Ethalfluralin impairs implantation by aggravation of mitochondrial viability and function during early pregnancy.

Ethalfluralin, a dinitroaniline-type herbicide, has been used for decades. As a result, its residues are detected on some farmlands. To determine the molecular mechanisms underlying the detrimental effects of ethalfluralin on early pregnancy, porcine luminal epithelium and trophectoderm cell lines were used. Ethalfluralin was found to inhibit the viability, proliferation, and migration of porcine luminal epithelial (pLE) and porcine trophectoderm (pTr) cells. Additionally, ethalfluralin induced apoptotic cell death by means of an imbalance in calcium homeostasis in both pLE and pTr cells. Ethalfluralin decreased mitochondrial membrane potential (ΔΨm) and impaired mitochondrial respiration by downregulating the mitochondrial respiratory complex-related genes. Ethalfluralin also activated endoplasmic reticulum stress signals and autophagy pathways, increased the phosphorylation of P38 MAPK and NF-κB, and suppressed the PI3K/AKT signaling pathway. Taken together, this study elucidated the molecular mechanisms by which ethalfluralin impedes the viability and mitochondrial function in fetal trophectoderm and maternal endometrial cells during early pregnancy.

Dinitroaniline herbicide resistance in a multiple-resistant Lolium rigidum population.

BACKGROUND: The pre-emergence dinitroaniline herbicides (such as trifluralin and pendimethalin) are vital to Australian no-till farming systems. A Lolium rigidum population collected from the Western Australian grain belt with a 12-year trifluralin use history was characterised for resistance to dinitroaniline, acetyl CoA carboxylase (ACCase)- and acetolactate synthase (ALS)-inhibiting herbicides. Target-site resistance mechanisms were investigated. RESULTS: This L. rigidum population exhibited 32-fold resistance to trifluralin, as compared with the susceptible population. It also displayed 12- to 30-fold cross-resistance to other dinitroaniline herbicides (pendimethalin, ethalfluralin and oryzalin). In addition, this population showed multiple resistance to commonly used post-emergence ACCase- and ALS-inhibiting herbicides. Two target-site α-tubulin gene mutations (Val-202-Phe and Thr-239-Ile) previously documented in other dinitroaniline-resistant weed species were identified, and some known target-site mutations in ACCase (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg) and ALS (Pro-197-Gln/Ser) were found in the same population. An agar-based Petri dish screening method was established for the rapid diagnosis of resistance to dinitroaniline herbicides. CONCLUSION: Evolution of target-site resistance to both pre- and post-emergence herbicides was confirmed in a single L. rigidum population. The α-tubulin mutations Val-202-Phe and Thr-239-Ile, documented here for the first time in L. rigidum, are likely to be responsible for dinitroaniline resistance in this population. Early detection of dinitroaniline herbicide resistance and integrated weed management strategies are needed to maintain the effectiveness of dinitroaniline herbicides. © 2017 Society of Chemical Industry.

Occurrence and distribution of trifluralin, ethalfluralin, and pendimethalin in soils used for long-term intensive cotton cultivation in central Greece.

In the present study, a soil monitoring program was undertaken in Greek cotton cultivated areas in 2012. Twenty-seven soil samples were collected from the entire Thessaly plain in early summer of 2012, corresponding to approximately three months (current use of pendimethalin), up to one year (for the banned ethalfluralin), and three years (for the also banned trifluralin), after the last dinitroaniline application. Low but not negligible levels of dinitroanilines were detected, ranging from 0.01 to 0.21 µg g-1 d.w. for trifluralin and 0.01-0.048 µg g-1 d.w. for pendimethalin, respectively. Trifluralin was the herbicide most frequently detected (44.4%). The high historic application of trifluralin and its high persistence and accumulation potential is in line with the abundance of the detected residues. The present data indicate that soil samples contain extractable residues of banned trifluralin, but based on the comparison of the theoretical PECplateau for trifluralin (0.277 µg g-1) and the maximum Measured Environmental Concentration, it was concluded that the detected residues should be attributed to previous years' application. The latter suggested the need for continual monitoring of the dinitroaniline family of pesticides, including the banned substances, aiming thus to an improved environmental profile for agricultural areas.

