Facile green approach towards the synthesis of some phenyl piperazine based dithiocarbamates as potent hemolytic and thrombolytic agents.

The facile and efficient protocol for the synthesis of N-phenyl piperazine based di-thio-carbamates has been reported under neat conditions. A library of novel piperazine based di-thio-carbamates (3a-h) in excellent yields has been prepared. Solvent free, catalyst free and easy work up conditions make this protocol an attractive synthetic protocol to achieve novel biologically active di-thio-carbamates. The synthesized molecules have been characterized by FT-IR, 1HNMR and 13CNMR spectroscopic techniques. The pharmacological aspects of these derivatives have been evaluated via hemolysis and thrombolysis. All the target molecules (3a-h) exhibit mild to medium potential as hemolytic and thrombolytic agents. Among the synthesized derivatives, compound 3c showed least cytotoxicity and better thrombolytic potential.

Metam sodium exposure during pregnancy and lactation in mice caused behavioral abnormalities and oxidative stress in offspring.

Metam sodium (MS) is a widespread biocide with a broad-spectrum activity. Here, we addressed the behavioral impact of MS by exposing female mice to 50, 100 and 150 mg/kg of MS during both pregnancy and lactation, and evaluated the oxidative stress as a potential mechanism of MS-induced neurotoxicity. The results showed that MS affected fertility and reproduction parameters as well as some aspects of maternal behavior, especially at high doses. In offspring, MS caused a significant delay in the ontogeny of sensorimotor functions. In addition, treated mice exhibited during adulthood an increase of anxiety-like, depression-like behaviors as well as learning and memory impairment. These alterations were accompanied by an increase of the superoxide dismutase activity, and a significant decreased catalase and malondialdehyde activities in specific brain areas. The present work revealed that early exposure to MS induced sensorimotor and behavioral impairments in offspring likely associated with onset of oxidative stress.

Studies on oral subacute toxicity of cartap in male mice.

S-[3-carbamoylsulfanyl-2-(dimethylamino)propyl] carbamothioate (Cartap) (CAS number: 15263-52-2) is a synthetic insecticide of thiocarbamates group that is extensively used in field of agriculture for controlling of several pests like rice stem borer, leaf folder pests in paddy field and diamond back moth, aphids in cabbage and cauliflower crops. Cartap, as a pesticide has not been investigated yet for its effect on vital organs and biochemical stress in vivo and the present study was undertaken to evaluate the same in Swiss albino mice. For this purpose male mice were given three different dose levels of cartap, i.e. 5 mg/kg, 7.5 mg/kg and 15 mg/kg body weight respectively, for 28 days orally. Water was used as vehicle to dissolve cartap. Oral administration of cartap caused significant increase in serum biomarkers, tissue oxidants and decrease in antioxidants along with histopathological findings in liver, kidney and brain tissues. Thus, present study showed that in vivo exposure to cartap induces tissue damage probably via oxidative stress in important vital organs of mice.

Anti-Inflammatory and Antioxidative Potential of Aloe vera on the Cartap and Malathion Mediated Toxicity in Wistar Rats.

Aloe vera has been the most useful medicinal herb in the world since ancient times due to its vast biological effects. The presence of high content of bioactive compounds make Aloe vera a promising complementary and alternative agent in disease prevention. The effectiveness of A. vera-based medicines against pesticide toxicity has never been evaluated. It was therefore envisaged to develop an A. vera-based strategy to protect the non-target animals from adverse effects of the pesticides. This article illustrates the ameliorating effect of aqueous extract (AE) of A. vera leaves against the cartap and malathion toxicity. To evaluate the protective impact of A. vera against cartap (Ctp), malathion (Mtn) and a mixture of both pesticides, the animals were divided in eight groups, each containing six rats: Group 1- C (control), Group 2- AE + C, Group 3- Ctp, Group 4- Mtn, Group 5- Ctp + Mtn, Group 6- AE + Ctp, Group 7- AE + Mtn, Group 8- AE + Ctp + Mtn. Wistar rats exposed to Ctp, Mtn and Ctp + Mtn, displayed significant change in body weight. It was observed that the WBC level increased significantly in Mtn and Ctp + Mtn challenged groups. The contents of TNF-α and IL-6 in serum increased expressively in the Ctp, Mtn and Ctp + Mtn challenged groups. Rats treated with Ctp, Mtn and Ctp + Mtn displayed significant alterations in the levels of antioxidative indices (MDA, GSH, GST, GPx, SOD and CAT). Significant alterations were recorded in the activities of AST, ALT, ACP and ALP in Ctp, Mtn and Ctp + Mtn challenged groups. The histopathological results of liver supported the biochemical data. The pre-treatment of rats with the aqueous extract of A. vera leaves significantly protected them from the toxicity of pesticides. These results suggested that A. vera extract may be used as a promising natural agent for the management of pesticide induced toxicity.

