The environmental toxicant ziram enhances neurotransmitter release and increases neuronal excitability via the EAG family of potassium channels.

Environmental toxicants have the potential to contribute to the pathophysiology of multiple complex diseases, but the underlying mechanisms remain obscure. One such toxicant is the widely used fungicide ziram, a dithiocarbamate known to have neurotoxic effects and to increase the risk of Parkinson's disease. We have used Drosophila melanogaster as an unbiased discovery tool to identify novel molecular pathways by which ziram may disrupt neuronal function. Consistent with previous results in mammalian cells, we find that ziram increases the probability of synaptic vesicle release by dysregulation of the ubiquitin signaling system. In addition, we find that ziram increases neuronal excitability. Using a combination of live imaging and electrophysiology, we find that ziram increases excitability in both aminergic and glutamatergic neurons. This increased excitability is phenocopied and occluded by null mutant animals of the ether a-go-go (eag) potassium channel. A pharmacological inhibitor of the temperature sensitive hERG (human ether-a-go-go related gene) phenocopies the excitability effects of ziram but only at elevated temperatures. seizure (sei), a fly ortholog of hERG, is thus another candidate target of ziram. Taken together, the eag family of potassium channels emerges as a candidate for mediating some of the toxic effects of ziram. We propose that ziram may contribute to the risk of complex human diseases by blockade of human eag and sei orthologs, such as hERG.

Delayed Puberty by Ziram Is Associated with Down Regulation of Testicular Phosphorylated AKT1 and SIRT1/PGC-1α Signaling.

Ziram is a dimethyldithiocarbamate fungicide, which may influence the male reproductive system as a potential endocrine disruptor. We interrogated the disruption of ziram on rat progenitor Leydig cell development. Prepubertal male Sprague-Dawley rats were orally treated with 0, 2, 4, or 8 mg/kg ziram for 2 weeks. We investigated the effects of ziram on serum testosterone levels, Leydig cell number, and Leydig and Sertoli cell gene and protein expression, SIRT1/PGC-1α levels, and phosphorylation of AKT1, ERK1/2, and AMPK in vivo. We also interrogated the effects of ziram on reactive oxidative species (ROS) level, apoptosis rate, and mitochondrial membrane potential of progenitor Leydig cells in vitro. Ziram decreased serum testosterone and follicle-stimulating hormone levels, the down-regulated Leydig cell-specific gene ( Lhcgr, Scarb1, Star, Cyp17a1, and Hsd17b3), and their protein expression. However, ziram stimulated anti-Müllerian hormone production. Ziram lowered SIRT1/PGC-1α and phosphorylated protein levels of AKT1. Ziram induced ROS and apoptosis and lowered the mitochondrial membrane potential of progenitor Leydig cells in vitro. In conclusion, ziram disrupts Leydig cell development during the prepubertal period potentially through the SIRT1/PGC-1α and phosphorylated AKT1 signaling.

Effects on metabolic parameters in young rats born with low birth weight after exposure to a mixture of pesticides.

Pesticide exposure during fetal life can lead to low birth weight and is commonly observed in reproductive toxicology studies. Associations have also been found in low birth weight babies born from pesticide-exposed gardeners. Since low birth weight is also linked to metabolic disorders, it can be speculated that early life exposure to pesticides could increase the risk of becoming obese or developing diabetes later in life. We have analyzed potential long-term effects of gestational and lactational exposure to a low dose mixture of six pesticides that individually can cause low birth weight: Cyromazine, MCPB, Pirimicarb, Quinoclamine, Thiram, and Ziram. Exposed male offspring, who were smaller than controls, displayed some degree of catch-up growth. Insulin and glucagon regulation was not significantly affected, and analyses of liver and pancreas did not reveal obvious histopathological effects. Efforts towards identifying potential biomarkers of metabolic disease-risk did not result in any strong candidates, albeit leptin levels were altered in exposed animals. In fat tissues, the key genes Lep, Nmb and Nmbr were altered in high dosed offspring, and were differentially expressed between sexes. Our results suggest that early-life exposure to pesticides may contribute to the development of metabolic disorders later in life.

