A cell-free testing platform to screen chemicals of potential neurotoxic concern across twenty vertebrate species.

There is global demand for new in vitro testing tools for ecological risk assessment. The objective of the present study was to apply a set of cell-free neurochemical assays to screen many chemicals across many species in a relatively high-throughput manner. The platform assessed 7 receptors and enzymes that mediate neurotransmission of γ-aminobutyric acid, dopamine, glutamate, and acetylcholine. Each assay was optimized to work across 20 vertebrate species (5 fish, 5 birds, 7 mammalian wildlife, 3 biomedical species including humans). We tested the screening assay platform against 80 chemicals (23 pharmaceuticals and personal care products, 20 metal[loid]s, 22 polycyclic aromatic hydrocarbons and halogenated organic compounds, 15 pesticides). In total, 10 800 species-chemical-assay combinations were tested, and significant differences were found in 4041 cases. All 7 assays were significantly affected by at least one chemical in each species tested. Among the 80 chemicals tested, nearly all resulted in a significant impact on at least one species and one assay. The 5 most active chemicals were prochloraz, HgCl2 , Sn, benzo[a]pyrene, and vinclozolin. Clustering analyses revealed groupings according to chemicals, species, and chemical-assay combinations. The results show that cell-free assays can screen a large number of samples in a short period of time in a cost-effective manner in a range of animals not easily studied using traditional approaches. Strengths and limitations of this approach are discussed, as well as next steps. Environ Toxicol Chem 2017;36:3081-3090. © 2017 SETAC.

In ovo exposure to organophosphorous flame retardants: survival, development, neurochemical, and behavioral changes in white leghorn chickens.

Organophosphorous flame retardants (OPFRs) are contaminants of emerging concern. There is growing evidence of environmental contamination and exposures to both humans and wildlife. Here, the objective was to increase understanding of the potential neurodevelopmental effects of two relevant OPFRs, TMPP (tri (methylphenyl) phosphate; a non-halogen-containing OPFR) and TDCIPP (tris (1,3-dichloro-isopropyl) phosphate; a halogen-containing OPFR) in an avian embryo/chick model. We injected white leghorn chicken eggs with a range of TMPP (0, 10, 100, and 1000 ng/g) or TDCIPP (0, 10, 100, 1000, 50,000 ng/g) concentrations at incubation day 0 exposing embryos throughout the ~21-day in ovo period. Hatching success was unaffected by TMPP, but TDCIPP-exposed chicks had higher early-incubation mortality in 100 and 50,000 ng/g groups. On 7-9-day-old chicks, we assessed behavior via tests concerning righting reflex, angled balance beams, gait patterns, wing flap reflex, and open field movements. Chicks exposed to 100 ng/g TDCIPP achieved 40% lower maximum velocity in the open field test than vehicle-exposed controls, while those exposed to 1000 ng/g TDCIPP achieved 20% higher maximum velocity than vehicle-exposed controls. Chicks exposed to 50,000 ng/g TDCIPP showed reduced righting response success. There were no dose- or treatment-related differences in angled beam, gait analysis, or wing flap reflex tests. Cerebrum hemispheres from 10-day-old chicks were examined for neurochemistry (acetylcholinesterase [AChE] activity and both nicotinic [nACh] and muscarinic [mACh] acetylcholine receptor levels) and cerebellums were examined for histopathology. TDCIPP-exposed chicks had reduced number of degenerate Purkinje cells (TDCIPP, 1000 ng/g), possibly indicating disruption of neurodevelopment. No neurochemical effects were found in TMPP- or TDCIPP-exposed chicks. In general this study shows some possible neurodevelopmental effects in chicks exposed to TDCIPP when levels greatly exceeded those measured in wild bird eggs and no clear changes in TMPP-exposed chicks. This study builds upon previous in vitro studies as well as work on adult birds showing that toxic responses in avian models can vary among species and OPFRs.

Hepatic polybrominated diphenyl ether (PBDE) levels in Wisconsin river otters (Lontra canadensis) and Michigan bald eagles (Haliaeetus leucocephalus).

