Thiram Toxicity Research Articles

Resveratrol alleviates thiram-induced tibial dyschondroplasia by regulating BMP-2, RUNX-2 and HIF-1α expressions in broiler chickens

Tetramethylthiuram disulfide (thiram) is a pesticide and fungicide, widely used in agriculture, and is a recognized cause of tibial dyschondroplasia (TD), a skeletal abnormality in fast-growing broiler chickens. This study aimed to investigate the effect of resveratrol (RSV) against TD induced by thiram in broilers. A total of 240 broilers were evenly divided into three groups (n = 80) i.e., Control, TD and RSV. All groups received feed and water ad libitum, whereas the TD and RSV groups received standard diet with an addition of thiram (50 mg/kg of feed) from 4–7 days to induce TD. After the cessation of thiram on day 8, the RSV group was administered RSV at 400 mg/kg body weight orally from 8–18 days. Histopathological changes in tibia were assessed using hematoxylin and eosin (H&E) staining. In addition, the mRNA and protein expressions of targeted genes were analyzed using RT-qPCR, immunohistochemistry, and western blotting. Thiram feeding resulted in an increased growth plate (GP) width, decreased vascularization, irregular shaped chondrocytes with pyknotic nuclei, lameness, poor production performance, reduced levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), alkaline phosphatase (ALP) and elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA). The mRNA and protein expressions of BMP-2 and RUNX-2 were downregulated, whereas HIF-1α was upregulated in TD affected broilers (p < 0.05). RSV administration resulted in a significant increase in mRNA and protein levels of BMP-2 and RUNX-2 (p < 0.05) and a decrease in the levels of HIF-1α. In addition, RSV improved vascularization, angiogenesis, alleviated lameness, and enhanced production efficiency. Furthermore, it restored the GP width, serum biochemical levels and liver antioxidant enzymes. In conclusion, this study indicated that resveratrol mitigated the adverse effects of thiram toxicity in broiler chickens.

Baicalin inhibits apoptosis and enhances chondrocyte proliferation in thiram-induced tibial dyschondroplasia in chickens by regulating Bcl-2/Caspase-9 and Sox-9/Collagen-II expressions

Avian tibial dyschondroplasia (TD) is a skeletal disease affecting fast growing chickens, resulting in non-mineralized avascular cartilage. This metabolic disorder is characterized by lameness and reduced growth performance causing economic losses. The aim of this study was to investigate the protective effects of baicalin against TD caused by thiram exposure. A total of two hundred and forty (n = 240) one day-old broiler chickens were uniformly and randomly allocated into three different groups (n = 80) viz. control, TD, and baicalin groups. All chickens received standard feed, however, to induce TD, the TD and baicalin groups received thiram (tetramethylthiuram disulfide) at a rate of 50 mg/kg feed from days 4–7. The thiram induction in TD and baicalin groups resulted in lameness, high mortality, and enlarged growth-plate, poor production performance, reduction in ALP, GSH-Px, SOD, and T-AOC levels, and increased AST and ALT, and MDA levels. Furthermore, histopathological results showed less vascularization, and mRNA and protein expression levels of Sox-9, Col-II, and Bcl-2 showed significant downward trend, while caspase-9 displayed significant up-regulation in TD-affected chickens. After the TD induction, the baicalin group was orally administered with baicalin at a rate of 200 mg/kg from days 8–18. Baicalin administration increased the vascularization, and chondrocytes with intact nuclei, alleviated lameness, decreased GP size, increased productive capacity, and restored the liver antioxidant enzymes and serum biochemical levels. Furthermore, baicalin significantly up-regulated the gene and protein expressions of Sox-9, Col-II, and Bcl-2, and significantly down-regulated the expression of caspase-9 (p < 0.05). Therefore, the obtained results suggest that baicalin could be a possible choice in thiram toxicity alleviation by regulating apoptosis and chondrocyte proliferation in thiram-induced tibial dyschondroplasia.

Ginsenoside Rg1 regulates thiram-induced chondrocytes' apoptosis and angiogenesis in broiler chickens.