Zinc- and oxidative property-dependent degradation of pro-caspase-1 and NLRP3 by ziram in mouse macrophages.

The NLRP3 inflammasome, composed of caspase-1, NLRP3 and ASC, plays a critical role in the clearance of microbial pathogens. Here, we found that the treatment of mouse macrophages with the zinc-containing dithiocarbamate ziram, a widely used fungicide in agriculture, caused a decrease in pro-caspase-1 and NLRP3 levels while not affecting ASC level. Ziram did not affect levels of pro-caspase-1 and NLRP3 mRNA, and no cleavage products of pro-caspase-1 including p10 subunit, which is an autocleavage product of pro-caspase-1, were detected, indicating that the decrease was associated with degradation of these proteins. The decrease was inhibited by SH-type antioxidants, N-acetyl cysteine, dithiothreitol and 2-mercaptoethanol, or a metal chelator EDTA but not by inhibitors of proteasome, lysosomes, autophagy and matrix metalloproteases. Thiram, a comparator for ziram that does not contain zinc, showed a weaker decrease in protein levels. Furthermore, the zinc-containing dithiocarbamate, zinc diethyldithiocarbamate, efficiently decreased the levels of pro-caspase-1 and NLRP3, whereas dithiocarbamates, dimethyldithiocarbamate and diethyldithiocarbamate without zinc, were less active. The organic zinc compound [3,4-toluenedithiolato(2-)]zinc hydrate did not induce a decrease in protein levels. Ziram also inhibited IL-1ß production by macrophages in response to lipopolysaccharide and bacterial clearance during Salmonella infection of macrophage cells. These results indicate that ziram causes degradation of pro-caspase-1 and NLRP3 in a zinc- and oxidative property-dependent manner and suggest that exposure to ziram may compromise the clearance of microbial pathogens.

Ziram and sodium N,N-dimethyldithiocarbamate inhibit ubiquitin activation through intracellular metal transport and increased oxidative stress in HEK293 cells.

Ubiquitin activating enzyme E1 plays a pivotal role in ubiquitin based protein signaling through regulating the initiating step of the cascade. Previous studies demonstrated that E1 is inhibited by covalent modification of reactive cysteines contained within the ubiquitin-binding groove and by conditions that increase oxidative stress and deplete cellular antioxidants. In this study, we determined the relative contribution of covalent adduction and oxidative stress to E1 inhibition produced by ziram and sodium N,N-dimethyldithiocarbamate (DMDC) in HEK293 cells. Although no dithiocarbamate-derived E1 adducts were identified on E1 using shotgun LC/MS/MS for either ziram or DMDC, both dithiocarbamates significantly decreased E1 activity, with ziram demonstrating greater potency. Ziram increased intracellular levels of zinc and copper, DMDC increased intracellular levels of only copper, and both dithiocarbamates enhanced oxidative injury evidenced by elevated levels of protein carbonyls and expression of heme oxygenase-1. To assess the contribution of intracellular copper transport to E1 inhibition, coincubations were performed with the copper chelator triethylenetetramine hydrochloride (TET). TET significantly protected E1 activity for both of the dithiocarbamates and decreased the associated oxidative injury in HEK293 cells as well as prevented dithiocarbamate-mediated lipid peroxidation assayed using an ethyl aracidonate micelle system. Because TET did not completely ameliorate intracellular transport of copper or zinc for ziram, TET apparently maintained E1 activity through its ability to diminish dithiocarbamate-mediated oxidative stress. Experiments to determine the relative contribution of elevated intracellular zinc and copper were performed using a metal free incubation system and showed that increases in either metal were sufficient to inhibit E1. To evaluate the utility of the HEK293 in vitro system for screening environmental agents, a series of additional pesticides and metals was assayed, and eight agents that produced a significant decrease and five that produced a significant increase in activated E1 were identified. These studies suggest that E1 is a sensitive redox sensor that can be modulated by exposure to environmental agents and can regulate downstream cellular processes.

