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Persulfate/Fe 2+ -based advanced oxidation processes are widely used to treat water contaminated with 2,4-dinitrotoluene (DNT). However, the oxidation of DNT by persulfate/Fe 2+ in the presence of the chloride ion (Cl⁻) has not been addressed, and the transformation pathways and toxicities of the intermediate products remain unclear. In this study, the effect of different Cl⁻ concentrations on the oxidation of DNT was investigated by persulfate/Fe 2+ . After the addition of 1.0 mM Cl⁻ and 6 h of oxidation, the removal efficiency of DNT increased by 68.5%. Scavenging experiments and an electron spin resonance analysis suggested that Cl⁻ caused hydroxyl radicals to increase in content in the persulfate/Fe 2+ system, thus promoting the removal of DNT. Eight intermediate products of DNT were accurately detected using high-resolution mass spectrometry, and the transformation pathways of DNT were proposed, including hydroxylation/oxidation, elimination of the nitro group, and chlorination process. The acute and chronic toxicities of the intermediate products decreased during the oxidation process, but chlorinated by-products posed a higher toxicological risk. This result is vital for the practical application and environmental safety evaluation of persulfate/Fe 2+ -based advanced oxidation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In response to the EU cosmetics directive regulation and REACH legislation which encourage cell culture methods in order to reduce or replace the use of animals in toxicology studies, we settled the culture of prepubertal domestic cat seminiferous tubules in our validated BioAlter® model, usually used with prepubertal rat, called here BioAlter®-rat, by opposition to BioAlter®-cat settled here. We carried out a comparative study on the effects of 3 testicular toxicants, 1,3-dinitrobenzene at 60 μM, 2-methoxyacetic acid at 2.5 mM and carbendazim at 50 nM or 500 nM in both BioAlter®-cat and BioAlter®-rat over a 3-week culture period. Sertoli cell or each germ cell populations as well as the levels of Sertoli cell or germ cell specific mRNAs were studied. The harmful effects of the 3 toxicants on pre-meiotic, meiotic and post-meiotic cell numbers and on Sertoli or germ cell specific mRNAs were clearly observed in the two species, even if there might be some small differences in the intensity of the effects on some of the studied parameters. Hence, BioAlter®-cat might be a solution to the requirements of the EU cosmetics directive and REACH legislation for male reproductive toxicology studies., (Copyright © 2022 INSERM U 1208, 18 avenue du Doyen Lépine, 69500 Bron France. Published by Elsevier Ltd.. All rights reserved.)

The dinitrotoluene isomers 2,4 and 2,6-dinitrotoluene (DNT) represent highly toxic, mutagenic, and carcinogenic compounds used in explosive manufacturing and in commercial production of polyurethane foam. Bioremediation, the use of microbes to degrade residual DNT in industry wastewaters, represents a promising, low cost and environmentally friendly alternative technology to landfilling. In the present study, the effect of different bioremediation strategies on the degradation of DNT in a microcosm-based study was evaluated. Biostimulation of the indigenous microbial community with sulphur phosphate (2.3 g/kg sludge) enhanced DNT transformation (82% transformation, from 300 g/L at Day 0 to 55 g/L in week 6) compared to natural attenuation over the same period at 25 °C. The indigenous microbial activity was found to be capable of transforming the contaminant, with around 70% transformation of DNT occurring over the microcosm study. 16S rDNA sequence analysis revealed that while the original bacterial community was dominated by Gammaproteobacteria (30%), the addition of sulphur phosphate significantly increased the abundance of Betaproteobacteria by the end of the biostimulation treatment, with the bacterial community dominated by Burkholderia (46%) followed by Rhodanobacter , Acidovorax and Pseudomonas . In summary, the results suggest biostimulation as a treatment choice for the remediation of dinitrotoluenes and explosives waste.

A major challenge in regulatory developmental neurotoxicity (DNT) assessment is lack of toxicological information for many compounds. Therefore, the Test Guidelines programme of the Organisation for Economic Cooperation and Development (OECD) took the initiative to coordinate an international collaboration between diverse stakeholders to consider integration of alternative approaches towards improving the current chemical DNT testing. During the past few years, a series of workshops was organized during which a consensus was reached that incorporation of a DNT testing battery that relies on in vitro assays anchored to key neurodevelopmental processes should be developed. These key developmental processes include neural progenitor cell proliferation, neuronal and oligodendrocyte differentiation, neural cell migration, neurite outgrowth, synaptogenesis and neuronal network formation, as well key events identified in the existing Adverse Outcome Pathways (AOPs). AOPs deliver mechanistic information on the causal links between molecular initiating event, intermediate key events and an adverse outcome of regulatory concern, providing the biological context to facilitate development of Integrated Approaches to Testing and Assessment (IATA) for various regulatory purposes. Developing IATA case studies, using mechanistic information derived from AOPs, is expected to increase scientific confidence for the use of in vitro methods within an IATA, thereby facilitating regulatory uptake. This manuscript summarizes the current state of international efforts to enhance DNT testing by using an in vitro battery of assays focusing on the role of AOPs in informing the development of IATA for different regulatory purposes, aiming to deliver an OECD guidance document on use of in vitro DNT battery of assays that include in vitro data interpretation., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Atopic dermatitis is a chronic inflammatory skin disease in humans. Although Olea europaea leaf extract (OLE) and Spirodela polyrhiza extract (SPE) have been used to protect against skin damage, the effects of their combined administration on atopic dermatitis have yet to studied., Purpose: In this study, we evaluated the potential therapeutic effects of an OLE and SPE combination on the progression of atopic dermatitis and the possible mechanisms underlying these effects in 1-chloro-2,4-dinitrobenzene (DNCB)-treated NC/Nga mice., Methods: Atopic dermatitis was induced by topical application of 0.2% w/v DNCB prepared in an olive oil:acetone solution (1:3), and thereafter OLE, SPE and OLE + SPE were administered orally for 5 weeks. We determined atopic dermatitis symptoms, serum IgE levels, and levels of cytokine- and gene expression in the dorsal skin and splenocytes, and performed histological and immune cell subtype analyses. The expression of skin barrier-related proteins (filaggrin, sirtuin 1, and claudin 1) was also evaluated., Results: The OLE + SPE combination significantly ameliorated atopic dermatitis symptoms, including dermatitis scores, and reduced epidermal thickness and infiltration of different inflammatory cells in mice with DNCB-induced atopic dermatitis. It also significantly reduced the number of CD4 + , CD8 + , and CD4 + /CD69 + T cells; immunoglobulin E-producing B cells (CD23 + /B220 + ) in the axillary lymph nodes; CD3 + T-cell eosinophils (chemokine-chemokine receptor 3 + /CD11b + ) in the skin; and CD3 + T cells, immunoglobulin E-producing B cells (CD23 + /B220 + ), and eosinophils in peripheral blood mononuclear cells. Additionally, the experimental combination lowered levels of serum immunoglobulin E and histamine, as well as Th2-mediated cytokines, and interleukin-4, -5, and -13, whereas it increased the levels of Th1-mediated cytokine interferon-γ in splenocytes. Furthermore, the preparation significantly restored expression of the skin barrier-related proteins filaggrin, sirtuin 1, and claudin 1, and also reduced the expression of the inflammatory cytokine interleukin-6 and chemokine-chemokine receptor 3, as well as the pruritus-related cytokine interleukin-31 and interleukin-31 receptor, in atopic dermatitis skin lesions., Conclusion: Taken together, our findings indicate that administration of a combination of OLE and SPE can alleviate atopic dermatitis symptoms by regulating immune balance and skin barrier function and may be an effective therapeutic option for the treatment of atopic dermatitis., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Testicular injury is often observed in drug development. Serum hormones are usually used as noninvasive biomarkers for testicular injury; however, their sensitivities are low. Therefore, it is difficult to monitor testicular injury in drug development. In recent years, molecules in body fluid exosomes have attracted attention as biomarkers for diseases. In this study, small RNAs in serum exosomes were analyzed to identify noninvasive biomarkers of testicular injury in rats, which are often used in preclinical drug development. The rat models of testicular injury were prepared by a single oral administration of 2000 mg/kg ethylene glycol monomethyl ether, in which spermatocyte degeneration and Sertoli cell vacuolation were observed, or 400 mg/kg carbendazim, in which Sertoli cell vacuolation and seminiferous tubule dilation were observed. Serum exosomal small RNA-seq analysis of these models was performed. The analysis identified 3 small RNAs that fluctuated in common between the models, and miR-423-5p and miR-128-3p were selected as candidate markers. For evaluating these candidate markers in other testicular injury models, the models were prepared by a single oral administration of 60 mg/kg 1,3-dinitrobenzene or 500 mg/kg nitrofurazone, and spermatocyte degeneration and Sertoli cell vacuolation were observed. In qPCR analysis, these exosomal miRNAs were upregulated in all models except for the 1,3-dinitrobenzene model, in which severe hemolysis was observed. By contrast, these miRNAs in whole serum extracts did not significantly change in any of the models. In conclusion, we identified miR-423-5p and miR-128-3p in serum exosomes as noninvasive biomarkers for testicular injury in rats., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

