Identifying Toxic Biotransformation Products of the Insensitive

Dec 9, 2016 - The library was comprised of DNAN and the commercially available ... product monitored in the selected parent ion list in increasing ord...
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Chapter 9

Identifying Toxic Biotransformation Products of the Insensitive Munitions Compound, 2,4-Dinitroanisole (DNAN), Using Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS) Christopher I. Olivares,*,1 Leif Abrell,2,3 Jon Chorover,2 Michael Simonich,4 Robert L. Tanguay,4 Reyes Sierra-Alvarez,1 and Jim A. Field1 1Chemical

and Environmental Engineering, University of Arizona, Tucson, Arizona 85721, United States 2Soil, Water, and Environmental Science, University of Arizona, Tucson, Arizona 85721, United States 3Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, United States 4Environmental and Molecular Toxicology, Sinnhuber Aquatic Research Laboratory and the Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97333, United States *E-mail: [email protected].

As insensitive munitions (IMs) replace conventional explosives, releases of 2,4-dinitronanisole (DNAN) –an IM compound– are expected to increase in the environment. DNAN is readily biotransformed in soils, and while toxicity studies have evaluated DNAN, little attention has been given to its biotransformation products. In this work, we elucidated and semiquantitated product mixtures formed during anaerobic biotransformation of DNAN using high-resolution mass spectrometry techniques. DNAN underwent nitroreduction, and at later bioconversion stages, formed azo-dimers, accounting for the majority of end-products from DNAN anaerobic biotransformation. The chemical analyses © 2016 American Chemical Society

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were complemented by microbial (methanogenesis and Aliivibrio fischeri bioluminescence) and zebrafish embryo toxicity assays on mixtures of products formed, as well as individual compounds (biotransformation products and model azo-oligomers). Methanogens were severely inhibited during nitroreduction stages, but recovered at longer incubation times when azo-dimers were predominant. On the other hand, A. fischeri bioluminescence decreased at later biotransformation stages. When tested individually, the most toxic products to the microbial targets were the azo-oligomer models, while the least toxic species were 2,4-diaminoanisole and its N-acetylated analog. Zebrafish embryos showed few active developmental endpoints for the individual compounds tested, but 3-nitro-4-methoxyaniline and the model azo-dimer 2,2′-dimethoxy-4,4′-azodianiline had a lowest observable adverse effect level (LOAEL) of 6.4 μM. Overall, the interdisciplinary experimental design allowed to identify key transformation processes and products that alter toxicity beyond the parent compound.

Introduction 2,4-dinitroanisole (DNAN) is a compound used in insensitive munition (IM) formulations. IMs are replacing traditional explosives, such as 2,4,6-trinitrotoluene (TNT), due to a lower risk of accidental detonation (1, 2). With increased manufacturing and use in testing sites and battlefields, DNAN will inevitably be released into the environment. DNAN, although slightly more water soluble than TNT, is expected to remain in soils tainted with the explosives for years, while other ingredients in IM formulations are expected to dissolve faster (3). Therefore, biotic and abiotic reactions, such as biotransformation (4–6) and photolysis (7), are expected to be the main mechanisms affecting the fate of DNAN in the environment. Soil catalyzes abiotic and biotic (5, 8) nitro group reductions of DNAN, forming 2-methoxy-5-nitroaniline (MENA), and subsequently 2,4-diaminoanisole (DAAN). The reduction of DNAN to its transformation products increases the compound mobility because MENA and DAAN are more hydrophilic than DNAN, the parent compound (9). Moreover, during the course of nitroreduction, reduced amino metabolites can couple forming azo-dimers, that are expected to be long-term end-products in most natural soil scenarios (5, 6). While there have been recent reports on DNAN toxicity effects (10, 11) there is a need to characterize changing adverse effects posed to ecotoxicological targets from its multiple biotransformation products, as well as to identify potential key chemical species that drive toxicity. For instance, preliminary findings reported nitro group reduction in DNAN to occur with decreased inhibition towards methanogens (12), as well as reduced mortality to zebrafish after a 48 h exposure 134