Inhibition of the in vitro growth of Babesia bigemina, Babesia caballi and Theileria equi parasites by trifluralin analogues.

Bovine and equine babesiosis caused by Babesia bovis, Babesia bigemina and Babesia caballi, along with equine theileriosis caused by Theileria equi are global tick-borne hemoprotozoan diseases characterized by fever, anemia, weight losses and abortions. A common feature of these diseases are transition from acute to chronic phases, in which parasites may persist in the hosts for life. Antiprotozoal drugs are important for managing infection and disease. Previous research demonstrated that trifluralin analogues, designated (TFLAs) 1-15, which specifically bind to regions of alpha-tubulin protein in plants and protozoan parasites, have the ability to inhibit the in vitro growth of B. bovis. The inhibitory activity of TFLAs 1-15 minus TFLA 5 was tested in vitro against cultured B. bigemina, B. caballi and T. equi. The four TFLAs with greatest inhibitory activity were then analyzed for hemolytic activity and toxicity against erythrocytes. All TFLAs tested in the study showed inhibitory effects against the three parasite species. TFLA 2, TFLA 11, TFLA 13 and TFLA 14 were the most effective inhibitors for the three species tested, with estimated IC50 between 5.1 and 10.1µM at 72h. The drug's solvent (DMSO/ethanol) did not statistically affect the growth of the parasites nor cause hemolysis. Also, TFLA 2, 13 and 14 did not cause statistically significant hemolytic activity on bovine and equine erythrocytes at 15µM, and TFLA 2, 11 and 13 had no detectable toxic effects on bovine and equine erythrocytes at 15µM, suggesting that these drugs do not compromise erythrocyte viability. The demonstrated ability of the trifluralin analogues to inhibit in vitro growth of Babesia spp. and Theileria equi, and their lack of toxic effects on erythrocytes supports further in vivo testing and eventually their development as novel alternatives for the treatment of babesiosis and theileriosis.

Lipid-based nanoformulations of trifluralin analogs in the management of Leishmania infantum infections.

AIM: To improve the potential of trifluralin (TFL) in the management of Leishmania infantum infections through the synthesis of analogs (TFLA) and incorporation in nanoparticulate drug delivery systems (NanoDDS), liposomes and solid lipid nanoparticles, for selective targeting to leishmania infection sites. MATERIAL & METHODS: In vitro screening of 18 TFLA was performed by flow cytometry. NanoDDS were loaded with active TFLA and evaluated for antileishmanial efficacy in mice through determination of parasite burden in liver and spleen. RESULTS: The in vitro testing revealed the most active and nontoxic TFLAs, which were selected for the in vivo studies based on high incorporation in liposomes and lipid nanoparticles (>90%). Selected TFLA nanoformulations showed superior antileishmanial activity in mice (parasite burden >80%), over free TFLA and Glucantime. CONCLUSION: The modification of TFL structure to obtain active TFLA, together with their incorporation in NanoDDS, improved their in vivo performance against L. infantum infection.

Gas-phase degradation of the herbicide ethalfluralin under atmospheric conditions.

The gas-phase degradation of ethalfluralin, N-ethyl-α,α,α-trifluoro-N-(2-methylallyl)-2,6-dinitro-p-toluidine, a widely used herbicide, was investigated under atmospheric conditions at the large outdoor European simulation chamber (EUPHORE) in Valencia, Spain. The photolysis of ethalfluralin was investigated under solar radiation and the mean photolysis rate coefficient was determined: J(ethalfluralin)=(1.3±0.2)×10(-3) s(-1) (JNO2=8×10(-3) s(-1)). The rate coefficients for the reactions of hydroxyl radicals and ozone with ethalfluralin in the dark were also measured under atmospheric conditions using the relative rate and the absolute rate technique, respectively. The rate coefficients values for the reactions of kOH(ethalfluralin)=(3.5±0.9)×10(-11)cm(3)molecule(-1)s(-1), and kO3(ethalfluralin)=(1.6±0.4)×10(-17) cm(3) molecule(-1) s(-1) were determined at 300±5 K and atmospheric pressure. The results show that removal of ethalfluralin from the atmosphere by reactions with OH radicals (τ ~ 4 h) or ozone (τ ~ 25 h) is slow compared to loss by photolysis. The available kinetic data suggest that the gas-phase tropospheric degradation of ethalfluralin will be controlled mainly by photolysis and provide an estimate for the tropospheric lifetime of approximately 12 min. The atmospheric implications of using ethalfluralin as a herbicide are discussed.