2,4-dinitrotoluene (2,4-DNT) is a common environmental pollutant, and was classified as a group 2B human carcinogenic compound by the International Agency for Research on Cancer. This study determined the toxic effects of 2,4-DNT exposure on zebrafish at the embryo-larvae stage, in terms of organ morphogenesis and the expression pattern of selected target genes related to lipid metabolism and oxygen transportation. The results showed that the 120-h post-fertilization LC 50 of 2,4-DNT was 9.59 mg/L with a 95% confidence interval of 8.89-10.44 mg/L. The larvae treated with 2,4-DNT showed toxic symptoms including smaller body, less skin pigment production, yolk malabsorption, and disordered liver development. Further studies on the expression of genes related to lipid transport and metabolism, and respiration indicated that they were significantly affected by 2,4-DNT. It is concluded that 2,4-DNT exposure perturbed liver development and yolk absorption in early-life zebrafish, and disturbed the lipid metabolism /oxygen transport gene expression.

Buried explosive material, e.g., landmines, represent a severe issue for human safety all over the world. Most explosives consist of environmentally hazardous chemicals like 2,4,6-trinitrotoluene (TNT), carcinogenic 2,4-dinitrotoluene (2,4-DNT) and related compounds. Vapors leaking from buried landmines offer a detection marker for landmines, presenting an option to detect landmines without relying on metal detection. 2,4-Dinitrotoluene (DNT), an impurity and byproduct of common TNT synthesis, is a feasible detection marker since it is extremely volatile. We report on the construction of a wireless, handy and cost effective 2,4-dinitrotoluene biosensor combining recombinant bioluminescent bacterial cells and a compact, portable optical detection device. This biosensor could serve as a potential alternative to the current detection technique. The influence of temperature, oxygen and different immobilization procedures on bioluminescence were tested. Oxygen penetration depth in agarose gels was investigated, and showed that aeration with molecular oxygen is necessary to maintain bioluminescence activity at higher cell densities. Bioluminescence was low even at high cell densities and 2,4-DNT concentrations, hence optimization of different prototypes was carried out regarding radiation surface of the gels used for immobilization. These findings were applied to sensor construction, and 50 ppb gaseous 2,4-DNT was successfully detected.

Thiols, such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH), hydrogen sulfide (H 2 S), and thiophenol are metabolically correlated with each other via redox reactions. As a result of the similarity of chemical properties between Cys, Hcy, GSH, H 2 S, and thiophenol, it is very challenging to develop an effective methodology to differentiate them. In this work, a triple-emission fluorescent probe, NCQ, was reported for the simultaneous detection of Cys/Hcy, GSH/H 2 S, and thiophenol with high sensitivity and selectivity. The solution of NCQ displayed distinct fluorescent signals toward Cys/Hcy, GSH/H 2 S, and thiophenol: blue and green for Cys/Hcy, blue for GSH/H 2 S, blue and red for thiophenol. Through the blue-green-red emission color combination, Cys/Hcy, GSH/H 2 S, and thiophenol could be discriminatively detected in solution and in living cells.

Increased industrial processes have introduced emerging toxic pollutants into the environment. Phytoremediation is considered to be a very useful, economical and ecofriendly way of controlling these pollutants, however, certain pollutants can potentially travel through the food chain and accumulate at hazardous levels. Four isomers of dinitrotoluenes (DNT) were investigated and observed their potential toxicity towards A. thaliana. Two different aphid species (generalist and specialist) were allowed to feed on plants treated with DNTs and toxicity to aphids determined. Reduced metabolites of DNT (in both plant and aphids) were recovered and quantified through GC-MS analyses. 2,6-DNT was observed to be the toxic of the DNTs tested. Complete metabolism of DNTs to their reduced products was never achieved for higher concentrations. Regioselectivity was observed in the case of 2,4-DNT, with 4A2NT as the dominant isomer. Feeding aphids showed a similar toxicity pattern for DNT isomers as host plants. Metabolites were recovered from the body of aphids, demonstrating the potential transport of metabolites through the food chain. Plants show varied toxicity responses towards the DNT isomers. Aphids fed on A. thaliana plants treated with DNTs were shown to have ANTs present, which reflects the propagation of DNT metabolites through the food chain., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Musk ambrette (4-tert-butyl-3-methoxy-2,6-dinitrotoluene) is a nitro musk, a cheap substitute for natural musk and a potential environmental pollutant based on its persistence, accumulation in human organisms. We investigated the acute toxicity of musk ambrette using wild-type AB and transgenic Tg(fli1a:EGFP) y1 zebrafish. Different concentrations were delivered to zebrafish by direct soaking from 6 to 72 h post-fertilization (hpf). The LC 50 of musk ambrette was 76.4 µg mL -1 . As musk ambrette concentration increased, zebrafish embryos showed developmental delays (50 µg mL -1 , 22 hpf), pericardial edema (5 µg mL -1 , 48 hpf), circulatory disturbances, curved body axis (1 µg mL -1 , 72 hpf) and death (100 µg mL -1 , 22 hpf). Target organ toxicity was evaluated by a zebrafish angiogenesis model. Musk ambrette induced cardiovascular morphological changes, vessel permeability variation, angiogenic changes and cardiotoxicity (10 µg mL -1 , 48 hpf). The disappearance of caudal vein plexus confirmed the vascular development toxicity. Musk ambrette negatively affects early life-stage survival and demonstrates various toxicities in zebrafish.

Individual effects of nitrogen-based energetic materials (EMs) 2,4-dinitrotoluene (2,4-DNT), 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), nitroglycerin (NG), and 2,4,6,8,10,12-hexanitrohexaazaisowurtzitane (CL-20) on litter decomposition, an essential biologically-mediated soil process, were assessed using Orchard grass (Dactylis glomerata) straw in Sassafras sandy loam (SSL) soil, which has physicochemical characteristics that support "very high" qualitative relative bioavailability for organic chemicals. Batches of SSL soil were separately amended with individual EMs or acetone carrier control. To quantify the decomposition rates, one straw cluster was harvested from a set of randomly selected replicate containers from within each treatment, after 1, 2, 3, 4, 6, and 8 months of exposure. Results showed that soil amended with 2,4-DNT or NG inhibited litter decomposition rates based on the median effective concentration (EC50) values of 1122 mg/kg and 860 mg/kg, respectively. Exposure to 2-ADNT, 4-ADNT or CL-20 amended soil did not significantly affect litter decomposition in SSL soil at ≥ 10,000 mg/kg. These ecotoxicological data will be helpful in identifying concentrations of EMs in soil that present an acceptable ecological risk for biologically-mediated soil processes., (Published by Elsevier Inc.)

