Toxicity and Environmental Risk Assessment of Polycarbamate and Its Main Components to Marine Algae and Crustaceans.

Polycarbamate is commonly used as an antifoulant coating on fishing nets in Japan. Although its toxicity to freshwater organisms has been reported, its toxicity to marine organisms is currently unknown. We conducted algal growth inhibition and crustacean immobilization tests to assess the effects of polycarbamate on marine organisms. We also evaluated the acute toxicity of the main components of polycarbamate, namely, dimethyldithiocarbamate and ethylenebisdithiocarbamate, to algae, which are the most sensitive tested organisms to polycarbamate. The toxicities of dimethyldithiocarbamate and ethylenebisdithiocarbamate partially explain that of polycarbamate. To assess the primary risk, we derived the predicted no-effect concentration (PNEC) for polycarbamate in a probabilistic manner using species sensitivity distributions. The 72 h no observed effect concentration (NOEC) of polycarbamate to the alga Skeletonema marinoi-dohrnii complex was 0.45 µg/L. The toxicity of dimethyldithiocarbamate may have contributed up to 72% of the toxicity observed for polycarbamate. The fifth percentile of hazardous concentration (HC5) derived from the acute toxicity values was 0.48 µg/L. Comparison of previously reported environmental polycarbamate concentrations in Hiroshima Bay, Japan, to the PNEC estimated using the minimum NOEC and HC5 suggest that polycarbamate currently poses a high ecological risk. Therefore, reducing the risk by restricting polycarbamate use is necessary.

Demonstration of N,N-Dimethyldithiocarbamate as a Copper-Dependent Antibiotic against Multiple Upper Respiratory Tract Pathogens.

Transition metals are necessary cofactors and structural elements in living systems. Exposure to high concentrations of biologically important transition metals, such as zinc and copper, results in cell toxicity. At the infection site, the immune system deploys metal sorbent proteins (e.g., lactoferrin and calprotectin) to starve pathogens of necessary metals (such as iron), while phagocytes expose engulfed pathogens to high levels of other metals, such as copper and zinc. The opportunistic pathogen Streptococcus pneumoniae (the pneumococcus) encounters macrophages during initial and protracted infections. The pneumococcus employs a copper export pathway, which improves colonization and persistent infection of the nasopharynx and the upper respiratory tract. Because copper is tightly regulated in the host, we instead sought to leverage the localized power of nutritional immunity by identifying small molecules with copper-dependent toxicity (CDT) through a targeted screen of compounds for antibiotic efficacy. We chose to include dithiocarbamates, based on the copper synergy observed in other organisms with 1-(diethylthiocarbamoyldisulfanyl)-N,N-diethyl-methanethioamide (tetraethylthiuram disulfide, disulfiram). We observed CDT of some dithiocarbamates in S. pneumoniae. Only N,N-dimethyldithiocarbamate (DMDC) was consistently toxic across a range of concentrations with copper both in vitro and in vivo against the pneumococcus. We also observed various degrees of CDT in vitro using DMDC in Staphylococcus aureus, Coccidioides posadasii, and Schistosoma mansoni. Collectively, we demonstrate that the compound DMDC is a potent bactericidal compound against S. pneumoniae with antimicrobial efficacy against bacterial and fungal pathogens. IMPORTANCE With the rise of antibiotic resistance, approaches that add new antimicrobials to the current repertoire are vital. Here, we investigate putative and known copper ionophores in an attempt to intoxicate bacteria and use ionophore/copper synergy, and we ultimately find success with N,N-dimethyldithiocarbamate (DMDC). We show that DMDC has in vitro efficacy in a copper-dependent manner and kills pathogens across three different kingdoms, Streptococcus pneumoniae, Coccidioides posadasii, and Schistosoma mansoni, and in vivo efficacy against S. pneumoniae. As such, dithiocarbamates represent a new potential class of antimicrobials and thus warrant further mechanistic investigation.

Prenatal treatment of mosaic mice (Atp7a mo-ms) mouse model for Menkes disease, with copper combined by dimethyldithiocarbamate (DMDTC).