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(13). Moreover, recent research on the ecotoxicity of DNAN have suggested transformation reactions occur during toxicity assays (10, 11, 14, 15). In this study, an integrated chemical and toxicological characterization of DNAN biotransformation is reported, that correlates the biological activity of specific products with their abundance and identifies key drivers of changes in toxicity potency. Our objectives were (1) to develop a high resolution mass spectrometry method to elucidate and semiquantitate biotransformation products, and (2) to integrate the chemical characterization of product mixtures with quantitative toxicity assays that probe (i) inhibition of methanogens, (ii) bioluminescence of the bacterium Aliivibrio fischeri, and (iii) embryo development in zebrafish.

Materials and Methods Chemicals and Biological Materials 2,4-dinitroanisole (CAS# 119-27-7, DNAN, Alfa-Aesar, 98%), 2methoxy-5-nitroaniline (CAS# 99-59-2, MENA, Sigma-Aldrich, 98%), 3-nitro-4-methoxyaniline (CAS# 577-72-0, denoted “iMENA", Accela Chem Bio, 97% ), N-(5-amino-2-methoxyphenyl) acetamide (CAS# 64353-88-4, denoted “Ac-DAAN”, Chem-Bridge Corporation, 95%), 2,2′-dimethoxy-4,4′-azodianiline (CAS# 6364-31-4, denoted “dimer L”, MolMall Sarl, >90%), and Bismarck Brown Y (m-Bis(2,4-diaminophenylazo)-benzene, CAS# 8005-77-4, denoted “BBY”, Chem-Impex International, 46%) were used in this study without further purification. Camp Navajo (water content = 9.3%), a surface soil with surrounding military activity described previously (5, 16), was used for biotransformation assays. The soil was sieved (2 mm) and stored at 4 °C before use. Microbial toxicity analyses were performed with methanogenic microorganisms in anaerobic sludge and with the marine bioluminescent bacterium Aliivibrio. fischeri (NRRL-B-11177, Modern Water Inc., New Castle, DE, USA). Anaerobic sludge (dry weight (dwt) solids = 11.2 %, volatile suspended solids (VSS) = 7.9 % per wet weight) was procured from a full-scale upflow anaerobic sludge blanket reactor treating industrial wastewater from a brewery (Mahou, Guadalajara, Spain). Developmental zebrafish (Danio rerio) embryo toxicity was also evaluated (17). Zebrafish aquaculture was performed at the Sinnhuber Aquatic Research Laboratory (18).

Staggered Anaerobic Biotransformation Assays The composition of the biotransformation products at different stages of DNAN (500 μM) bioconversion in soil and sludge was measured in a series of anaerobic microcosms that were incubated for up to 50 days to identify and semiquantitate biotransformation products with high resolution mass spectrometry and to evaluate toxicity (Figure 1). Briefly, 500 μM DNAN was added to a basal mineral medium (19) containing a phosphate buffer (18 mM, pH 7.2) that was 135

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amended with 10 mM pyruvate. Soil (100 mg) or sludge (75 mg) were added before sealing the tubes and flushing with He/CO2 (80/20%) to achieve anaerobic conditions. Triplicate tubes were incubated for 0, 1, 5, 10, 20, 30, 40, and 50 days, for destructive sampling upon completion of each incubation time. For microbial toxicity evaluation and LC-QToF-MS analysis, samples from the liquid phase were retrieved inside an anaerobic glovebox to avoid autoxidation of products that were potentially unstable under prolonged air exposure. Liquid samples were centrifuged (10 min, 9,600×g) to remove suspended particulate matter and the supernatant solutions were frozen (−20 °C) until chromatographic analysis.

Figure 1. Overview of experimental approach to integrate biotransformation product elucidation and characterization with toxicity evaluation. Anaerobic biotransformation assays were incubated for different time periods (0 to 50 days) upon sampling of the liquid phase. Biotransformation products were elucidated with non-targeted LC-QToF-MS and semiquantified based on mass chromatograms for each incubation period. Toxicity of profile mixtures was evaluated in microbial targets and to zebrafish embryos.