Immunoglobulin E (IgE) is involved in the onset of allergic reaction, and the suppression of IgE production leads to alleviation of allergic symptoms. We found that mango peel ethanol extract (MPE) significantly suppresses IgE production by human myeloma cell line U266 cells, suggesting that MPE has an anti-allergic effect by inhibiting the production of IgE. Although mangiferin is contained in mango, which suppresses IgE production by U266 cells, it was not contained in MPE. We investigated the suppressive effect of MPE in 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis model mice. The elevation of serum IgE level was significantly suppressed by oral administration of MPE. Intake of MPE also suppressed the expression level of IL-4 in the DNFB-challenged ears, suggesting that MPE suppresses the IL-4-mediated maturation into IgE-producing cells. Our findings indicate that MPE has a potential to alleviate the increase in serum IgE level that is feature of type I allergy.

2,6-Dicyano-4-nitroaniline and 2-cyano-4-nitroaniline (CNNA; 2-amino-5-nitrobenzonitrile) are potent mutagens in the Ames test, even though unsubstituted nitroanilines (NAs) are no more than weak mutagens. These compounds are putative reduction products of many commercial azo dyes, including Disperse Blue 165, Disperse Blue 337, Disperse Red 73, Disperse Red 82, Disperse Violet 33, and Disperse Violet 63. We have examined the mutagenicity in strains TA98 and YG1024 of a series of commercially-available isomers of CNNA, and some related compounds, to probe the relationship between structure and genotoxic activity in this class of compounds. The potentiating effect of the cyano substituent is seen in many cases; e.g. 2-amino-4-nitrobenzonitrile is a much more potent mutagen than 3-NA. 2,4-Dinitrobenzonitrile is also highly mutagenic. Possible mechanisms for the "cyano effect" are considered, with respect to the likely structures of cyanonitroaniline-DNA adducts and the roles of the enzymes (nitroreductase and acetyl CoA:arylamine N-acetyltransferase) believed to be involved in the activation of nitroaromatic compounds. Environ. Mol. Mutagen. 59:114-122, 2018. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: In search of safer treatments for inflammatory bowel disease in subjects not responding to, or showing adverse effects to TNF-α antagonists, we tested three novel indoline carbamates in the 2,4-dinitrobenzene sulfonic acid (DNBS) model of colitis in rats. The compounds have anti-inflammatory activity in other disease models in mice., Methods: AN827 (3-(2-(methoxy carbonyl) ethyl) indolin-4-ylethyl methyl) carbamate (0.1 or 1mg/kg), AN680 (3-(2-(methoxy carbonyl) ethyl) indolin-6-ylethyl methyl) carbamate (1.25 or 2.5mg/kg) and AN917 (3-(3-amino propyl) indolin-4-ylethyl methyl) carbamate (1 or 2mg/kg), 5-aminosalycilic acid (5-ASA) (1 or 100mg/kg) or saline (1ml/kg) were administered rectally 1h after intracolonic administration of DNBS, (35mg/kg in 30% alcohol). Disease severity was assessed four days after DNBS administration by change in body weight, colon weight, area of ulceration, myeloid peroxidase (MPO) activity, colonic TNF-α, IL-6 and IL-1β levels. Histopathological scoring was performed after staining colon sections with hematoxylin and eosin and with antibodies to CD68 and CD11b., Results: AN827 (0.1 and 1mg/kg), AN680 (2.5mg/kg) and AN917 (2.0mg/kg) significantly reduced all macroscopic and microscopic parameters of colitis, colonic pro-inflammatory cytokines, TNF-α, IL-1β and IL-6 and MPO activity by about 80%., Conclusions: The indoline derivatives largely prevented the symptoms of colitis and were 500-50 times more potent and more effective than 5-ASA. It may be worth evaluating them in models of established colitis. Since AN827 is strongly bound by plasma proteins no adverse effects are expected if compound is absorbed into the circulation after rectal administration., (Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Explosive-contaminated soil is harmful to people's health and the local ecosystem. The acute toxicity of its extracting solution was tested by bacterial luminescence assay using three kinds of luminescent bacteria to characterize the toxicity of the soil. An orthogonal test L 16 (4 5 ) was designed to optimize the soil extracting conditions. The optimum extracting conditions were obtained when the ultrasonic extraction time, ultrasonic extraction temperature, and the extraction repeat times were 6 h, 40 °C, and three, respectively. Fourier transform infrared spectroscopy (FTIR) results showed that the main components of the contaminated soil's extracting solution were 2,4-dinitrotoluene-3-sulfonate (2,4-DNT-3-SO 3 - ); 2,4-dinitrotoluene-5-sulfonate (2,4-DNT-5-SO 3 - ); and 2,6-dinitrotoluene (2,6-DNT). Compared with Photobacterium phosphoreum and Vibrio fischeri, Vibrio qinghaiensis sp. Nov. is more suitable for assessing the soil extracting solution's acute toxicity. Soil washing can remove most of the contaminants toxic to luminescent bacterium Vibrio qinghaiensis sp. Nov., suggesting that it may be a potential effective remediation method for explosive-contaminated soil.

Three water contaminants were selected to be tested in the zebrafish embryo toxicity test (DarT) in order to investigate the sensitivity of the zebrafish embryo toxicity test with respect to mixture effect detection. The concentration-response curves for the observed effects lethality and hypo-pigmentation were calculated after an exposure of the embryos for 96 h with a fungicide (carbendazim), a plasticizer or propellent precursor (2,4-DNT: 2,4- dinitrotoluene) and an aromatic compound (AαC: 2-amino-9H-pyrido[2,3-b]indol), respectively. Follow-up mixture tests were based on the calculated LC50 or EC50 of the single compounds and combined effects were predicted according to the mixture concepts of concentration addition (CA) and independent action (IA). The order of toxicity for the single substances was carbendazim (LC50 = 1.25 μM) < AαC (LC50 = 8.16 μM) < 2,4-DNT (LC50 = 177.05 μM). For AαC and 2,4 DNT hypo-pigmentation was observed in addition (AαC EC50 = 1.81 μM; 2,4-DNT EC50 = 8.81 μM). Two binary and one ternary mixture were studied on lethality and one on hypo-pigmentation: 2,4-DNT/AαC (LC50 = 119.21 μM, EC50 = 5.37 μM), carbendazim/AαC (LC50 = 4.49 μM) and AαC/Carbendazim/2,4 DNT (LC50 = 108.62 μM). Results showed that the effects were in agreement with the CA model when substances were tested in mixtures. Therefore, in a reasonable worst case scenario substance combination effects in fish embryos were at maximum only prone to overestimation when using CA as the mixture concept., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Due to the complex physiology of the testes, in vitro models have been largely unsuccessful at modeling testicular toxicity in vivo. We conducted a pilot study to evaluate the utility of the Durand ex vivo rat seminiferous tubule culture model [1-3] that supports spermatogenesis through meiosis II, including the formation of round spermatids. We used this system to evaluate the toxicity of four known testicular toxicants: 1,3-dinitrobenzene (DNB), 2-methoxyacetic acid (MAA), bisphenol A (BPA), and lindane over 21 days of culture. This organotypic culture system demonstrated the ability to successfully model in vivo testicular toxicity (Sertoli cell toxicity and disruption of meiosis) for all four compounds. These findings support the application of this system to study molecules and evaluate mechanisms of testicular toxicity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Age-related disturbances in astrocytic mitochondrial function are linked to loss of neuroprotection and decrements in neurological function. The immortalized rat neocortical astrocyte-derived cell line, DI-TNC1, provides a convenient model for the examination of cellular aging processes that are difficult to study in primary cell isolates from aged brain. Successive passages in culture may serve as a surrogate of aging in which time-dependent adaptation to culture conditions may result in altered responses to xenobiotic challenge. To investigate the hypothesis that astrocytic mitochondrial homeostatic function is decreased with time in culture, low passage DI-TNC1 astrocytes (LP; #2-8) and high passage DI-TNC1 astrocytes (HP; #17-28) were exposed to the mitochondrial neurotoxicant 1,3-dinitrobenzene (DNB). Cells were exposed in either monoculture or in co-culture with primary cortical neurons. Astrocyte mitochondrial membrane potential, morphology, ATP production and proliferation were monitored in monoculture, and the ability of DI-TNC1 cells to buffer K(+)-induced neuronal depolarization was examined in co-cultures. In HP DI-TNC1 cells, DNB exposure decreased proliferation, reduced mitochondrial membrane potential and significantly decreased mitochondrial form factor. Low passage DI-TNC1 cells effectively attenuated K(+)-induced neuronal depolarization in the presence of DNB whereas HP counterparts were unable to buffer K(+) in DNB challenge. Following DNB challenge, LP DI-TNC1 cells exhibited greater viability in co-culture than HP. The data provide compelling evidence that there is an abrupt phenotypic change in DI-TNC1 cells between passage #9-16 that significantly diminishes the ability of DI-TNC1 cells to compensate for neurotoxic challenge and provide neuroprotective spatial buffering. Whether or not these functional changes have an in vivo analog in aging brain remains to be determined., (Copyright © 2016. Published by Elsevier B.V.)

