Alejandro J. Ramirez

Determination of select antidepressants in fish from an effluent‐dominated stream

Increasing evidence indicates widespread occurrence of pharmaceuticals and personal care products (PPCPs) in municipal effluent discharges and surface waters. Studies that characterize the fate and effects of PPCPs in aquatic systems are limited, and to our knowledge, data regarding pharmaceutical accumulation in fish of effluent-dominated ecosystems have not been previously reported. In the present study, fish populations were sampled from a reference stream and an effluent-dominated stream in north Texas, USA. Lepomis macrochirus, Ictalurus punctatus, Cyprinus carpio, and Pomoxis nigromaculatus were killed; the liver, brain, and lateral filet tissues dissected; and the tissues stored at -80 degrees C until analysis. Fish tissues were extracted using solid-phase extraction and then analyzed by gas chromatography-mass spectrometry in the negative chemical ionization mode. The selective serotonin reuptake inhibitors (SSRIs) fluoxetine and sertraline and the SSRI metabolites norfluoxetine and desmethylsertraline were detected at levels greater than 0.1 ng/g in all tissues examined from fish residing in a municipal effluent-dominated stream. To our knowledge, the present study is the first report of SSRI residues in fish residing within municipal effluent-dominated systems.

Occurrence of pharmaceuticals and personal care products in fish: Results of a national pilot study in the united states

Pharmaceuticals and personal care products are being increasingly reported in a variety of biological matrices, including fish tissue; however, screening studies have presently not encompassed broad geographical areas. A national pilot study was initiated in the United States to assess the accumulation of pharmaceuticals and personal care products in fish sampled from five effluent-dominated rivers that receive direct discharge from wastewater treatment facilities in Chicago, Illinois; Dallas, Texas; Orlando, Florida; Phoenix, Arizona; and West Chester, Pennsylvania, USA. Fish were also collected from the Gila River, New Mexico, USA, as a reference condition expected to be minimally impacted by anthropogenic influence. High performance liquid chromatography-tandem mass spectrometry analysis of pharmaceuticals revealed the presence of norfluoxetine, sertraline, diphenhydramine, diltiazem, and carbamazepine at nanogram-per-gram concentrations in fillet composites from effluent-dominated sampling locations; the additional presence of fluoxetine and gemfibrozil was confirmed in liver tissue. Sertraline was detected at concentrations as high as 19 and 545 ng/g in fillet and liver tissue, respectively. Gas chromatography-tandem mass spectrometry analysis of personal care products in fillet composites revealed the presence of galaxolide and tonalide at maximum concentrations of 2,100 and 290 ng/g, respectively, and trace levels of triclosan. In general, more pharmaceuticals were detected at higher concentrations and with greater frequency in liver than in fillet tissues. Higher lipid content in liver tissue could not account for this discrepancy as no significant positive correlations were found between accumulated pharmaceutical concentrations and lipid content for either tissue type from any sampling site. In contrast, accumulation of the personal care products galaxolide and tonalide was significantly related to lipid content. Results suggest that the detection of pharmaceuticals and personal care products was dependent on the degree of wastewater treatment employed.

Comparison of contaminants of emerging concern removal, discharge, and water quality hazards among centralized and on-site wastewater treatment system effluents receiving common wastewater influent.

A comparative understanding of effluent quality of decentralized on-site wastewater treatment systems, particularly for contaminants of emerging concern (CECs), remains less understood than effluent quality from centralized municipal wastewater treatment plants. Using a novel experimental facility with common influent wastewater, effluent water quality from a decentralized advanced aerobic treatment system (ATS) and a typical septic treatment system (STS) coupled to a subsurface flow constructed wetland (WET) were compared to effluent from a centralized municipal treatment plant (MTP). The STS did not include soil treatment, which may represent a system not functioning properly. Occurrence and discharge of a range of CECs were examined using isotope dilution liquid chromatography-tandem mass spectrometry during fall and winter seasons. Conventional parameters, including total suspended solids, carbonaceous biochemical oxygen demand and nutrients were also evaluated from each treatment system. Water quality of these effluents was further examined using a therapeutic hazard modeling approach. Of 19 CECs targeted for study, the benzodiazepine pharmaceutical diazepam was the only CEC not detected in all wastewater influent and effluent samples over two sampling seasons. Diphenhydramine, codeine, diltiazem, atenolol, and diclofenac exhibited significant (p<0.05) seasonal differences in wastewater influent concentrations. Removal of CECs by these wastewater treatment systems was generally not influenced by season. However, significant differences (p<0.05) for a range of water quality indicators were observed among the various treatment technologies. For example, removal of most CECs by ATS was generally comparable to MTP. Lowest removal of most CECs was observed for STS; however, removal was improved when coupling the STS to a WET. Across the treatment systems examined, the majority of pharmaceuticals observed in on-site and municipal effluent discharges were predicted to potentially present therapeutic hazards to fish.