Menkes disease is a fatal neurodegenerative disorder in infants caused by mutations in the gene ATP7A which encodes a copper (Cu) transporter. Defects in ATP7A lead to accumulated copper in the small intestine and kidneys and to copper deficiencies in the brain and the liver. The copper level in the kidney in postnatal copper-treated Menkes patients may reach toxic levels. The mouse model, mosaic Atp7a (mo-ms) recapitulates the Menkes phenotype and die about 15.75±1.5 days of age. In the present study we found that prenatal treatment of mosaic murine fetuses throughout gestation days 7, 11, 15 and 18 with a combination of CuCl(2) (50 mg/kg) and dimethyldithiocarbamate (DMDTC) (280 mg/kg) leads to an increase in survival to about 76±25.3 days, whereas treatment with CuCl(2) alone (50 mg/kg) only leads to survival for about 21 days ±5 days. These copper-DMDTC treated mutants showed an improved locomotor activity performance and a gain in body mass. In contrast to treatment with CuCl(2) alone, a significant increase in the amount of copper was observed in the brain after prenatal copper-DMDTC treatment as well as a decrease in the amount of accumulated copper in the kidney, both leading towards a normalization of the copper level. Although copper-DMDTC prenatal treatment only leads to a small increase in the sub-normal copper concentration in the liver and to an increase of copper in the already overloaded small intestine, the combined results suggest that prenatal copper-DMDTC treatment also should be considered for humans.

Chemical treatment of chelated metal finishing wastes.

This study evaluated two chemical approaches for treatment of commingled cadmium-cyanide (Cd-CN) and zinc-nickel (Zn-Ni) wastewaters. The first approach, which involved application of sodium hypochlorite (NaOCl), focused on elimination of chelating substances. The second approach evaluated the use of sodium dimethyldithiocarbamate (DMDTC) to specifically target and precipitate regulated heavy metals. Results demonstrated that by maintaining a pH of 10.0 and an oxidation-reduction potential (ORP) value of +600 mV, NaOCl treatment was effective in eliminating all chelating substances. Cadmium, chromium, nickel, and zinc solution concentrations were reduced from 0.27, 4.44, 0.06, and 0.10 ppm to 0.16, 0.17, 0.03, and 0.06 ppm, respectively. Similarly, a 1% DMDTC solution reduced these same metal concentrations in commingled wastewater to 0.009, 1.142, 0.036, and 0.320 ppm. Increasing the DMDTC concentration to 2% improved the removal of all regulated heavy metals except zinc, the removal of which at high pH values is limited by its amphotericity.

Highly cytotoxic substitutionally inert rhodium(III) tris(chelate) complexes: DNA binding modes and biological impact on human cancer cells.

The antiproliferative properties and cellular impact of novel substitutionally inert rhodium(III) complexes of the types [Rh{(CH3)2 NCS2}2(pp)]Cl 3-5 (pp=5,6-Me2phen, dpq, dppz) and OC-6-23-[Rh(2-S-py)2(pp)]Cl 6 and 7 (2-S-py=pyridine-2-thiolate; pp=dpq, dppz) have been investigated for the adherent human cancer cell lines MCF-7 and HT-29 and for non-adherent Jurkat cells. Whereas CD and viscosity measurements indicate that the polypyridyl ligands of 4 and 5 intercalate into CT DNA, this is not the case for the analogous pyridine-2-thiolate complexes 6 and 7. Complexes 3-7 all exhibit a high antiproliferative activity towards MCF-7 and HT-29 cells, with IC(50) values in the range 0.055-0.285 µM. As established by online monitoring with a cell-based sensor chip, the highly cytostatic complex 6 (IC(50)=0.059 and 0.078 µM) invokes an immediate concentration-dependent reduction of MCF-7 cell respiration and a time-delayed decrease in cellular impedance, which can be ascribed to the induction of cell death. Annexin V/PI assays demonstrated that 6 also has a pronounced antiproliferative activity towards Jurkat cells and that it invokes extensive apoptosis and high concentrations of reactive oxygen species in these leukemia cells. The observation of a dose-dependent inhibition of the oxygen consumption of isolated mice mitochondria indicates the involvement of an intrinsic mitochondrial pathway in this process.

Ziram causes dopaminergic cell damage by inhibiting E1 ligase of the proteasome.

The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated alpha-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target.

An updated study of mortality among North American synthetic rubber industry workers.

