Metabolomics Water Treatment Analytical Chemistry

Metabolomics- Water Treatment- Analytical Chemistry

Metabolic Profiling of Waste-Water Steroids & Their Disinfection Byproducts in Embryonic Zebrafish via GC-QToF Spectrometry

Steroids are a broad class of chemical compounds known to have significant metabolic effects on humans and other organisms. Their use in pharmaceutical, agricultural, and industrial applications, is, however, likely to result in contamination of surface water. Subsequent waste-water processing is intended to safely degrade steroids and other xenobiotics; yet presently employed water refinement methodology may be insufficient to wholly remove such compounds. Even extremely minuscule levels of steroidal compounds, on the order of ppm or lower concentrations, are potentially likely to have deleterious metabolic effects. As well, the treatment process itself may yield additional degradation products each with their own negative metabolic impact.

The proposed project will analyze and evaluate waste-water influent and effluent streams to determine the removal efficiency for known steroids. As well, waste-water supplies will be evaluated for the presence of novel degradation by-products arising as a consequence of in-house water treatment and disinfection protocols. Embryonic zebrafish, a known model for human developmental and metabolic processes, will be used to probe the effects of steroidal waste-water contaminants on health of humans and other organisms. With a goal of protecting consumer health, this study may potentially provide useful data for local water treatment facilities to upgrade treatment processes as necessary.

The project objectives will be achieved through the following approaches: (a) High accuracy in identification of targeted and un-targeted steroids in pre-treated waste-water samples, using Gas Chromatographic High-Resolution Time of Flight Mass Spectrometry (GC-QToF); (b) Evaluation of removal efficiency for such steroids following in-house treatment; (c) Analysis of treated water samples to assess the formation of by-products following disinfection processes; (d) Profiling of metabolic response following exposure to treated and untreated water using an embryonic zebrafish model to target human health-relevant endpoints.

1. Rationale

1.1. Steroids in water

The widespread prevalence of pharmaceutical drugs, personal care products and carcinogenic and/or pre-carcinogenic compounds produced by residential, industrial, and agricultural applications using public water has drawn considerable interest with a concomitant focus on health concerns. This is due to the potential impact of these compounds on drinking water if they are not fully removed from the water by standard water-treatment protocols. Synthetic steroids are pharmaceuticals that are widely used for treatment of inflammation, auto-immune disorders such as asthma and rheumatoid arthritis, and other conditions. [1, 2] These compounds, both endogenous (from the adrenal gland) and exogenous, from pharmaceutical and veterinary applications, as well as industrial processes and treatment of livestock, are excreted into the environment and ultimately into water. Glucocorticoids such as prednisolone, can be measured in natural aquifers and sludge at parts per trillion (ppt) and parts per billion (ppb) concentrations, respectively. These relatively high levels of steroids in drinking water have raised concerns about the impact of such contaminants on human health. As well, steroidal contaminants may impact the health of aquatic organisms. [3]

At present, there are no significant governmental regulations limiting the use of steroids in industrial, livestock, or agricultural settings. Furthermore, steroidal water contaminants are also a by-product of human and veterinary use of pharmaceuticals. The potentially deleterious health effects of such water contaminants have not been fully addressed. The goal of waste-water management is to remove these and other toxins. However, in the absence of detailed research on steroids and their degradation by-products, effectiveness of such removal protocols has not been measured. In consequence, active research has begun to address methods of efficient removal of steroids in waste-water treatment. These protocols include adsorption, chloramination, chlorination, oxidation, and ozonation. [4,5]

1.2. Transformation Products

The treatment of glucocorticoid steroids in source water by 'disinfection', also known as chemical treatment, has the potential to produce new, sometimes more harmful compounds known as disinfection byproducts. These transformation products are formed by interactions between the chemical precursors, organic matter, and chemicals used in water treatment. There are few studies of glucocorticoid removal from waste-water, and even less is known about glucocorticoid by-products formed through adventitious reaction with waste-water treatment chemicals. To date, it appears that neither advanced nor conventional waste-water treatment, including chemical means, has the ability to fully remove glucocorticoids from the final effluent. [6] Studies have revealed that toxic chlorinated and brominated steroidal by-products formed by disinfection processes tend to be more estrogenic in character than the precursor compound(s). Because there is 'cross-talk' between glucocorticoids and estrogenic receptors, it is possible that chlorinated and other degradation by-products of glucocorticoid compounds could exhibit similar estrogenic behavior. [4,7]

