Stratum corneum levels of inflammatory mediators and natural moisturizing factor in patch test reactions to thiurams and fragrances and their possible role in discrimination between irritant and allergic reactions to hapten mixtures.

BACKGROUND: Patch test (PT) reactions to thiuram mix (TM) and fragrance mix (FM) I or II without concomitant reactions to their single constituents are potentially caused by the irritant properties of the mixes. OBJECTIVE: Comparing inflammatory profiles of PT reactions to TM, FM I, FM II, and their constituents and assessing their potential in discrimination of irritant and allergic reactions. PATIENTS AND METHODS: Levels of 14 cytokines and natural moisturizing factor (NMF) were determined in stratum corneum samples collected from PT reactions to TM, FM I or II, their constituents, and petrolatum (pet.) control sites in 36 individuals. RESULTS: Levels of interleukin (IL)-16, chemokine (CXC motif) ligand (CXCL) 8, CXCL10, chemokine (CC motif) ligand (CCL) 17, and CCL22 were significantly increased in reactions (+, ++) to thiurams and fragrances compared to their petrolatum. controls, except for PT reactions to FM I/II with negative breakdown testing in which, however, decreased levels of NMF were observed. In doubtful reactions to FM I/II with negative breakdown testing, NMF was significantly lower than in petrolatum controls. CONCLUSIONS: PT reactions to thiurams and fragrances indicate a Th2-skewed inflammation. The inflammatory profiles suggest that weak or doubtful FM I/II reactions without accompanying reaction to a constituent were irritant. IL-16 might be suitable to distinguish irritant from allergic reaction.

Experimental exposure of red-legged partridges (Alectoris rufa) to seeds coated with imidacloprid, thiram and difenoconazole.

Pesticide coated seeds are commonly used in agriculture, and may be an important source of food for some birds in times of scarcity, as well as a route of pesticide ingestion. We tested the lethal and sub-lethal effects of treated seed ingestion by the red-legged partridge (Alectoris rufa), a game bird of high socio-economic value in Spain. One year-old partridges (n = 42 pairs) were fed for 10 days in spring (prior to breeding) with wheat treated with difenoconazole (fungicide), thiram (fungicide) or imidacloprid (insecticide), using two doses for each pesticide (the one recommended, and its double to represent potential cases of abuse of pesticides). We investigated the direct and indirect effects on the body condition, physiology, immunology, coloration and subsequent reproduction of exposed partridges. For the latter, eggs were collected, measured and incubated and the growth and survival of chicks were monitored. Thiram and imidacloprid at high exposure doses produced mortalities of 41.6 and 58.3 %, respectively. The first death was observed at day 3 for imidacloprid and at day 7 for thiram. Both doses of the three pesticides caused sublethal effects, such as altered biochemical parameters, oxidative stress and reduced carotenoid-based coloration. The high exposure doses of imidacloprid and thiram also produced a decrease in cellular immune response measured by the phytohemagglutinin test in males. Bearing in mind the limitation of the small number of surviving pairs in some treatments, we found that the three pesticides reduced the size of eggs and imidacloprid and difenoconazole also reduced the fertilization rate. In addition, both thiram and imidacloprid reduced chick survival. These experiments highlight that the toxicity of pesticide-treated seeds is a factor to consider in the decline of birds in agricultural environments.

Changes in antioxidant defense systems induced by thiram in V79 Chinese hamster fibroblasts.

The role of antioxidant defence systems in protection against oxidative damage of lipids and proteins induced by fungicide thiram during in vitro exposure was investigated in cultured Chinese hamster V79 cells with normal, depleted, and elevated glutathione (GSH) levels. We analyzed the catalytic activities of superoxide dismutases (SOD1 and SOD2), Se-dependent and Se-independent glutathione peroxidases (GSH-Px), glutathione reductase (GR), and catalase (CAT), as well as total glutathione/glutathione disulfide ratio (GSH(total)/GSSG). Thiram treatment resulted in an increase in activities of SOD1, Se-dependent GSH-Px, and GR at the highest tested dose (150 microM). On the contrary, inhibition of CAT and Se-independent GSH-Px activities, and no significant changes in the level of SOD2 activity was observed at any tested doses (100-150 microM). GSH(total)/GSSG ratio in the 100 microM thiram treated cells was not significantly changed comparing to the control, despite significant decrease of GSH total (50%). In 150 microM thiram treated cells the ratio falls to 43% of control value. Pretreatment with l-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, significantly enhanced decrease in CAT and Se-independent GSH-Px activities, as well as GSH(total)/GSSG ratio, and reduced Se-dependent GSH-Px activity, following exposure to thiram. Simultaneously, L-BSO pretreatment enhanced increase in SOD1 activity, and had no effect on SOD2, following thiram exposure. Pretreatment with N-acetyl cysteine (NAC), a GSH precursor, prevented enzymatic changes in CAT, Se-dependent GSH-Px, GR, SOD1 activities, and significantly decreased SOD2 activity following exposure to thiram. GSH(total)/GSSG ratio was restored to the control value. This study suggests that following the changes in antioxidant defense systems thiram can act through the production of free radicals.