CO2, as the typical greenhouse gas causing the greenhouse effect, is a major global environmental problem and has attracted increasing attention from governments. Using algae to eliminate CO2, which has been proposed as an effective way to reduce the greenhouse effect in the past decades, can be disturbed by a growing number of artificial chemicals. Thus, seven types of chemicals and Selenastrum capricornutum (algae) were examined in this study, and the good consistency between the toxicity of artificial chemicals to algae and the disturbance of carbon fixation by the chemicals was revealed. This consistency showed that the disturbance of an increasing number of artificial chemicals to the carbon fixation of algae might be a "malware" worsening the global greenhouse effect. Therefore, this study proposes an original, promising index to assess the risk of deepening the greenhouse effect by artificial chemicals before they are produced and marketed., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Glutathione (GSH) is the most prominent antioxidant in cells and the co-factor of an important set of enzymes involved in the skin metabolic clearance system, glutathione S-transferases (GST). Here, we describe an LC-MS (liquid chromatography-mass spectroscopy) method to measure GSH and its disulfide form (GSSG) in HaCaT cells and a 3D Reconstructed Human Epidermis (RHE) model. In our assay, the basal level of GSH in both systems was in the low nmol/mg soluble protein range, while the level of GSSG was systematically below our limit of quantification (0.1 μM). We found that 2,4-dinitrohalobenzenes deplete the GSH present in HaCaT cells within the first hour of exposure, in a dose dependent manner. The level of GSH in HaCaT cells treated with a single non-toxic dose of 10 μM of dinitrohalobenzene was also shown to increase after two hours. While cells treated with 1-chloro-2,4-dinitrobenzene (DNCB) and 1-fluoro-2,4-dinitrobenzene (DNFB) repleted GSH to levels similar to untreated control cells within 24h, 1-bromo-2,4-dinitrobenzene (DNBB) seemed to prevent such a repletion and appeared to be the most toxic compound in all assays. A mathematical modelling of experimental results was performed to further rationalise the differences observed between test chemicals. For this purpose the biological phenomena observed were simplified into two sequential events: the initial depletion of the GSH stock after chemical treatment followed by the repletion of the GSH once the chemical was cleared. Activation of the nuclear factor E2-related factor 2 (Nrf2) pathway was observed with all compounds within two hours, and at concentrations less than 10 μM. These data show that GSH depletion and repletion occur rapidly in skin cells and emphasize the importance of conducting kinetic studies when performing in vitro experiments exploring skin sensitization., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

With m-Dinitrobenzene (m-DNB) as a selected model neurotoxicant, we demonstrate how to assess neurotoxicity, using morphology based measurement of neurite degeneration, in a conventional "full-contact" and a modern "restricted-contact" co-culture of rat cortical neurons and astrocytes. In the "full-contact" co-culture, neurons and astrocytes in complete physical contact are "globally" exposed to m-DNB. A newly emergent "restricted-contact" co-culture is attained with a microfluidic device that polarizes neuron somas and neurites into separate compartments, and the neurite compartment is "selectively" exposed to m-DNB. Morphometric analysis of the neuronal area revealed that m-DNB exposure produced no significant change in mean neuronal cell area in "full-contact" co-cultures, whereas a significant decrease was observed for neuron monocultures. Neurite elaboration into a neurite exclusive compartment in a compartmentalized microfluidic device, for both monocultures (no astrocytes) and "restricted" co-cultures (astrocytes touching neurites), decreased with exposure to increasing concentrations of m-DNB, but the average neurite area was higher in co-cultures. By using co-culture systems that more closely approach biological and architectural complexities, and the directionality of exposure found in the brain, this study provides a methodological foundation for unraveling the role of physical contact between astrocytes and neurons in mitigating the toxic effects of chemicals such as m-DNB., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The liver micronucleus assay using young adult rats has the potential to detect liver carcinogens by repeated dosing, and could be expected to be integrated into repeated-dose toxicity studies using a hepatocyte isolation method without the traditional in situ collagenase perfusion. In this study, to assess the performance of the repeated-dose liver micronucleus assay, 2,4-dinitrotoluene (DNT), which is a rodent liver carcinogen, was administered orally to male rats at doses of 50, 100 and 200 mg/kg/day once daily for 14 or 28 consecutive days, and the frequencies of micronucleated hepatocytes (MNHEPs) and micronucleated immature erythrocytes (MNIMEs) were examined. Significant increases in the MNHEPs were observed at 50 mg/kg/day or more in the 14-day treatment, and 50 and 100 mg/kg/day in the 28-day treatment. These increases were dependent on both the dose and the number of administrations, which indicates the possibility that the MNHEPs accumulate as a result of repeated dosing. In contrast, no increase in the MNIMEs was observed. In conclusion, the repeated-dose liver micronucleus assay using young adult rats is sufficiently sensitive to detect the genotoxicity of 2,4-DNT at a low dose.

As part of a collaborative study by the Mammalian Mutagenicity Study Group of the Environmental Mutagen Society of Japan, we examined micronucleus induction in hepatocytes following oral administration of 2,6-dinitrotoluene (2,6-DNT) at 30, 40, and 50mg/kg/day for 14 days or at 20, 30, and 40mg/kg/day for 28 days to young adult male rats. This compound is known to be a rat liver carcinogen. The formation of micronucleated hepatocytes was confirmed to be dose-dependent with statistically significant increases observed in both treatments. In contrast, no statistically significant changes in the percentage of micronucleated immature erythrocytes were observed in any dose group in the bone marrow micronucleus assay. These results indicated that the repeated-dose liver micronucleus assay has the potential to detect genotoxic hepatocarcinogens and can be integrated into general toxicological studies., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Exposure to 2,4,6-trinitrotoluene (TNT) causes methemoglobin (metHb) formation, hemolysis and negative heme balance in vivo, but the mechanistic details are poorly understood. In the present study, we examined the participation of metabolic activation in TNT-mediated hematotoxicity. Exposure of rats with TNT (300 mg/kg, i.p.) for 4 days resulted in a decrease of hematocrit value coupled to an increase in metHb formation. The red blood cells treated with 4-hydroxylamino-2,6-dinitrotoluene (HADNT), a metabolite of TNT, underwent readily hemolysis in vitro, whereas such a phenomenon was not seen with TNT. Consistent with this, HADNT is active toward metHb formation and the decrease in thiol content of the globin moiety compared with TNT and its metabolites 4-amino-2,6-dinitrotoluene (ADNT) and 4-acetylamino-2,6-dinitrotoluene (AADNT). Furthermore, interaction of purified rat oxyhemoglobin (oxyHb) with HADNT, but not TNT, ADNT, and AADNT, caused a concentration-dependent production of H2O2 and ferrylhemoglobin (ferrylHb) which is a highly oxidizing state formed by reaction of oxyHb with H2O2. Notably, hemin was released during interaction of oxyHb with HADNT. Taken together, these findings suggest that HADNT is an active metabolite that mediates TNT-induced hematotoxicity via formation of prooxidants such as H2O2 and ferrylHb.

