Toward Less Hazardous Industrial Compounds: Coupling Quantum Mechanical Computations, Biomarker Responses, and Behavioral Profiles To Identify Bioactivity of SN2 Electrophiles in Alternative Vertebrate Models.

Sustainable molecular design of less hazardous chemicals promises to reduce risks to public health and the environment. Computational chemistry modeling coupled with alternative toxicology models (e.g., larval fish) present unique high-throughput opportunities to understand structural characteristics eliciting adverse outcomes. Numerous environmental contaminants with reactive properties can elicit oxidative stress, an important toxicological response associated with diverse adverse outcomes (i.e., cancer, diabetes, neurodegenerative disorders, etc.). We examined a common chemical mechanism (bimolecular nucleophilic substitution (SN2)) associated with oxidative stress using property-based computational modeling coupled with acute (mortality) and sublethal (glutathione, photomotor behavior) responses in the zebrafish (Danio rerio) and the fathead minnow (Pimephales promelas) models to identify whether relationships exist among biological responses and molecular attributes of industrial chemicals. Following standardized methods, embryonic zebrafish and larval fathead minnows were exposed separately to eight different SN2 compounds for 96 h. Acute and sublethal responses were compared to computationally derived in silico chemical descriptors. Specifically, frontier molecular orbital energies were significantly related to acute LC50 values and photomotor response (PMR) no observed effect concentrations (NOECs) in both fathead minnow and zebrafish. This reactivity index, LC50 values, and PMR NOECs were also significantly related to whole body glutathione (GSH) levels, suggesting that acute and chronic toxicity results from protein adduct formation for SN2 electrophiles. Shared refractory locomotor response patterns among study compounds and two alternative vertebrate models appear informative of electrophilic properties associated with oxidative stress for SN2 chemicals. Electrophilic parameters derived from frontier molecular orbitals were predictive of experimental in vivo acute and sublethal toxicity. These observations provide important implications for identifying and designing less hazardous industrial chemicals with reduced potential to elicit oxidative stress through bimolecular nucleophilic substitution.

Burn pit-related smoke causes developmental and behavioral toxicity in zebrafish: Influence of material type and emissions chemistry.

Combustion of mixed materials during open air burning of refuse or structural fires in the wildland urban interface produces emissions that worsen air quality, contaminate rivers and streams, and cause poor health outcomes including developmental effects. The zebrafish, a freshwater fish, is a useful model for quickly screening the toxicological and developmental effects of agents in such species and elicits biological responses that are often analogous and predictive of responses in mammals. The purpose of this study was to compare the developmental toxicity of smoke derived from the burning of 5 different burn pit-related material types (plywood, cardboard, plastic, a mixture of the three, and the mixture plus diesel fuel as an accelerant) in zebrafish larvae. Larvae were exposed to organic extracts of increasing concentrations of each smoke 6-to-8-hr post fertilization and assessed for morphological and behavioral toxicity at 5 days post fertilization. To examine chemical and biological determinants of toxicity, responses were related to emissions concentrations of polycyclic hydrocarbons (PAH). Emissions from plastic and the mixture containing plastic caused the most pronounced developmental effects, including mortality, impaired swim bladder inflation, pericardial edema, spinal curvature, tail kinks, and/or craniofacial deformities, although all extracts caused concentration-dependent effects. Plywood, by contrast, altered locomotor responsiveness to light changes to the greatest extent. Some morphological and behavioral responses correlated strongly with smoke extract levels of PAHs including 9-fluorenone. Overall, the findings suggest that material type and emissions chemistry impact the severity of zebrafish developmental toxicity responses to burn pit-related smoke.

Inconsistencies in variable reporting and methods in larval zebrafish behavioral assays.

New approaches in developmental neurotoxicity (DNT) screening are needed due to the tens of thousands of chemicals requiring hazard assessments. Zebrafish (Danio rerio) are an alternative vertebrate model for DNT testing, but without a standardized protocol for larval behavioral assays, comparison of results among laboratories is challenging. To evaluate the congruence of protocols across laboratories, we conducted a literature review of DNT studies focusing on larval zebrafish behavior assays and cataloged experimental design consistencies. Our review focused on 51 unique method variables in publications where chemical exposure occurred in early development and subsequent larval locomotor evaluation focused on assays that included a light/dark photoperiod transition. We initially identified 94 publications, but only 31 exclusively met our inclusion criteria which focused on parameters that are important to an assay employed by our laboratory. No publication reported 100% of the targeted variables; only 51 to 86% of those variables were reported in the reviewed publications, with some aspects of the experimental design consistent among laboratories. However, no protocol was exactly the same for any two publications. Many of these variables had more than one parameter/design reported, highlighting the inconsistencies among methods. Overall, there is not only a strong need for the development of a standardized testing protocol for larval zebrafish locomotor assays, but there is also a need for a standardized protocol for reporting experimental variables in the literature. Here we include an extensive guideline checklist for conducting larval zebrafish developmental behavior assays.

Influence of Methylene Blue or Dimethyl Sulfoxide on Larval Zebrafish Development and Behavior.