Orbencarb induces lethality and organ malformation in zebrafish embryos during development.

Thiocarbamates are one of the components of pesticides that target weeds by inhibiting adenosine triphosphate (ATP) synthesis. Orbencarb, one of the isomeric thiocarbamates applied to wheat, maize, and soybean, has been found to have toxic effects on mammals and marine ecosystems. Although the toxicity ranges of orbencarb in different organisms are known, specific studies on the environmental contamination and harmful effects of orbencarb on non-target organisms are scarce. In this study, we observed that orbencarb induced embryotoxicity during zebrafish development as well as apoptosis and reactive oxygen species (ROS) production in the intestine. It was further observed that orbencarb decreased the viability of the embryos and simultaneously affected the heart rate and vessel formation. Orbencarb decreased the mRNA levels of ccnd1, ccne1, cdk2, and cdk6 and induced abnormal development of the eyes, brain, yolk sac, and spinal cord in zebrafish embryos. Orbencarb also hampered vasculogenesis in the zebrafish embryos by inhibiting the mRNA expression of flt1, flt4, kdr, and vegfc. Collectively, these results suggested that orbencarb is embryotoxic and disrupts the normal growth of zebrafish embryos by inducing the generation of ROS and hampering vasculogenesis.

Preliminary Study of Multiple Stress Response Reactions in the Pond Snail Lymnaea stagnalis Exposed to Trace Metals and a Thiocarbamate Fungicide at Environmentally Relevant Concentrations.

Gastropod mollusks have achieved an eminent importance as biological indicators of environmental quality. In the present study, we applied a multibiomarker approach to evaluate its applicability for the pond snail Lymnaea stagnalis, exposed to common industrial and agricultural pollutants at environmentally relevant concentrations. The snails were exposed to copper (Cu2+, 10 µg L-1), zinc (Zn2+, 130 µg L-1), cadmium (Cd2+, 15 µg L-1), or the thiocarbamate fungicide "Tattoo" (91 µg L-1) during 14 days. Metal treatment and exposure to "Tattoo" caused variable patterns of increase or decrease of metal levels in the digestive gland, with a clear accumulation of only Cd and Zn after respective metal exposure. Treatment with Cu and "Tattoo" caused an increase of cytochrome P450-related EROD activity. Glutathione S-transferase was inhibited by exposure to Cu, Zn, and "Tattoo." Treatment with the "Tattoo" led to an inhibition of cholinesterase activity, whereas Cu and Cd increased its activity. Caspase-3 activity was enhanced by up to 3.3 times in all treatments. A nearly uniform inhibitory effect for oxidative stress response parameters was observed in all kinds of exposure, revealing an inhibition of superoxide dismutase (Mn-SOD) activity, a depression of glutathione (GSH and GSSG) and of protein carbonyl levels. Pollutant-specific effects were observed for the catalase activity, superoxide anion production, and lipid peroxidation levels. Due to the high response sensitivity of Lymnaea stagnalis to chemical impacts, we suggest our study as a contribution for biomarker studies with this species under field conditions.

Prenatal exposure to the pesticide metam sodium induces sensorimotor and neurobehavioral abnormalities in mice offspring.

The present study has investigated developmental neurotoxicity of Metam sodium (MS), from gestational day 6 and throughout the gestation period until delivery. Therefore, mated female mice were orally exposed on a daily basis to 0 (control), 50, 100 or 150 mg of MS/kg of body weight and their standard fertility and reproductive parameters were assessed. The offspring were examined for their sensorimotor development, depression and cognitive performance. Our results showed that MS exposure during pregnancy led to one case of mortality, two cases of abortion and disturbed fertility and reproductive parameters in pregnant dams. In offspring, MS induced an overall delay in innate reflexes and sensorimotor performances. Furthermore, all prenatally treated animals showed an increased level of depression-like behavior as well as a pronounced cognitive impairment in adulthood. These results demonstrated that prenatal exposure to MS causes a long-lasting developmental neurotoxicity and alters a wide range of behavioral functions in mice.

