Differential immunomodulatory effects of six pesticides of different chemical classes on human monocyte-derived macrophage functions.

Exposure to pesticides through eyes, skin, ingestion and inhalation may affects human health by interfering with immune cells, such as macrophages. We evaluated, in vitro, the effect of six pesticides widely used in apple arboriculture on the functions of human monocyte-derived macrophages (hMDMs). hMDMs were cultured for 4 or 24 h with or without pesticides (0.01, 0.1, 1, 10 µmol.L-1). We showed that chlorpyrifos, thiacloprid, thiophanate, boscalid, and captan had little toxic effect at the tested concentrations, while dithianon had low-cytotoxicity at 10 µmol.L-1. While boscalid showed no effect on hMDMs function, thiophanate (0.01 µmol.L-1) stimulated with TPA and thiacloprid (1, 10 µmol.L-1) stimulated with zymosan activated ROS production. Chlorpyrifos, dithianon, and captan inhibited ROS production and TNF-α, IL-1ß pro-inflammatory cytokines. We established that dithianon (0.01-1 µmol.L-1) and captan (0.1, 1 µmol.L-1) induced mRNA expression of NQO1 and HMOX1 antioxidant enzymes. Dithianon also induced the mRNA expression of catalase, superoxide dismutase-2 at 10 µmol.L-1. Together, these results show that exposure to chlorpyrifos, dithianon, and captan induce immunomodulatory effects that may influence the disease fighting properties of monocytes/macrophages while pesticides such as thiacloprid, thiophanate and boscalid have little influence.

Immunomodulatory effect of mancozeb, chlorothalonil, and thiophanate methyl pesticides on macrophage cells.

Mancozeb (MZ), chlorothalonil (CT), and thiophanate methyl (TM) are pesticides commonly used in agriculture due to their efficacy, low acute toxicity to mammals, and short environmental persistence. Although the toxic effects of these pesticides have been previously reported, studies regarding their influence on the immune system are limited. As such, this study focused on the immunomodulatory effect of MZ, CT, and TM pesticides on macrophage cells. RAW 264.7 cells were exposed to a range of concentrations (0.1-100 µg/mL) of these pesticides. CT exposure promoted an increase in reactive oxygen species (ROS) and nitric oxide (NO) levels. The MTT and ds-DNA assay results demonstrated that MZ, CT, and TM exposure induced macrophage proliferation. Moreover, MZ, CT, and TM promoted cell cycle arrest at S phase, strongly suggesting macrophage proliferation. The levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, and IFN-γ) and caspases (caspase 1, 3, and 8) in macrophages exposed to MZ, CT, and TM pesticides increased, whereas the anti-inflammatory cytokine levels decreased. These results suggest that MZ, CT, and TM exert an immunomodulatory effect on the immune system, inducing macrophage activation and enhancing the inflammatory response.

Methyl thiophanate-induced toxicity in liver and kidney of adult rats: a biochemical, molecular and histopathological approach.

The aim of this study was to elucidate the redox effects of Thiophanate methyl (MT) in the rat liver and kidney. Our results showed, after 3 days of MT injection (700 mg/kg), an increase in malondialdehyde (MDA), hydrogen peroxide and advanced oxidation protein products levels. Glutathione peroxidase and superoxide dismutase activities were also remarkably increased in the liver but decrease in the kidney. Glutathione and vitamin C values were significantly reduced. The changes in biochemical parameters were substantiated by histological and molecular data. A smear without ladder formation on agarose gel was shown, indicating random DNA degradation in the liver and the kidney of MT treated rats. The increase in cyclooxygenase-2 gene expression, marker of inflammation, and an increase in genes expression of glutathione peroxidase and superoxide dismutase in liver and their decrease in the kidney were also occurred after MT exposure. These data confirmed the pro-oxidant and genotoxic effects of this fungicide.

The impact of pesticides on the pathogen Batrachochytrium dendrobatidis independent of potential hosts.

Amphibians around the world are experiencing the greatest organismal decline in recent history. Xenobiotics, such as pesticides, and pathogenic biotic perturbations, including the fungus Batrachochytrium dendrobatidis (Bd), have played major roles in amphibian decreases. We conducted laboratory culture studies to determine the effects of three pesticides {carbaryl, glyphosate, and thiophanate-methyl [TM; Topsin-M(R) (Cerexagri-Nisso LLC)]} on Bd zoospore production and zoosporangia growth. We applied Bd to pesticides mixed in an agar culture to simulate pathogen introduction to a system with pre-existing pesticides (Bd addition). Alternatively, pesticides were applied to pre-established Bd to simulate pesticide introduction after Bd establishment (pesticide addition). We then measured Bd zoosporangia and zoospore production. All pesticides significantly inhibited zoospore production; however, glyphosate and TM were more effective at doing so than carbaryl. In addition, only carbaryl and glyphosate inhibited zoosporangia production. Our data suggest that carbaryl and glyphosate are equally effective at inhibiting both zoosporangia and zoospore production; however, TM is selectively toxic to zoospores but not zoosporangia. One possible explanation for this observation could be that TM is toxic to zoospores but not the protective zoosporangia. In the case of pesticides applied to established Bd cultures, all pesticides caused significant mortality in both zoosporangia and zoospores, and no differences were found among pesticides. We conclude that examining pesticide and pathogen interactions independent of hosts provides mechanistic understanding of such interactions before and after host infection or contamination.