This study evaluated mortality rates from leukemia and other diseases during the time period 1944 through 1998 among 17,924 men employed in the synthetic rubber industry. In this group, there were 6237 deaths, which is 14% fewer than the 7242 deaths expected based on general population rates. Numbers of observed versus expected deaths (shown hereafter as observed/expected) were 1608/1741 for all cancers combined, including 71/61 for leukemia, 53/53 for non-Hodgkin lymphoma (NHL*), and 26/27 for multiple myeloma. The higher than expected number of deaths from leukemia (16% increase) was concentrated in workers paid hourly who had started work 20 to 29 years earlier, had worked 10 or more years in the industry, and had worked in subgroups employed in polymerization, coagulation, maintenance labor, and laboratory operations. The overall higher leukemia mortality rate, as well as the higher rate in the subgroup of hourly workers who had 20 or more years since hire and 10 or more years worked, was not limited to a particular form of leukemia. Cumulative exposure to 1,3-butadiene (BD) was associated positively with all leukemias, with chronic myelogenous leukemia and, to a lesser extent, with chronic lymphocytic leukemia (CLL). Exposure to styrene or to dimethyldithiocarbamate (DMDTC) also was associated positively with leukemia. Exposures to these two agents were correlated with exposure to BD; data were limited on the independent effects of each of the three chemicals on leukemia. After controlling for the effects of BD, we found no consistent exposure-response relation between either styrene or DMDTC and all leukemias, chronic myelogenous leukemia, or CLL. However, a positive association between any exposure to DMDTC and leukemia persisted. The data from this study indicate that employment in the synthetic rubber industry is related causally to leukemia. Uncertainty remains about the specific agent or agents responsible for the association. The carcinogenic mechanisms through which BD, styrene, or DMDTC could cause leukemia in humans have not been established, and epidemiologic support for a leukemogenic role is limited for these agents. Styrene and DMDTC were associated positively with NHL. External support for this relation has not been reported from other epidemiologic studies. The study did not find any clear relation between exposure to BD, styrene, or DMDTC and multiple myeloma. Some subgroups of subjects had more than the expected number deaths from colorectal cancer, prostate cancer, and other diseases. These increases did not appear to be related to occupational exposure in the industry.

Cisplatin stabilizes a multimeric complex of the human Ctr1 copper transporter: requirement for the extracellular methionine-rich clusters.

Cisplatin is a highly effective cancer chemotherapy agent. However, acquired resistance currently limits the clinical utility of this drug. The human high affinity copper importer, hCtr1, and its yeast and murine orthologues have been shown to mediate the uptake of cisplatin. This transporter is located at the plasma membrane under low copper conditions, and excess copper concentrations stimulate its endocytosis and degradation. In this study we further examined the link between cisplatin and hCtr1 by examining whether cisplatin can also stimulate the endocytosis and degradation of hCtr1. The steady-state location of hCtr1 and its endocytosis from the plasma membrane were not altered by cisplatin treatment. Unexpectedly, cisplatin treatment of a cell line expressing hCtr1 revealed the time- and concentration-dependent appearance of a stable hCtr1 multimeric complex, consistent with a homotrimer, which was not observed following copper treatment of these same cells. Mutagenesis studies identified two methionine-rich clusters in the extracellular amino-terminal region of hCtr1 that were required for stabilization of the hCtr1 multimer by cisplatin, suggesting that these sequences bind cisplatin and form crosslinks between hCtr1 polypeptides. Treatment with the metal chelator dimethyldithiocarbamate disassembled the hCtr1 multimer following cisplatin exposure, suggesting that platinum was an integral component of this complex. These studies provide the first evidence for a direct interaction between cisplatin and the hCtr1 protein and establish that cisplatin and copper have distinct biochemical consequences on this transporter.

Photoinhibition of photosystem I is accelerated by dimethyldithiocarbamate, an inhibitor of superoxide dismutase, during light-chilling of spinach leaves.

In vivo photoinhibition of photosystem I (PS I) was investigated at chilling temperature using the leaves of the chilling-resistant spinach plant treated with an inhibitor of superoxide dismutase, diethyldithiocarbamate (DDC). When spinach leaves were treated with DDC during chilling at 4 degrees C for 12 h with a light intensity of 120 micromol m(-2) s(-1), the activity of PS I and the content of iron-sulfur centers declined to about 50% and 25% of the non-DDC-treated controls, respectively. A native green gel analysis of thylakoid membranes isolated from the DDC-treated leaves resolved a novel chlorophyll-protein complex, which was identified as the light-harvesting complex I (LHC I)-deficient PS I complex when examined by 77 K fluorescence spectroscopy and two-dimensional sodium dodecyl sulfate gel electrophoresis. The possible dissociation of LHC I as an early structural change in the PS I complex after DDC-induced photoinhibition of PS I is discussed.