2,4-dinitrotoluene (2,4-DNT) is a nitroaromatic used in industrial dyes and explosives manufacturing processes that is found as a contaminant in the environment. Previous studies have implicated antagonism of PPARα signaling as a principal process affected by 2,4-DNT. Here, we test the hypothesis that 2,4-DNT-induced perturbations in PPARα signaling and resultant downstream deficits in energy metabolism, especially from lipids, cause organism-level impacts on exercise endurance. PPAR nuclear activation bioassays demonstrated inhibition of PPARα signaling by 2,4-DNT whereas PPARγ signaling increased. PPARα (-/-) and wild-type (WT) female mice were exposed for 14 days to vehicle or 2,4-DNT (134 mg/kg/day) and performed a forced swim to exhaustion 1 day after the last dose. 2,4-DNT significantly decreased body weights and swim times in WTs, but effects were significantly mitigated in PPARα (-/-) mice. 2,4-DNT decreased transcript expression for genes downstream in the PPARα signaling pathway, principally genes involved in fatty acid transport. Results indicate that PPARγ signaling increased resulting in enhanced cycling of lipid and carbohydrate substrates into glycolytic/gluconeogenic pathways favoring energy production versus storage in 2,4-DNT-exposed WT and PPARα (-/-) mice. PPARα (-/-) mice appear to have compensated for the loss of PPARα by shifting energy metabolism to PPARα-independent pathways resulting in lower sensitivity to 2,4-DNT when compared with WT mice. Our results validate 2,4-DNT-induced perturbation of PPARα signaling as the molecular initiating event for impaired energy metabolism, weight loss, and decreased exercise performance., (Published by Oxford University Press on behalf of Toxicological Sciences 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

1,3-Dinitrobenzene (DNB) is an industrial intermediate and testicular toxicant that has been shown to target Sertoli cells. The mechanism of action of DNB in the testis, however, is unclear. To investigate global alterations in gene or protein expression during testicular toxicity, testes from rats treated orally with DNB were subjected to microarray and two-dimensional gel electrophoresis (2-DE) analyses. Histopathological abnormalities were detected in the testes of the DNB-treated rats. Microarray analysis revealed that, during early testicular toxicity, several genes involved in apoptosis, germ cell/Sertoli cell junction, and tight junction signaling pathways were differentially expressed. Based on 2-DE analysis, 36 protein spots showing significantly different expression during early testicular toxicity were selected and identified. Network analysis of the identified proteins revealed that these proteins are associated with cellular development or reproductive system diseases. Collectively, these data will help clarify the molecular mechanism underlying testicular toxicity in DNB-exposed rats., (Copyright © 2013. Published by Elsevier Inc.)

The aim of the study was to evaluate the potential protective effect of ozonized olive oil (OZO) in 2,4-dinitrobenzene sulphuric acid (DNBS) induced colitis in rats and to elucidate the role of some antioxidant defense system (superoxide dismutase "SOD," glutathione peroxidase "GSH-Px," and catalase "CAT") in these effects. The physicochemical parameters including viscosity, peroxide, and acid values of olive oil and OZO were evaluated. The animals were divided into several groups and the colitis was induced in the rats by intracolonic instillation of DNBS at dose of 15 mg/rat. Olive oil (OO) at dose of 6 mg/kg and OZO at doses of 3 and 6 mg/kg was administered orally for 7 days, starting the day before induction of colitis. Our results showed that macroscopic and microscopic damage scores were significantly reduced in a dose response manner in rats pretreated with OZO only. In contrast, CAT, GSH-Px, and SOD activities were significantly increased in the distal colon of inflamed animals pretreated with OZO with respect to control group dose dependently. Results demonstrate that OZO pretreatment exerts protective effects in DNBS induced colitis in rats and provide evidence that the protective effects of OZO are mediated by stimulation of some antioxidant enzymes.

This comparative evaluation of neurotoxicants previously identified as models of chemical-induced mitochondrial dysfunction and energy deprivation demonstrated that subtoxic concentrations of 1,3-dinitrobenzene (1,3-DNB), 3-nitropropionic acid (3-NPA), and 3-chloropropanediol (3-CPD) each led to concentration-dependent loss of the mitochondrial membrane potential (ΔΨm) associated with similar patterns of protein carbonylation. Subtoxic concentrations of each neurotoxicant were determined by measuring DI TNC1 cell viability using the MTS cell proliferation assay. Although exposure 1 μM, 10 μM, and 100 μM concentrations of each toxicant did not result in loss of cell viability after 48 h, exposure to each toxicant at these concentrations led to concentration-dependent loss of tetramethyl rhodamine methyl ester (TMRM) fluorescence over the same exposure period. Preincubation with the antioxidant, deferoxamine, was effective in preventing loss of TMRM flurorescence. Through the combined use of two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and Oxyblot analysis, this study demonstrated that exposure to each toxicant resulted in the formation of distinctly similar patterns of protein carbonylation comprised of specific proteins identified with tandem MS/MS. Our results provide insight as to how exposure to different neurotoxicants that enhance oxidative stress may, in fact, lead to mitochondrial injury and subsequent toxicity through selective, yet shared, pathways of protein modification by oxidative carbonylation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: The objective of this study was to evaluate the utility of plasma Inhibin B (InhB) as a biomarker of testicular injury in adult rats following short-term exposure to the known Sertoli cell toxicants mono-2-ethylhexyl phthalate (MEHP), 1,3 dinitrobenzene (DNB), or carbendazim (CBZ)., Methods: Following oral gavage administration of the compounds for 2 or 7 days, the rats were evaluated for clinical signs, body weight, food consumption, organ weights, plasma hormone levels, and gross and microscopic pathology., Results: MEHP, DNB, and CBZ produced a range of testicular toxicity characterized by minimal exfoliation of germ cells as demonstrated by increased cellular debris in the epididymis (MEHP) to more severe and dose/duration responsive Sertoli cell vacuolation, germ cell degeneration, and multinucleated giant cells of germ cell origin (DNB and CBZ). The slight to moderate Sertoli and germinal cell injuries did not correlate with significant changes in plasma InhB levels following 2- or 7-day exposures. However, more severe injury to germinal epithelium following up to 7 days of exposure, but not after 2 days of exposure, correlated with decreased plasma InhB levels and less consistently with increases in plasma follicle stimulating hormone., Conclusion: In conclusion, under the conditions of these studies, changes in InhB were not an effective early onset marker of testicular toxicity or an effective marker for slight to moderate levels of acute injury, and only reflected more severe disruption of spermatogenesis. Changes in plasma InhB and follicle stimulating hormone were poorly correlated except in some instances of moderate to marked testicular toxicity., (© 2013 Wiley Periodicals, Inc.)

Background: This study was conducted as part of an ILSI-HESI International Life Sciences Institute-Health & Environmental Sciences Institute consortium effort to assess the utility of circulating Inhibin B as an early biomarker of Sertoli cell-specific testicular toxicity in rats. 1, 3-Dinitrobenzene (1,3-DNB) was selected as a testicular toxicant in this study as it is known to target Sertoli cells., Methods: 1,3-DNB (2 and 6 mg/kg/day) or control (corn oil) was administered orally to male rats for two or five consecutive days. Blood was collected from rats treated for 2 days on days 1 and 2 and from rats treated for 5 days on days 1, 3, and 5. The resulting serum was evaluated for Inhibin B and follicle stimulating hormone. At the end of the treatment periods, the testes were removed, weighed, and examined histopathologically., Results: Daily administration of 1,3-DNB resulted in decreased testis weight only on day 5 and only at the high dose (6 mg/kg/day). There was a time-dependent increase in incidence and severity of testicular findings characterized by degeneration of the germinal epithelium with loss of pachytene spermatocytes and vacuolization of the Sertoli cells in the seminiferous tubules at the high dose. Inhibin B levels in 1,3-DNB-treated animals were decreased with treatment only on day 5 at the high dose; there were no associated changes in follicle stimulating hormone., Conclusions: Changes in serum Inhibin B levels were detected only in association with moderate or severe testicular toxicity as evidenced by histopathology and is therefore considered to be of limited value as a biomarker for Sertoli cell toxicity., (© 2013 Wiley Periodicals, Inc.)