Tibial dyschondroplasia (TD) is a developmental cartilaginous disease due to thiram toxicity. The abnormity of chondrocytes and insufficient angiogenesis within the growth plate are the major factors leading to the occurrence of TD in most cases. In the current study, we evaluated the beneficial effects of ginsenoside (Rg1) against thiram-induced TD for knowing the possible underlying mechanisms in broiler chickens through in vivo and in vitro assessment. Arbor acres broilers (1-day-old, n = 120) were randomly divided for the in vivo evaluation. The control broilers were fed under normal conditions during the whole experiment cycle (18days). The TD broilers were fed with 50mg/kg thiram, while the treatment group was given 40mg/kg of Rg1. According to our findings, thiram caused a decrease in production performance and tibia parameters (p < 0.05), which were significantly reversed by Rg1 administration. In addition, the results from the histological evaluation showed that the proliferative zone had a smaller number of blood vessels, surrounded by inviable chondrocytes, proving apoptosis during the occurrence of TD, while Rg1 treatment significantly increased blood vessels and decreased apoptotic cells. Furthermore, it was found that Rg1 effectively ameliorated the angiogenesis by regulation of HIF-1α/VEGFA/VEGFR2 signaling pathway and the chondrocytes' apoptosis via the mitochondrial pathway. Hence, these findings suggest that Rg1 might be a perfect choice in the prevention and treatment of TD via regulating chondrocytes apoptosis and angiogenesis. Also, it might be a potential therapeutic drug for humans to overcome different bone disorders, involving chondrocytes.

Fabrication of a label-free electrochemical cell-based biosensor for toxicity assessment of thiram

Thiram has been widely used in agriculture and may invades the food chain, posing a threat to human health. In this research, a label-free electrochemical cell-based biosensor was presented for in vitro toxicity assessment of thiram. HepG2 cells were cultured on poly-l-lysine@gold nano-flowers functionalized indium tin oxide coated glass electrode (PLL@AuNFs/ITO) to serve as biorecognition elements. AuNFs were electrodeposited on ITO to provide an enlarged specific surface area and benefited the output signal amplification. PLL was selected as an effective biocompatible coating material to facilitate cell adhesion and proliferation, thereby realizing one-step recording of electrochemical signals from thiram-treated cells. With the aid of the differential pulse voltammetry method, the fabricated biosensor was applied to assess the cytotoxicity of thiram. Results showed that the cytotoxicity measured by the fabricated biosensor exhibited a linear relationship related to thiram concentration ranging from 5 to 50 μM with a detection limit of 2.23 μM. The IC50 of thiram obtained by the biosensor was 29.5 μM, which was close to that of conventional MTT assay (30.8 μM). The effects of thiram on HepG2 cells were also investigated via SEM and flow cytometry. Meanwhile, the proposed biosensor was used to evaluate the toxicity of thiram in fruit samples. Results indicated that the toxicity of thiram cannot be ignored even at a low residual concentration in food (≤5 mg/kg). In conclusion, the developed sensor showed excellent sensitivity, stability, and reliability, which provided a great capacity for the convenient toxicity evaluation of thiram residue in food.

Chlorogenic acid suppresses miR-460a in the regulation of Bcl-2, causing interleukin-1β reduction in thiram exposed chondrocytes via caspase-3/caspase-7 pathway