Enzymological characterization of EpoA, a laccase-like phenol oxidase produced by Streptomyces griseus.

Laccase is an enzyme that catalyzes the oxidation of phenolic compounds by coupling the reduction of oxygen to water. While many laccases have been identified in plant and fungal species, enzymes of prokaryotic origin are poorly known. Here we report the enzymological characterization of EpoA, a laccase-like extracytoplasmic phenol oxidase produced by Streptomyces griseus. EpoA was expressed and purified with an Escherichia coli host-vector system as a recombinant protein fused with a C-terminal histidine-tag (rEpoA). Physicochemical analyses showed that rEpoA comprises a stable homotrimer containing all three types of copper (types 1-3). Various known laccase substrates were oxidized by rEpoA, while neither syringaldazine nor guaiacol served as substrates. Among the substrates examined, rEpoA most effectively oxidized N,N-dimethyl-p-phenylenediamine sulphate with a Km value of 0.42 mM. Several metal chelators caused marked inhibition of rEpoA activity, implying the presence of a metal center essential for the oxidase activity. The pH and temperature optima of rEpoA were 6.5 and 40 degrees C, respectively. The enzyme retained 40% activity after preincubation at 70 degrees C for 60 min. EpoA-like activities were detected in cell extracts of 8/40 environmental actinomycetes strains, which suggests that similar oxidases are widely distributed among this group of bacteria.

A new 2D-TLC bioautography method for the discovery of novel antifungal agents To control plant pathogens.

A new bioassay has been developed combining the simplicity of direct bioautography with the improved chromatographic resolution of 2D-TLC. Mixtures of structurally diverse antifungal agents were tested to establish the validity and utility of this method in the discovery of new natural products with activity against agriculturally important fungal pathogens.

Dithiocarbamates inhibit hematopoiesis via a copper-dependent mechanism.

Dithiocarbamates (DTC), an important class of therapeutic and industrial chemicals, have alternatively been reported to be either beneficial or toxic to the hematopoietic and immune systems. In the present study, we investigated the potential of dimethyl- and diethyl-dithiocarbamate to alter clonogenic response of primary human CD34(+) bone marrow cells in vitro. Our results demonstrate that both compounds are potent inhibitors of clonogenic response in human CD34(+) bone marrow cells, suppressing cytokine-induced colony formation at concentrations between 100 and 500 nM. Pretreatment of bone marrow cells for 1 h with very high doses of DTC (30 microM) had no effect on colony formation. DTCs are known inhibitors of nuclear factor-kappa B (NF-kappa B); however, data presented herein demonstrate that DTC do not inhibit cytokine activation of NF-kappa B in CD34(+) bone marrow cells. Additional experiments demonstrate that DTCs induce a dose-related increase in apoptosis, potentially acting via a cytotoxic mechanism. We further demonstrate that the addition of copper sulfate greatly potentiates the hematotoxicity of DTC and that the addition of a copper-specific chelator completely abrogates DTC clonogenic suppression. These data support a role for copper in DTC-induced hematotoxicity.

Thiram and dimethyldithiocarbamic acid interconversion in Saccharomyces cerevisiae: a possible metabolic pathway under the control of the glutathione redox cycle.