A novel alkyl phosphocholine-dinitroaniline hybrid molecule exhibits biological activity in vitro against Leishmania amazonensis.

Parasitic protozoa of the Leishmania genus cause leishmaniasis, an important complex of tropical diseases that affect about 12 million people around the world. The drugs used to treat leishmaniasis are pentavalent antimonials, miltefosine, amphotericin B and pentamidine. In the present study, we evaluated the effect of a novel alkyl phosphocholine-dinitroaniline hybrid molecule, TC95, against Leishmania amazonensis promastigotes and intracellular amastigotes. Antiproliferative assays indicated that TC95 is a potent inhibitor of promastigotes and intracellular amastigotes with IC50 values of 2.6 and 1.2 µM, respectively. Fluorescence microscopy with anti-α-tubulin antibody revealed changes in the cytoskeleton, whilst scanning electron microscopy showed alterations in the shape, plasma membrane, length of the flagellum, and cell cycle. Flow cytometry confirmed the cell cycle arrest mainly in G1 phase, however a significant population appeared in sub G0/G1 and super-G2. The alterations in the plasma membrane integrity were confirmed by fluorometric analysis using Sytox Blue. Transmission electron microscopy also revealed an accumulation of lipid bodies, confirmed by fluorescence microscopy and fluorometric analysis using Nile Red. Important lesions were also observed in organelles such as mitochondrion, endoplasmic reticulum and Golgi complex. In summary, our study suggests that TC95, an alkyl phosphocholine-trifluralin hybrid molecule, is a promising novel compound against L. amazonensis.

Enhanced dissipation of oxyfluorfen, ethalfluralin, trifluralin, propyzamide, and pendimethalin in soil by solarization and biosolarization.

This study was conducted to assess the effects of solarization and biosolarization on the degradation of oxyfluorfen, ethalfluralin, trifluralin, propyzamide, and pendimethalin. The experimental design consisted of 17 L pots filled with clay-loam soil, which were contaminated with the studied herbicides. Then, soil disinfection treatments were applied during the summer season, including a control without disinfection (C), solarization (S), and biosolarization (BS). Soil from five pots per treatment was sampled periodically up to 90 days. Herbicide dissipation rates were higher in both S and BS treatments with regard to the control. Similar dissipation rates were observed under S and BS for most of the herbicides studied, except oxyfluorfen and pendimethalin, which were degraded to a greater extent in the BS than in the S treatment. The obtained results showed that both solarization and biosolarization can be considered, in addition to soil disinfection techniques, such as bioremediation tools for herbicide-polluted soils.

Characterization of trifluralin binding with recombinant tubulin from Trypanosoma brucei.

The binding kinetics of five novel trifluralin analogs with recombinant alpha- and beta-tubulin proteins from Trypanosoma brucei rhodesiense was determined. Native tubulin from rats was used to determine the extent of binding of each analog to mammalian tubulin. The results of this study clearly demonstrate two important characteristics of the binding of these trifluralins to tubulin. Firstly, they have specific affinity for trypanosomal tubulin compared with mammalian tubulin irrespective of the chemical composition of the trifluralin analog tested. Secondly, they have a stronger affinity for trypanosomal alpha-tubulin compared with trypanosomal beta-tubulin. In addition, compounds 1007, 1008, 1016, and 1017 have strong binding affinities for alpha-tubulin, with limited binding affinity for mammalian tubulin, which indicates that these compounds selectively bind to trypanosomal tubulin.

Study of the voltammetric behaviour of the ethalfluralin and methalpropalin and its determination in environmental matrices at hanging mercury drop electrode.