BackgroundApoptosis is thought to be involved in all processes, including normal cell cycle, immune system, atrophy, embryonic development, and chemical-induced cellular damage. However, if the normal apoptotic process fails, the results might be disastrous, e.g., chondrocytes damage in tibial dyschondroplasia (TD). TD is a worldwide issue in the poultry sector due to thiram toxicity. Thiram (Tetramethyl thiuram disulfide) is a dithiocarbamate pesticide and fungicide commonly used in horticulture to treat grains meant for seed protection and preservation. PurposeAccording to prior studies, chlorogenic acid (CGA) is becoming essential for regulating apoptosis. But still, the specific role of CGA in chondrocyte cells remains unclear. The present study explored the molecular mechanism of CGA on chondrocytes' apoptosis with B-cell lymphoma 2 signaling under the effect of miR-460a. MethodsAn in vivo and in vitro study was performed according to our previously developed methodology. Flow cytometry, western blotting, reverse transcription-quantitative polymerase chain reaction, and immunofluorescence assay were used to investigate the involvement of apoptosis and inflammasome related pathways. ResultsThe CGA decreased the apoptosis rate with the deactivation of miR-460a, accompanied by the activation of Bcl-2. The high expression of miR-460a reduced the cell viability of chondrocytes in vitro and in vivo, that led to the interleukin-1β production. While the apoptotic executioners (caspase-3 and caspase-7) acted upstream in miR-460a overexpressing cells, and its depletion downgraded these executioners. The CGA administrated cells negatively regulated miR-460a expression and thus indicating the deactivation of the apoptotic and inflammasome related pathways. ConclusionChlorogenic acid had a negative effect on miR-460a, setting off specific feedback to regulate apoptotic and inflammasome pathways, which might be a key feature for chondrocytes' survival.

Residue of thiram in food, suppresses immune system stress signals and disturbs sphingolipid metabolism in chickens

Thiram, a well-known sulfur containing organic compound is frequently and extensively used in agriculture because of high biological activity to control different pests. In certain cases, due to long persistence in the environment pesticides and other environmental contaminants induce undesirable toxic impacts to public health and environment. To ascertain the potential mechanisms of toxicity of thiram on different immune organs of broilers, a total of 100 one-day-old chicks were obtained and randomly divided into two groups including thiram group (50 mg/kg) and untreated control group. Thymus and spleen tissues were collected at the age of 14 days from the experimental birds. At necropsy level, thymus was congested, enlarged and hyperemic while spleen had no obvious lesions. The results on mechanisms (apoptosis and autophagy) of immunotoxicity showed significantly increased expression of bax, caspase3, cytc, ATG5, beclin1 and p62 in spleen of treated mice. Results indicated significantly decreased expression of m-TOR and bcl2 to activate apoptosis and autophagy. The expressions of bax, p53 and m-TOR were up-regulated in the thymus while the expressions of ATG5 and Beclin1 were down-regulated to mediate cell apoptosis and inhibit autophagy. The results on different metabolome investigation showed that the sphingolipid metabolism in the thymus of chicks exposed to thiram was disrupted resulting in up-regulation of metabolites related to cell membrane components such as SM, galactosylceramide and lactosylceramide. The results of our experimental research suggest that thiram can interfere with the sphingolipid metabolism in thymus and angiogenesis, inhibit the proliferation of vascular endothelial cells to induce potential toxic effects in chicken.

Cluster of differentiation 147 (CD147) expression is linked with thiram induced chondrocyte’s apoptosis via Bcl-2/Bax/Caspase-3 signalling in tibial growth plate under chlorogenic acid repercussion

Tibial dyschondroplasia (TD) is a metabolic disease of young poultry that affects bone andcartilage's growth. It mostly occurs in broilers due to thiram toxicity in the feed. In this disease, tibial cartilage is not yet ripe for ossification, but it also results in lameness, death, and moral convictions of commercial poultry due to numerous apoptotic changes on cell level. These changes serve a cardinal role in this situation. Many potential problems indicate that chlorogenic acid (CGA) performs an extensive role in controlling apoptosis's perception. However, the actual role of CGA in TD affected chondrocytes in-vitro is still unidentified. The current study investigates the imperceptible insight of CGA on chondrocyte’s apoptosis via B-cell lymphoma 2 (Bcl-2), Bcl-2 associated x-protein (Bax), and Caspase-3 with CD147 signalling. The expression of these markers was investigated by Immunofluorescence, western blot analysis, and reverse transcription-quantitative polymerase chain (RT-qPCR). Chondrocytes from the growth plate of tibia were isolated, cultured, and processed. A sub-lethal thiram (2.5 μg/mL) was used to induce cytotoxicity and then treated with an optimum dose (40 μg/ mL) of CGA. According to the results, thiram distorted chondrocyte cells with enhanced apoptotic rate. But, in case of CGA, high expression of CD147 enhanced cell viability of chondrocytes, accompanied by downregulation of Bax/Caspase-3 signalling with the upregulation of Bcl-2. The first possibility has ruled out in the present study by the observation that the cells apoptosis marker, Caspase-3 showed a significant change in CD147 overexpressing cells. Conversely, immunodepletion of CD147 with enhanced cleavage of Caspase-3, indicating the activation of apoptosis in chondrocytes cells. Therefore, these findings suggest a novel insight about CD147 in thiram induced TD about the regulation of Bcl-2/Bax/Caspase-3 apoptosis-signalling axis.