1,3-Dinitrobenzene (DNB) causes testicular injury, particularly to Sertoli cells, and induces apoptosis in the surrounding germinal cells in rodents; however, the mechanisms causing this toxicity are poorly understood. Our studies, using standard and molecular tools, were conducted to better understand the pathogenesis of the testicular effects. Four daily oral doses of 0.1-8mg/kg/day caused marked testicular lesions in rats from 4mg/kg/day. Global transcriptomics revealed cell cycle and cell death as the major biological processes affected with the expression of genes associated with cell cycle progression ("mitotic roles of polo-like kinase") being particularly altered. In a single dose time course study (4mg/kg), no adverse changes were recorded; however, in contrast to the data from the multiple dose study, plasma testosterone and testicular steroidogenesis-related gene expression were affected. These steroid hormone effects were confirmed in vitro using the H295R steroidogenesis assay. With this global approach we show that DNB not only induces apoptosis and interferes with cell cycle in the testes but that DNB can also modulate steroid hormone biosynthesis, suggesting an interference with the endocrine system. However, the contribution of the endocrine changes to the severe testicular lesions is presently unknown and requires further investigation., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Dinitrotoluene (DNT) is a nitroaromatic explosive used in propellant mixtures and in the production of plastics. Isomers of DNT were administered daily via oral gavage to male Sprague-Dawley rats for 14 days to determine the subacute toxicity of individual isomers of DNT. The 3,5-DNT isomer was the most toxic isomer, inducing weight loss and mortality within 3 days. Cyanosis and anemia were observed for all isomers. Exposure to 2,4-, 2,6-, and 3,5-DNT resulted in decreased testes mass and degenerative histopathological changes. Increased splenic mass was observed for 2,4-, 2,6-, and 2,5-DNT. Extramedullary hematopoiesis of the spleen was noted for all isomers, while lymphoid hyperplasia of the spleen was noted for all isomers except 2,5-DNT. Increased liver mass was observed for 2,3-DNT and 3,4-DNT. Hepatocellular lesions were observed for 2,6-DNT and 2,4-DNT. Neurotoxic effects were noted for 3,4-DNT, 2,4-DNT, and 3,5-DNT.

Dinitrotoluene (DNT) is a nitroaromatic explosive that exists as six isomers; two major isomers (2,4- and 2,6-DNT) and four minor isomers (2,3-, 2,5-, 3,4-, and 3,5-DNT). DNT has been found in soil, surface water, and groundwater near ammunition production plants. The major isomers of DNT are classified as "likely to cause cancer in humans."In vitro studies have provided conflicting data regarding the genotoxicity of the minor isomers. Studies indicate that metabolism in the gut and liver are necessary to convert DNT to genotoxic compounds. As such, in the present study the genotoxicity of isomers of DNT was assessed using two in vivo genotoxicity assays. The Comet assay was used to detect DNA damage in liver cells from male Sprague-Dawley rats following oral exposure (14-day) to individual isomers of DNT. The micronucleus assay was conducted using flow cytometric analysis to detect chromosomal damage in peripheral blood. Treatment with 2,3-, 3,4-, 2,4-, 2,5- and 3,5-DNT did not induce DNA damage in liver cells or increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood at the doses tested. Treatment with 2,6-DNT induced DNA damage in liver tissue at all doses tested, but did not increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood. Thus, 2,4-DNT and the minor isomers were not genotoxic under these test conditions, while 2,6-DNT was genotoxic in the target tissue, the liver. These results support previous research which indicated that the hepatocarcinogenicity of technical grade DNT (TG-DNT) could be attributed to the 2,6-DNT isomer., (Published by Elsevier B.V.)

In this study relative condition factor (RCF) and hepatosomatic index (HSI) as well as the available energy reserves of common carp (Cyprinus carpio) by 2,4-DNT semi-static bioassay were determined and linked to effects of enzymes in liver tissues. Fish were exposed at sublethal concentrations of 2,4-DNT (0.13 μg/L, 0.1, 0.5, 1.0mg/L) for 7 and 15 d. Based on the results, there was no significant change in all parameters measured in fish exposed to 2,4-DNT at environmental related concentration, but 2,4-DNT stress in fish exposed to higher concentrations reflected the significant changes of physiological and biochemical responses. 2,4-DNT stress resulted in EROD activity induction in the liver, and the levels of EROD activity ranged from 0.39- to 1.83-fold higher than control. For GK, Na(+)/K(+)-ATPase, and GST, these enzyme activity continued to decline after exposure to 0.1, 0.5 and 1.0mg/L 2,4-DNT, whereas the trend on GK and Na(+)/K(+)-ATPase was more obvious than GST. Through principal component analysis, effects by 2,4-DNT-stress in each test group were distinguished. Additionally, indications of a trade-off between metabolic cost of toxicant exposure and processes vital to the survival of the organism were seen at the enzyme activity level as well as on higher levels of biological organization., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Astrocytes are acutely sensitive to 1,3-dinitrobenzene (1,3-DNB) while adjacent neurons are relatively unaffected, consistent with other chemically-induced energy deprivation syndromes. Previous studies have investigated the role of astrocytes in protecting neurons from hypoxia and chemical injury via adenosine release. Adenosine is considered neuroprotective, but it is rapidly removed by extracellular deaminases such as adenosine deaminase (ADA). The present study tested the hypothesis that ADA is inhibited by 1,3-DNB as a substrate mimic, thereby preventing adenosine catabolism. ADA was inhibited by 1,3-DNB with an IC(50) of 284 μM, Hill slope, n = 4.8 ± 0.4. Native gel electrophoresis showed that 1,3-DNB did not denature ADA. Furthermore, adding Triton X-100 (0.01-0.05%, wt/vol), Nonidet P-40 (0.0015-0.0036%, wt/vol), or bovine serum albumin (0.05 mg/ml or changing [ADA] (0.2 and 2 nM) did not substantially alter the 1,3-DNB IC(50) value. Likewise, dynamic light scattering showed no particle formation over a (1,3-DNB) range of 149-1043 μM. Kinetics revealed mixed inhibition with 1,3-DNB binding to ADA (K(I) = 520 ± 100 μM, n = 1 ± 0.6) and the ADA-adenosine complex (K(IS) = 262 ± 7 μM, n = 6 ± 0.6, indicating positive cooperativity). In accord with the kinetics, docking predicted binding of 1,3-DNB to the active site and three peripheral sites. In addition, exposure of DI TNC-1 astrocytes to 10-500 μM 1,3-DNB produced concentration-dependent increases in extracellular adenosine at 24 h. Overall, the results demonstrate that 1,3-DNB is a mixed inhibitor of ADA and may thus lead to increases in extracellular adenosine. The finding may provide insights to guide future work on chemically-induced energy deprivation.