Involvement of the sodium-calcium exchanger 3 (NCX3) in ziram-induced calcium dysregulation and toxicity.

Ziram is a dimethyldithiocarbamate fungicide which can cause intraneuronal calcium (Ca(2+)) dysregulation and subsequently neuronal death. The signaling mechanisms underlying ziram-induced Ca(2+) dyshomeostasis and neurotoxicity are not fully understood. NCX3 is the third isoform of the sodium-calcium exchanger (NCX) family and plays an important role in regulating Ca(2+) homeostasis in excitable cells. We previously generated a mouse model deficient for the sodium-calcium exchanger 3 and showed that NCX3 is protective against ischemic damage. In the present study, we aim to examine whether NCX3 exerts a similar role against toxicological injury caused by the pesticide ziram. Our data show baby hamster kidney (BHK) cells stably transfected with NCX3 (BHK-NCX3) are more susceptible to ziram toxicity than cells transfected with the empty vector (BHK-WT). Increased toxicity in BHK-NCX3 was associated with a rapid rise in cytosolic Ca(2+) concentration [Ca(2+)]i induced by ziram. Profound mitochondrial dysfunction and ATP depletion were also observed in BHK-NCX3 cells following treatment with ziram. Lastly, primary dopaminergic neurons lacking NCX3 (NCX3(-/-)) were less sensitive to ziram neurotoxicity than wildtype control dopaminergic neurons. These results demonstrate that NCX3 genetic deletion protects against ziram-induced neurotoxicity and suggest NCX3 and its downstream molecular pathways as key factors involved in ziram toxicity. Our study identifies new molecular events through which pesticides (e.g. ziram) can lead to pathological features of degenerative diseases such as Parkinson's disease and indicates new targets to slow down neuronal degeneration.

Ziram activates mitogen-activated protein kinases and decreases cytolytic protein levels in human natural killer cells.

Human natural killer (NK) cells are central in immune defense with their ability to lyse tumor cells and virally infected cells. Tumor formation and viral infection may increase if NK cytotoxic function is disrupted. Ziram (zinc dithiocarbamate) is used as an accelerating agent in the production of latex and to protect various fruits and vegetables from fungal infection. Previously, we have shown that exposure to ziram inhibits NK lytic function. Butyltin environmental contaminants, which also inhibit NK lytic function, cause rapid activations of mitogen-activated protein kinases (MAPKs) and decreases in expression of the cytolytic proteins granzyme B and perforin (after 24 h) in exposed NK cells. MAPKs are important regulators of the lytic response of NK cells, and spurious activation of these enzymes by contaminants would leave the NK cells unable to respond to appropriate targets. This study examined the effects of ziram exposures on MAPKs (p44/42, p38, and c-jun-N-terminal kinase) and on levels of cytolytic proteins. Ten-minute to 6-h exposures of NK cells to ziram caused activation of MAPKs, p44/42, and p38. Exposure to ziram for 24 h caused a decrease in granzyme B and perforin levels. MAPK inhibitors were able to prevent these ziram-induced decreases in granzyme B and perforin. These results suggest that ziram-induced MAPK activation is at least in part responsible for decreased cytolytic function in ziram-exposed NK cells. Furthermore, the results indicate that these changes are in common with other environmental contaminants that have been shown to decrease NK lytic function.

Parkinson's disease risk from ambient exposure to pesticides.

