Pathway-Based Approaches for Assessing Biological Hazards of Complex Mixtures of Contaminants: A Case Study in the Maumee River.

Assessment of ecological risks of chemicals in the field usually involves complex mixtures of known and unknown compounds. We describe the use of pathway-based chemical and biological approaches to assess the risk of chemical mixtures in the Maumee River (OH, USA), which receives a variety of agricultural and urban inputs. Fathead minnows (Pimephales promelas) were deployed in cages for 4 d at a gradient of sites along the river and adjoining tributaries in 2012 and during 2 periods (April and June) in 2016, in conjunction with an automated system to collect composite water samples. More than 100 industrial chemicals, pharmaceuticals, and pesticides were detected in water at some of the study sites, with the greatest number typically found near domestic wastewater treatment plants. In 2016, there was an increase in concentrations of several herbicides from April to June at upstream agricultural sites. A comparison of chemical concentrations in site water with single chemical data from vitro high-throughput screening (HTS) assays suggested the potential for perturbation of multiple biological pathways, including several associated with induction or inhibition of different cytochrome P450 (CYP) isozymes. This was consistent with direct effects of water extracts in an HTS assay and induction of hepatic CYPs in caged fish. Targeted in vitro assays and measurements in the caged fish suggested minimal effects on endocrine function (e.g., estrogenicity). A nontargeted mass spectroscopy-based analysis suggested that hepatic endogenous metabolite profiles in caged fish covaried strongly with the occurrence of pesticides and pesticide degradates. These studies demonstrate the application of an integrated suite of measurements to help understand the effects of complex chemical mixtures in the field. Environ Toxicol Chem 2021;40:1098-1122. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Metabolomics for informing adverse outcome pathways: Androgen receptor activation and the pharmaceutical spironolactone.

One objective in developing adverse outcome pathways (AOPs) is to connect biological changes that are relevant to risk assessors (i.e., fecundity) to molecular and cellular-level alterations that might be detectable at earlier stages of a chemical exposure. Here, we examined biochemical responses of fathead minnows (Pimephales promelas) to inform an AOP relevant to spironolactone's activation of the androgen receptor, as well as explore other biological impacts possibly unrelated to this receptor. Liquid chromatography with high resolution mass spectrometry (LC-MS) was used to measure changes in endogenous polar metabolites in livers of male and female fish that were exposed to five water concentrations of spironolactone (0, 0.05, 0.5, 5, or 50µgL-1) for 21days. Metabolite profiles were affected at the two highest concentrations (5 and 50µgL-1), but not in the lower-level exposures, which agreed with earlier reported results of reduced female fecundity and plasma vitellogenin (VTG) levels. We then applied partial least squares regression to assess whether metabolite alterations covaried with changes in fecundity, VTG gene expression and protein concentrations, and plasma 17ß-estradiol and testosterone concentrations. Metabolite profiles significantly covaried with all measured endpoints in females, but only with plasma testosterone in males. Fecundity reductions occurred in parallel with changes in metabolites important in osmoregulation (e.g., betaine), membrane transport (e.g., l-carnitine), and biosynthesis of carnitine (e.g., methionine) and VTG (e.g., glutamate). Based on a network analysis program (i.e., mummichog), spironolactone also affected amino acid, tryptophan, and fatty acid metabolism. Thus, by identifying possible key events related to changes in biochemical pathways, this approach built upon an established AOP describing spironolactone's androgenic properties and highlighted broader implications potentially unrelated to androgen receptor activation, which could form a basis for the development of an AOP network.

Metabolite profiling of fish skin mucus: a novel approach for minimally-invasive environmental exposure monitoring and surveillance.

The application of 'omics tools to biologically based monitoring and surveillance of aquatic environments shows considerable promise for complementing chemical monitoring in ecological risk assessments. However, few of the current approaches offer the ability to sample ecologically relevant species (e.g., fish) in a way that produces minimal impact on the health of the organism(s) under study. In the current study we employ liquid chromatography tandem mass spectrometry (LC-MS/MS) to assess the potential for skin mucus-based metabolomics for minimally invasive sampling of the fathead minnow (FHM; Pimephales promelas). Using this approach we were able to detect 204 distinct metabolites in the FHM skin mucus metabolome representing a large number of metabolite classes. An analysis of the sex specificity of the skin mucus metabolome showed it to be highly sexually dimorphic with 72 of the detected metabolites showing a statistically significant bias with regard to sex. Finally, in a proof-of-concept fashion we report on the use of skin mucus-based metabolomics to assess exposures in male and female fathead minnows to an environmentally relevant concentration of bisphenol A, a nearly ubiquitous environmental contaminant and an established endocrine active chemical.

