Effects of Metformin and its Metabolite Guanylurea on Fathead Minnow (Pimephales promelas) Reproduction.

Metformin, along with its biotransformation product guanylurea, is commonly observed in municipal wastewaters and subsequent surface waters. Previous studies in fish have identified metformin as a potential endocrine-active compound, but there are inconsistencies with regard to its effects. To further investigate the potential reproductive toxicity of metformin and guanylurea to fish, a series of experiments was performed with adult fathead minnows (Pimephales promelas). First, explants of fathead minnow ovary tissue were exposed to 0.001-100 µM metformin or guanylurea to investigate whether the compounds could directly perturb steroidogenesis. Second, spawning pairs of fathead minnows were exposed to metformin (0.41, 4.1, and 41 µg/L) or guanylurea (1.0, 10, and 100 µg/L) for 23 days to assess impacts on reproduction. Lastly, male fathead minnows were exposed to 41 µg/L metformin, 100 µg/L guanylurea, or a mixture of both compounds, with samples collected over a 96-h time course to investigate potential impacts to the hepatic transcriptome or metabolome. Neither metformin nor guanylurea affected steroid production by ovary tissue exposed ex vivo. In the 23 days of exposure, neither compound significantly impacted transcription of endocrine-related genes in male liver or gonad, circulating steroid concentrations in either sex, or fecundity of spawning pairs. In the 96-h time course, 100 µg guanylurea/L elicited more differentially expressed genes than 41 µg metformin/L and showed the greatest impacts at 96 h. Hepatic transcriptome and metabolome changes were chemical- and time-dependent, with the largest impact on the metabolome observed at 23 days of exposure to 100 µg guanylurea/L. Overall, metformin and guanylurea did not elicit effects consistent with reproductive toxicity in adult fathead minnows at environmentally relevant concentrations. Environ Toxicol Chem 2022;41:2708-2720. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Untargeted Lipidomics for Determining Cellular and Subcellular Responses in Zebrafish (Danio rerio) Liver Cells Following Exposure to Complex Mixtures in U.S. Streams.

Surface waters often contain a variety of chemical contaminants potentially capable of producing adverse outcomes in both humans and wildlife due to impacts from industrial, urban, and agricultural activity. Here, we report the results of a zebrafish liver (ZFL) cell-based lipidomics approach to assess the potential ecotoxicological effects of complex contaminant mixtures using water collected from eight impacted streams across the United States mainland and Puerto Rico. We initially characterized the ZFL lipidome using high resolution mass spectrometry, resulting in the annotation of 508 lipid species covering 27 classes. We then identified lipid changes induced by all streamwater samples (nonspecific stress indicators) as well as those unique to water samples taken from specific streams. Subcellular impacts were classified based on organelle-specific lipid changes, including increased lipid saturation (endoplasmic reticulum stress), elevated bis(monoacylglycero)phosphate (lysosomal overload), decreased ubiquinone (mitochondrial dysfunction), and elevated ether lipids (peroxisomal stress). Finally, we demonstrate how these results can uniquely inform environmental monitoring and risk assessments of surface waters.

Cell-Based Metabolomics for Untargeted Screening and Prioritization of Vertebrate-Active Stressors in Streams Across the United States.

The U.S. Geological Survey and the U.S. Environmental Protection Agency have assessed contaminants in 38 streams across the U.S., using an extensive suite of target-chemical analysis methods along with a variety of biological effects tools. Here, we report zebrafish liver (ZFL) cell-culture based NMR metabolomic analysis of these split stream samples. We used this untargeted approach to evaluate the sites according to overall impact on the ZFL metabolome and found that neither the total number of organics detected at the sites, nor their cumulative concentrations, were good predictors of these impacts. Further, we used partial least squares regression to compare ZFL endogenous metabolite profiles to values for 455 potential stressors (organics, inorganics, and physical properties) measured in these waters and found that the profiles covaried with at most 280 of the stressors, which were subsequently ranked into quartiles based on the strength of their covariance. While contaminants of emerging concern (CECs) were well represented in the top, most strongly covarying quartile-suggesting considerable potential for eliciting biological responses at these sites-there was even higher representation of various well-characterized legacy contaminants (e.g., PCBs). These results emphasize the importance of complementing chemical analysis with untargeted bioassays to help focus regulatory efforts on the most significant ecosystem threats.