Gas chromatography-mass spectrometry screening methods for select UV filters, synthetic musks, alkylphenols, an antimicrobial agent, and an insect repellent in fish.

Two screening methods have been developed for simultaneous determination of ten extensively used personal care products (PCPs) and two alkylphenol surfactants in fish. The methods consisted of extraction, clean-up, derivatization and analysis by gas chromatography-mass spectrometry with selected ion monitoring (GC-SIM-MS) or gas chromatography-tandem mass spectrometry (GC-MS/MS) techniques. Among solvents tested to assess recovery of target compounds from 1-g tissue homogenates, acetone was selected as optimal for extracting compounds with dissimilar physicochemical properties from fish tissue. Initial experiments confirmed that GC-SIM-MS could be applied for analysis of lean fillet tissue (<1% lipid) without gel-permeation chromatography (GPC), and this approach was applied to assess the presence of target analytes in fish fillets collected from a regional effluent-dominated stream in Texas, USA. Benzophenone, galaxolide, tonalide, and triclosan were detected in 11 of 11 environmental samples at concentrations ranging from; 37 to 90, 234 to 970, 26 to 97, and 17 to 31 ng/g, respectively. However, performance of this analytical approach declined appreciably with increasing lipid content of analyzed tissues. Successful analysis of samples with increased lipid content was enabled by adding GPC to the sample preparation protocol and monitoring analytes with tandem mass spectrometry. Both analytical approaches were validated using fortified fillet tissue collected from locations expected to be minimally impacted by anthropogenic influences. Average analyte recoveries ranged from 87% to 114% with RSDs <11% and from 54% to 107% with RSDs <20% for fish tissue containing <1% and 4.9% lipid, respectively. Statistically derived method detection limits (MDLs) for GC-SIM-MS and GC-MS/MS methodologies ranged from 2.4 to 16 ng/g, and 5.1 to 397 ng/g, respectively.

Enantiospecific toxicity of the β-blocker propranolol to Daphnia magna and Pimephales promelas

Propranolol is a widely prescribed, nonselective beta-adrenergic receptor-blocking agent. Propranolol has been detected in municipal effluents from the ng/L to the low-microg/L range. Like many therapeutics and other aquatic contaminants, propranolol is distributed as a racemic mixture ((R,S)-propranolol hydrochloride). Although the (S)-enantiomer is the most active form in mammals (up to 100-fold difference), no information is available regarding the enantiospecific toxicity of propranolol to aquatic organisms. Acute and chronic studies were conducted with Daphnia magna and Pimephales promelas to determine enantiospecific toxicity of propranolol to a model aquatic invertebrate and vertebrate, respectively. Also, enantiospecific effects of propranolol on D. magna heart rate were examined. Propranolol treatment levels were verified using high-performance liquid chromatography/mass spectrometry. Acute (48-h) responses of both organisms were similar for all enantiomer treatments. Chronic P. promelas responses to propranolol enantiomers followed the hypothesized relationship of (S)-propranolol being more toxic than (R)-propranolol, but chronic D. magna responses did not. This is potentially the result of a lack of beta-type receptors in cladocerans. No enantiospecific effects on daphnid heart rate were observed in acute exposures. Interestingly, some propranolol enantiomer treatments produced significant increases in reproduction before causing reproduction to decrease at higher treatment levels. To our knowledge, this research represents the first study of enantiospecific toxicity of chiral pharmaceutical pollutants.