Metabolomics for in situ environmental monitoring of surface waters impacted by contaminants from both point and nonpoint sources.

We investigated the efficacy of metabolomics for field-monitoring of fish exposed to wastewater treatment plant (WWTP) effluents and nonpoint sources of chemical contamination. Lab-reared male fathead minnows (Pimephales promelas, FHM) were held in mobile monitoring units and exposed on-location to surface waters upstream and downstream of the effluent point source, as well as to the actual effluent at three different WWTP sites in Minnesota. After four days of exposure, livers were collected, extracted, and analyzed by (1)H NMR spectroscopy and GC-MS to characterize responses of the hepatic metabolome. Multivariate statistical analysis revealed distinct metabolite profile changes in response to effluent exposure from each of the three WWTPs. Differences among locations (i.e., upstream, downstream, and effluent) within each of the three sites were also identified. These observed differences comport with land-use and WWTP characteristics at the study sites. For example, at one of the sites, the metabolomic analyses suggested a positive interactive response from exposure to WWTP effluent and nearby nonpoint (likely agricultural related) contamination. These findings demonstrate the utility of metabolomics as a field-based technique for monitoring the exposure of fish to impacted surface waters.

Field-based approach for assessing the impact of treated pulp and paper mill effluent on endogenous metabolites of fathead minnows (Pimephales promelas).

A field-based metabolomic study was conducted during a shutdown of a pulp and paper mill (PPM) to assess the impacts of treated PPM effluent on endogenous polar metabolites in fathead minnow (FHM; Pimephales promelas) livers. Caged male and female FHMs were deployed at a Great Lakes area of concern during multiple periods (pre-, during, and post-shutdown) near the outflow for a wastewater treatment plant. Influent to this plant is typically 40% PPM effluent by volume. Additional FHMs were exposed to reference lake water under laboratory conditions. A bioassay using T47D-KBluc cells showed that estrogenic activity of receiving water near the outflow declined by 46% during the shutdown. We then used (1)H NMR spectroscopy and principal component analysis to profile abundances of hepatic endogenous metabolites for FHMs. Profiles for males deployed pre-shutdown in receiving water were significantly different from those for laboratory-control males. Profiles were not significantly different for males deployed during the shutdown, but they were significant again for those deployed post-shutdown. Impacts of treated effluent from this PPM were sex-specific, as differences among profiles of females were largely nonsignificant. Thus, we demonstrate the potential utility of field-based metabolomics for performing biologically based exposure monitoring and evaluating remediation efforts occurring throughout the Great Lakes and other ecosystems.

Metabolite profiling and a transcriptional activation assay provide direct evidence of androgen receptor antagonism by bisphenol A in fish.

Widespread environmental contamination by bisphenol A (BPA) has created the need to fully define its potential toxic mechanisms of action (MOA) to properly assess human health and ecological risks from exposure. Although long recognized as an estrogen receptor (ER) agonist, some data suggest that BPA may also behave as an androgen receptor (AR) antagonist. However, direct evidence of this activity is deficient. To address this knowledge gap, we employed a metabolomic approach using in vivo exposures of fathead minnows (FHM; Pimephales promelas ) to BPA either alone or in a binary mixture with 17ß-trenbolone (TB), a strong AR agonist. Changes in liver metabolite profiles in female FHM in response to these exposures were determined using high resolution (1)H NMR spectroscopy and multivariate and univariate statistics. Using this approach, we observed clear evidence of the ability of BPA to mitigate the impact of TB, consistent with an antiandrogenic MOA. In addition, a transcriptional activation assay with the FHM AR was used to confirm the AR antagonistic activity of BPA in vitro. The results of these in vivo and in vitro analyses provide strong and direct evidence for ascribing an antiandrogenic MOA to BPA in vertebrates.

Investigating compensation and recovery of fathead minnow (Pimephales promelas) exposed to 17alpha-ethynylestradiol with metabolite profiling.