Rats were treated with a single oral dose of 10, 25 and 50mg/kg of 1,3-dinitrobenzene (DNB), and the testis was subjected to a GeneChip microarray analysis. A total of 186 and 304 gene probe sets were up- and down-regulated, respectively, by the DNB treatment, where spermatocyte death and Sertoli cell vacuolation in testis and increased debris of spermatogenic cell in epididymis were noted. The expression profile for four sets of genes were investigated, whose expressions are reported to localize in specific cell types in the seminiferous epithelium, namely Sertoli cells, spermatogonia plus early spermtocytes, pachytene spermatocytes and round spermatids. The data demonstrated that pachytene spermatocyte-specific genes elicited explicit down-regulation in parallel with the progression of spermatocyte death, while other gene sets did not show characteristic expression changes. In addition, Gene Ontology analysis indicated that genes associated with cell adhesion-related genes were significantly enriched in the up-regulated genes following DNB treatment. Cell adhesion-related genes, namely Cdh2, Ctnna1, Vcl, Zyx, Itgb1, Testin, Lamc3, Pvrl2 and Gsn, showed an increase in microarray and the up-regulation of Cdh2 and Testin were confirmed by real time RT-PCR. The gene expression changes of pachytene spermatocyte-specific genes and cell adhesion-related genes were thought to reflect a decrease in the number of spermatocytes and dysfunction of Sertoli-germ cells adhesion junction, and therefore these genes would be potential genomic biomarkers for assessing DNB-type testicular toxicity., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

This study demonstrated that 1,3-dinitrobenzene-induced (1,3-DNB) oxidative stress led to the oxidative carbonlyation of specific protein targets in DI TNC1 cells. 1,3-DNB-induced mitochondrial dysfunction, as indicated by loss of tetramethyl rhodamine methyl ester (TMRM) fluorescence, was initially observed at 5h and coincided with peak reactive oxygen species (ROS) production. ROS production was inhibited in cells pre-treated with the mitochondrial permeability transition (MPT) inhibitor, bonkrekic acid (BkA). Pre-incubation with the antioxidant deferoxamine inhibited loss of TMRM fluorescence until 24h after initial exposure to 1,3-DNB. Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and subsequent Oxyblot analysis were used to determine if 1,3-DNB exposure led to the formation of protein carbonyls. Exposing DI TNC1 cells to 1,3-DNB led to marked protein carbonylation 45 min following initial exposure. Pre-treatment with deferoxamine or Trolox reduced the intensity of protein carbonylation in DI TNC1 cells exposed to 1mM 1,3-DNB. Tandem MS/MS performed on protein samples isolated from 1,3-DNB-treated cells revealed that specific proteins within the mitochondria, endoplasmic reticulum (ER), and cytosol are targets of protein carbonylation. The results presented in this study are the first to suggest that the molecular mechanism of 1,3-DNB neurotoxicity may occur through selective carbonylation of protein targets found within specific intracellular compartments of susceptible cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Prolonged exposure to the chemical intermediate, 1,3-dinitrobenzene (1,3-DNB), produces neuropathology in the central nervous system of rodents analogous to that observed in various conditions of acute energy deprivation including thiamine deficiency and Leigh's necrotizing encephalopathy. Increased production of reactive intermediates in addition to induction of oxidative stress has been implicated in the neurotoxic mechanism of 1,3-DNB, but a clear metabolic target has not been determined. Here we propose that similar to thiamine deficiency, the effects of 1,3-DNB on metabolic status may be due to inhibition of the thiamine-dependent α-ketoacid dehydrogenase complexes. The effects of 1,3-DNB on astroglial metabolic status and α-ketoacid dehydrogenase activity were evaluated using rat C6 glioma cells. Exposure to 1,3-DNB resulted in altered morphology and biochemical dysfunction consistent with disruption of oxidative energy metabolism. Cotreatment with acetyl-carnitine or acetoacetate attenuated morphological and metabolic effects of 1,3-DNB exposure as well as increased cell viability. 1,3-DNB exposure inhibited pyruvate dehydrogenase complex (PDHc) and the inhibition correlated with the loss of lipoic acid (LA) immunoreactivity, suggesting that modification of LA is a potential mechanism of inhibition. Treatment with antioxidants and thiol-containing compounds failed to protect against loss of LA. Alternatively, inhibition of dihydrolipoamide dehydrogenase, the E3 component of the complex attenuated loss of LA. Collectively, these data suggest that 1,3-DNB impairs oxidative energy metabolism through direct inhibition of the PDHc and that this impairment is due to perturbations in the function of protein-bound LA.

Chronic aqueous exposures were conducted using bullfrog (Rana catesbeiana) tadpoles (8 d old) exposed to TNT (0-4 mg/L), 2,4-DNT (0-4 mg/L), and 2,6-DNT (0-8 mg/L) for 90 d. Survival of tadpoles examined using Cox proportional hazard models was reduced at all concentrations tested. Percent of abnormal swimming and other morphological abnormalities after sublethal exposure to TNT, 2,4-DNT, and 2,6-DNT at 2 mg/L were also evaluated. The effects of TNT, 2,4-DNT, and 2,6-DNT on wet body mass, snout vent length (SVL), and developmental stage of surviving tadpoles were examined. Only 2,4-DNT did not have a significant effect on body mass or SVL, but all three compounds tested had significant effects on survival. Long-term continuous exposure to these compounds at concentrations of 0.25 mg/L could lead to significant changes in growth and survival of larval amphibians., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Infrared microspectroscopy (IMS) is emerging as an important analytical tool for the structural analysis of biological tissue. This report describes the use of IMS coupled to a synchrotron source combined with principal components analysis (PCA) to monitor the fate and effect of dinitrotoluenes in the roots of maize and sunflower plants. Infrared imaging revealed that maize roots metabolized 2,4-dinitrotoluene (DNT) and 2,6-DNT. The DNTs and their derivative aromatic amines were predominantly associated with epidermis and xylem. Both isomers of DNT altered the structure and production of pectin and pectic polysaccharides in maize and sunflower plant roots. Infrared peaks diagnostic for aromatic amines were seen at the 5 mg L concentrations for both DNTs in maize and sunflower treated tissue. However, only infrared peaks for nitro groups, not aromatic amines, were present in the maize treated at 10 mg L For sunflower, the 10 mg L level was toxic and also produced very dark root systems making spectra difficult to obtain. Maize and sunflower seem unable to metabolize effectively at concentrations higher than about 5 mg L DNT in hydroponic solution. Based on the results of this study, IMS combined with PCA can be an effective means of determining the fate and metabolism of organic contaminants in plant tissue when isotopically labeled compounds are not available., (American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America.)

Objective: To review the literature concerning the risks associated with the main xenobiotics contained in military ammunition and explosive residues and damage to human and environmental health., Methodology: Using "ammunition", "military", "environmental", "health", "explosive", "metal", "TNT", "RDX", "pollution", and "contamination" as search terms, a large database, namely ISI Web of Knowledge and PubMed, was searched for studies on military ammunition and explosive residues from 1989 to 2010. Other sources used to conduct the search included the library of the Toxicology Laboratory of the Center for Workers' Health and Human Ecology (CESTEH) at the National School of Public Health., Results: In total, 15 different combinations were used with the search words above and 708 papers were found. Among them, 76 papers concerned this review. More than 12 references of interest were discovered in the library of the CESTEH. The results were organized into metals, dinitrotoluene, trinitrotoluene (TNT), and royal demolition explosive (RDX), showing their main uses, occurrence in the environment, the current toxic effects to human and environmental health, and remediation possibilities., Conclusion: Because military activities can cause the acute and chronic exposure of human beings, the public administration must aim politics towards suitable environmental management.

Background: Military and industrial activities have lead to reported release of 2,4-dinitrotoluene (2,4DNT) into soil, groundwater or surface water. It has been reported that 2,4DNT can induce toxic effects on humans and other organisms. However the mechanism of 2,4DNT induced toxicity is still unclear. Although a series of methods for gene network construction have been developed, few instances of applying such technology to generate pathway connected networks have been reported., Results: Microarray analyses were conducted using liver tissue of rats collected 24h after exposure to a single oral gavage with one of five concentrations of 2,4DNT. We observed a strong dose response of differentially expressed genes after 2,4DNT treatment. The most affected pathways included: long term depression, breast cancer regulation by stathmin1, WNT Signaling; and PI3K signaling pathways. In addition, we propose a new approach to construct pathway connected networks regulated by 2,4DNT. We also observed clear dose response pathway networks regulated by 2,4DNT., Conclusions: We developed a new method for constructing pathway connected networks. This new method was successfully applied to microarray data from liver tissue of 2,4DNT exposed animals and resulted in the identification of unique dose responsive biomarkers in regards to affected pathways.