A highly selective and ultrafast near-infrared fluorescent turn-on and colorimetric probe for hypochlorite in living cells.

Hypochlorous acid (HOCl)/hypochlorite (OCl-), important reactive oxygen species, play essential roles in many physiological and pathological progresses. Accordingly, we developed a novel dicyanomethylene-4H-pyran (DCM)-based probe DCM-OCl for colorimetric and near-infrared fluorescent turn-on detection of OCl-. The probe exhibited excellent selectivity and sensitivity for OCl- over other bio-related analytes with a detection limit of 80 nM. The excellent selectivity of DCM-OCl for OCl- was ascribed to specific oxidative cleavage of the dimethylthiocarbamate (DMTC) recognition unit by OCl-. Moreover, DCM-OCl exhibited an ultrafast turn-on response (<3 s) to OCl-, potentially allowing real-time detection of OCl-. Furthermore, DCM-OCl was successfully used to image endogenous/exogenous OCl- in living cells.

In vitro assessment of mechanistic events induced by structurally related chemical rubber sensitizers.

Allergic contact dermatitis (ACD) is one of the most common forms of immunotoxicity, and increased understanding of how chemicals trigger these adverse reactions is needed in order to treat or design testing strategies to identify and subsequently avoid exposure to such substances. In this study, we investigated the cellular response induced by rubber chemicals in a dendritic cell (DC) model, focusing on the structurally similar chemicals diethylthiocarbamylbenzothiazole sulfide and dimethylthiocarbamylbenzothiazole sulfide, with regard to regulation of microRNA, and messenger RNA expression. Only a few miRNAs were found to be commonly regulated by both rubber chemicals, among them miR1973, while the overall miRNA expression profiles were diverse. Similarly, out of approximately 500 differentially regulated transcripts for each chemical, about 60% overlapped, while remaining were unique. The pathways predicted to be enriched in the cell model by stimulation with the rubber chemicals were linked to immunological events, relevant in the context of ACD. These results suggest that small structural differences can trigger specific activation of the immune system in response to chemicals. The here presented mechanistic data can be valuable in explaining the immunotoxicological events in DC activation after exposure to skin sensitizing chemicals, and can contribute to understanding, preventing and treating ACD.

The PFOS disturbed immunomodulatory functions via nuclear Factor-κB signaling in liver of zebrafish (Danio rerio).

Excessive perfluorooctane sulfonate (PFOS) in natural water ecosystem has the potential to detrimentally affect immune system, but little is known of such effects or underlying mechanisms in fish. In the present study, we evaluated the effects of PFOS on growth performance, organizational microstructure, activities of immune-related enzymes and expressions of immune-related genes in male zebrafish (Danio rerio) exposed to different concentrations of 0, 0.02, 0.04 and 0.08 mg/L of PFOS for 7, 14, and 21 days or cotreatment with PFOS and PDTC to investigate the effects of PFOS on immune system and the potential toxic mechanisms caused by PFOS. The results indicated that PFOS accumulated in livers after exposure, and remarkably elevations were found in three exposure groups compared with the control group at three stages. The growth of the adult zebrafish in the experiments was significantly inhibited, the microstructures of liver were serious damaged. The ROS levels were remarkably increased. The activities of ACP, AKP, and lysozyme were obviously decreased, while the activities of MPO and NF-κB were significantly increased. The expressions of immune-related mRNA were significantly affected. After co-treatment with PFOS and PDTC, the growth inhibition, the morphological damage, the ROS induction, and the expressions of immune-related mRNA were reversed. Taken together, the results indicated that PFOS can significantly inhibit the growth, disturb the immune system by changing the normal structure of liver, the activities of immune-related enzymes, and a series of gene transcriptions involved in immune regulation in liver of male zebrafish. PFOS-induced pro-inflammatory effect of hepatocytes was observed, and the involvement of NF-κB signaling pathway was participated in its action mechanism. These findings provide further evidence that PFOS interferes with the immune regulation of liver of male zebrafish under in vivo conditions.