1.3. Health Impacts

Endogenous steroids such as glucocorticoids are normally excreted from the adrenal cortex into the body, where they function to control physiological development, stress management, and other normal human processes. Glucocorticoids are named for their function in metabolic regulation of glucose. These steroids act as agonists, initiating cellular response by activation of receptors that regulate transcription factors. As well, evidence has shown the potential of synthetic steroids to mimic natural steroid hormones, through binding at the target receptors. Reports indicate that steroids and their by-products inhibit immunological response and interfere with reproduction and development, through processes mediated by the glucocorticoid receptor. [9]

Thus, potentially deleterious health implications associated with glucocorticoid steroid exposure in drinking water involve changes in physiological state of organisms, through metabolic processes regulated by glucocorticoid and estrogenic cellular receptors. [8] Induced feminization in aquatic organisms, and a more extreme phenomenon known as sex reversal, has occurred with cortisol exposure during differentiation stages of fish development. These phenomena are believed to have been mediated by interactions between synthetic glucocorticoids and estrogen receptors. A study of female flathead fish revealed the impact of synthetic glucocorticoid- and estrogen receptor-mediated interactions, resulting in a decrease in natural estradiol levels induced by estrogen receptor binding. [10, 11] The link between exposure to glucocorticoid steroids, their transformation products, and associated adverse health effects is presently unclear. This has not only prompted considerable attention from regulatory groups, but has also shifted the focus of research towards a better understanding of the mechanisms of steroidal contaminant formation, health implications and mitigation solutions. [9, 12]

2. Methodology

2.1. Methodology for Gas Chromatographic High-Resolution Time of Flight Mass Spectrometry (GC-QToF)

Gas chromatographic high-resolution time of flight mass spectrometric analysis (GC-QToF) provides a highly sensitive experimental methodology. GC-QToF provides rapid data acquisition as well as accurate mass measurements of various compounds and metabolites. [13] Recently, GC-QToF has begun to emerge as a powerful tool for metabolomics research, with the potential to characterize and identify endogenous biomarkers of disease, while monitoring variations in metabolic profiles. GC-QToF is capable of measuring relatively low levels of metabolites produced via normal biochemical pathways in response to environmental stressors. [14] In addition to applications in analytical chemistry, cellular biochemistry, and toxicology, advantages of GC-QToF include statistical analysis capability and significant reduction of sample collection volumes, thus decreasing potentially negative or invasive impact of research sampling. [14-17] As a result, the techniques of mass spectrometry provide a highly valuable and thorough method for structure elucidation and investigation of metabolites in complex matrices.

2.2. Methodology for Embryonic Zebrafish Model

Embryonic zebrafish (Danio rerio) are an in vivo model widely used to study the impact of toxicity upon vertebrate development; this model is also an emerging tool for glucocorticoid receptor research. [18] The zebrafish model is an experimentally simple, yet ethically acceptable alternative to other more complex animal models, while simultaneously offering the capability to provide useful and relevant experimental data concerning metabolic effects upon the human corticosteroid system. Zebrafish rapidly produce embryos in large numbers; these undergo developmental phases that can successfully model precise cellular and molecular processes of humans. [19] An ELISA bioassay for embryonic zebrafish was recently developed to address the role of glucocorticoid receptors in adaptive physiological response to external stresses. These experiments also suggested a potential for experimental manipulation of embryonic zebrafish, particularly for toxicological and environmental studies. [20] Similar research using embryonic zebrafish cells focused on cellular mechanisms for toxicity induced by micro- and sub-micro-pollutants in waste-water. [21] While several studies of embryonic zebrafish have addressed pharmacological effects on glucocorticoid receptors, these data have not to date been integrated with more precise data accessible from other bio-analytical tools. Thus, this work will incorporate high-resolution gas chromatography (GC) and mass spectrometry (MS), using modern and efficient GC-QToF methodology as an experimental platform, to more precisely map the alteration of metabolic pathways arising from the presence of steroids, steroidal by-products and other xenobiotics in the waste-water stream.