Due to the heavy and expanding agricultural use of neurotoxic pesticides suspected to affect dopaminergic neurons, it is imperative to closely examine the role of pesticides in the development of Parkinson's disease (PD). We focus our investigation on pesticide use in California's heavily agricultural central valley by utilizing a unique pesticide use reporting system. From 2001 to 2007, we enrolled 362 incident PD cases and 341 controls living in the Central Valley of California. Employing our geographic information system model, we estimated ambient exposures to the pesticides ziram, maneb, and paraquat at work places and residences from 1974 to 1999. At workplaces, combined exposure to ziram, maneb, and paraquat increased risk of PD three-fold (OR: 3.09; 95% CI: 1.69, 5.64) and combined exposure to ziram and paraquat, excluding maneb exposure, was associated with a 80% increase in risk (OR:1.82; 95% CI: 1.03, 3.21). Risk estimates for ambient workplace exposure were greater than for exposures at residences and were especially high for younger onset PD patients and when exposed in both locations. Our study is the first to implicate ziram in PD etiology. Combined ambient exposure to ziram and paraquat as well as combined ambient exposure to maneb and paraquat at both workplaces and residences increased PD risk substantially. Those exposed to ziram, maneb, and paraquat together experienced the greatest increase in PD risk. Our results suggest that pesticides affecting different mechanisms that contribute to dopaminergic neuron death may act together to increase the risk of PD considerably.

Molecular mechanisms underlying mancozeb-induced inhibition of TNF-alpha production.

Mancozeb, a polymeric complex of manganese ethylenebisdithiocarbamate with zinc salt, is widely used in agriculture as fungicide. Literature data indicate that ethylenebisdithiocarbamates (EBDTCs) may have immunomodulatory effects in humans. We have recently found in agricultural workers occupationally exposed to the fungicide mancozeb a statistically significant decrease in lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF) production in leukocytes. TNF is an essential proinflammatory cytokine whose production is normally stimulated during an infection. The purpose of this work was to establish an in vitro model reflecting in vivo data and to characterize the molecular mechanism of action of mancozeb. The human promyelocytic cell line THP-1 was used as in vitro model to study the effects of mancozeb and its main metabolite ethylenthiourea (ETU) on LPS-induced TNF release. Mancozeb, but not ETU, at non-cytotoxic concentrations (1-100 microg/ml), induced a dose- and time-dependent inhibition of LPS-induced TNF release, reflecting in vivo data. The modulatory effect observed was not limited to mancozeb but also other EBDTCs, namely zineb and ziram, showed similar inhibitory effects. Mancozeb must be added before or simultaneously to LPS in order to observe the effect, indicating that it acts on early events triggered by LPS. It is known that nuclear factor-kappaB (NF-kappaB) tightly regulates TNF transcription. We could demonstrate that mancozeb, modulating LPS-induced reactive oxygen species generation, prevented IkappaB degradation and NF-kappaB nuclear translocation, which in turn resulted in decreased TNF production. To further understand the mechanism of the effect of mancozeb on TNF transcription, THP-1 cells were transfected with NF-kappaB promoter-luciferase construct, and the effect of mancozeb on luciferase activity was measured. Cells transfected with promoter constructs containing kappaB site showed decreased LPS-induced luciferase activity relative to control after mancozeb treatment, confirming NF-kappaB binding as an intracellular target of mancozeb. Overall, this study contributes to our understanding of the mechanism underlying mancozeb-induced immunotoxicity.

Persistent inhibition of human natural killer cell function by ziram and pentachlorophenol.

Ziram is a currently used agricultural fungicide. It is also used as an additive in the production of latex gloves. Because of these uses, there is a potential for human exposure to this compound. Pentachlorophenol (PCP) has been used as an insecticide, fungicide, disinfectant, and ingredient in antifouling paints. Currently, it is used as a wood preservative for power-line poles and fence posts. Measurable levels of PCP have been detected in human blood and urine. In previous studies we demonstrated that both these compounds could cause very significant inhibition of the tumor-killing function of human natural killer (NK) cells. NK lymphocytes play a central role in immune defense against viral infection and the formation of primary tumors. So interference with their function could increase the risk of tumor development. In the present study we examined the effects of exposure to ziram or PCP of brief duration (1 h) on the ability of NK cells to destroy tumor cells. NK cells were exposed to either ziram (5-0.5 microM) or PCP (10-5 microM) for 1 h followed by 0 h, 24 h, 48 h, or 6 days in compound-free media and then were tested for the ability to lyse as well as to bind tumor cells. A 1-h exposure to as little as 2.5 microM ziram decreased the ability of NK cells to lyse target tumor cells, which persisted up to 6 days following exposure. The loss of lytic function for from 24 h to 6 days following exposure was accompanied by a comparable loss of NK capacity to bind tumor cells. Exposure to 10 microM PCP for 1 h caused a progressive loss (greater than 80%) of lytic function within 6 days of exposure. In contrast to ziram, PCP exposure caused no accompanying loss of binding function.