1H NMR spectroscopy was used to profile metabolite changes in the livers of fathead minnows (Pimephales promelas) exposed to the synthetic estrogen 17alpha-ethynylestradiol (EE2) via a continuous flow water exposure. Fish were exposed to either 10 or 100 ng EE2/L for 8 days, followed by an 8 day depuration phase. Livers were collected after days 1, 4, and 8 of the exposure, and at the end of the depuration phase. Analysis of polar extracts of the liver revealed a greater impact of EE2 on males than females, with metabolite profiles of the former assuming similarities with those of the females (i.e., feminization) early in the exposure. Biochemical effects observed in the males included changes in metabolites relating to energetics (e.g., glycogen, glucose, and lactate) and liver toxicity (creatine and bile acids). In addition, amino acids associated with vitellogenin (VTG) synthesis increased in livers of EE2-exposed males, a finding consistent with increased plasma concentrations of the lipoprotein in the fish. Using partial least-squares discriminant analysis (PLS-DA), the response trajectories of the males at both exposure concentrations were compared. This revealed an apparent ability of the fish to compensate for the presence of EE2 later in the exposure, and to partially recover from its effects after the chemical was removed.

NMR analysis of male fathead minnow urinary metabolites: a potential approach for studying impacts of chemical exposures.

The potential for profiling metabolites in urine from male fathead minnows (Pimephales promelas) to assess chemical exposures was explored using nuclear magnetic resonance (NMR) spectroscopy. Both one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy was used for the assignment of metabolites in urine from unexposed fish. Because fathead minnow urine is dilute, we lyophilized these samples prior to analysis. Furthermore, 1D 1H NMR spectra of unlyophilized urine from unexposed male fathead minnow and Sprague-Dawley rat were acquired to qualitatively compare rat and fish metabolite profiles and to provide an estimate of the total urinary metabolite pool concentration difference. As a small proof-of-concept study, lyophilized urine samples from male fathead minnows exposed to three different concentrations of the antiandrogen vinclozolin were analyzed by 1D 1H NMR to assess exposure-induced changes. Through a combination of principal components analysis (PCA) and measurements of 1H NMR peak intensities, several metabolites were identified as changing with statistical significance in response to exposure. Among those changes occurring in response to exposure to the highest concentration (450 microg/L) of vinclozolin were large increases in taurine, lactate, acetate, and formate. These increases coincided with a marked decrease in hippurate, a combination potentially indicative of hepatotoxicity. The results of these investigations clearly demonstrate the potential utility of an NMR-based approach for assessing chemical exposures in male fathead minnow, using urine collected from individual fish.

Comparison and evaluation of laboratory performance on a method for the determination of perchlorate in fertilizers.

This report details the interlaboratory validation of a method for the determination of perchlorate in fertilizers. In this method (EPA/600/R-01/026), a solid sample of fertilizer is ground. Subsequently, the ground material is either leached with deionized water to dissolve any perchlorate salt in the case of minimally soluble fertilizers (e.g. supertriplephosphate or timed-release products), or simply dissolved in the case of highly soluble fertilizers (e.g. urea, NaNO3 or KCl). The resulting aqueous solution is then subjected to ion chromatography with suppressed conductivity detection. Four laboratories applied the method to field samples of 48 different products (commodity chemicals) and to seven quality control samples prepared by the Environmental Protection Agency (EPA). Additional tests were conducted by three other laboratories on specific materials. Recovery (81-111%) was demonstrated on spikes of known concentration, and a preliminary assured reporting level was determined for each fertilizer matrix by each laboratory. Injection-to-injection precision was satisfactory: generally less than 15% difference, and always less than 25%. All of the laboratories used Dionex IonPac AG16 guard and AS16 separation columns with NaOH or KOH as eluent. Detection was by suppressed conductivity. The method was shown to be sufficiently robust for the screening of fertilizers for perchlorate, but performance was low on a mixture of siliceous minerals (kaolinite and bentonite). Both laboratory performance and method performance are validated.

Identification of drinking water contaminants in the course of a childhood cancer investigation in Toms River, New Jersey.

Using a combination of gas chromatography/mass spectrometry (GC/MS) and gas chromatography/infrared spectroscopy (GC/IR) spectroscopic techniques, chemical contaminants and their hydrolysis products were identified in well water sampled in connection with a suspected childhood cancer cluster located in Dover Township, Ocean County, New Jersey. The drinking water contamination resulted from the leaching of industrial waste chemicals from drums that were disposed of at the site known as Reich Farm. Contaminants identified include dinitrile-tetralin compounds, known as 'trimers,' that are by-products of a polymerization process widely used by several polymer manufactures during the 1970s and 1980s (and still used today). Also identified were 'trimer' hydrolysis products, formed by the hydrolysis of their nitrile groups to amides. These industrial contaminants were not present in any of the mass or IR spectral library databases, and their identification required unconventional spectroscopic methods (including high resolution mass spectrometry, chemical ionization mass spectrometry, and IR spectroscopy), along with scientific reasoning and interpretation. It is currently not known whether these chemical contaminants are responsible for the childhood cancers observed in this area.