Testicular toxicity of methyl thiophanate in the Italian wall lizard (Podarcis sicula): morphological and molecular evaluation.

The effects of the fungicide methyl thiophanate (MT) on testis were determined in the Italian wall lizard (Podarcis sicula) using morphological and molecular analyzes. Three experimental trials were performed: an acute test using six doses, a two-week chronic test, and "ecotoxicological" exposure (3 weeks). The minimal lethal dose (LD(50)) of pure MT, reached by the acute test, was 100 mg/kg body weight. Testicular histopathology of surviving animals showed a reduced lumen and several multinucleated giant cells 24 h after injection followed by large decreases in spermatogonia (72%) and secondary spermatocytes (58%) and a loss of spermatids and sperms 7 days after. In the chronic test, a dose equivalent to 1/100 of LD(50) was injected on alternate days. Complete shutting of the lumen and a great decrease in spermatogonia (82%) were observed. In "ecotoxicological" exposure, achieved with a commercial MT compound, testis showed a decrease in primary spermatocytes (20%) and several vacuoles. An increase in germ cell apoptosis was observed in all experimental groups using TUNEL assay. A decrease in expression of androgen and estrogen receptor (AR and ER) mRNAs was seen in all experimental groups. The reduction in AR and ER mRNAs was correlated to exposure time. Indeed, in the "ecotoxicological" treatment (30 days), the decrease reached 82 and 90% for AR and ER mRNAs, respectively. These data strongly indicate that treatment with MT, damaging the seminiferous epithelium and decreasing steroid receptor expression, might render exposed lizards infertile.

Genotoxic fungicide methyl thiophanate as an oxidative stressor inducing 8-oxo-7,8-dihydro-2' -deoxyguanosine adducts in DNA and mutagenesis.

Dimethyl 4,4' -(O-phenylene)bis(3-thioallophanate), commonly known as methyl thiophanate (MT), is a systemic fungicide and suspected carcinogen to humans. In this study, the oxidative potential of this category-III acute toxicant has been ascertained based on its capacity of inducing reactive oxygen species (ROS) and promutagenic 8-oxo-7,8-dihydro-2' -deoxyguanosine (8-oxodG) adducts in DNA. The discernible MT dose-dependent reduction in fluorescence intensity of a cationic dye rhodamine (Rh-123) in human lymphocytes and increased fluorescence intensity of 2',7'-Dichlorodihydro fluorescein diacetate (DCFH-DA) treated cells signifies decreased mitochondrial membrane potential (Delta Psi m) due to intracellular ROS generation. The (32)P-post-labeling assay demonstrated the MT-induced 8-oxodG adduct formation in calf thymus DNA. Thus, it is concluded that MT, as a potent oxidative stressor, produces ROS leading to mitochondrial dysfunction, oxidative DNA damage and mutagenesis.

Flower-inhabiting Frankliniella Thrips (Thysanoptera: Thripidae), pesticides, and Fusarium hardlock in cotton.

Cotton hardlock caused by Fusarium verticillioides (Sacc. Nirenberg) can reduce cotton, Gossypium hirsutum L., yields > 70% in the southeastern United States. The spores infect flowers on the day of pollination, resulting in hardlock, which is the failure of the fiber to fluff as the boll opens at maturity. Frankliniella spp. Thrips (Thysanoptera: Thripidae) inhabiting the flowers are hypothesized to increase hardlock by spreading the conidia or by creating entranceways for the germinating Fusarium conidia. Experiments were conducted at Marianna and Quincy in Florida in 2006 and 2007 to determine whether there was a relationship between the number of adult and larval thrips inhabiting the flowers of cotton and the incidence of cotton hardlock. Frankliniella tritici (Fitch) was > 98% of the adult thrips in the samples at both locations each year. The adults of Frankliniella bispinosa (Morgan) and Frankliniella occidentalis (Pergande) also were collected. There were no significant regression relationships between weekly mean densities of thrips in the flowers and the incidence of cotton hardlock at harvest in any of the experiments. Additional experiments were conducted at each location in 2006 and 2007 to determine whether weekly applications during flowering of the insecticide lambda-cyhalothrin, the fungicide thiophanate methyl, and the combination of the two reduced the incidence of cotton hardlock at harvest. Applications of the insecticide significantly reduced the numbers of adult F. tritici, the number of thrips larvae, and the incidence of hardlock at harvest. Applications of the insecticide were as affective as applications of the insecticide plus fungicide. In one experiment, applications of the fungicide reduced the incidence of hardlock at harvest. Applications of the insecticide usually significantly increased the number of adult F. occidentalis. None of the pesticide treatments significantly affected the numbers of the key thrips predator Orius insidiosus (Say). We conclude that insecticidal control of the adults and larvae of F. tritici during flowering reduced the incidence of cotton hardlock. However, there were no significant regression relationships between the incidence of cotton hardlock at harvest and the number of thrips in the flowers.