3.1 Sampling campaign

The sampling campaign involved collection of the following samples: (a) source water from the Agua Nueva Wastewater Reclamation Plant; and (b) finished water released into the Santa Cruz River. Sampling also included untreated influent water, primary (pre-disinfection) and secondary (disinfected) effluent, and water from a point in the Santa Cruz River downstream from the Agua Nueva outfall. *[Suggest distance downstream be inserted]* The secondary effluent is known to have undergone conventional waste-water treatment with final chloramination and dechloramination steps. The collected samples followed a tiered testing protocol.

3.2 The identification of glucocorticoid compounds present in waste-water will require the following stages (Figure 1):

In order to analyze targeted precursors and untargeted transformation products, a subset of water samples will be spiked with three glucocorticoid steroid pharmaceuticals (dexamethasone, prednisolone, and triamcinolone) at a concentration of 5 ?M for each compound. A control sample is also present (See Figure 1).

Disinfected samples will be produced via addition of 4 ppm chloramine; following a 24-hr incubation with chloramine, the samples will be dechlorinated with 20 ppm sodium bisulfite.

The 'raw' and finished water samples, as well as the fortified effluent samples, will undergo solid phase extraction, followed by derivatization and analysis using GC-QToF spectrometry, operated in scan mode to identify both targeted and untargeted compounds.

All samples will be analyzed with the appropriate quality analysis and quality control (QA/QC) methodologies.

The physical characteristics of the water, such as total organic carbon (TOC), pH, and turbidity, will be measured and reported.

Figure 1. Matrix for glucocoriticoid steroid and disinfection byproduct analysis by GC-QToF *5 uM in text body-50 uM in Figure*

3.3. The metabolic profiling of glucocorticoids using an embryonic zebrafish assay will be accomplished through the following stages as shown in the Figure 2 matrix:

Zebrafish embryos will be collected according to the protocols of Huang et al. [24] These are in accordance with the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore (Protocol 081/10). Fifty embryos will be collected per data set and subsequently exposed to a specified volume of the water samples, produced according to the methodology described above in section 3.2 of this proposal. The embryos will be treated with corticosteroid concentrations and disinfection treatment doses based on the findings in section 3.2. Following treatment, samples will be snap-frozen and freeze-dried overnight.

*consider definition of 'snap-frozen* *consider testing for effects of freezing protocol on identity/integrity of steroids*

Using a modification of the Huang et al. [24] procedure, the targeted and untargeted compounds will be extracted from treated embryos.

Metabolic profiles for the zebrafish embryos exposed to treated/untreated and spiked/unspiked wastewater will be determined based on GC-QToF results.

Figure 2. Matrix for embryonic zebrafish metabolomic analysis by GC-QToF

3. Preliminary Results

Preliminary experimentation was conducted to evaluate the manner in which direct exposure of zebrafish embryos to glucocorticoid steroids affected the metabolic regulation of protein systems. The zebrafish embryos were subjected to a matrix of three drugs and three doses (shown in Figure 3), followed by incubation, extraction and subsequent analysis by GC-QToF.

Figure 3. Matrix of zebrafish embryo treatment with glucocorticoid steroids

Interpretation of the data from Dexamethasone doping revealed 210 common compounds in the various dosed samples, as illustrated in Figure 4. After further inspection, the amino acid glycine was identified with high mass accuracy and matched using the NIST library (Figure 5). Dexamethasone has previously been shown to inhibit glycine uptake in rat hepatoma cells. [25] Additional data processing is necessary to fully evaluate the impact of the three glucocorticoid steroids on amino acid enhancement and/or suppression (and thus protein bio-synthesis) during cellular regulation.