Puerarin enhance vascular proliferation and halt apoptosis in thiram-induced avian tibial dyschondroplasia by regulating HIF-1α, TIMP-3 and BCL-2 expressions.

Tetramethyl thiuram disulfide (thiram) is a dithiocarbamate pesticide used for crop protection and storage. But, it's widespread utilization is associated with deleterious growth plate cartilage disorder in broilers termed as avian tibial dyschondroplasia (TD). TD results in non-mineralized and less vascularized proximal tibial growth plate cartilage causing lameness and poor growth performance. This study investigated the therapeutic potential of puerarin against thiram toxicity in TD affected chickens. One-day-old broiler chickens (n=240) were alienated into three equal groups i.e. control, TD and puerarin (n=80) and were offered standard feed. Additionally, TD and puerarin groups were offered thiram at 50mg/kg of feed from 4 to 7 days for TD induction followed by puerarin therapy at 120mg/kg to puerarin group only from 8 to 18 days for TD treatment. Thiram feeding to TD and puerarin group chickens caused lameness, mortality, and increased the aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA) levels and growth plate (GP) size and upregulated HIF-1α expression. Besides, the production parameters, alkaline phosphatase (ALP), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels and the expressions of TIMP-3 and BCL-2 were decreased (p<0.05). Puerarin alleviated lameness, enhanced angiogenesis and growth performance and serum and antioxidant enzymes, decreased apoptosis and recuperated GP width by significantly downregulating HIF-1α and upregulating the TIMP-3 and BCL-2 mRNA and protein expressions in puerarin group chickens (p<0.05). In conclusion, the toxic effects associated with thiram can be mitigated using puerarin.

Identification and expression analysis of microRNAs in tibial growth plate of chicken through thiram toxicity.

Thiram is a widely known tibial dyschondroplasia (TD) inducer. TD, a common metabolic cartilage disease, presents in rapidly growing poultry birds. There are evidences that miRNAs are involved in diverse aspects of normal skeletal development, but very less is known about the role of miRNAs in TD. Therefore, this study aimed to determine which genes and pathways show differential expression between TD suffered chickens and normal chickens. We collected growth plates from ten-days-old TD chickens and control chickens and performed high-throughput RNA sequencing (RNA-Seq). Afterwards, target prediction, GO annotation and KEGG pathway analysis were carried out to understand the role of DEMs (differentially expressed microRNAs). We obtained 96,884,760 and 94,574,290 clean reads and identified 17 significant DEMs between the TD and control groups. Functional enrichment analysis of DEMs indicated that the putative targets of miRNAs were remarkably enriched in bone-related pathways, such as Notch, MAPK and Autophagy. Overall, this study provides detailed understanding about the pathogenesis of thiram induced TD and new insights towards the molecular mechanism of miRNAs.

Plastrum Testudinis Extract Mitigates Thiram Toxicity in Broilers via Regulating PI3K/AKT Signaling.