Effects of in vitro exposure to low levels of organotin and carbamate pesticides on human natural killer cell cytotoxic function.

Human natural killer (NK) lymphocytes play a central role in immune system defense against viral infection and against the formation of primary tumors. Organotin (OT) pesticides have been used in industrial and agricultural applications, and OT contamination has been reported in water, sediment, and fish. Carbamate pesticides are currently used in agricultural chemicals. Two specific carbamates used in agriculture are ziram and maneb; ziram also is used as an additive in rubber products including latex gloves. In previous studies we demonstrated that at concentrations in the 150-200 nM range, the OTs tributyltin (TBT) and triphenyltin (TPT) were capable of disrupting the function of human NK cells after incubations to as short as 24 h. Previously, we also examined the effects of ziram and maneb at higher concentrations on the cytotoxic function of human NK cells. The current study examined the effects of exposure of up to 6 days to lower concentrations of each of these compounds on the cytotoxic function of NK cells. The OTs were studied at concentrations ranging from 200 to 10 nM; ziram was studied at concentrations of 2.5 microM-125 nM and maneb at concentrations of 10-1 microM. These conditions were studied both in highly purified NK cells and in a mixture of lymphocytes containing both T and NK cells. As little as 25 nM TBT decreased the function of purified NK cells after 24 and 48 h, whereas 10 nM TBT was effective after 6 days. The lowest level of TPT that was effective at 24 h was 50 nM whereas the results after 48 h and 6 days were similar to those seen with TBT. The presence of T lymphocytes diminished the effects of both TBT and TPT on NK cytotoxic function. A concentration of ziram as low as 125 nM produced significant loss of cytotoxic function in highly purified NK cells (65% decrease in function after 6 days). The toxicity of each of the compounds studied increased very significantly with length of exposure.

Risk assessment of dithiocarbamate accelerator residues in latex-based medical devices: genotoxicity considerations.

The Medical Devices Agency (MDA) has investigated potential human health hazards arising from the presence of dithiocarbamate vulcanization accelerators in latex products (mainly gloves). After collection of manufacturer's data on usage and residues of these accelerators, an independent investigation of solvent extractable residues and dithiocarbamate migration into aqueous simulants was commissioned, to complement equivalent "in-house" test data from two major manufacturers. The presence of extractable accelerator residues in commercial products was confirmed. Potential human health hazards associated with dithiocarbamates include genotoxicity and possible carcinogenicity: a review of published data was conducted to evaluate the evidence for this, with particular reference to three zinc dithiocarbamates with significant commercial usage (ZDMC, ZDEC and ZDBC: see Fig. 1). Data gaps were identified, and mutagenicity studies commissioned to fill these. These studies comprised tests both in vitro (bacterial and L5178Y cell gene mutation, cultured lymphocyte chromosome aberration) and in vivo (mouse bone marrow micronucleus, rat liver UDS). It is concluded that ZDMC must be considered a genotoxin (and thus a probable carcinogen): residues of this substance in latex medical devices should be minimized. ZDEC proved genotoxic in vitro but was not clearly genotoxic in vivo, and may have activity intermediate between that of ZDMC and that of ZDBC, which showed at most weak activity in a single in vitro (chromosome aberration) test. It is proposed that the use of ZDBC as a vulcanization accelerator in the manufacture of latex gloves, rather than ZDEC, ZDMC or their precursors, would reduce or remove the health concerns arising from accelerator residues.

Effect of Ziram, Thiram, and Dithane M-45 on bone marrow cells of mice-assessed by micronucleus test.