Discovery of 2,4-diarylaminopyrimidine derivatives bearing dithiocarbamate moiety as novel FAK inhibitors with antitumor and anti-angiogenesis activities.

A series of 2,4-diarylaminopyrimidine derivatives containing dithiocarbamate moiety were designed by molecular hybridization strategy and synthesized for screening as inhibitors of focal adhesion kinase (FAK). Most of these compounds exhibit significant antiproliferative activities on human cancer cell lines expressing high levels of FAK at nanomolar concentrations. The compound 14z was identified as the most potent FAK inhibitor among these candidates. 14z has excellent anti-proliferative effect with IC50 values from 0.001 µM to 0.06 µM on HCT116, PC-3, U87-MG and MCF-7 cell lines and relatively less cytotoxicity to a nonmalignant cell line MCF-10A compared with MCF-7 cells (SI value > 10). 14z also exhibits significant FAK inhibitory activity (IC50 = 0.07 nM). In addition, compound 14z causes cell cycle arrest at G2/M and prompted apoptosis in both HCT116 and MCF-7 cells in a dose-dependent manner. Further studies show that compound 14z inhibits migration of MCF-7 and has anti-angiogenesis effect on HUVEC cells.

Endpoint sensitivity in Amphibian Metamorphosis Assay.

The Amphibian Metamorphosis Assay (AMA) is a screening test for detecting chemicals with thyroid activity. There is little experience in data interpretation and in using AMA data for screening, testing and identifying endocrine disruptors. To investigate the sensitivity of different endpoints of the AMA, the publically available data for 57 thyroid active and inactive chemicals were compiled and analyzed. Endpoints body weight and length appeared as sensitive as apical thyroid responsive endpoints hind limb length (HLL) and developmental stage (DS) for 12 thyroid active chemicals. The sensitivity of body weight, length and HLL was comparable, which is higher than that of DS for 45 thyroid inactive chemicals. The decision logic of the AMA suggests that an advanced development alone indicates thyroid activity. The analysis here showed that advanced development at day 7 could indicate thyroid activity of a chemical. However, advanced development at day 21 may be influenced by thyroid inactive chemicals. Among 39 thyroid inactive chemicals, which affected one or more endpoints, 33% and 77% induced changes in HLL and/or DS at day 7 and 21, respectively; only 10% influenced thyroid histology. These results showed that apical thyroid responsive endpoints HLL and DS are influenced by thyroid active chemicals as well as thyroid inactive chemical. Both endpoints should be combined with thyroid histology for the identification of thyroid active chemicals. The use of the AMA in a testing strategy to identify chemicals with thyroid activity is discussed.

Coumarin-dithiocarbamate hybrids as novel multitarget AChE and MAO-B inhibitors against Alzheimer's disease: Design, synthesis and biological evaluation.

A series of new coumarin-dithiocarbamate hybrids were designed and synthesized as multitarget agents for the treatment of Alzheimer's disease. Most of them showed potent and clearly selective inhibition towards AChE and MAO-B. Among these compounds, compound 8f demonstrated the most potent inhibition to AChE with IC50 values of 0.0068 µM and 0.0089 µM for eeAChE and hAChE, respectively. Compound 8g was identified as the most potent inhibitor to hMAO-B, and it is also a good and balanced inhibitor to both hAChE and hMAO-B (0.114 µM for hAChE; 0.101 µM for hMAO-B). Kinetic and molecular modeling studies revealed that 8g was a dual binding site inhibitor for AChE and a competitive inhibitor for MAO-B. Further studies indicated that 8g could penetrate the BBB and exhibit no toxicity on SH-SY5Y neuroblastoma cells. More importantly, 8g did not display any acute toxicity in mice at doses up to 2500 mg/kg and could reverse the cognitive dysfunction of scopolamine-induced AD mice. Overall, these results highlighted 8g as a potential multitarget agent for AD treatment and offered a starting point for design of new multitarget AChE/MAO-B inhibitors based on dithiocarbamate scaffold.

Toxicity of the biocide polycarbamate, used for aquaculture nets, to some marine fish species.