Rinsing and management of pesticides' containers.

In order to reduce the effects on the environment, it is necessary to improve the management of pesticides' containers. Usually, users burn or bury empty containers. These methods, even though decreasing must be avoided or even forbidden. Since 1996, empty containers are systematically collected in Belgium and are specifically removed by the firm Phytofar Recover created by the Belgian Federation of pesticides' manufacturers. Since the beginning, the recovery rate (percentage of containers recovered compared with the containers sold) goes on increasing to exceed 85% in 2001. These action and results are a world first (more than 500 tons of empty containers are collected yearly). Once collected, empty containers are subjected to the European Policy about toxic wastes since they contained dangerous products. Their removal must follow a specific removal process by incineration at very high temperature (> 1200 degrees C) with a specific filtration of the smoke. The treatment cost is high and reaches 2 Euros per kg of container. If the container is rinsed and the residue does not exceed 1000, 10,000 or 30,000 mg per kg of container (depending on the dangerousness of product: very toxic, corrosive or toxic), it will be considered as domestic waste and will therefore follow a much more economical energy production process. The study aims at determining the quantities of residue contained in empty containers and the parameters reducing the rinsing efficiency: the formulation (EC, WP, WG), the container's size, packaging's type (plastic container or paper bag), the rinsing technique. Almost 150 tests and analyses of residue have been carried out. A manual rinsing procedure has been set up in order to meet the standards about residue. Rinsing three times with an average volume of water (20 to 30%) allows to reach the lowest residue level. As bags containing powder (WG or WP) container not be rinsed, it is necessary to empty them completely. It is however difficult to reach the 1000 ppm residue limit.

Testicular toxicity in rat to repeated oral administration of tetramethylthiuram disulfide (Thiram).

Thiram was administered to male rats through gavage at doses 5, 10 and 25 mg/kg/day for 180 and 360 days. Thiram has caused marginal increase in the relative weight of testes and epididymis and decrease in the weight of seminal vesicle and prostate. Marked degenerative changes were observed in seminiferous tubules together with alterations in testicular enzyme profile. The activity of testicular enzymes such as ACP, SDH and ATPase (Na+ + K+ dependent) was decreased whereas activity of LDH, G-6-PDH and ALP increased. The levels of serum cholesterol and testicular free sialic acid were enhanced, while the level of testicular protein was lowered. It is evident from the present study that long term treatment of thiram at tested dose levels has resulted in dose and time dependent morphological and biochemical changes in testes of rat.

Analysis of micronuclei and DNA single-strand breaks in mouse splenocytes and peripheral lymphocytes after oral administration of tetramethylthiuram disulfide (thiram).

The fungicide thiram (tetramethylthiuram disulfide, TMTD) was administered by repeated oral intubations to groups of male B6C3F1 mice at 100, 300 and 900 mg/kg body weight for 4 consecutive days, or at 300 mg/kg for 8 and 12 days. 24 hr after the last treatment animals were killed, and splenocyte cultures were set up for the analysis of micronuclei by the cytokinesis-block method. DNA single strand breaks (ssb) and alkali labile sites were also analysed by the single cell gel electrophoresis (Comet) assay in splenocytes and lymphocytes of animals receiving the 8- and 12-day treatments. Parallel experiments with human peripheral lymphocytes were carried out to assess the ability of thiram to induce micronuclei and DNA ssb and alkaline labile sites under in vitro conditions. No significant increase of micronucleated splenocytes was observed in treated animals, despite some evidence of treatment-related cellular toxicity. A borderline excess of DNA damage was suggested by the Comet assay on circulating lymphocytes, whereas negative results were obtained with splenocytes. In vitro, positive results with both genetic end points were obtained in assays with human lymphocytes in the dose ranges 0.5-24 microg/ml and 0.1-8 microg/ml for micronucleus and Comet assays, respectively. These results suggest that thiram, despite its established genotoxicity in vitro, is devoid of appreciable clastogenic and/or aneugenic activity in vivo after oral administration to mice at the maximum tolerated dose.