Tibial dyschondroplasia (TD) negatively affects broilers all over the world, in which the accretion of the growth plate (GP) develops into tibial proximal metaphysis. Plastrum testudinis extract (PTE) is renowned as a powerful antioxidant, anti-inflammatory, and bone healing agent. The current study was conducted to evaluate the efficacy of PTE for the treatment of thiram-induced TD chickens. Broilers (day old; n = 300) were raised for 3 days with normal feed. On the 4th day, three groups (n = 100 each) were sorted, namely, the control (normal diet), TD, and PTE groups (normal diet+ thiram 50 mg/kg). On the 7th day, thiram was stopped in the TD and PTE group, and the PTE group received a normal diet and PTE (30 mg/kg/day). Plastrum testudinis extract significantly restored (p < 0.05) the liver antioxidant enzymes, inflammatory cytokines, serum biochemicals, GP width, and tibia weight as compared to the TD group. The PTE administration significantly increased (p < 0.05) growth performance, vascularization, AKT (serine/threonine-protein kinase), and PI3K expressions and the number of hepatocytes and chondrocytes with intact nuclei were enhanced. In conclusion, PTE has the potential to heal TD lesions and act as an antioxidant and anti-inflammatory drug in chickens exposed to thiram via the upregulation of AKT and PI3K expressions.

Salicylic acid alleviates thiram toxicity by modulating antioxidant enzyme capacity and pesticide detoxification systems in the tomato (Solanum lycopersicum Mill.)

In this study, we investigated how 6.6 mM thiram induces to stress response in tomato and evaluated the possible protective role of different concentration of salicylic acid (0.01, 0.1 and 1 mM SA) against thiram toxicity by analyzing tomato leaf samples taken on the 1st, 5th, 11th day of the treatment. The thiram treatment resulted in oxidative stress through an increase in hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels in a time-dependent manner and led to a decline in the total chlorophyll and carotenoid levels. However, thiram-treated plants induced antioxidant enzyme activities, including catalase (CAT; EC 1.11.1.6), glutathione reductase (GR; EC 1.6.4.2), and ascorbate peroxidase (APX; EC 1.11.1.11), as well as pesticide detoxification enzymes such as peroxidase (POX; EC 1.11.1.7) and glutathione S-transferase (GST; EC.2.5.1.18). In addition, three genes (GST1, GST2, GST3) that encode for glutathione S-transferase and one gene (P450) that encodes for cytochrome P-450 monooxygenases were upregulated. SA showed a positive effect on the plants treated with thiram thanks to the decrease in the H2O2 and MDA levels, the enhancement of photosynthetic pigments, and the regulation in antioxidant enzyme activities in the tomato leaves. In addition, the SA-pretreatment triggered the activity and expression of pesticide detoxification enzymes in the thiram-treated leaves. Particularly the pretreatment with 1 mM SA significantly improved the activity of GST and led to the upregulation of GST1, GST2, GST3, and P450 expression levels. These results indicate that the application of thiram fungicide causes toxicity; however, the damaging effect could be mitigated through pretreatment with SA.

The case for thyroid disruption in early life stage exposures to thiram in zebrafish (Danio rerio)

Thiram, a pesticide in the dithiocarbamate chemical family, is widely used to prevent fungal disease in seeds and crops. Its off-site movement to surface waters occurs and may place aquatic organisms at potential harm. Zebrafish embryos were used for investigation of acute (1 h) thiram exposure (0.001–10 µM) at various developmental stages. Survival decreased at 1 µM and 10 µM and hatching was delayed at 0.1 µM and 1 µM. Notochord curvatures were seen at 0.1 and 1 μM thiram when exposure was initiated at 2 and at 10 hpf. Similar notochord curvatures followed exposure to the known TPO inhibitor, methimazole (MMI). Changes were absent in embryos exposed at later stages, i.e., 12 hpf. In embryos exposed to 0.1 or 1 μM at 10 hpf, levels of the thyroid enzyme, Deiodinase 3, increased by 12 hpf. Thyroid peroxide (TPO), important in T4 synthesis, decreased by 48 hpf in embryos exposed to 1 µM at 10 hpf. Thiram toxicity was stage-dependent and early life stage exposure may be responsible for adverse effects seen later. These effects may be due to impacts on the thyroid via regulation of specific thyroid genes including TPO and Deiodinase 3.