Sex-dependent effects of bisphenol A on type 1 diabetes development in non-obese diabetic (NOD) mice.

Type 1 diabetes (T1D) is an autoimmune disease caused by immune-mediated pancreatic ß-cell destruction. The endocrine disrupting chemical bisphenol A (BPA) has widespread human exposure and can modulate immune function and the gut microbiome (GMB), which may contribute to the increasing T1D incidence worldwide. It was hypothesized that BPA had sex-dependent effects on T1D by modulating immune homeostasis and GMB. Adult female and male non-obese diabetic (NOD) mice were orally administered BPA at environmentally relevant doses (30 or 300 µg/kg). Antibiotic-treated adult NOD females were exposed to 0 or 30 µg/kg BPA. BPA accelerated T1D development in females, but delayed males from T1D. Consistently, females had a shift towards pro-inflammation (e.g., increased macrophages and Bacteroidetes), while males had increases in anti-inflammatory immune factors and a decrease in both anti- and pro-inflammatory GMB. Although bacteria altered during sub-acute BPA exposure differed from bacteria altered from chronic BPA exposure in both sexes, the GMB profile was consistently pro-inflammatory in females, while males had a general decrease of both anti- and pro-inflammatory gut microbes. However, treatment of females with the antibiotic vancomycin failed to prevent BPA-induced glucose intolerance, suggesting changes in Gram-positive bacteria were not a primary mechanism. In conclusion, BPA exposure was found to have sex dimorphic effects on T1D with detrimental effects in females, and immunomodulation was identified as the primary mechanism.

Assessing the impact of wastewater treatment plant effluent on downstream drinking water-source quality using a zebrafish (Danio Rerio) liver cell-based metabolomics approach.

Cell-based metabolomics was used in a proof-of-concept fashion to investigate the biological effects of contaminants as they traveled from a wastewater treatment plant (WWTP) discharge to a drinking water treatment plant (DWTP) intake in a surface-water usage cycle. Zebrafish liver (ZFL) cells were exposed to water samples collected along a surface-water flowpath, where a WWTP was located ∼14.5 km upstream of a DWTP. The sampling sites included: 1) upstream of the WWTP, 2) the WWTP effluent discharging point, 3) a proximal location downstream of the WWTP outfall, 4) a distal location downstream of the WWTP outfall, 5) the drinking water intake, and 6) the treated drinking water collected prior to discharge to the distribution system. After a 48-h laboratory exposure, the hydrophilic and lipophilic metabolites in ZFL cell extracts were analyzed by proton nuclear magnetic resonance (1H NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS), respectively. Multivariate statistical analysis revealed distinct changes in metabolite profiles in response to WWTP effluent exposure. These effects on the hydrophilic metabolome gradually diminished downstream of the WWTP, becoming non-significant at the drinking water intake (comparable to upstream of the WWTP, p = 0.98). However, effects on the lipophilic metabolome increased significantly as the river flowed from the distal location downstream of the WWTP to the drinking water intake (p < 0.001), suggesting a source of bioactive compounds in this watershed other than the WWTP. ZFL cells exposed to treated drinking water did not exhibit significant changes in either the hydrophilic (p = 0.15) or lipophilic metabolome (p = 0.83) compared to the upstream site, suggesting that constituents in the WWTP effluent were efficiently removed by the drinking water treatment process. Impacts on ZFL cells from the WWTP effluent included disrupted energy metabolism, a global decrease in amino acids, and altered lipid metabolism pathways. Overall, this study demonstrated the utility of cell-based metabolomics as an effective tool for assessing the biological effects of complex pollutant mixtures, particularly when used as a complement to conventional chemical monitoring.

Derivation and Evaluation of Putative Adverse Outcome Pathways for the Effects of Cyclooxygenase Inhibitors on Reproductive Processes in Female Fish.