Human pancreatic thread protein, an exocrine thread protein with possible implications to Alzheimer's disease: secondary structure in solution at acid pH.

The secondary structure of human pancreatic thread protein (HPTP) in solution at acid pH was derived using Fourier transform infrared (FT-IR) and laser Raman spectroscopic studies. The experimentally derived secondary structure of HPTP was compared with the secondary structure obtained by the Chou-Fasman algorithm. Pancreatic thread protein is a major exocrine secretory protein that in vitro forms filamentous bundles reminiscent of the paired helical filaments of Alzheimer's disease (AD). PTP immunoreactivity in brains afflicted with AD has been demonstrated previously and high levels of its mRNA in the developing human brain have also been reported in the literature. The above studies suggest that AD is associated with enhanced expression of PTP-related transcripts with interneuronal accumulation of PTP-like proteins. The experimentally derived secondary structure of HPTP consists of a significant proportion of beta-sheets and beta-turns and lesser amounts of alpha-helical structures. The beta-sheet component presumably plays an important role in the pH-dependent globule-fibril transformation of HPTP leading to antiparallel beta-sheet structure in the aggregated state. The secondary structure of HPTP and its globule-fibril transformation lend credence to the belief that AD may be viewed as a conformational disease.

Non-uniform triple helical structure in chick skin type I collagen on thermal denaturation: Raman spectroscopic study.

The individual chains in the triple helix of collagen occur in a conformation related to polyproline II because of the presence of large number of imino peptide bonds. However, these residues are not evenly distributed in the collagen molecule which also contains many non-imino residues. These non-imino regions of collagen may be expected to show preference for other than triple helical conformations. The appearance of several Raman bands in solution phase at 65 degrees C raises the possibility of non-uniform triple helical structure in collagen. Raman spectroscopic studies on collagen in the solid state and in solution at a temperature greater than its denaturation temperature, reported here suggest that denatured collagen may exhibit an ensemble of conformational states with yet unknown implications to the biochemical interactions of this important protein component of connective tissues.

Multispectral identiftcation of alkyl and chloroalkyl phosphates from an industrial effluent.

Multispectral techniques (gas chromatography combined with low and high resolution electron-impact mass spectrometry, low and high resolution chemical ionization mass spectrometry, and Fourier transform infrared spectroscopy) were used to identify 13 alkyl and chloroalkyl phosphates in a water sample taken from the effluent of a plant that manufactures fire-retardant chemicals. Of the 13 phosphates identified, only 4 were located in hbrary mass spectral data bases; thus, techniques other than conventional low resolution electron-impact mass spectrometry with data base matching were required. Several of the Identified phosphates are commonly used ftre retardants; however, three exhibited chemical structures different from those of the commercially manufactured fire retardants and the reactants used in their synthesis.

Microbial degradation of seven amides by suspended bacterial populations.

Microbial transformation rate constants were determined for seven amides in natural pond water. A second-order mathematical rate expression served as the model for describing the microbial transformation. Also investigated was the relationship between the infrared spectra and the second-order rate constants for these amides. Second-order rate constants (k2) ranged from a low of 2.0 X 10(-14) to a high of 1.1 X 10(-9) liters organism-1 h-1 for niclosamide (2',5-dichloro-4'-nitrosalicylanilide) and propachlor (2-chloro-N-isopropylacetanilide), respectively. The mechanism of degradation (i.e., microbially mediated hydrolysis) of the amides was consistent with that of other organic chemicals previously studied in a variety of natural waters. Preliminary investigations indicate that temporal variations in measured second-order rate constants are small. A simple linear regression of the infrared carbonyl-stretching frequency with log K2 gave a correlation coefficient (r2) of 0.962.

Conformational features of dynorphin A-(1-13), Laser Raman spectroscopic studies.

Conformational features of dynorphin A-(1-13) were examined by laser Raman spectroscopy. Dynorphin A-(1-13) appears to have a mixture of extended beta-pleated sheet and "random" structure.

Conformational states of Leu5- and Met5-enkephalins in solution.

The molecular conformations of Leu5- and Met5-enkephalins in aqueous and DMSO solutions were investigated by FT-IR and laser Raman spectroscopic methods. The amide I, II, and III regions in the FT-IR spectra of Leu5- and Met5-enkephalins in aqueous solution were analyzed by performing Fourier self-deconvolution of the bands. Leu5-enkephalin in aqueous solution is found to exist in both type II beta-turn and beta-sheet structures, whereas Met5-enkephalin has a lesser tendency to form beta-turn structure in aqueous solution. It is likely that these different conformers of enkephalins might bind to different receptor types.