LC-QToF-MS Elucidation of Biotransformation Products Characterization of bioconversion products soluble in the aqueous phase was performed on an UltiMate 3000 UHPLC-DAD (Dionex, Sunnyvale, CA) coupled to a TripleTOF 5600 quadrupole time-of-flight mass spectrometer (Q-ToF-MS) (AB Sciex, Framingham, MA, USA). Samples (10 μL) were run in the UHPLCwith an isocratic 60/40 % H2O/MeOH eluent (0.25 mL min-1, run time 15 min) at room temperature through a reversed phase C18 column, Acclaim RSLC Explosives E2 (2.1 x 100 mm, 2.2 μm) (Thermo Fisher Scientific, Waltham, WA, USA). DNAN, MENA, and DAAN were monitored by UV detection (wavelength in nm: retention time in min): 300:9, 254:5, and 210:2.3, respectively. The QToF-MS was operated with an electrospray ionization source (ESI) in positive 136

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mode kept at 450 °C with a capillary setting of 5.5 kV, and declustering potential of 80 V. N2 was used as curtain, desolvation, and nebulizer gas at 30, 35, and 35 psi, respectively. A non-targeted approach was used to obtain high-resolution accurate masses from information dependent acquisition (IDA) mass spectra; 0.1 sec cycle time, 6 triggered ions per cycle, mass range 35-1000 Da. Instrument calibration was performed by automated infusion of a solution periodically, over a mass range of 35-1000 Da. Analyst TF 1.6 with PeakView 1.2.0.3 and Formula Finder 1.1.0.0 were used to process spectral data and to propose molecular formulae for the biotransformation product candidates. Molecular formulae corresponding to measured molecular ions within 5 ppm of calculated monoisotopic ion (exact) mass were considered, but in most cases measurements were within 1 ppm. Once a molecular formula was identified, it was evaluated by manual inspection of product ion mass spectral fragments was conducted. Exact product ion masses corresponding to proposed fragment losses were calculated and matched with molecular formulae using ChemCalc (20), and were required to be within 5 ppm of calculated (exact) monoisotopic masses. Common fragmentation reaction patterns emerged among the biotransformation products such as: [M-CH3], M-15.0235; [M-NH3], M-17.0265; [M-OCH3], M-31.0164; [M-CH4O], M-32.0262; [M-NO2], M-45.9929; [M-C2H4O2], M-46.0419; [M-C2H3NO2], M-73.0164; and [M-C6H6N2O], M-122.0480. Finally, the cohort of biotransformation products was compared with related studies with DNAN and literature on biological reactions with nitroaromatics, aromatic amines, and phenyl-azo compounds

LC-QToF-MS Semiquantitation of Biotransformation Products In order to study the change in the composition of biotransformation products correlated with incubation time, the elucidated products were semiquantitated by integrating accurate, selected parent ion [M+H]+ and product ion [M+H-Y]+ mass chromatogram peaks with reproducible retention times. In the absence of pure standard calibrants, extracted ion chromatogram peak areas provided semiquantitative evaluation of the abundance of the biotransformation products. Selected ion chromatograms were extracted using a 10 mDa window that was centered on the calculated (exact) mass. The same UHPLC and QToF-MS parameters were used for biotransformation product semiquantitation and elucidation (described above).

Microbial and Zebrafish Embryo Toxicity Evaluation of Chemical Library A chemical library was built for toxicology evaluation based on the biotransformation products elucidated with LC-QToF-MS. The library was comprised of DNAN and the commercially available biotransformation products (MENA, iMENA, DAAN, Ac-DAAN), as well as azo-oligomer model compounds (dimer L, BBY). Microbial activity inhibition assays were performed for 137

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acetoclastic methanogenesis and bioluminescence, described in detail previously (12), in order to determine concentrations resulting in 50% inhibition (IC50) correlating to inhibition potency of individual compounds in the chemical library. Zebrafish embryo developmental toxicity was evaluated following a previously reported high-throughput protocol (18, 21) for exposures of individual toxicants (0.0064-64 μM, 32 replicates for each concentration and toxicant-free control) to embryos from 6 to 120 hours post-fertilization (hpf), with development assessment at 24 and 120 hpf for a total of 22 developmental endpoints and mortality. Lowest observable adverse effect levels (LOAELs) were determined for compounds exhibiting developmental toxicity endpoints above threshold statistical significance (18, 21).