Elicitation thresholds for thiuram mix using petrolatum and ethanol/sweat as vehicles.

An estimate of amounts of thiurams that may be released from rubber gloves into synthetic sweat, has previously been generated. These amounts should be compared to elicitation thresholds of patch tests performed with serial dilutions of thiuram mix using synthetic sweat as vehicle. Because of solubility properties of thiurams in aqueous media, such dilutions cannot directly be prepared. In this study, a stem solution was prepared in ethanol. This solution was then further diluted with synthetic sweat. Thiuram mix 0.5 w/v% was the most concentrated solution in ethanol achievable. The patch test reactions were compared to reactions to serial dilutions using petrolatum as vehicle. The experiment revealed that endpoint dilution with synthetic sweat was not achieved in this study. The threshold for elicitation of positive patch test reactions seemed to be lower for ethanol/sweat as vehicle compared to petrolatum: 32% reacted to ethanol/synthetic sweat 0.001 mg/cm2 compared to 14% reacting to thiuram in pet. 0.002 mg/cm2. Based on these results, synthetic sweat may be considered a more relevant medium for threshold finding studies than petrolatum. Because of expected instability of the aqueous solutions, petrolatum is probably a more suitable vehicle for routine testing. The study does not permit final conclusions concerning acceptable thresholds for leachable thiurams in rubber gloves, but it is likely that an acceptable threshold would be substantially less than 0.001 mg/ cm2.

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).

Comparative effects of two dithiocarbamates disulfiram and thiram, on adrenal catecholamine content and on plasma dopamine-beta-hydroxylase activity.

Both disulfiram (tetraethylthiuram disulfide), an alcohol aversive drug, and thiram (tetramethyl-thiuram disulfide), a widely used pesticide, significantly increased the dopamine pool in the adrenal glands of dosed rats. The dopamine increase was detectable within 4 h of oral dosing with 100 mg/kg of either dithiocarbamate and peaked 24 h later at 10 times control values. In control rats the dopamine turnover was 0.51 h-1 as calculated by the assumed first order decline of dopamine after a single injection of alpha-methyl-p-tyrosine (alpha-MT, 400 mg/kg i.p.) resulting in a dopamine-beta-hydroxylase (DBH) activity of 0.73 nmol/h per pair of adrenals. In the adrenals of rats pretreated with thiram and then injected with alpha-MT, the adrenal dopamine content did not significantly decline, indicating that thiram reduced the conversion of dopamine to noradrenaline, eventually leading to the observed dopamine increase. Plasma DBH activity was significantly reduced 4 h and 24 h after dosing with thiram, but was unchanged after treatment with disulfiram. The determination of plasma DBH activity could be a marker to monitor the effect of thiram on catecholamine metabolism in occupationally exposed workers but not that of disulfiram in abstinent alcoholics.

Chronic toxicity studies with thiram in Wistar rats and beagle dogs.

Groups of 64 male and 64 female Wistar rats were given thiram at constant dietary doses of 0, 3, 30, and 300 ppm (0, 0.1, 1.2, and 11.6 mg/kg/day for males and 0, 0.1, 1.4, and 13.8 mg/kg/day for females) for 104 weeks. Eight males and eight females in each group were killed after Weeks 13, 26, and 52. For the dog study, four male and four female beagle dogs were alloted to each group and treated with the compound at 0, 0.4, 4, and 40 mg/kg/day for 104 weeks. The dogs in the 40 mg/kg/day group had severe toxic signs, including nausea or vomiting, salivation, and occasional clonic convulsion, and all were subjected to unscheduled necropsy before Day 203 of treatment. The dogs also had ophthalmological changes such as fundal hemorrhage, miosis, and desquamation of the retina which were consistent with the retinal lesions shown by histopathology. The rats of the high-dose group had retarded growth with a slightly decreased food intake. Anemia was evident in high-dose female rats and in middle- and high-dose dogs. Liver failure in male and female dogs and kidney damage in female dogs were detected in middle- and high-dose groups by blood biochemistry and/or histopathology. Regressive changes of the sciatic nerve accompanied by atrophy of the calf muscle were seen in female rats of the high-dose group but not in male rats. In high-dose rats, progression of myocardial lesions of the heart and chronic nephrosis of the kidney were depressed in males and females, respectively. Female rats of the middle- and high-dose groups had decreased occurrences of mammary fibroadenoma and decreased development of skin masses.