We investigated toxic effects of the antifouling biocide polycarbamate (PC) on marine fish by conducting acute, early-life stage toxicity (ELS), and embryo toxicity tests. Mummichog (Fundulus heteroclitus) 96-h LC50 values for hatched larvae (body weight about 2.0 mg) and juveniles (660 ±â€¯36 mg) were about 12 and 630 µg/L, respectively. The ELS test using mummichog embryos yielded a lowest-observed-effect concentration of 3.9 µg/L and a no-observed-effect concentration of 2.1 µg/L with growth as the most sensitive endpoint. The embryo toxicity test for spotted halibut (Verasper variegatus) revealed a 10-d EC50 of 8.1 µg/L with abnormality as an endpoint. During the ELS and embryo toxicity tests, morphological abnormalities (notochord undulation) were induced in the embryos. Biochemical and gene-expression analysis suggest that PC-induced morphological abnormalities involve disruption of lysyl oxidase-mediated collagen fiber organization, essential for notochord formation, and inhibition of gene expression related to notochord formation.

Dithiocarbamates: Efficient metallo-ß-lactamase inhibitors with good antibacterial activity when combined with meropenem.

The activity of ß-lactam antibiotics is compromised by metallo-ß-lactamases (MBLs). Herein, a series of dithiocarbamate derivatives were designed and synthesized. Their antibacterial activities were tested in combination with meropenem (MEM) against several MBL (NDM and IMP type)-producing clinical isolates. Clinical isolates harboring NDM-1 and IMP-4 became susceptible to MEM when it was combined with dithiocarbamate compounds 4a, 4b or 4f synthesized in this work. Compounds 4a and 4b increased the effectiveness of MEM by up to 2560 times against strains. In vitro bactericidal dynamics tests showed that bacteria died within 24 h when they were treated with compound 4f + MEM. Compounds 4a, 4b and 4f were non-hemolytic and exhibited low toxicity toward HeLa cells in vitro. These data show that compounds containing dithiocarbamate functional group may be helpful in the development of MBL inhibitors.

Effects of water temperature and light intensity on the acute toxicity of herbicide thiobencarb to a green alga, Raphidocelis subcapitata.

The present study investigated how principal environmental factors such as temperature and light intensity change the toxicological properties of thiobencarb (TB) herbicide to the green alga, Raphidocelis subcapitata. At first, we investigated the inhibitory effect of TB (0, 15.6, 31.2, 62.4, and 125 µg L-1) on growth of R. subcapitata at five temperatures (10, 15, 20, 25, or 30 °C) for 144 h exposure and calculated 72- and 144-h effective concentration values (EC10, 20, and EC50) for growth rate. All EC values significantly decreased with an increasing temperature. The maximum quantum yield of photosystem II in R. subcapitata exposed to 125 µg L-1 of TB was also significantly inhibited with increased temperature. These physiological effects could explain the lower EC values at high temperatures. Then, single and interactive effects of TB, temperature, and light intensity on growth rate were investigated by three-way of analysis of variance. As a result, single and interactive effects were detected in all explanatory variables. These results suggest that temperature and light intensity change the acute toxicity parameter in R. subcapitata exposed to TB and must be considered in evaluating the risk of TB.

Probabilistic dietary risk assessment of triazole and dithiocarbamate fungicides for the Brazilian population.

Residue data for triazoles (TR) and dithiocarbamates (DT) in 30,786 samples of 30 foods were obtained from mainly two national monitoring programs, and consumption data from a national survey conducted among persons aged 10 years or older. About 16% of the samples contained TR, mainly grape (53.5%), and 16.2% contained DT, mainly apple (59.3%). Flusilazole was the index compound used for the acute effects of TR for women of child-bearing-age (cranium-facial malformation and skeletal variation), cyproconazole for the chronic effects of TR (hepatoxicity), and ethylene-bis-dithitiocarbamates (EBDC) for DT (thyroid toxicity). Exposures were estimated using the Monte Carlo Risk Assessment software. Different models were tested, and a Model-Then-Add approach was found to best estimate the chronic exposures to DT and TR. At the 99.9th percentile (P99.9), the cumulative acute TR intakes accounted for up to 0.5% of the flusilazole ARfD, mainly from beans and rice consumption. The chronic TR and DT intakes accounted for 1 and 6.7% of the respective index compound ADIs, with beans and rice accounting for most of the TR intake (∼70%), and apple for about 51-56% of the DT intake. The estimated risks from the exposure to TR and DT indicate no health concern for the Brazilian population.