Micronucleus test is an extensively used protocol to assess the mutagenicity of environmental chemicals. This was developed by Schmid and his co-workers (Matter and Schmid, 1971; Ledebur and Schmid, 1973). The micronucleus test is simple, quick and as sensitive as the chromosome aberration analysis. It is based on the principle that during anaphase, acentric chromatid and chromosome fragments lag behind, where as centric elements move towards the spindle pole. After telophase both the undamaged chromosomes and the centric fragments give rise to the daughter nuclei. The lagging elements are transferred into one or several secondary nuclei, which are as a rule much smaller than the main nucleus, and therefore called micronucleus (Schmid, 1973). The clastogenic effect of various chemicals is measured by micronucleus test. Erythrocytes are two types, the younger ones are polychromatic erythrocytes (PCE), which stain bluish and the older, the normo chromatic erythrocytes (NCE) which stain reddish. A few hours after the completion of last mitosis the erythroblasts expel their nucleus for unknown reasons and the micronucleus alone remains in the cytoplasm of the Polychromatic erythrocytes, and they are easily recognisable. Erythrocyte micronucleus represents the consequence of chromosomal aberrations induced during preceding mitotic division of erythrocytes (Matter and Grauwiler, 1974).

Genotoxicity of dithiocarbamates and their metabolites.

Dithiocarbamate fungicides are widely used in agriculture for protection of vegetable crops and seeds. The mutagenicity spectra of ziram, thiram, zineb S-65 and ETU were determined by employing a battery of test systems included the bacterium Salmonella typhimurium (strains TA98, TA100, TA102, TA104, TA1535, TA1538), the yeast Saccharomyces cerevisiae (strain D61.M) and the shallot Allium ascalonicum somatic cells. Plate incorporation assay with S. typhimurium demonstrated direct mutagenicity of ziram in TA100 and thiram in TA100 and TA98 whereas zineb S-65 and ETU were ineffective. Tests for mitotic chromosome malsegregation in S. cerevisiae D61.M gave positive results with thiram, zineb S-69 and ETU. In shallot somatic root-tip cells ziram, thiram and ETU induced different genetic damages e.g. mitotic disturbance, polyploidy and micronuclei.

Clastogenic effects of the dithiocarbamate fungicides thiram and ziram in Chinese hamster cell lines cultured in vitro.

We report here the results obtained using the dithiocarbamate fungicides thiram and ziram to investigate the induction of chromosomal aberrations (CAs) in Chinese hamster ovary (CHO) cells both in the absence and presence of S9 metabolism, and in a Chinese hamster epithelial liver (CHEL) cells which retain metabolic competence to activate different classes of promutagens/procarcinogens. Both thiram and ziram proved to be strong chromosome breaking agents in the CHEL cells and CHO cells in the presence of S9 metabolism. These findings suggest that thiram and ziram require metabolic conversion to become genetically active, and corroborate the evidence that CHEL cells are suitable to activate and detect a broad spectrum of chemical procarcinogens including these two pesticides.

Toxicological effects of ziram, thiram, and dithane M-45 assessed by sperm shape abnormalities in mice.

The three commonly used dithiocarbamate fungicides ziram, thiram, and dithane M-45 were investigated for their mutagenic and carcinogenic potency using sperm shape abnormalities in mice. The fungicides were administered intraperitoneally in single and cumulative doses. All three of the fungicides tested were found to induce significant increase in the frequency of abnormal sperm at all the doses, and a linear dose effect was observed.

Further in vitro and in vivo mutagenicity assays with thiram and ziram fungicides: bacterial reversion assays and mouse micronucleus test.