An electroanalytical method has been developed for the determination of the herbicides ethalfluralin[N-ethyl-N-(2-methyl-2-propenyl)-2,6-dinitro-4-(trifluoromethyl) bezenamine] and methalpropalin [N-(2-methyl-2-propenyl)-2, 6-dinitro-N-propyl-4 (trifluoromethyl) benzenamine] by differential pulse adsorptive stripping voltammetry (DP-AdSV) on a hanging mercury drop electrode (HMDE) with universal buffer as supporting electrolyte. The optimum adsorption conditions were found to be pH 6.0, an accumulation potential of -0.6 V (HMDE vs SCE), an accumulation time of 80 s. and scan rate 45 mVs(-1). Calibration curve is linear in the range 1.30 x 10(-9) to 1.32 x 10(-5) M of ethalfluralin and 1.13 x 10(-5) to 2.0 x 10(-8) M of methalpropalin with detection limits of 1.08 x 10(-9) and 1.87 x 10(-8) M, respectively. The relative SD and correlation coefficients were found to be 1.24%, 0.998 and 1.34%, 0.995, respectively for ten replicates. The method is applied to the determination of the ethalfluralin and methalpropalin in formulations and environmental matrices.

Secondary mutations correct fitness defects in Toxoplasma gondii with dinitroaniline resistance mutations.

Dinitroanilines (oryzalin, trifluralin, ethafluralin) disrupt microtubules in protozoa but not in vertebrate cells, causing selective death of intracellular Toxoplasma gondii parasites without affecting host cells. Parasites containing alpha1-tubulin point mutations are dinitroaniline resistant but show increased rates of aberrant replication relative to wild-type parasites. T. gondii parasites bearing the F52Y mutation were previously demonstrated to spontaneously acquire two intragenic mutations that decrease both resistance levels and replication defects. Parasites bearing the G142S mutation are largely dependent on oryzalin for viable growth in culture. We isolated 46 T. gondii lines that have suppressed microtubule defects associated with the G142S or the F52Y mutations by acquiring secondary mutations. These compensatory mutations were alpha1-tubulin pseudorevertants or extragenic suppressors (the majority alter the beta1-tubulin gene). Many secondary mutations were located in tubulin domains that suggest that they function by destabilizing microtubules. Most strikingly, we identified seven novel mutations that localize to an eight-amino-acid insert that stabilizes the alpha1-tubulin M loop, including one (P364R) that acts as a compensatory mutation in both F52Y and G142S lines. These lines have reduced dinitroaniline resistance but most perform better than parental lines in competition assays, indicating that there is a trade-off between resistance and replication fitness.

Body mass index and bromoxynil exposure in a sample of rural residents during spring herbicide application.

Bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), a phenolic herbicide, is widely used in production of cereals and other crops. Little is known, however, about bromoxynil exposure in humans. Results of previous research suggest a longer residence time in the body for bromoxynil compared to phenoxy herbicides [e.g., (2,4-dichlorophenoxy)acetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA)] and that bromoxynil would tend to partition into fatty tissue more so than 2,4-D. In previous research, body mass index (BMI) was found to be an independent predictor of plasma concentrations of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), the persistent lipophilic metabolite of the chlorinated pesticide bis(4-chlorophenyl)-1,1,1-trichloroethane (DDT). As part of the Prairie Ecosystem Study, gas chromatography/mass spectrometry analysis was used to measure concentrations of bromoxynil and seven other herbicides (2,4-D, dicamba, fenoxaprop, MCPA, ethalfluralin, triallate, and trifluralin) in plasma from residents (104 men, 88 women, 24 youths age 12-17 yr) of a cereal-producing region in Saskatchewan, Canada, during spring herbicide application, 1996. Multiple logistic regression analysis was used to explore whether BMI predicted detection of bromoxynil in plasma from the adults. The prevalence of detection (detection limits: 2-50 microg/L) was markedly higher for bromoxynil (men, 44.2%; women, 14.8%; youths, 20.8%) compared to each of the other herbicides including 2,4-D (men, 16.5%; women, 3.4%; youths, 12.5%) and MCPA (men, 6.8%; women, 1.1%; youths, 4.2%), although bromoxynil is commonly formulated or tank mixed with these herbicides. In the multiple logistic regression analysis, the variables BMI, exposure group [bromoxynil applicators, non-applicator family members of bromoxynil applicators, all others (reference group)], and days elapsed since the last use of bromoxynil were found to be independent predictors of detection of bromoxynil, while age, gender, and farm residency were not statistically significant. With adjustment for exposure group [bromoxynil applicators: odds ratio (OR = 24.30, 95% confidence interval (CI) = 9.59-61.58; nonapplicator family members of bromoxynil applicators: OR = 3.53, 95% CI = 1.19-10.44; all others (reference group)], the OR for detection of bromoxynil was 2.35 (95% CI = 0.87-6.33) for participants in the middle (25.53-29.00 kg/m2) tertile (men: OR = 2.85, 95% CI = 0.75-10.82; women: OR = 1.63, 95% CI = 0.36-7.40) of BMI and 4.01 (95% CI = 1.46-11.03) for participants in the highest (> 29.00 kg/m2) tertile (men: OR = 4.67, 95% CI = 1.17-18.58; women: OR = 2.20, 95% CI = 0.44-10.99) with participants in the lowest (< 25.53 kg/m2) tertile as the reference group. Similar ORs were observed for BMI with adjustment for days elapsed since the last use of bromoxynil. In conclusion, further research is needed to investigate whether adiposity is an important modifying factor for persistence of bromoxynil in the body.

Determination of ethalfluralin in canola seed, meal, and refined oil by capillary gas chromatography with mass selective detection.

Ethalfluralin is a herbicide that is effective for weed control on a wide variety of crops, including canola. A method is described for the determination of ethalfluralin residues in canola seed, meal, and refined oil. Residues are extracted from canola sample matrixes with acetonitrile. An aliquot of the extract is diluted with water and purified by C(18) solid-phase extraction prior to analysis by capillary gas chromatography with mass selective detection. For all three sample matrixes, the method has a validated limit of quantitation of 0.02 microg/g and a limit of detection of 0.006 microg/g. Recoveries averaged 96 +/- 7% for canola seed, 87 +/- 6% for canola meal, and 89 +/- 5% for refined oil. In a magnitude-of-residue study, canola seed from field plots that had been treated with ethalfluralin at one to three times the maximum label rate for weed control were found to contain no detectable residue of the herbicide.

Inhibition of Toxoplasma gondii replication by dinitroaniline herbicides.

Submicromolar concentrations of several dinitroaniline herbicides have been found to specifically inhibit intracellular replication of the protozoan parasite Toxoplasma gondii. IC50 concentrations for T. gondii survival were approximately 100 nM for ethalfluralin and oryzalin and approximately 300 nM for trifluralin. Primary human fibroblasts employed as host cells for parasite culture were unaffected at > 100-fold higher concentrations. Extracellular parasites were unaffected by these drugs, but within 8 hr after treatment of infected cell cultures, intracellular tachyzoites formed large amorphous bodies containing distorted nuclei. Parasite cytokinesis was completely blocked by drug treatment; nucleic acid synthesis, however, continued at near-normal levels for several days in the continuous presence of drug. All dinitroanilines appear to block nuclear division by inhibition of intranuclear spindle formation, but other cytoskeletal components were differentially affected by the various drugs tested. Subpellicular microtubules were absent in oryzalin-treated parasites, and large fragments of the inner membrane complex were observed throughout the parasite cytoplasm. In contrast, subpellicular microtubules and the inner membrane complex remained intact in ethalfluralin-treated parasites, but the endoplasmic reticulum and nuclear envelope were highly distended. Cytoskeletal elements associated with the conoid were not affected by any of the dinitroanilines tested, and treatment with the Ca2+ ionophore A23187 failed to trigger release of drug-treated parasites from infected cells. Mutant parasites resistant to oryzalin, ethalfluralin, or trifluralin were selected by chemical mutagenesis and examined for cross-resistance. An ethalfluralin-resistant mutant displayed cross-resistance to both oryzalin and trifluralin, while a trifluralin-resistant mutant was sensitive to oryzalin and only partially resistant ethalfluralin; an oryzalin-resistant mutant exhibited higher resistance to ethalfluralin and trifluralin than to oryzalin itself. Similarities between Apicomplexan and plant tubulin are discussed.

