In vitro neurotoxic hazard characterisation of dinitrophenolic herbicides.

Dinitrophenolic compounds are powerful toxicants with a long history of use in agriculture and industry. While (high) human exposure levels are not uncommon, in particular for agricultural workers during the spraying season, the neurotoxic mechanism(s) that underlie the human health effects are largely unknown. We therefore investigated the in vitro effects of two dinitrophenolic herbicides (DNOC and dinoseb) on a battery of neurotoxicity endpoints in (dopaminergic) rat PC12 cells. Cell viability, mitochondrial activity, oxidative stress and caspase activation were assessed using fluorescence-based bioassays (CFDA, alamar Blue, H2DCFDA and Ac-DEVD-AMC, respectively), whereas changes in intracellular [Ca(2+)]i were assessed using single-cell fluorescence microscopy with Fura-2AM. The combined results demonstrate that exposure to both DNOC and dinoseb is linked to calcium release from the endoplasmic reticulum and activation of caspase-mediated apoptotic pathways. In subsequent experiments, immunofluorescent labelling with specific antibodies was used to determine changes in intracellular α-synuclein levels, demonstrating that both DNOC and dinoseb increase levels of intracellular α-synuclein. The combined results indicate that in vitro exposure to DNOC and dinoseb activates pathways that are not only involved in acute neurotoxicity but also in long-term effects as seen in neurodegeneration.

Quenching of tryptophan fluorescence in the presence of 2,4-DNP, 2,6-DNP, 2,4-DNA and DNOC and their mechanism of toxicity.

Although they are widely used as insecticides, acaricides and fungicides in the agriculture or as raw materials in the dye industry, dinitrophenols (DNPs) are extremely noxious, death cases having been registered. These compounds produce cataracts, lower leucocyte levels, disturb the general metabolism and can cause cancer. It is also assumed that DNPs hinder the proton translocation through the mitochondrial inner membrane and therefore inhibit oxidative phosphorylation. Their fluorescence quenching properties can help understand and explain their toxicity. Fluorescence quenching of tryptophan was tested using different dinitrophenols such as 2,4-dinitrophenol (2,4-DNP), 4,6-dinitro-orthocresol (DNOC), 2-[(2,4-dinitrophenyl)amino]acetic acid (GlyDNP), 2-(1-methyl-heptyl)-4.6-dinitrophenyl crotonate (Karathan), 2-amino-5-[(1-((carboxymethyl)amino)-3-((2,4-dinitrophenyl)thio)-1-oxopropan-2-yl)amino]-5-oxopentanoic acid (SDN GSH), 2,4-dinitroanisole (2,4-DNA) and 2,4-dinitrobenzoic acid (2,4-DNB). 2,4-DNP and DNOC showed the highest tryptophan fluorescence quenching constant values, these being also the most toxic compounds. The electronic chemical potential value of the most stable complex of 2,4-DNP-with tryptophan is higher than the values of the electronic chemical potentials of complexes corresponding to the derivatives.

Dinitro-o-cresol induces apoptosis-like cell death but not alternative oxidase expression in soybean cells.

In plants, programmed cell death is thought to be activated during differentiation and in response to biotic and abiotic stresses. Although its mechanisms are far less clear, several morphological and biochemical features have been described in different experimental systems, including DNA laddering and cytosolic protease activation. Moreover, plant mitochondria have an alternative terminal oxidase (AOX), which is thought to be involved in protection against increased reactive oxygen species production, perhaps representing a mechanism to prevent programmed cell death. In this study, we analysed cell death induced by the herbicide dinitro-o-cresol (DNOC) in soybean (Glycine max) suspension cell cultures and evaluated biochemical and molecular events associated with programmed cell death. AOX capacity and expression were also determined. DNOC-treated cells showed fragmented nuclear DNA as assessed by an in situ assay that detects 3'-OH ends. In addition, specific colorimetric assays and immunoblot analysis revealed activation of caspase-3-like proteins and release of cytochrome c from mitochondria, respectively, confirming the apoptotic-like phenotype. Surprisingly, AOX capacity and protein levels decreased in DNOC-treated cells, suggesting no association between cell death and AOX under these experimental conditions. In conclusion, the results show that DNOC induces programmed cell death in soybean cells, suggesting that plants and animals might share similar pathways. Further, the role of AOX in cell death has not been confirmed, and may depend on the nature and intensity of stress conditions.