A rapid decrease of intracellular glutathione (GSH) was observed when exponentially growing cells of Saccharomyces cerevisiae were treated with sublethal concentrations of either dimethyldithiocarbamic acid or thiram [bis(dimethylthiocarbamoyl) disulfide]. The underlying mechanism of this effect possibly involves the intracellular oxidation of dimethyldithiocarbamate anions to thiram, which in turn oxidizes GSH. Overall, a linear relationship was found between thiram concentrations up to 21 microM and production of oxidized GSH (GSSG). Cytochrome c can serve as the final electron acceptor for dimethyldithiocarbamate reoxidation, and it was demonstrated in vitro that NADPH handles the final electron transfer from GSSG to the fungicide by glutathione reductase. These cycling reactions induce transient alterations in the intracellular redox state of several electron carriers and interfere with the respiration of the yeast. Thiram and dimethyldithiocarbamic acid also inactivate yeast glutathione reductase when the fungicide is present within the cells as the disulfide. Hence, whenever the GSH regeneration rate falls below its oxidation rate, the GSH:GSSG molar ratio drops from 45 to 1. Inhibition of glutathione reductase may be responsible for the saturation kinetics observed in rates of thiram elimination and uptake by the yeast. The data suggest also a leading role for the GSH redox cycle in the control of thiram and dimethyldithiocarbamic acid fungitoxicity. Possible pathways for the handling of thiram and dimethyldithiocarbamic acid by yeast are considered with respect to the physiological status, the GSH content, and the activity of glutathione reductase of the cells.

Comparative evaluation of chelating agents on the mobilization of cadmium: a mechanistic approach.

A comparative evaluation of chelating agents, namely, diethyl dithiocarbamate (DDC), dimethyl dithiocarbamate (DMDC), 1,4,8,11-tetraazacyclotetradecane (CYCLAM), 1,4,8,12-tetraazacyclopentadecane (TACPD), 2,3-dimercaptosuccinic acid (DMSA), and 2,3-dimercapto-1-propane sulfonate (DMPS) was conducted to assess their efficacy against acute cadmium (Cd) toxicity. DMSA and DMPS appeared to be most effective in reducing mortality as well as Cd burden of liver, kidneys, and brain in cadmium intoxicated mice. DMDC reduced Cd levels only in liver and kidneys, while DDC significantly enhanced its level in brain. CYCLAM and TACPD significantly increased the Cd level in liver and kidneys and were ineffective in brain. The therapeutic index as well as therapeutic efficacy was highest for DMSA followed by DMPS and DMDC. A fair degree of correlation was found to exist between (1) stability constant of Cd chelates and percent survival (r = .438, (2) stability constant and percent transport (r = .479), and (3) percent survival and percent transport (r =.447). However, the lipophilicity did not show any appreciable correlation with percent survival and stability constant of Cd chelates.

Dialkyldithiocarbamates inhibit tyrosine hydroxylase activity in PC12 cells and in fibroblasts that express tyrosine hydroxylase.

Dithiocarbamates and CS2 have been associated with neurobehavioural changes suggestive of central dopaminergic dysfunction. Diethyldithiocarbamate (DEDC), dimethyldithiocarbamate (DMDC), and methyldithiocarbamate (MDC) were examined for their ability to inhibit tyrosine hydroxylase (TH) activity in PC12 cells and transfected CHO fibroblasts that expressed TH (CHO/TH) activity when tetrahydrobiopterin (BH4) was added to medium. DEDC or DMDC did not significantly alter viability of PC12 cells or CHO/TH cells at < or = 100 microM for 18 h; the EC50 for each compound was approximately 5 mM in both cell lines. In contrast, the EC50 for MDC was 41 or 74 microM in PC12 or CHO/TH cultures, respectively. There was no change in immunodetectable levels of TH in PC12 or CHO/TH cells following exposure to subcytotoxic concentrations of dithiocarbamates. DEDC and DMDC (5 to 100 microM) produced concentration-dependent reductions in PC12 cell dopamine and dopac levels as well as in dopa levels in CHO/TH cultures. Reduction of PC12 catechols was not due to altered vesicular storage. In vitro PC12 TH activity was 80.2 +/- 3.4% or 82.4 +/- 2.9% of control following exposure to 100 microM DEDC or DMDC, respectively, and was not fully restored by incubation with Fe2+. These results show that DEDC and DMDC, but not MDC, are low potency cytotoxins that decrease TH activity in cultured cells through mechanisms other than inhibition of BH4 biosynthesis or iron chelation.

Protection against alloxan-induced diabetes by various dialkyldithiocarbamates.

Dialkyldithiocarbamates injected into mice 0.5 h prior to alloxan protected dose-dependently against the diabetogenic action of alloxan, and increased blood glucose levels at the time of alloxan injection. Furthermore, they exhibited anti-oxidative properties in vitro such as inhibition of lipid peroxidation, removal of hydrogen peroxide and reduction of the stable free radical, 1, 1-diphenyl-2-picrylhydrazyl (DPPH). These results suggest that dialkyldithiocarbamates protect against the development of alloxan-induced diabetes by the indirect mechanism of producing hyperglycemia at the time of alloxan injection and possibly by their anti-oxidative effects as well.