[Study of combined effects of mixtures of pesticides from various chemical groups on calcium absorption in the rat intestine. I. Dichlorvos and thiram]. / Badanie skojarzonego dzialania mieszanin pestycydów z róznych grup chemicznych na wchlanianie wapnia w jelicie szczura CZ. I. Dichlorfos i tiuram.

Investigation was made of the effect of many-times repeated treatment with single pesticides: tiuram and dichlorfos (administered in a daily dose corresponding to 5% of LD50), and with their mixture, on calcium uptake by rat duodenum sections. Calcium uptake was examined by the method of Papworth and Patrick, as modified by the present authors. The amount of calcium transported by the sections was determined by the liquid scintillation method. Total transport was expressed in microM Ca2+/g tissue/h; subsequently, the participation of active and passive transport in total transport was determined. It was shown that administration of DDVP to rats during 14 days caused disturbances in active absorption of calcium, manifesting themselves by a significant decrease in the constant Jm (maximal transport rate) and in the constant Kt, this pointing to a rise of calcium affinity to the carrier. The effect of DDVP on passive transport was only slight. Under the same experimental conditions tiuram exerted an effect on both active and passive calcium transport. It caused a drop in the constant Jm and Kt (active transport), and to inhibition of passive transport. According to statistical treatment of the results, the effect of both pesticides combined on active transport does not differ from the effect of DDVP itself. Thus, the differences in diffusion probably result from the action of tiuram.

[Neurochemical disorders in rats caused by combined action of Primor and thiram. I. Changes in catecholamine and serotonin levels in the brain, heart and adrenal glands of rats with acute poisoning]. / Zaburzenia neurochemiczne w ustroju szczura przy lacznym oddzialywaniu primoru i tiuramu. CZ. I. Zmiany poziomów amin katecholowych i serotoniny w mózgu, sercu i nadnerczach szczura w warunkach zatrucia ostrego.

Study was made of the effect of acute poisoning with primor or with an equitoxic mixture of primor/tiuram (oral dose amounting to 50% of LD50) on the dynamic equilibrium of catecholamines and serotonin in rat whole brain, anatomic parts of the brain, heart and suprarenal glands. Both primor and an equitoxic mixture of primor/tiuram were found to cause changes in the levels of the investigated neurotransmitters in the central and peripheral nervous system. The mixture, as compared with primor alone, displayed the same direction of action, but the changes proceeded with enhanced dynamics and lasted throughout the whole experimental period (12h). The action of primor alone usually manifested itself only during the first hour after poisoning. Both primor and an equitoxic mixture of primor/tiuram exhibited no selective affinity to any separate areas of the brain, and lowered the contents of dopamine and norepinephrine in all brain structures studied.

The effect of thiram on the germ cells of male mice.

The effects of thiram or tetramethylthiuram disulphide on the germ cells of Swiss albino male mice were evaluated by analysing spermatocytes (derived from treated spermatogonia) for chromosomal aberrations and by the sperm-head morphology assay. The total doses tested were 80, 200 and 320 mg/kg body weight given by gavage in three consecutive daily doses, the top dose being slightly below the LD50 of thiram. There was a significant increase in the frequency of numerical chromosomal aberrations and abnormal sperms in mice treated with thiram at all dose levels. Such results could have implications for man in that they suggest that undue exposure to thiram could result in the birth of human infants with numerical chromosomal aberrations.

Influence of dietary tetramethylthiuram disulfide (a fungicide) on growth and incidence of tibial dyschondroplasia in single comb White Leghorn chicks.

The effect of dietary tetramethylthiuram disulfide (TMTD) (0, 30, or 60 mg/kg diet) on the performance and incidence and severity of tibial dyschondroplasia (TD) in Single Comb White Leghorn (SCWL) chicks was studied in a 6-week experiment. Body weights (3 weeks of age) and bone ash (4 and 6 weeks of age) of chicks fed either 30 or 60 mg TMTD/kg diet were significantly lower compared to controls. Dietary TMTD, however, significantly increased the incidence and severity of TD in layer chicks with the highest incidence (69%) occurring in 6-week old-birds. This study showed that SCWL chicks were susceptible to TMTD-induced TD as early as 2-weeks of age and that the condition persisted throughout the growing phase.