of Cd mobilization from liver, kidney, spleen, and intestine over that which was observed with DDTC alone. When DTPA was administered with DDTC, it did not prevent accumulation of Cd in lung and brain which was observed upon treatment with DDTC alone. Combined treatment did produce a more marked depletion of total body 109Cd burden than did the administration of DDTC alone. A more rapid rate of both fecal and urinary excretion of Cd was observed when the chelators were coadministered. It was concluded that at least an additive or possibly supraadditive effect may be obtained by combining a dithiocarbamate chelator with one of the aminocarboxylate class in total body Cd decorporation.

Factors influencing cadmium mobilization by diethyldithiocarbamate: chelator dose, cadmium burden, and sex.

A study was made of the effects of diethyldithiocarbamate (DDTC) on organ distribution and excretion of cadmium (Cd) as influenced by DDTC dose, Cd burden, and sex of mice. One hundred to 250 mg per kg of DDTC did not promote mobilization of hepatic Cd, but it was effective at 500 mg per kg. All doses tested were significantly effective in mobilizing renal and splenic Cd, and the response was dose-dependent, as was the increase in brain levels of Cd. Cadmium mobilization by DDTC following Cd loads over a three-fold range appeared to be first order in nature; i.e., a given regimen of DDTC treatment promoted mobilization of a virtually constant percentage of the Cd administered, rather than a constant amount of Cd. The response to DDTC was markedly sex-dependent, being more effective in females than males. In organs of control Cd-loaded mice one sex-related difference was noted; spleens of females retained a considerably greater percentage of administered Cd than those of males.

Dithiocarbamates and cadmium metabolism: further correlations of cadmium chelate partition coefficients with pharmacologic activity.

N,N-bis(beta-Hydroxyethyl)dithiocarbamate (DHDC), N-methyl-N-hydroxyethyldithiocarbamate (MHDC), and N-ethyl-N-hydroxyethyldithiocarbamate (EHDC) were compared for their relative efficacies in promoting organ mobilization and excretion of metallothionein-bound cadmium (Cd). An attempt was made to correlate certain aspects of their pharmacodynamic properties with the octanol/aqueous partition coefficient of each Cd-dithiocarbamate complex. All compounds assessed were effective in mobilizing renal, intestinal, and testicular Cd. Those analogs with slightly greater lipid than aqueous solubility were more effective in this regard (DHDC less than MHDC less than EHDC). However, those analogs with higher lipid solubility (log10P greater than 0) promoted redistribution of Cd to liver. Only DHDC, with the lowest lipid solubility (log10P = -0.3 to -0.6), promoted Cd excretion. None of the congeners effected any redistribution of Cd to brain. Methods of preparation of the sodium salts of these analogs are described in detail.