Stimulatory effect of cooling tower biocides on amoebae.

Two species of amoebae were isolated from the cooling tower of an air-conditioning system and examined for effects of exposure to four cooling tower biocides, a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds, another quaternary ammonium compound alone, and an isothiazolin derivative. The amoebae isolated were Acanthamoeba hatchetti and a Cochliopodium species. Two other amoeba cultures, an A. hatchetti culture and Cochliopodium bilimbosum, were obtained from the American Type Culture Collection (ATCC) and were also tested. The cooling tower isolates were more resistant to most of the biocides than the ATCC isolates were. The isothiazolin derivative was the least inhibitory to all four amoeba isolates, and tributyltin neodecanoate mixed with quaternary ammonium compounds was the most inhibitory to three of the four isolates. After exposure to lower concentrations of the biocides, including for one strain the manufacturer's recommended concentration of one biocide, the cooling tower amoeba populations increased significantly compared with unexposed controls, whereas the ATCC isolates were not stimulated at any of the concentrations tested. In some cases, concentrations which stimulated cooling tower amoebae inhibited the growth of the ATCC isolates. These results suggest that cooling tower amoebae may adapt to biocides, underscoring the need to use freshly isolated cooling tower organisms rather than organisms from culture collections for testing the efficacy of such biocides. The stimulatory effect of biocides on amoeba populations is an alarming observation, since these organisms may be reservoirs for legionellae. Biocides used to control microbial growth may actually enhance populations of host organisms for pathogenic bacteria.

Tetramethylthiuram disulfide or dimethyldithiocarbamate induces the synthesis of cadystins, heavy metal chelating peptides, in Schizosaccharomyces pombe.

Tetramethylthiuram disulfide (TMTD) or dimethyldithiocarbamate (DMDTC) induces the synthesis of cadystins, a family of heavy metal chelating isopeptides with the formula (gamma-Glu-Cys)n-Gly (n = 2,3,4,...), in the fission yeast Schizosaccharomyces pombe. Amount of cadystins synthesized in TMTD or DMDTC treated cells is less than that synthesized in CdCl2 treated cells but much more than that synthesized in ZnCl2 or CuSO4 treated cells.

Enhancement of cytotoxicity of bleomycin by dithiocarbamates: formation of bis(dithiocarbamato) cu(II).

Properties of the reactions of dithiocarbamates and their Cu(II) or Fe(III) complexes with Ehrlich cells were determined and related to their effects on the inhibition of cell proliferation caused by bleomycin and Cu bleomycin. In complete culture medium containing Eagle's minimal essential medium plus Earles salts and 2.5% fetal calf serum, dimethyl- and diethyldithiocarbamates and their copper complexes inhibit cell proliferation and cause cell death. The copper complexes are more effective agents. Ferric tris-diethyldithiocarbamate is also a cytotoxic species. In contrast, when cells are exposed to dimethyldithiocarbamate or its copper complex in Ringer's buffer under metal-restricted condition, washed, and then placed in complete medium, the copper complex is much more active in inhibiting cell growth. The difference is magnified when dihydroxyethyldithiocarbamate and N-methylglucamine dithiocarbamate and their copper complexes are compared in complete media. Incubation of bleomycin or copper bleomycin with Ehrlich cells in Ringer's buffer with or without dimethyldithiocarbamate or bis-dimethyldithiocarbamato Cu(II) leads to no enhancement of cytotoxicity from combinations of agents, except when the two copper complexes are present. Diethyl- or dimethyldithiocarbamate readily extracts copper from Cu(II)bleomycin and iron from Fe(III)bleomycin when ethylacetate is present to remove the tris-dithiocarbamato Fe(III) complex from aqueous solution. When bis-dimethyldithiocarbamato Cu(II) is incubated with Ehrlich cells, copper is released from the complex and bound to high molecular weight and metallothionein fractions. A reductive mode of dissociation of the copper complexes in cells is supported by ESR experiments. Reactions of diethyl- and dimethyldithiocarbamato Cu(II) with thiol compounds demonstrates one possible mechanism of reduction of these complexes.