Effect of glutathione depletion on apoptosis induced by thiram in Chinese hamster fibroblasts

Fungicide thiram, which is also known as an inducer of allergic contact dermatitis (ACD), was used as a model compound of thiuram chemicals, and its cellular effects were investigated in cultured Chinese hamster V79 cells. The level of intracellular reduced glutathione (GSH), protein sulfhydryl (PSH) groups, protein carbonyls (PC), membrane lipid peroxidation reflected by enhanced thiobarbituric acid reactive substrates (TBARS) production, as well as apoptotic effect were determined. The apoptosis induction was determined by assessing DNA fragmentation by TUNEL, annexin V binding, and caspases activation assays, using fluorescent microscope or flow cytometry, respectively. The concentrations of thiram required to induce cellular GSH depletion (by 40–50%), protein, and membrane lipid peroxidation (2-fold, and 1.7-fold, respectively), as well as to induce apoptosis in V79 Chinese hamster fibroblasts without causing necrosis through cytotoxic effects were between 50–100 μM. To investigate the role of decreased GSH content in the toxicity of thiram, GSH level was modified prior to exposure. Pretreatment of V79 cells with N-acetyl- l-cysteine (NAC), a GSH biosynthesis precursor, prevented GSH decrease, PC and TBARS production, as well as caspases activation induced by thiram exposure. On the other hand, thiram effects were enhanced by the previous depletion of cellular GSH by l-buthionine-(S,R)-sulfoximine (BSO).

Thiram-induced cytotoxicity is accompanied by a rapid and drastic oxidation of reduced glutathione with consecutive lipid peroxidation and cell death

The toxic effect of thiram, a widely used dithiocarbamate fungicide, was investigated in cultured human skin fibroblasts. Cell survival assays demonstrated that thiram induced a dose-dependent decrease in the viable cell recovery. Thiram exposure resulted in a rapid depletion of intracellular reduced glutathione (GSH) content with a concomitant increase in oxidized glutathione (GSSG) concentration. Alteration of glutathione levels was accompanied by a dose-dependent decrease in the activity of glutathione reductase (GR), a key enzyme for the regeneration of GSH from GSSG. Thiram-exposed cells exhibited increased lipid peroxidation reflected by enhanced thiobarbituric acid reactive substances (TBARS) production, suggesting that GSH depletion and the lower GR activity gave rise to increased oxidative processes. To investigate the role of decreased GSH content in the toxicity of thiram, GSH levels were modulated prior to exposure. Pretreatment of fibroblasts with N-acetyl- l-cysteine (NAC), a GSH biosynthesis precursor, prevented both lipid peroxidation and cell death induced by thiram exposure. In contrast, thiram cytotoxicity was exacerbated by the previous depletion of cellular GSH by l-buthionine-(S,R)-sulfoximine (BSO). Taken together, these results strongly suggest that thiram induces GSH depletion, leading to oxidative stress and finally cell death.

An evaluation of thiram toxicity on cultured human skin fibroblasts

Thiram is widely used in agriculture as a fungicide and, to a lesser extent, as a vulcanizing agent in the rubber industry. In spite of the extensive use of thiram, knowledge on its toxicity and health risk remains limited, and few investigations have been performed to assess specific damage at the cellular and subcellular level. We report here the cytotoxic effects of thiram on cultured human skin fibroblasts. Our results demonstrated that thiram exposure induced a dose- and time-dependent decrease in the viable cell recovery with 100% cell death observed with a concentration of 5.0 mg/l. As judged by morphological changes and biochemical criteria, thiram-mediated cell death was not of the apoptotic but seemed to be of the necrotic type. This cell death was not associated with a modification of gene expression of different constituents of the extracellular matrix. A late increase of lactate production was evident after thiram treatment, suggesting a mitochondrial metabolic pathway dysfunction as reported by other authors using similar compounds. However, this phenomenon appeared as a secondary response to the toxic action of thiram. The cytotoxic effect of thiram is possibly due to an oxidant effect inherent to the structure of thiram and the interaction between thiram and vital cellular molecules.