The use of larval zebrafish developmental testing and assessment, specifically larval zebrafish locomotor activity, has been recognized as a higher throughput testing strategy to identify developmentally toxic and neurotoxic chemicals. There are, however, no standardized protocols for this type of assay, which could result in confounding variables being overlooked. Two chemicals commonly employed during early-life stage zebrafish assays, methylene blue (antifungal agent) and dimethyl sulfoxide (DMSO, a commonly used vehicle) have been reported to affect the morphology and behavior of freshwater fish. In this study, we conducted developmental toxicity (morphology) and neurotoxicity (behavior) assessments of commonly employed concentrations for both chemicals (0.6-10.0 µM methylene blue; 0.3%-1.0% v/v DMSO). A light-dark transition behavioral testing paradigm was applied to morphologically normal, 6 days postfertilization (dpf) zebrafish larvae kept at 26°C. Additionally, an acute DMSO challenge was administered based on early-life stage zebrafish assays typically used in this research area. Results from developmental toxicity screens were similar between both chemicals with no morphological abnormalities detected at any of the concentrations tested. However, neurodevelopmental results were mixed between the two chemicals of interest. Methylene blue resulted in no behavioral changes up to the highest concentration tested, 10.0 µM. By contrast, DMSO altered larval behavior following developmental exposure at concentrations as low as 0.5% (v/v) and exhibited differential concentration-response patterns in the light and dark photoperiods. These results indicate that developmental DMSO exposure can affect larval zebrafish locomotor activity at routinely used concentrations in developmental neurotoxicity assessments, whereas methylene blue does not appear to be developmentally or neurodevelopmentally toxic to larval zebrafish at routinely used concentrations. These results also highlight the importance of understanding the influence of experimental conditions on larval zebrafish locomotor activity that may ultimately confound the interpretation of results.

Developmental Neurotoxicity and Behavioral Screening in Larval Zebrafish with a Comparison to Other Published Results.

With the abundance of chemicals in the environment that could potentially cause neurodevelopmental deficits, there is a need for rapid testing and chemical screening assays. This study evaluated the developmental toxicity and behavioral effects of 61 chemicals in zebrafish (Danio rerio) larvae using a behavioral Light/Dark assay. Larvae (n = 16-24 per concentration) were exposed to each chemical (0.0001-120 µM) during development and locomotor activity was assessed. Approximately half of the chemicals (n = 30) did not show any gross developmental toxicity (i.e., mortality, dysmorphology or non-hatching) at the highest concentration tested. Twelve of the 31 chemicals that did elicit developmental toxicity were toxic at the highest concentration only, and thirteen chemicals were developmentally toxic at concentrations of 10 µM or lower. Eleven chemicals caused behavioral effects; four chemicals (6-aminonicotinamide, cyclophosphamide, paraquat, phenobarbital) altered behavior in the absence of developmental toxicity. In addition to screening a library of chemicals for developmental neurotoxicity, we also compared our findings with previously published results for those chemicals. Our comparison revealed a general lack of standardized reporting of experimental details, and it also helped identify some chemicals that appear to be consistent positives and negatives across multiple laboratories.

Sunlight concurrently reduces Prymnesium parvum elicited acute toxicity to fish and prymnesins.

Prymnesium parvum continues to spread globally, producing harmful algal blooms that release toxins known to cause fish kills. While previous work has identified possible P. parvum toxin(s) (e.g., prymnesins, fatty acids, fatty acid amides) and investigated treatment strategies targeted at minimizing cell abundance, studies examining efficacy of treatment approaches to remove toxins are lacking. To understand influences of sunlight on toxins stability and toxicity to fish, acutely toxic P. parvum cultures were exposed to three light scenarios (lab dark control, field dark, and field light) and then evaluated for acute toxicity to fish and prymnesins abundance. Previous work showed acute toxicity to fathead minnow larvae was ameliorated after 2 h of sunlight exposure, and results observed herein found an identical trend. Acute toxicity disappeared in light exposed filtrate, but filtrate exposed to 35 °C without sunlight remained acutely toxic to fish. Additionally, six prymnesins were identified through high-resolution mass spectrometry and abundance corresponded to acute toxicity levels. Prymnesins were present at the highest level in filtrate that was acutely toxic but diminished in filtrate that was exposed to light and correspondingly ameliorated acute toxicity to fish. These findings suggest prymnesins are responsible for measured acute toxicity and are photo-labile, which represents an important implication for treatment strategies.

Nutrients and salinity influence Prymnesium parvum (UTEX LB 2797) elicited sublethal toxicity in Pimephales promelas and Danio rerio.

The magnitude, frequency, and duration of harmful algal blooms (HABs) are increasing worldwide, primarily due to climate change and anthropogenic activities. Prymnesium parvum is a euryhaline and eurythermal HAB forming species that has expanded throughout North America, resulting in massive fish kills. Previous aquatic ecology and toxicology efforts supported an understanding of conditions resulting in P. parvum HABs and fish kills; however, the primary endpoint selected for these studies was acute mortality. Whether adverse sublethal responses to P. parvum occur in fish are largely unknown. To begin to address this question, molecular and biochemical oxidative stress (OS) biomarker responses and photomotor behavioral alterations were investigated in two common fish models, the fathead minnow (Pimephales promelas) and zebrafish (Danio rerio). Varying nutrient and salinity conditions influenced P. parvum related OS biomarkers and fish behavioral responses in zebrafish and fathead minnows, which were heightened by nonoptimal conditions for P. parvum growth. Such sublethal observations present important considerations for future aquatic assessments and management of P. parvum HABs.