Abnormal SDS‐PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding

The amino acid substitution or post‐translational modification of a cytosolic protein can cause unpredictable changes to its electrophoretic mobility during SDS‐PAGE. This type of “gel shifting” has perplexed biochemists and biologists for decades. We identify a mechanism for “gel shifting” that predominates among a set of ALS (amyotrophic lateral sclerosis) mutant hSOD1 (superoxide dismutase) proteins, post‐translationally modified hSOD1 proteins, and homologous SOD1 proteins from different organisms. By first comparing how 39 amino acid substitutions throughout hSOD1 affected SDS‐PAGE migration, we found that substitutions that caused gel shifting occurred within a single polyacidic domain (residues ∼80–101), and were nonisoelectric. Substitutions that decreased the net negative charge of domain 80–101 increased migration; only one substitution increased net negative charge and slowed migration. Capillary electrophoresis, circular dichroism, and size exclusion chromatography demonstrated that amino acid substitutions increase migration during SDS‐PAGE by promoting the binding of three to four additional SDS molecules, without significantly altering the secondary structure or Stokes radius of hSOD1‐SDS complexes. The high negative charge of domain 80–101 is required for SOD1 gel shifting: neutralizing the polyacidic domain (via chimeric mouse‐human SOD1 fusion proteins) inhibited amino acid substitutions from causing gel shifting. These results demonstrate that the pattern of gel shifting for mutant cytosolic proteins can be used to: (i) identify domains in the primary structure that control interactions between denatured cytosolic proteins and SDS and (ii) identify a predominant chemical mechanism for the interaction (e.g., hydrophobic vs. electrostatic).

Perspectives on Human Pharmaceuticals in the Environment

Human interaction with the environment remains one of the most pervasive facets of modern society. Whereas the anthropocene is characterized by rapid population growth, unprecedented global trade and digital communications, energy security, natural resource scarcities, climatic changes and environmental quality, emerging diseases and public health, biodiversity and habitat modifications are routinely touted by the popular press as they canvas global political agendas and scholarly endeavors. With a concentration of human populations in urban areas unlike any other time in history, the coming decades will be defined by “A New Normal,” as proposed by Sandra Postel, where the interplay among sustainable human activities and natural resource management will inherently determine the regional fates of human societies. In recent years, few topics have captured the public’s attention like the presence of human pharmaceuticals in environment. But why have citizens been so engaged by fish on Prozac and drug residues in drinking water? Because pharmacotherapy is now entrenched in everyday life, a realization that common drugs are found in the water we drink or the fish we eat likely produces a boomerang effect, where our daily reliance on well-accepted therapies is concretely linked in a new way with their potential consequences to the natural world. On an increasingly urban planet, pharmaceutical residues and traces of other contaminants of emerging concern represent signals of the rapidly urbanizing water cycle and harbingers of the “New Normal.” This volume examines current issues and provides timely future perspectives on human pharmaceuticals in the environment. We further provide a novel prediction that 10% of pharmaceuticals may result in internal fish plasma concentrations equaling the human Cmax value at or below an environmentally relevant concentration of 29 ng/L.

Nitrosyl hydride (HNO) replaces dioxygen in nitroxygenase activity of manganese quercetin dioxygenase

Quercetin dioxygenase (QDO) catalyzes the oxidation of the flavonol quercetin with dioxygen, cleaving the central heterocyclic ring and releasing CO. The QDO from Bacillus subtilis is unusual in that it has been shown to be active with several divalent metal cofactors such as Fe, Mn, and Co. Previous comparison of the catalytic activities suggest that Mn(II) is the preferred cofactor for this enzyme. We herein report the unprecedented substitution of nitrosyl hydride (HNO) for dioxygen in the activity of Mn-QDO, resulting in the incorporation of both N and O atoms into the product. Turnover is demonstrated by consumption of quercetin and other related substrates under anaerobic conditions in the presence of HNO-releasing compounds and the enzyme. As with dioxygenase activity, a nonenzymatic base-catalyzed reaction of quercetin with HNO is observed above pH 7, but no enhancement of this basal reactivity is found upon addition of divalent metal salts. Unique and regioselective N-containing products (14N/15N) have been characterized by MS analysis for both the enzymatic and nonenzymatic reactions. Of the several metallo-QDO enzymes examined for nitroxygenase activity under anaerobic condition, only the Mn(II) is active; the Fe(II) and Co(II) substituted enzymes show little or no activity. This result represents an enzymatic catalysis which we denote nitroxygenase activity; the unique reactivity of the Mn-QDO suggests a metal-mediated electron transfer mechanism rather than metal activation of the substrate’s inherent base-catalyzed reactivity.