Testicular Toxicity of Thiram in Rat: Morphological and Biochemical Evaluations.

Tetramethylthiuram disulphide (Thiram), a dithiocarbamate fungicide was administered to male rats through gavage at dose 5, 10 and 25 mg/kg/day for 30, 60 and 90 days. A significant increase in relative testes weight (GSI) was observed at dose (25 mg/kg/d) after 90 days exposure, associated with mild pathomorphological changes. This included degeneration of seminiferous tubules with sloughing of germinal cells and accumulation of edematous fluid in tubular lumen. The activity of testicular enzymes such as lactate dehydrogenase (LDH), alkaline phosphatase (ALP), glucose 6-phosphate dehydrogenase (G-6-PDH) were increased significantly in dose dependent manner. The activities of succinate dehydrogenase (SDH) and acid phosphatase (ACP) decreased significantly while serum cholesterol and testicular free sialic acid were increased at all dose levels. The alterations in the activity of testicular enzymes associated with marked increase in GSI and pathomorphological changes suggest that thiram induces testicular dysfunctions in rats.

Prevention of Thiram-induced dyschondroplasia by copper

Broiler chicks were dietarily intoxicated with Thiram. The toxic effects observed included reduced ceruloplasmin activity and bone strength, and suppressed primary humoral immunity. In addition, Thiram induced a lesion characterized by abnormal cartilage formation in the proximal end of the tibiotarsus known as tibial dyschondroplasia. The incidence and severity of dyschondroplasia induced by Thiram was reduced by supplementing the diet with CuSO 4, but not with ZnSO 4 or MnSO 4. However, all three trace elements prevented the adverse effect of Thiram on primary humoral immunity. The prevention of toxicity of Thiram by supplementing the diets with trace elements strongly suggested that either Thiram interfered with the metabolism of trace elements or the trace elements rendered Thiram less toxic. Based on these observations, we conclude that the etiology of Thiram-induced dyschondroplasia is distinct from dyschondroplasia which is not prevented by dietary copper supplementation.

Toxicity of thiram (tetramethylthiuram disulfide) to mink and European ferrets.

Toxicity of thiram (tetramethylthiuram disulfide) to mink and European ferrets.

An example of interaction between environmental pollutants: Modification of thiram toxicity to freshwater organisms by nitrites or nitrates in relation to nitrosamine synthesis

Thiram, a dithiocarbamate fungicide, is known to evolve to dimethylnitrosamine (DMNA) when associated with nitrites. Conditions of appearance of that carcinogenic compound have been studied in short-term experiments by association of the fungicide, nitrates or nitrites, and species representative of freshwater biota. DMNA has been estimated by GLC equipped with a specific detector. Chlorella vulgaris can rapidly produce nitrites from nitrates and DMNA is obtained in presence of thiram. Daphnia magna can also synthesize DMNA but nitrites have to be added to the medium. Increased toxicity of thiram is observed. The same results are obtained on Cyprinus carpio and for a part on Brachydanio rerio. When the species are associated in a 15-day experimental food chain, and intoxicated algae feed the two other levels, no significant transfer is observed. Nevertheless, some DMNA hazard may exist for particular species exposed to thiram associated with nitrites or even nitrates if algae are present.

Thiram toxicity to non-target organisms: a comparative study with protozoan and mammalian cells

The toxicity of the dithiocarbamate fungicide thiram was studied on KB 3 cells (by ATP content) on two strains of vacuole-forming and four strains of non-vacuole-forming mutants of Tetrahymena (by growth rates) and on 33 strains of amoebae (by growth test). The sensitivity range is 0·1 mg litre −1 for mammalian cells, 0.5–1 mg litre −1 for T. thermophila and 2–16 mg litre −1 for amoebae, which are the most resistant organisms. The results suggest that protozoa are probably affected by thiram in soil and when accidental water contamination occurs. The results obtained with vacuoleless mutants of T. thermophila also suggest that the cell membrane is the essential site of uptake of the toxicant.