Cyclooxygenase (COX) inhibitors are ubiquitous in aquatic systems and have been detected in fish tissues. The exposure of fish to these pharmaceuticals is concerning because COX inhibitors disrupt the synthesis of prostaglandins (PGs), which modulate a variety of essential biological functions, including reproduction. In this study, we investigated the effects of well-characterized mammalian COX inhibitors on female fathead minnow reproductive health. Fish (n = 8) were exposed for 96 h to water containing indomethacin (IN; 100 µg/l), ibuprofen (IB; 200 µg/l) or celecoxib (CX; 20 µg/l), and evaluated for effects on liver metabolome and ovarian gene expression. Metabolomic profiles of IN, IB and CX were not significantly different from control or one another. Exposure to IB and CX resulted in differential expression of comparable numbers of genes (IB = 433, CX = 545). In contrast, 2558 genes were differentially expressed in IN-treated fish. Functional analyses (canonical pathway and gene set enrichment) indicated extensive effects of IN on PG synthesis pathway, oocyte meiosis, and several other processes consistent with physiological roles of PGs. Transcriptomic data were congruent with PG data; IN-reduced plasma PG F2α concentration, whereas IB and CX did not. Five putative AOPs were developed linking the assumed molecular initiating event of COX inhibition, with PG reduction and the adverse outcome of reproductive failure via reduction of: (1) ovulation, (2) reproductive behaviors mediated by exogenous or endogenous PGs, and (3) oocyte maturation in fish. These pathways were developed using, in part, empirical data from the present study and other publicly available data.

Cytosol-Localized UDP-Xylose Synthases Provide the Major Source of UDP-Xylose for the Biosynthesis of Xylan and Xyloglucan.

Xylan and xyloglucan are the two major cell wall hemicelluloses in plants, and their biosynthesis requires a steady supply of the sugar donor, UDP-xylose. UDP-xylose is synthesized through conversion of UDP-glucuronic acid (UDP-GlcA) by the activities of UDP-xylose synthase (UXS). There exist six UXS genes in the Arabidopsis thaliana genome; three of them (UXS1, UXS2 and UXS4) encode Golgi-localized enzymes and the other three (UXS3, UXS5 and UXS6) encode cytosol-localized enzymes. In this report, we investigated the contributions of these UXS genes in supplying UDP-xylose for the biosynthesis of xylan and xyloglucan. Expression analyses revealed that the six UXS genes exhibited distinct and overlapping expression patterns in different cell types of stems, root-hypocotyls and young seedlings, and that the relative enzymatic activity of UXS in the cytosol was 17 times higher than that in the Golgi. Among the six UXS genes, UXS3, UXS5 and UXS6 showed the highest expression in stems and were expressed predominantly in xylem cells and interfascicular fibers. Their predominant expression in secondary wall-forming cells was consistent with the finding that the expression of UXS3, UXS5 and UXS6 was directly activated by the secondary wall NAC master switches. Although simultaneous mutations of UXS1, UXS2 and UXS4 did not cause any apparent effects on plant growth and xylan biosynthesis, simultaneous down-regulation/mutations of UXS3, UXS5 and UXS6 led to a drastic reduction in secondary wall thickening, a severe deformation of xylem vessels, a significant decrease in xylan content without an apparent reduction in its chain length and an absence of GlcA side chains in xylan, which are reminiscent of the phenotypes of some known xylan-deficient mutants. Moreover, Immunolocalization with two xyloglucan monoclonal antibodies, LM15 and LM25, revealed a significant reduction in the amount of xylogulcan in the primary walls. These results demonstrate that the cytosol-localized UXS3, UXS5 and UXS6 play a predominant role in the supply of UDP-xylose for the biosynthesis of xylan and xyloglucan.

An Integrated Experimental and Computational Approach for Characterizing the Kinetics and Mechanism of Triadimefon Racemization.

Enantiomers of chiral molecules commonly exhibit differing pharmacokinetics and toxicities, which can introduce significant uncertainty when evaluating biological and environmental fates and potential risks to humans and the environment. However, racemization (the irreversible transformation of one enantiomer into the racemic mixture) and enantiomerization (the reversible conversion of one enantiomer into the other) are poorly understood. To better understand these processes, we investigated the chiral fungicide, triadimefon, which undergoes racemization in soils, water, and organic solvents. Nuclear magnetic resonance (NMR) and gas chromatography / mass spectrometry (GC/MS) techniques were used to measure the rates of enantiomerization and racemization, deuterium isotope effects, and activation energies for triadimefon in H2 O and D2 O. From these results we were able to determine that: 1) the alpha-carbonyl carbon of triadimefon is the reaction site; 2) cleavage of the C-H (C-D) bond is the rate-determining step; 3) the reaction is base-catalyzed; and 4) the reaction likely involves a symmetrical intermediate. The B3LYP/6-311 + G** level of theory was used to compute optimized geometries, harmonic vibrational frequencies, nature population analysis, and intrinsic reaction coordinates for triadimefon in water and three racemization pathways were hypothesized. This work provides an initial step in developing predictive, structure-based models that are needed to identify compounds of concern that may undergo racemization. Chirality 28:633-641, 2016. © 2016 Wiley Periodicals, Inc.