Suspect and non-target screening of acutely toxic Prymnesium parvum.

Harmful algal blooms (HABs) are increasing in frequency, magnitude, and duration around the world. Prymnesium parvum is a HAB species known to cause massive fish kills, but the toxin(s) it produces contributing to this acute toxicity to fish have not been confirmed. In the present study, a 2 × 2 factorial design was employed to examine influences of salinity (2.4 or 5 ppt) and nutrient limitation (f/2 or f/8) on P. parvum acute toxicity to fish and produced molecules. Acute toxicity (LC50) of these cultures, following a 48-h mortality assay, ranged from 10,213 to 96,816 cells mL-1. Non-targeted analysis was performed using liquid chromatography high-resolution mass spectrometry (LC-HRMS) to investigate compounds contributing to the differential toxicological responses. When P. parvum elicited toxicity to fish, suspect screening confirmed the presence of several prymnesins, and the peak area of PRM-A (3 Cl; prymnesin2aglycone) was significantly (p < 0.05) and positively related to acute toxicity. In addition, a non-targeted approach to highlighting peaks that differ between two chemical fingerprints was developed, termed a relative difference plot, and used to search for peaks co-varying with P. parvum induced acute toxicity to fish. Several peaks were highlighted along with the prymnesins identified through suspect screening when acute toxicity to fish was observed.

Low dissolved oxygen increases uptake of a model calcium channel blocker and alters its effects on adult Pimephales promelas.

Human population growth accompanied with urbanization is urbanizing the water cycle in many regions. Urban watersheds, particularly with limited upstream dilution of effluent discharges, represent worst case scenarios for exposure to multiple environmental stressors, including down the drain chemicals (e.g., pharmaceuticals) and other stressors (e.g., dissolved oxygen (DO)). We recently identified the calcium channel blocker diltiazem (DZM) to accumulate in fish plasma exceeding human therapeutic doses (e.g., Cmin) in coastal estuaries impaired due to nonattainment of DO water quality standards. Thus, we examined whether DO influences DZM uptake by fish, and if changes in DO-dependent upatke alter fish physiological and biochemical responses. Low DO (3.0 mg DO/L) approximately doubled diltiazem uptake in adult fathead minnows relative to normoxic (8.2 mg DO/L) conditions and were associated with significant (p < 0.05) increases in fish ventilation rate at low DO levels. Decreased burst swim performance (Uburst) of adult fathead minnows were significantly (p < 0.05) altered by low versus normal DO levels. DO × DZM studies reduced Uburst by 13-31% from controls, though not significantly (p = 0.06). Physiological responses in fish exposed to DZM alone were minimal; however, in co-exposure with low DO, decreasing trends in Uburst appeared inversely related to plasma lactate levels. Such physiological responses to multiple stressors, when paired with internal tissue concentrations, identify the utility of employing biological read across approaches to identify adverse outcomes of heart medications and potentially other cardiotoxicants impacting fish cardiovascular function across DO gradients.

Prymnesium parvum differentially triggers sublethal fish antioxidant responses in vitro among salinity and nutrient conditions.

Significant fish kills have been attributed to Prymnesium parvum in coastal and inland waters around the world. However, specific mechanisms responsible for adverse outcomes resulting from this harmful algal bloom (HAB) species remain unclear, though the gill has previously been identified as an important target organ. In the present study, an in vitro approach was used to examine cytotoxicity and antioxidant responses in fish liver (Hepa-E1 and PLHC-1) and gill (G1B and RTgill-W1) cell lines, following exposure to P. parvum grown at different salinities and nutrient concentrations, which can influence the magnitude of acute toxicity. Cultures from high salinity compromised survival of hepatic cell lines exposed to high dilutions, whereas no significant cytotoxicity was observed for gill cell lines. With respect to control groups, catalase showed significant activity in both gill cell lines, especially RTgill-W1, following exposure to high salinity cultures. High levels of superoxide dismutase were measured in Hepa-E1 cells exposed to all experimental treatment combinations and in RTgill-W1 cells following exposure to high salinity conditions, with respect to non-exposed cells Glutathione peroxidase activity was also detected at significant levels in Hepa-E1 cells after exposure to cultures from high salinity and the low salinity X low nutrients. Slight GPx increases were only observed in PLHC-1 and G1B exposed to P. parvum grown at high salinity. These results suggest that: 1. specific combinations of salinity and nutrient levels may contribute to production and potency of P. parvum toxins resulting in sub-lethal effects, and 2. sub-lethal responses are more prominent than cytotoxicity, and that oxidative stress may be a significant adverse effect of toxins produced by P. parvum.