Toxic effects, at three pH levels, of two reference molecules on common carp embryo.

Early life-stage survival, motility, and growth toxicity tests were carried out on common carp (Cyprinus carpio L.) endotrophic embryo, with two reference molecules (captafol and DNOC), from fertilization to the end of the first two-thirds period of mortalities by starvation. Thirteen days duration exposure was performed in daily renewed medium, at 24.5 degrees C, in standard synthetic water, at three pHs (6.9, 7.8, 9.0), in the presence of 10 mM/liter nontoxic pH buffer. Nominal concentrations of toxicants were 0.0, 0.25, 0.5, 1.0, and 2.0 mg/liter. Toxic effects on survival, motility, and growth decreased with increasing pH. From pH 6.9 to 9.0, the "no-observed-effect concentrations" differed by a factor greater than 8. Motility and growth cannot be considered, at population level, as true sublethal embryo-larval toxicity criteria. The toxicological interpretation of these results is discussed. They confirm the need of multifactorial methods for toxic risks and effects assessment on fish early life stages in the environment.

Genetic safety evaluation of pesticides in different short-term tests.

Cyanazine, cyhexatin, dicamba and DNOC are pesticides commonly and broadly used in agriculture pest control. However, there is little information on their toxicity and mutagenicity in human cells and in whole animals. Therefore, UDS assay and SCE assay in human peripheral lymphocytes, and chromosome aberration analysis in bone marrow of rats have been used to assess the DNA-damaging activity of the above pesticides. Cyanazine proved non-genotoxic in all the test systems. Cyhexatin showed only weakly positive results for SCE induction in human lymphocytes, providing no concern for genotoxicological hazard. While dicamba did not show clastogenic effects in rodents, DNOC gave significant dose-related increases of structural chromosome aberrations in rat bone marrow cells. Female animals showed increased sensitivity to the toxic effects by DNOC at the highest dose. The results provide further information on the intrinsic genotoxic activity of the tested pesticides, which may contribute to the toxicological assessment of the risk associated with human exposure.

In vivo unwinding fluorimetric assay as evidence of the damage induced by fenarimol and DNOC in rat liver DNA.

Five pesticides [amitraz, cyanazine, cyhexatin, dinitro-o-cresol (DNOC), and fenarimol] were tested as pure active ingredients for in vivo induction of DNA strand breaks on rat hepatocytes after intraperitoneal (ip) treatment. Two pesticides, fenarimol and DNOC, were capable of inducing DNA damage because they significantly increased the DNA unwinding rate. On the contrary, amitraz, cyanazine, and cyhexatin were not DNA-damaging agents.

[Circadian rhythm of the toxicokinetics and toxicodynamics of the plant protection substance dinitro-ortho-cresol in rabbits]. / Circadiane Rhythmik der Toxikokinetik und -dynamik des Pflanzenschutzmittels DNOC (Dinitro-ortho-kresol) beim Kaninchen.

Results from chronotoxicological investigations with the plant protection chemical DNOC has been described and judged. It has been studied hyperthermia caused by i.v. application of 7.5 mg/kg body weight on rabbits to 6 different times, starting at 4 a.m. with 4-hours-intervals as well as the dose-effect relation in each case with 3 doses to the time of application at 12 a.m., 6 p.m., 12 p.m. and 6 a.m. and the renal DNOC excretion after a dose of 10 mg/kg body weight. There was found a time dependent rhythm in all experiments. Acrophase of hyperthermia is placed in the middle of the phase of activity of the animals at 12 p.m., however it depends on the dose. The results are showing, that the position of acrophase by constant environmental factor is neither a substance specific nor an animal specific constant.

Induction of biotransformation in the liver of Eel (Anguilla anguilla L.) by sublethal exposure to dinitro-o-cresol: an ultrastructural and biochemical study.