Microtubule inhibitors: structure-activity analyses suggest rational models to identify potentially active compounds.

Trifluralin, a dinitroaniline microtubule inhibitor currently in use as an herbicide, has been shown to inhibit the proliferation of Plasmodium falciparum, Trypanosoma brucei, and several species of Leishmania, in vitro. As a topical formulation, trifluralin is also effective in vivo (in BALB/c mice) against Leishmania major and Leishmania mexicana. Although trifluralin and other dinitroaniline herbicides show significant activity as antiparasitic compounds, disputed indications of potential carcinogenicity will probably limit advanced development of these substances. However, researchers have suggested that the activity of trifluralin is due to an impurity or contaminant, not to trifluralin itself. We have pursued this lead and identified the structure of the active impurity. This compound, chloralin, is 100 times more active than trifluralin. On the basis of its structure, we developed a rational structure-activity model for chloralin. Using this model, we have successfully predicted and tested active analogs in a Leishmania promastigote assay; thus, we have identified the putative mechanism of action of this class of drugs in Leishmania species. Potentially, this will allow the design of noncarcinogenic, active drugs.

In vitro anticryptosporidial activity of dinitroaniline herbicides.

Despite the evaluation of over 100 antimicrobial drugs, the diarrheal disease cryptosporidiosis has remained refractory to treatment. We report the evaluation of five dinitroaniline herbicides including trifluralin, profluralin, nitralin, pendimethalin, and fluchloralin for anticryptosporidial activity in an in vitro cultivation model of Cryptosporidium parvum. All five compounds exhibited significant anticryptosporidial activities with no corresponding evidence of toxicity. The most active compound was pendimethalin with an IC50 of 0.19 microM while nitralin was the least active with an IC50 of 4.5 microM. These compounds should be evaluated further in an animal model of cryptosporidiosis.

Gas chromatographic determination of residue levels of the herbicides trifluralin, benefin, ethalfluralin, and isopropalin in soil with confirmation by mass selective detection.

A method is presented for the simultaneous or individual determination of the dinitroaniline herbicides trifluralin, benefin, ethalfluralin, and isopropalin in soil. The herbicides are extracted with acetonitrile-water (99 + 1), and the extracts are purified with small, disposable Florisil cartridges prior to analysis by gas chromatography using an electron capture detector or a mass selective detector. When electron capture detection is used as the primary detection system, confirmation with selective detection can be obtained using gas chromatography-mass spectrometry with a mass selective detector and a capillary column operated in the split mode. The limit of detection is 0.01 ppm, and recoveries averaged 95-112% for the 4 herbicides in several different soil types fortified at levels of 0.01-0.33 ppm.

Metabolism of profluralin in rats. 1. Identification of metabolites.

Fluorine NMR spectroscopy has been used to identify and quantitate metabolites of profluralin appearing in the urine of rats postingestion of the herbicide. The chemical shift of the trifluoromethyl group was shown to be a useful indicator of the chemical type of metabolite, and the shifts for the classes of materials previously identified as metabolites have been determined. By use of chemical shift criteria this work provides evidence for several metabolites that have not been recognized in previous work; some of these remain incompletely characterized. Though the number and types of metabolites detected in the urine were consistent from animal to animal, the time course for appearance of metabolites was found to be animal dependent.

Methodology for the determination of dinitroaniline herbicides in tissue and excreta.

A method is presented for the analysis of trace amounts of dinitroaniline herbicides in tissue and excreta. The method employs extraction of the tissue or excreta with organic solvent, clean up by liquid/liquid partitioning or silica gel chromatography, and ultimate analysis by gas chromatography using electron capture detection. Recoveries of greater than 80% were noted over the range of concentration 0.1 to 1.0 microgram/g.