Evolutionary Conservation of Xylan Biosynthetic Genes in Selaginella moellendorffii and Physcomitrella patens.

Xylan is a major cross-linking hemicellulose in secondary walls of vascular tissues, and the recruitment of xylan as a secondary wall component was suggested to be a pivotal event for the evolution of vascular tissues. To decipher the evolution of xylan structure and xylan biosynthetic genes, we analyzed xylan substitution patterns and characterized genes mediating methylation of glucuronic acid (GlcA) side chains in xylan of the model seedless vascular plant, Selaginella moellendorffii, and investigated GT43 genes from S. moellendorffii and the model non-vascular plant, Physcomitrella patens, for their roles in xylan biosynthesis. Using nuclear magentic resonance spectroscopy, we have demonstrated that S. moellendorffii xylan consists of ß-1,4-linked xylosyl residues subsituted solely with methylated GlcA residues and that xylans from both S. moellendorffii and P. patens are acetylated at O-2 and O-3. To investigate genes responsible for GlcA methylation of xylan, we identified two DUF579 genes in the S. moellendorffii genome and showed that one of them, SmGXM, encodes a glucuronoxylan methyltransferase capable of adding the methyl group onto the GlcA side chain of xylooligomers. Furthermore, we revealed that the two GT43 genes in S. moellendorffii, SmGT43A and SmGT43B, are functional orthologs of the Arabidopsis xylan backbone biosynthetic genes IRX9 and IRX14, respectively, indicating the evolutionary conservation of the involvement of two functionally non-redundant groups of GT43 genes in xylan backbone biosynthesis between seedless and seed vascular plants. Among the five GT43 genes in P. patens, PpGT43A was found to be a functional ortholog of Arabidopsis IRX9, suggesting that the recruitment of GT43 genes in xylan backbone biosynthesis occurred when non-vascular plants appeared on land.

Biomarker analysis of liver cells exposed to surfactant-wrapped and oxidized multi-walled carbon nanotubes (MWCNTs).

Carbon nanotubes (CNTs) have great potential in industrial, consumer, and mechanical applications, based partly on their unique structural, optical and electronic properties. CNTs are commonly oxidized or treated with surfactants to facilitate aqueous solution processing, and these CNT surface modifications also increase possible human and ecological exposures to nanoparticle-contaminated waters. To determine the exposure outcomes of oxidized and surfactant-wrapped multiwalled carbon nanotubes (MWCNTs) on biochemical processes, metabolomics-based profiling of human liver cells (C3A) was utilized. Cells were exposed to 0, 10, or 100ng/mL of MWCNTs for 24 and 48h; MWCNT particle size distribution, charge, and aggregation were monitored concurrently during exposures. Following MWCNT exposure, cellular metabolites were extracted, lyophilized, and buffered for (1)H NMR analysis. Acquired spectra were subjected to both multivariate and univariate analysis to determine the consequences of nanotube exposure on the metabolite profile of C3A cells. Resulting scores plots illustrated temporal and dose-dependent metabolite responses to all MWCNTs tested. Loadings plots coupled with t-test filtered spectra identified metabolites of interest. XPS analysis revealed the presence of hydroxyl and carboxyl functionalities on both MWCNTs surfaces. Metal content analysis by ICP-AES indicated that the total mass concentration of the potentially toxic impurities in the exposure experiments were extremely low (i.e. [Ni]≤2×10(-10)g/mL). Preliminary data suggested that MWCNT exposure causes perturbations in biochemical processes involved in cellular oxidation as well as fluxes in amino acid metabolism and fatty acid synthesis. Dose-response trajectories were apparent and spectral peaks related to both dose and MWCNT dispersion methodologies were determined. Correlations of the significant changes in metabolites will help to identify potential biomarkers associated with carbonaceous nanoparticle exposure.