Evaluation of testicular toxicity during drug development is currently based on histopathological evaluation. A sensitive biomarker for testicular toxicology could provide an in-life and "early warning" measurement. Previous studies suggested that disruption of spermatogenesis induced leakage of germ cell proteins from seminiferous tubules (STs) into interstitial fluid (IF); such proteins have potential for use as biomarkers. To investigate this possibility further, adult male rats were treated with three testicular toxicants thought to have differing sites of action; cadmium chloride affects the blood-testis barrier (BTB), methoxyacetic acid (MAA) disrupts pachytene spermatocytes, and 1,3-dinitrobenzene (DNB) targets Sertoli cells. IF proteins were assessed by Coomassie-based dye-stained gels. Immunostaining was used to identify toxicant-induced damage (DAZL) and BTB integrity (ZO-1, occludin, N-cadherin, and β-catenin) and function (biotin). Cadmium chloride induced dose-dependent leakage of proteins from STs into IF coincident with loss of integrity and function of the BTB. Two of the "leaked" proteins were identified on Westerns as being germ cell specific, namely VASA and fatty acid-binding protein 9 (FABP9). In contrast, similar protein leakage was not evident after either MAA-induced or DNB-induced disruption of spermatogenesis and neither of these treatments affected BTB integrity or function. These results suggest that loss of BTB integrity is required for germ cell-specific proteins to leak from STs into IF, implying that use of such biomarkers has very limited potential for noninvasive monitoring of compound-induced disruption to spermatogenesis.

The presence of energetic materials (used as explosives and propellants) at contaminated sites is a growing international issue, particularly with respect to military base closures and demilitarization policies. Improved understanding of the ecotoxicological effects of these materials is needed in order to accurately assess the potential exposure risks and impacts on the environment and its ecosystems. We studied the toxicity of the nitroaromatic energetic material 2,4-dinitrotoluene (2,4-DNT) on alfalfa (Medicago sativa L.), barnyard grass (Echinochloa crusgalli L. Beauv.), and perennial ryegrass (Lolium perenne L.) using four natural soils varying in properties (organic matter, clay content, and pH) that were hypothesized to affect chemical bioavailability and toxicity. Amended soils were subjected to natural light conditions, and wetting and drying cycles in a greenhouse for 13 weeks prior to toxicity testing to approximate field exposure conditions in terms of bioavailability, transformation, and degradation of 2,4-DNT. Definitive toxicity tests were performed according to standard protocols. The median effective concentration (EC(50)) values for shoot dry mass ranged from 8 to 229 mg kg(-1), depending on the plant species and soil type. Data indicated that 2,4-DNT was most toxic in the Sassafras (SSL) and Teller (TSL) sandy loam soils, with EC(50) values for shoot dry mass ranging between 8 to 44 mg kg(-1), and least toxic in the Webster clay loam soil, with EC(50) values for shoot dry mass ranging between 40 to 229 mg kg(-1). The toxicity of 2,4-DNT for each of the plant species was significantly (p < or = 0.05) and inversely correlated with the soil organic matter content. Toxicity benchmark values determined in the present studies for 2,4-DNT weathered-and-aged in SSL or TSL soils will contribute to development of an Ecological Soil Screening Level for terrestrial plants that can be used for ecological risk assessment at contaminated sites., (Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.)

The limited availability of genomic tools and data for nonmodel species impedes computational and systems biology approaches in nonmodel organisms. Here we describe the development, functional annotation, and utilization of genomic tools for the avian wildlife species Northern bobwhite (Colinus virginianus) to determine the molecular impacts of exposure to 2,6-dinitrotoluene (2,6-DNT), a field contaminant of military concern. Massively parallel pyrosequencing of a normalized multitissue library of Northern bobwhite cDNAs yielded 71,384 unique transcripts that were annotated with gene ontology (GO), pathway information, and protein domain analysis. Comparative genome analyses with model organisms revealed functional homologies in 8,825 unique Northern bobwhite genes that are orthologous to 48% of Gallus gallus protein-coding genes. Pathway analysis and GO enrichment of genes differentially expressed in livers of birds exposed for 60 days (d) to 10 and 60 mg/kg/d 2,6-DNT revealed several impacts validated by RT-qPCR including: prostaglandin pathway-mediated inflammation, increased expression of a heme synthesis pathway in response to anemia, and a shift in energy metabolism toward protein catabolism via inhibition of control points for glucose and lipid metabolic pathways, PCK1 and PPARGC1, respectively. This research effort provides the first comprehensive annotated gene library for Northern bobwhite. Transcript expression analysis provided insights into the metabolic perturbations underlying several observed toxicological phenotypes in a 2,6-DNT exposure case study. Furthermore, the systemic impact of dinitrotoluenes on liver function appears conserved across species as PPAR signaling is similarly affected in fathead minnow liver tissue after exposure to 2,4-DNT.

Aims: To study the impact of added 2,4-dinitrotoluene (DNT) on the anaerobic bacterial community in marine sediment collected from an unexploded ordnance dumping site in Halifax Harbour., Methods and Results: Marine sediment was spiked with 2,4-DNT and incubated under anaerobic conditions in the presence and absence of lactate. Indigenous bacteria in the sediment removed 2,4-DNT with subsequent formation of its mono- and diamino-derivatives under both conditions. PCR-DGGE and nucleotide sequencing were used to monitor the change in the bacterial population in sediment caused by the presence of 2,4-DNT. The results showed that denaturing gradient gel electrophoresis banding patterns of sediment microcosms treated with 2,4-DNT were different from controls that did not receive 2,4-DNT. Bacteroidetes, Firmicutes and delta-Proteobacteria were present in sediment incubated in the absence of 2,4-DNT. However, several gamma-Proteobacteria became dominant in sediment in the presence of 2,4-DNT, two of which were 99% similar to Shewanella canadensis and Shewanella sediminis. In the presence of both 2,4-DNT and lactate, two additional delta-Proteobacteria were enriched, one closely related (98% similarity) to Desulfofrigus fragile and the other affiliated (96% similarity) to Desulfovibrio sp. In contrast, none of the above four Proteobacteria were enriched in sediment incubated with lactate alone., Conclusions: Presence of 2,4-DNT led to a significant change in bacterial population of marine sediment with the enrichment of several gamma- and delta-Proteobacteria., Significance and Impact of the Study: Our results provided the first evidence on the impact of the pollutant 2,4-DNT on the indigenous bacterial community in marine sediment, and provided an insight into the composition of bacterial community that degrade 2,4-DNT.

Several studies have shown that 1,3-dinitrobenzene (1,3-DNB) causes injury to Sertoli cells and induces apoptosis in the surrounding germinal cells in male laboratory rats; however, the mechanism by which 1,3-DNB functions is not well understood. In this study, we investigated whether 1,3-DNB induces apoptosis and which pathways are undertaken in TM4 cells. When cells were treated with 1,3-DNB, a dose-dependent reduction in cell viability was observed by tetrazolium dye assay and LDH assay. The reduced cell viability by 1,3-DNB treatment appeared to involve necrosis as well as apoptosis, based on staining with annexin V-FITC and propidium iodide (PI) staining and Western blotting for PARP protein. 1,3-DNB treatment decreased total transcript and protein levels of the apoptosis inhibitory protein Bcl-2, and increased expression levels of the pro-apoptotic protein Bax. In addition, using FACS analysis we detected G2/M phase cell cycle arrest by 1,3-DNB, concurrent with a remarkable increase in p21 expression and decrease in cdc2 expression. Interestingly, we found that the phosphorylation of c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) was promoted by 1,3-DNB, furthermore, 1,3-DNB-induced cell death was significantly inhibited by the JNK inhibitor, but not by ERK inhibitor or the p38 inhibitor. Together, our results suggest that 1,3-DNB induces apoptotic cell death and G2/M phase cell cycle arrest, at least in part, via JNK signaling in TM4 mouse Sertoli cells.