Sepia Ink Oligopeptide Induces Apoptosis of Lung Cancer Cells via Mitochondrial Pathway.

BACKGROUND/AIMS: Our previous study suggested the anti-tumor activity of sepia ink oligopeptide (SIO). Here we sought to investigate the underlying molecular mechanism. METHODS: Cell proliferation was evaluated by cell counting kit-8 (CCK-8) assay. Cell apoptosis was determined by Annexin V/Propidium Iodide (PI) staining. The mitochondria pathway was characterized by quantification of Bcl-2, Bax, Caspase-9 and Cyto-C. The death receptor pathway was analyzed by determinement of Fas, Caspase-8 and NIK. The endoplasmic reticulum (ER)-dependent pathway was determined by measurement the expression of CHOP, Caspase-12, GRP78 and Calpain. The associated gene expression was quantified by RT-PCR and protein level was determined by immunoblotting. RESULTS: We demonstrated treatment with structurally modified SIO (CSIO, 5 µM) significantly inhibited cell proliferation and induced apoptosis in lung cancer cell line A549. The mitochondrial pathway, death receptor pathway and ER stress induced apoptosis were stimulated upon CSIO treatment. The administration with respective inhibitors including midiv-1 (50 µM for 2 h), PDTC (20 µM PDTC for 30 min) and ALLN (20 mM ALLN for 5 h) readily reversed the apoptosis inducing effect of CSIO. CONCLUSION: Our data demonstrates that CSIO is capable of induction apoptosis in lung cancer cell line, which is mediated by all three classical apoptotic pathways. Our results warrant further in vivo investigations of the anti-tumor potential of CSIO.

Design, synthesis, monoamine oxidase inhibition and docking studies of new dithiocarbamate derivatives bearing benzylamine moiety.

A new series of thirteen 2-[(4-fluorophenyl)(4-nitrobenzyl)amino]-2-oxoethyl-1-substituted-carbodithioate derivatives (4a-4m) were synthesized and tested for their human monoamine oxidase A and B (hMAO-A and hMAO-B) inhibitory potential by an in vitro fluorometric method. Most of the compounds have found to be selective towards MAO-B than MAO-A. Compound 4j that carrying 4-nitrophenyl piperazine moiety, was detected as the most active agent amongst all compounds with the IC50 value of 0.097 ±â€¯0.003 µM for MAO-B while that of selegiline was 0.040 ±â€¯0.002 µM. The enzyme kinetic study reported that compound 4j is a reversible and non-competitive inhibitor. Interaction modes between the hMAO-B and compound 4j were determined by docking studies. The study also revealed that compound 4j has the highest binding scores. Besides, compound 4j has not cytotoxicity at its effective concentration against hMAO-B.

Interaction patterns and toxicities of binary and ternary pesticide mixtures to Daphnia magna estimated by an accelerated failure time model.

Organisms in natural environments are often exposed to a broad variety of chemicals, and the multi-chemical mixtures exposure may produce significant toxic effects, even though the individual chemicals are present at concentrations below their no-observed-effect concentrations. This study represents the first attempt that uses the accelerated failure time (AFT) model to quantify the interaction and toxicity of multi-chemical mixtures in environmental toxicology. We firstly conducted the acute immobilization tests with Daphnia magna exposed to mixtures of diazinon (DZN), fenitrothion (MEP); and thiobencarb (TB) in single, binary, and ternary formulations, and then fitted the results to the AFT model. The 48-h EC50 (concentration required to immobilize 50% of the daphnids at 48h) values for each pesticide obtained from the AFT model are within a factor of 2 of the corresponding values calculated from the single pesticide exposure tests, indicating the methodology is able to provide credible toxicity values. The AFT model revealed either significant synergistic (DZN and MEP; DZN and TB) or antagonistic (MEP and TB) interactions in binary mixtures, while the interaction pattern of ternary mixture depended on both the concentration levels and concentration ratios of pesticides. With a factor of 2, the AFT model accurately estimated the toxicities for 78% of binary mixture formulations that exhibited significant synergistic effects, and the toxicities for all the ternary formulations. Our results showed that the AFT model can provide a simple and efficient way to quantify the interactions between pesticides and to assess the toxicity of their mixtures. This ability may greatly facilitate the ecotoxicological risk assessment of exposure to multi-chemical mixtures.