Toxicity Evaluations of Biotransformation Product Mixtures Mixtures of biotransformation products were obtained by destructive sampling of the staggered incubations of 500 μM DNAN from the incubation period 0-50 days. Mixtures exposed to microbial toxicity targets (methanogens and A. fischeri) were diluted (54-fold for methanogens, 36-fold for A. fischeri) to avoid complete inhibition of methanogenesis or bioluminescence that would prevent toxicity changes to be detected.

Results and Discussion DNAN Biotransformation Pathway Elucidation of DNAN biotransformation products and pathways was enabled by a non-targeted approach using UHPLC-QToF-MS with IDA experiments. Overall, as seen in Figure 2A, the DNAN biotransformation pathway involved regioselective nitroreduction of the ortho nitro group in DNAN, yielding MENA. However, nitroreduction of the para nitro group, leading to iMENA, was also detected at much lower levels. Further reduction of the remaining nitro group resulted in the aromatic diamine product, DAAN. During this process, reactive nitroso groups (not depicted in Figure 2A) condensed with DAAN, and formed azo-dimer m/z 273. The remaining amino moieties remained reactive in azo-dimers undergoing N-methyl substitutions to produce other dimeric products with m/z 285 and 269. Additionally, a parallel pathway involving acetylation of DAAN was also found (m/z 181).

DNAN Biotransformation and Microbial Toxicity of Product Mixtures DNAN underwent rapid nitroreduction in the anaerobic soil incubations. DNAN was removed within ten days of incubation, forming MENA and DAAN, as seen in Figure 3A. However, products formed at longer incubation times (30-50 days) could not be detected by HPLC-DAD, so a gap in mass balance from 138

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the initial DNAN in solution was evident over this time period. To understand this imbalance, a semiquantitative mass spectrometric analysis method, based on results from our initial non-targeted approach with IDA experiments, enabled characterization of additional transformation products monitored to obtain abundance estimates based on integrated peak areas from extracted mass chromatograms. As seen in Figure 3B, the majority of products from 30-50 d incubations were comprised of ions > m/z 200 attributed to a suite of azo-dimers, indicative of oligomerization reactions. While the majority of the dominant ions had been characterized during the non-targeted analyses, m/z 247 (Figure 3A) was putatively assigned the chemical formula C7H9N3O7, and was suspected to be a transformation product containing a moiety related to humic material (22).

Figure 2. Adapted with permission from reference (22). Copyright 2016. Elsevier. Panel A: Biotransformation pathway of DNAN in anaerobic soil incubations. DNAN undergoes nitroreduction, yielding MENA and DAAN. Reduced metabolites from nitroreduction lead to azo-dimer formation (m/z 273). The amino moieties in azo-dimers continue to react, forming N-alkyl groups (m/z 285, 269). Multiple arrows indicate multiple reaction steps. Panel B: Model dimer and trimer used for toxicity evaluations. 139

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Figure 3. DNAN biotransformation started with nitroreduction, while long-term incubation resulted in azo-dimer compounds. The changes in the composition of the product mixtures altered their toxicity potential to microbial targets. Panel A: HPLC-DAD resolvable products from DNAN (■) nitroreduction: MENA (●) and DAAN (▲). Panel B: LC-QToF-MS enabled semiquantitation of biotransformation products based on peak areas from mass chromatograms. Each shade of gray represents a transformation product monitored in the selected parent ion list in increasing order from dark (lower m/z) to light (higher m/z) gray shades [M+H]+: 139.0866, 165.0659, 169.0608, 181.0972, 185.0652, 193.0607, 228.0768, 243.0877, 243.1241, 245.1300, 247.0425, 259.1190, 267.0975, 269.1397, 273.1347, 274.0715, 275.1503, 285.1347, 299.1179, 301.1289, 313.1289, 325.1659, 327.1452, 431.1569. Ions in bold represent most abundant peak areas measured. Panel C: Normalized methanogenic (○) and A. fischeri bioluminescence (□) activity when exposed to transformation product mixtures formed at different times during DNAN biotransformation.