The fungicides thiram and ziram have been assayed in a battery of nine bacterial strains of different genetic specificity. The results obtained suggest the induction of excisable DNA lesion(s), and indicate similar mutability of strains with AT or GC base pairs at target sites. This mutagenic profile is clearly distinct from that of oxidative mutagens, and it does not support the proposed role of oxidative stress in the mechanism of dithiocarbamates mutagenicity in bacteria. Furthermore, the bone marrow micronucleus test has been carried out in B6C3F1 mice with intraperitoneal administration of high grade thiram (12.5-50 mg/kg) and ziram samples (2.5-10 mg/kg in males, and 5-20 mg/kg in females). Thiram produced a significant increase of micronucleated PCEs in male mice sampled 48 h after treatment with 25, 37.5, and 50 mg/kg. No significant increase was detected in treated females. Ziram, tested in a lower range of doses because of its higher toxicity, resulted negative in both sexes. Both the acute toxicity and the ratio polychromatic/normochromatic erythrocytes indicated some sex specificity in the toxic effects induced by these dithiocarbamates in the B6C3F1 mouse.

Effect of pretreatment with hepatic mixed-function oxidase inducers on the genotoxicity of four rat carcinogens in the hepatocyte/DNA repair assay.

Four genotoxic rat carcinogens, previously reported as negative in the standard hepatocyte/DNA repair assay, were studied in hepatocyte cultures derived from the livers of rats pretreated with the hepatic mixed-function oxidase (MFO) inducers, Aroclor (ARO) and 3-methylcholanthrene (3-MC). Cytembena and dimethyl hydrogen phosphite induced positive dose-dependent DNA repair responses in ARO- and 3-MC-pretreated cultures, while 2,4-, 2,6-toluene diisocyanate, and ziram were negative for unscheduled DNA synthesis in cultures from both pretreatments. The results from this study indicate that pretreatment with hepatic MFO inducers lowers the false-negative detection rate of various genotoxic rat carcinogens and other organ-specific genotoxins and significantly increases the sensitivity of the in vitro rat hepatocyte/DNA repair assay.

Cytogenetic effects of Cuman L, a dithiocarbamate fungicide.

Cuman L, a dithiocarbamate fungicide, was assessed for its effects in the germ cells and the bone marrow erythrocytes of Swiss Albino male mice. The 3 sublethal doses of 350, 700 and 1050 mg/kg b.w. of Cuman L induced a significant (P less than 0.01) increase in the number of chromosomal aberrations in the germ cells. A significant increase (P less than 0.01) in the percentage of micronuclei in the erythrocytes was also induced by the three doses.

Genotoxicity studies with a blend of zinc dialkyldithiophosphate lubricant additives.

The mutagenic activity of a blend of primary zinc dialkyldithiophosphate lubricant additives suspended in process oils and a blend of the process oils alone was investigated in agar layer cultures of Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100, both with and without the incorporation of a rat liver microsomal activation system (S9). Zinc dimethyldithiocarbamate was tested as a structurally-related model bacterial mutagen, and, in additional control experiments, the mutagenic activity of zinc dimethyldithiocarbamate and benzo[a]pyrene was investigated in combination with the blend of process oils in selected bacterial tester strains. Transformation frequencies of BHK cells were determined by colony growth in soft agar culture following treatment with a blend of the zinc dialkyldithiophosphate lubricant additives suspended in process oils, a blend of the process oils, 7,12-dimethylbenzanthracene, both in the presence and in the absence of a blend of the process oils, or zinc dimethyldithiocarbamate. All experiments incorporated a rat liver microsomal activation system (S9). Application of a blend of the zinc dialkyldithiophosphate additives or a blend of the process oils used in their manufacture did not increase the reverse mutation frequencies of Salmonella typhimurium TA1535, TA1537, TA1538, TA98 or TA100, or significantly increase the transformation frequency of BHK cells under the experimental conditions described. Zinc dimethyldithiocarbamate increased the reverse mutation frequency in some bacterial tester strains, but did not significantly increase the transformation frequency of BHK cells under the described experimental conditions. The addition of the blend of the process oils in combination with the control materials, zinc dimethyldithiocarbamate or benzo[a]pyrene had an inhibitory effect on the mutagenic activity at high doses of each in the bacterial assays, and in the BHK assay the transforming ability of 7,12-dimethylbenzanthracene was suppressed in the presence of the blend of the process oils. Thus, the additive materials showed no evidence of genotoxic activity in the bacterial mutation assays, or in the BHK transformation assay under the experimental conditions described.