l-Leucine Increases Skeletal Muscle IGF-1 but Does Not Differentially Increase Akt/mTORC1 Signaling and Serum IGF-1 Compared to Ursolic Acid in Response to Resistance Exercise in Resistance-Trained Men

Objective: Ursolic acid administration following resistance exercise increases mammalian target of rapamycin complex 1 (mTORC1) activity and skeletal muscle IGF-1 concentration in murines in a manner similar to l-leucine yet remains unexamined in humans. This study examined serum and skeletal muscle insulin-like growth factor-1 (IGF-1) and Akt/mTORC1 signaling activity following ingestion of either ursolic acid or l-leucine immediately after resistance exercise. Methods: Nine resistance-trained men performed 3 lower-body resistance exercise sessions involving 4 sets of 8–10 repetitions at 75%–80% one repetition maximum (1-RM) on the angled leg press and knee extension exercises. Immediately following each session, participants orally ingested 3 g cellulose placebo (PLC), l-leucine (LEU), or ursolic acid (UA). Blood samples were obtained pre-exercise and at 0.5, 2, and 6 hours postexercise. Muscle biopsies were obtained pre-exercise and at 2 and 6 hours postexercise. Results: Plasma leucine increased in LEU at 2 hours postexercise compared to PLC (p = 0.04). Plasma ursolic acid increased in UA at 2 h and 6 hours postexercise compared to PLC and LEU (p < 0.003). No significant differences were observed for serum insulin (p = 0.98) and IGF-1 (p = 0.99) or skeletal muscle IGF-1 receptor (IGF-1R; p = 0.84), Akt (p = 0.55), mTOR (p = 0.09), and p70S6K (p = 0.98). Skeletal muscle IGF-1 was significantly increased in LEU at 2 hours postexercise (p = 0.03) and 6 hours postexercise (p = 0.04) compared to PLC and UA. Conclusion: Three grams of l-leucine and ursolic acid had no effect on Akt/mTORC1 signaling or serum insulin or IGF-1; however, l-leucine increased skeletal muscle IGF-1 concentration in resistance-trained men.

Inhibition of Staphylococcus aureus cell wall biosynthesis by desleucyl-oritavancin: a quantitative peptidoglycan composition analysis by mass spectrometry

Oritavancin is a lipoglycopeptide antibiotic that exhibits potent activities against vancomycin-resistant Gram-positive pathogens. Oritavancin differs from vancomycin by a hydrophobic side chain attached to the drug disaccharide, which forms a secondary binding site to enable oritavancin binding to the cross-linked peptidoglycan in the cell wall. The mode of action of secondary binding site was investigated by measuring the changes in the peptidoglycan composition of Staphylococcus aureus grown in the presence of desleucyl-oritavancin at subinhibitory concentration using liquid chromatography-mass spectrometry (LC-MS). Desleucyl-oritavancin is an Edman degradation product of oritavancin that exhibits potent antibacterial activities despite the damaged d-Ala-d-Ala binding site due to its functional secondary binding site. Accurate quantitative peptidoglycan composition analysis based on 83 muropeptide ions determined that cell walls of S. aureus grown in the presence of desleucyl-oritavancin showed a reduction of peptidoglycan cross-linking, increased muropeptides with a tetrapeptide-stem structure, decreased O-acetylation of MurNAc, and increased N-deacetylation of GlcNAc. The changes in peptidoglycan composition suggest that desleucyl-oritavancin targets the peptidoglycan template to induce cell wall disorder and interferes with cell wall maturation.IMPORTANCE Oritavancin is a lipoglycopeptide antibiotic with a secondary binding site that targets the cross-linked peptidoglycan bridge structure in the cell wall. Even after the loss of its primary d-Ala-d-Ala binding site through Edman degradation, desleucyl-oritavancin exhibits potent antimicrobial activities through its still-functioning secondary binding site. In this study, we characterized the mode of action for desleucyl-oritavancins secondary binding site using LC-MS. Peptidoglycan composition analysis of desleucyl-oritavancin-treated S. aureus was performed by determining the relative abundances of 83 muropeptide ions matched from a precalculated library through integrating extracted ion chromatograms. Our work highlights the use of quantitative peptidoglycan composition analysis by LC-MS to provide insights into the mode of action of glycopeptide antibiotics.