In the absence of calibrants, this semiquantitative approach still provided valuable temporal concentration comparisons, since no significant changes in electrospray ionization of transformation products were anticipated between different incubation times – because the incubation medium matrix remained the same throughout the experiment (same basal medium and soil inoculum). The main limitation of this approach is based on the assumption that identical ionization potentials amongst all the biotransformation products do not exist, therefore an accurate interspecies chemical concentration comparison was not anticipated. In this respect, it is interesting to note that primary amines from various sources, such as polyurethane and azo dyes, measured by ESI LC-MS/MS, showed similar limits of detection (same order of magnitude) (23), suggesting similar ionization potentials between structurally similar amines. Finally, when overlaying microbial toxicity data with transformation product abundances (Figure 3C), it can be noted that methanogenic activity inhibition increased during DNAN nitroreduction. However, methanogenic activity recovered at longer incubation times, when DNAN was depleted and azo-dimers were predominant. This suggested that reactive intermediates formed during nitroreduction of DNAN might be responsible for further increases in inhibition, which lasted as long as there was a steady supply of these intermediates from DNAN. On the other hand, A. fischeri bioluminescence was not affected at the initial stages of DNAN biotransformation during nitroreduction. However, bioluminescence became inhibited at longer incubation times, when the predominant species included azo-dimers. Taken together, these results indicate that the changing anaerobic biotransformation product mixtures of DNAN exhibit fluctuating toxicity potency and mechanisms depending on the microbial target. 141

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Figure 4. Bioassay chemical library of DNAN, biotransformation products, and model compounds. Panel A: Concentrations resulting in 50% inhibition (IC50) of methanogenic (○) and A. fischeri bioluminescence (□) for individual chemical species. Gray, shaded area shows DNAN IC50 region as a reference. Panel B: Total number of developmental endpoint hits (out of 640 for 32 replicates with 20 possible endpoints each) for each chemical species evaluated at 6.4 μM. Asterisk denotes total hits above statistical significant threshold. No active endpoints were detected for MENA. DNAN was not tested (NT) (24). Panel A adapted with permission from reference (22). Copyright 2016. Elsevier.

Toxicity of Individual Biotransformation Products Toxicity to microbial targets and the zebrafish embryonic model was further evaluated to determine what types of biotransformation products (detected by LCMS) have the greatest changes in toxicity. Figure 4A shows the concentrations 142

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that result in 50% inhibition (IC50) of methanogenic or bioluminescent activity. The dimer and trimer model compounds (dimer L and BBY in Figure 2B), produced the lowest IC50 values (most toxic) in the chemical library tested. Complete nitroreduction to DAAN, and acetylation of DAAN, resulted in the formation of the least toxic species for both microbial targets. In comparison with the microbial toxicity assays, very little zebrafish developmental toxicity was measured in the tested range of 0.0064-64.0 μM. Only two chemical species (iMENA and dimer L) produced some active developmental toxicity endpoints to rise above a statistical threshold (Figure 4B) at 6.4 μM; the lowest observable adverse effect level (LOAEL) of for both compounds. Overall, zebrafish embryo development and microbial toxicity data suggest that dimers structurally similar to azo-dimers derived from DNAN biotransformation could have toxic effects on both prokaryotes and eukaryotes.

Conclusion and Implications of Experimental Design There has been recent growing interest in utilizing chemical analyses of emerging contaminants and their transformation products to better understand the biological activity of complex mixtures (25–27). The present work coupled mass spectrometry analyses of biotransformation product mixtures to toxicity evaluation. This interdisciplinary experimental design contributes to the study of xenobiotic transformation processes in the environment, and to identify key processes and products that alter toxicity beyond the parent compound. With further experimental iterations, biological activity tools could help guide research efforts on the information-rich data that derives from high-resolution mass spectrometry techniques.

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