Assessment of methyl thiophanate-Cu (II) induced DNA damage in human lymphocytes.

Dimethyl 4,4'-(O-phenylene)bis(3-thioallophanate), commonly known as methyl thiophanate (MT), is a category-III acute toxicant and suspected carcinogen to humans. Hence, the ability of this benzimidazole class of fungicide to engender DNA strand breaks was investigated using alkaline single cell gel electrophoresis (SCGE), alkaline unwinding and cytokinesis-blocked micronucleus (CBMN) assays. The SCGE of human lymphocytes treated with 1mM MT for 3h at 37 degrees C showed much higher Olive tail moment (OTM) value of 40.3+/-2.6 (p<0.001) vis-à-vis 3.3+/-0.09 in DMSO control. Treatment of cultured lymphocytes for 24h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). Stoichiometric data revealed the intrinsic property of MT to bind with Cu (II) and its reduction to Cu (I), which is known to form reactive oxygen species (ROS). We have detected the intracellular ROS generation in MT treated lymphocytes and observed an elevated level of MT-induced strand breaks per unit of calf thymus DNA in presence of Cu (II). Overall the data suggested that the formation of MT-Cu (II)-DNA ternary complex and consequent ROS generation, owing to Cu (II)/Cu (I) redox cycling in DNA proximity, is responsible for MT-induced DNA damage.

Molecular non-genetic biomarkers of effect related to methyl thiophanate cocarcinogenesis: organ- and sex-specific cytochrome P450 induction in the rat.

We used selective biochemical markers of effect to evaluate some non-genotoxic cocarcinogenic properties of methyl thiophanate (MTH) associated with cytochrome P450 (CYP) changes. Several CYP-dependent reactions were monitored in the liver, kidney and lung microsomes of male and female Sprague-Dawley rats treated (i.p.) with a single (285 or 570 mg/kg body weight) or repeated (daily 285 or 570 mg/kg body weight for three consecutive days) doses of this pesticide. No significant changes in absolute or relative liver, kidney and lung weights were observed after MTH injection. Highly specific substrates were used as probes of different isoforms, such as CYP1A1, 1A2, 2B1, 2E1 and 3A. A complex pattern of CYP induction, including organ- and sex-related differences, was observed, particularly in the liver (CYP3A, 2B1), kidney (CYP1A1, 2E1) and lung (CYP3A, 1A1). In the liver, an increase up to 29-fold in the 2B1-like activity, probed by the O-dealkylation of pentoxyresorufin, was observed at lower dose in both sexes, and the induction of CYP 1A2-mediated methoxyresorufin O-demethylase activity (up to 3.6-fold) was recorded at the higher dose in males. In the kidney, the O-deethylation of ethoxyresorufin (CYP1A1-linked) was increased up to 28.2-fold and the CYP2E1-dependent p-nitrophenol hydroxylases were enhanced up to 6.3-fold in females receiving higher multiple MTH administration. In the lung, the CYP3A-associated activity was the most induced oxidases, as exemplified by the marked increase in the O-demethylation of aminopyrine (up to 3.6-fold) in males. A weak, although significant, reduction of CYP2B1-linked oxidases was also observed in repeated treatment in the kidney (males) and lung (females). These results suggest that the induction of CYP-catalyzed drug metabolism by prolonged exposure to MTH may result in accelerated metabolism of coadministered drugs with important implications for their disposition Together with an alteration of endogenous metabolism, the adverse effects associated with CYP changes such as toxicity/cotoxicity, cocarcinogenicity and promotion may also have clinical consequences.

Cytogenetic effects of benzimidazoles in mouse bone marrow.

The cytogenetic effects of three benzimidazoles, i.e., benomyl, methyl thiophanate and methyl 2-benzimidazolecarbamate (MBC), were studied in mouse bone marrow cells by analyzing three genetic endpoints: micronuclei, structural chromosome aberrations plus or minus gaps, and aneugenic effects (hyperdiploidy or polyploidy). In general, the effects were small, but it was observed that benomyl and MBC significantly induced micronuclei as well as aneugenic effects, hyperdiploidy (no metaphases with more than one or two extra chromosomes, 2n + 1 or 2n + 2, were observed) and polyploidy (4n). The induction of chromosome gaps and breaks was less evident. Methyl thiophanate significantly induced micronuclei, but it was less effective than benomyl and MBC. Our results showed that micronuclei are a good indicator of aneugenic effects in mouse bone marrow cells. A curvilinear trend test has been devised to fit the curves originating from the time-dependent responses.