Structural and functional alterations in hepatocytes of the European eel, Anguilla anguilla, following a 4-week-exposure to 5, 50, and 250 micrograms/liter dinitro-o-cresol (DNOC) were investigated by means of electron microscopy and biochemistry and compared to liver pathology in eels exposed to the chemical spill into the Rhine river at Basle in November 1986. Whereas phenological parameters (growth, condition factor) are unaffected, ultrastructural and biochemical alterations are detectable at greater than or equal to 50 and 5 micrograms/liter DNOC, respectively. Structural modifications include: rounding-up of the nuclei; fractionation and reduction of the rough endoplasmic reticulum; proliferation of the smooth endoplasmic reticulum (SER), mitochondria, peroxisomes, and lysosomes; bundles of rod-shaped SER profiles; annulate lamellae; membrane whorls within mitochondria; crystallization of the peroxisomal matrix and glycogen bodies; glycogen depletion and lipid augmentation. Structural changes can be correlated to an increase in hepatic lipid and protein contents as well as stimulation of mitochondrial (cytochrome c oxidase), peroxisomal (catalase, allantoinase, uricase), lysosomal (arylsulfatase), and microsomal (esterase) enzymes. An increase in NADPH-cytochrome c reductase and cytochrome P450 as well as UDP-glucuronyltransferase and arylsulfotransferase activities in the microsomal fraction document an induction of hepatic biotransformation as a functional correlate to SER proliferation. Maximum inducibility of biotransformation enzymes at 50 micrograms/liter indicates a biphasic, concentration-dependent reaction of eel liver. Comparison of DNOC-induced effects with liver pathology in eel exposed to the chemical spill in 1986 reveals striking similarities so that DNOC may not be excluded as a possible factor in the fish kill in the Rhine river.

Effect of DNOC, Ferbam and Imidan exposure on mouse sperm morphology.

DNOC, Ferbam and Imidan were tested in (C3H X C57BL/6) F1 mice to assess their potential testicular toxicity. Chemicals were administered i.p. and per os at different doses for 5 consecutive days. After 35 days the testicular was toxicity was evaluated by measuring the testicular weights, the sperm counts and the percentage of abnormal sperm. DNOC and Imidan failed to induce teratospermia in mice treated by both routes of administration. Conversely Ferbam induced a statistically significant increase in teratospermia only following per os administration to mice at a dose of 1000 mg/kg b.w./day. These data indicate that per os administration of Ferbam succeeded in producing active metabolites able to interfere with the differentiation process of spermatogenic cells.

The relationship between uncoupling of oxidative phosphorylation and neuronal necrosis within the CNS in rats dosed with trihalogenated imidazoles.

The trihalogenated imidazoles, trichloroimidazole (TCI), tribromoimidazole (TBI), and triiodoimidazole (TII), are in vitro uncouplers of oxidative phosphorylation with similar activities. Although TCI and TBI are also uncouplers in vivo, some doubt exists for TII, which is much less toxic and produces atypical signs of poisoning. Dibromo- and monobromoimidazole do not uncouple oxidative phosphorylation either in vitro or in vivo. Dosing of TCI and TBI to rats resulted within 24-48 hr in neuronal necrosis within the CNS involving the vestibular nucleus, red nucleus, and outer parietal neocortex and ataxia of the hindlimbs. However, no neuronal necrosis or ataxia was observed after dosing of TII to rats, even when given at doses four times greater than for either TCI or TBI, resulting in much higher brain concentrations. Although TBI was equitoxic to rats, mice, hamsters, and gerbils, CNS damage and ataxia were observed only in the rat, even though comparable brain concentrations of TBI were found in the gerbil. Measurement of the concentration of TBI in the dissected rat brain gave no indication of localized concentrations of compound in the areas associated with neuronal damage. Doses of TBI and the classical uncoupler 3,5-dinitro-o-cresol (DNOC), matched for whole body O2 consumption, caused comparable changes in rat brain blood flow although DNOC does not cause brain damage. Changes in blood flow were not restricted to those brain areas susceptible to damage. Thus, although we were unable to completely dissociate CNS damage from uncoupling of oxidative phosphorylation produced by TBI and TCI in the rat, it is unlikely that such damage is primarily related to the uncoupling ability of these compounds.

Additional data on the mutagenic effect of dinitro-o-cresol-containing herbicides.

In in vivo experiments in mice it was studied, on the one hand, whether 1 year after treatment with dinitro-o-cresol (DNOC)-containing herbicide it was possible to detect any increase in chromosome aberrations in the bone marrow cells of the mouse, and on the other hand, to learn the frequency of chromosome aberrations in the subsequent generations when the treatment of the male animals with DNOC-containing herbicide was continued in each generation and when it was discontinued before mating. The chromosome aberrations of the bone marrow cells of the treated mice were demonstrated even 1 year after the treatment. After the treatment of the male animals was continued in each subsequent generation, the chromosome aberrations in the embryos increased, whereas when it was discontinued, it decreased in the subsequent generations.

The effects of five weeks treatment with dinitro-o-cresol- or trifluralin-containing pesticides on the germ cells of male mice.

The effects of two pesticides, the insecticide-herbicide Krezonit E, which contains 50% dinitro-o-cresol, and the herbicide Olitref, which contains 26% trifluralin (2,6-dinitro-N,N-dipropyl-4-trifluoromethylaniline), on the gonads and germ cells of male mice were studied. The pesticides were given twice a week for 5 weeks in i.p. doses of 0.6 mg kg-1 for Krezonit E and 6.0 mg kg-1 for Olitref. These doses are 1% of the i.p. LD50. Cytogenic analysis of germ cells carried out from 3 weeks onwards after the last treatment day showed that Olitref significantly increased the frequency of germinal chromosomal abnormalities at 6-7 weeks after treatment. This pesticide increased the frequency of autosomal univalents X/Y separations at meiotic metaphase and multivalent configurations. Krezonit E did not increase significantly the number of chromosomal abnormalities, although there was some increase at 3 weeks, mainly in the form of autosomal univalents.

Mutagenicity of DNOC in Drosophila melanogaster.

The insecticide/ascaricide dinitro-ortho-cresol (DNOC) was tested for mutagenicity in BASC or Muller-5 test with Drosophila melanogaster. The frequencies of induced sexlinked recessive lethals in premeiotic and postmeiotic germ cell stages of spermatogenesis were determined after exposure of adult wild-type males by oral application of DNOC. Sex-linked recessive lethals were scored in the F2 generation according to standard procedures. The mutagenic activity of DNOC at concentrations of 0.25 to 1.01 mM (50-200 ppm) in postmeiotic germ cell stages (11 lethals/1570 chromosomes tested = 0.70%) > premeiotic stages (3/1001 = 0.29%). The mutations rate in all germ cell stages was 0.59% (18/3140). The lowest effective concentration for the induction of recessive lethals in nature sperm was 0.25 mM which is 1/3 to 1/2 LD50 which is approximately 0.61 mM. The frequency of spontaneously occurring lethality was 0.06 (1/1718). The data presented here show significant differences in Kastenbaum-Bowman test. Dimethylnitrosamine (6.8 mM = 500 ppm) induced a rate of 21.0% of lethals (112/534).

[Effect of a dinitro-o-cresol-containing herbicide (Hedolit) on human reproduction]. / Uber den Einfluss eines Dinitro-o-kresol-haltigen Pflanzenschutzmittels (Hedolit) auf die menschliche Reproduktion.

After epicutaneous application of Hedolit to guinea pigs the above mentioned substance was found less concentrated in the semen and in the testis than in the serum. Hedolit concentrations, as they were found in the serum, lead already to motility limitations of human sperma and cause a small mutageneous effect in drosophila melanogaster. The chosen epicutaneous modus of application did not lead to such traceable concentrations of Hedolit in the semen and in the testis.

Antibacterial effect of some combinations of pesticides of pure cultures of microorganisms.

The investigations conducted showed that the antibacterial effect of dinitro-o-cresol, cuprozane, and trichlorfon is due to their chemical structure and to the species of microorganisms on which they act. Dinitro-o-cresol and Cuprozane possess selective toxicity with respect to B. anthracis and Rh. gracilis and cuprozane with respect to Ch. perfringens. The method of evaluating the effect of combinations of chemical compounds consisting of two or more components which have been developed made it possible to detect the antibacterial effect of mixtures consisting of the indicated pesticides. It was established that the combinations studied possess synergic and additive effects, which also depend on the chemical structure of the pesticides and the species of microorganisms.