Polybrominated diphenyl ethers (PBDEs) are persistent and toxic flame-retardant chemicals widespread in the Great Lakes ecosystem. These chemicals are now being regulated and phased-out of the region; therefore it remains important to understand the extent of contamination in order to track the efficacy of recent actions. Here, Σ4PBDE congeners (PBDE-47, 99, 100, 153;wetweight basis unless indicated)were determined in liver tissues from Wisconsin river otters (Lontra canadensis; n = 35; 2009-2010) and Michigan bald eagles (Haliaeetus leucocephalus; n = 33; 2009-2011). In otters, Σ4PBDE ranged from0.5 to 72.9 ng/g, with a mean (±SD) and median (25th-75th percentile inter-quartile range) of 16.3 ± 16.4 ng/g and 11.3 (5.6-18.9) ng/g, respectively. The mean lipid-adjusted Σ4PBDE was 1377 ± 1485 ng/g. In eagles, Σ4PBDE ranged from 0 to 1,538.8 ng/g, with a mean and median of 74.3 ± 266.7 ng/g and 21.2 (5.7-28.9) ng/g, respectively. The mean lipid-adjusted Σ4PBDE was 5274.5 ± 19,896.1 ng/g. In both species, PBDE-47 accounted for >50% of the Σ4PBDE, followed by PBDE-99 and PBDE-100 (each ~17-19% of the total). The PBDE levels reported here in otters are similar to mammalian wildlife elsewhere, though the levels in eagles are among the highest worldwide across studied birds. The findings indicate that apex Great Lakes wildlife remain exposed to appreciable levels of PBDEs and more work is needed to understand whether such exposures are associated with adverse health outcomes.

Methylmercury egg injections: part 1--Tissue distribution of mercury in the avian embryo and hatchling.

Methylmercury (MeHg) is transferred by female birds into their eggs thus leaving developing embryos exposed to MeHg from the time of fertilization through hatching. Although Hg is a developmental toxicant, little is known about how it distributes among embryonic tissues and subsequently affects neurodevelopment in birds. The main objective of the present study (Part 1 of 2) was to evaluate the distribution of Hg in tissues during different developmental stages in order to better understand potential targets of Hg in the embryo and hatchling. Eight independent, yet related, egg injection studies were conducted. In five studies, white leghorn chicken embryos were air cell injected with methylmercury chloride (MeHgCl; range injected: 0.17-6.4µg/g egg) and Hg concentrations were assessed in seven tissues. We found that soft tissue distribution in embryos and hatchlings was similar to that seen in older birds, with higher total Hg concentrations in liver and kidney than in heart, muscle, and brain (e.g., 5.1, 3.8, 1.9, 2.3, and 1.9µg/g wet weight, respectively, in day 19 embryos after injection with 6.4µg/g MeHgCl). Concentrations were highest in feathers and unabsorbed yolk (e.g., 24.1 and 13.0µg/g in day 19 embryos after injection with 6.4µg/g MeHgCl). Tissue concentrations rose through embryonic days 11, 14, 16, and 19 but generally leveled off at days 1 and 7 post-hatch. We also report on pilot studies that demonstrated that tissue Hg accumulation after MeHgCl injection is similar in chicken and Japanese quail embryos, and that tissue Hg accumulation in chicken embryos after methylmercury cysteine, but not mercury (2) chloride, injection is similar to accumulation after MeHgCl injection. These findings suggest that embryos may accumulate kidney and brain Hg concentrations known to cause renal and neurotoxicity seen in older birds, but that sequestration of Hg into liver and excretion into rapidly growing feathers may offer some protection. This work also demonstrates that air cell injection studies are potentially a useful tool for studies of Hg toxicity in the laboratory.

Methylmercury egg injections: part 2--pathology, neurochemistry, and behavior in the avian embryo and hatchling.

Methylmercury (MeHg) is a toxic metal that has been frequently linked to neurochemical alterations, brain lesions, neurobehavioral changes, and reproductive impairments in wild and captive birds. Much less is known about the effects of MeHg on the developing avian brain and resulting effects on hatchling behavior. The objective of this work was to use air cell injection studies to investigate the effect of in ovo MeHg exposure on brain pathology and four neurochemical biomarkers (N-methyl-d-aspartate (NMDA) receptor, γ-aminobutyric acid (GABA) receptor, glutamine synthetase (GS) and glutamic acid decarboxylase (GAD)) that have previously been studied in wild birds, and on hatchling righting response, balance, and startle response. In a series of six studies, we exposed white leghorn chicken and Japanese quail embryos to methylmercury chloride (MeHgCl) (range: 0-6.4µg/g egg) via egg injection on embryonic day (ED) 0 and measured receptor levels and enzyme activity at different stages of embryonic (days 11, 14, and 19 in chicken; day 15 in quail) and hatchling (day 1 and day 7) development, and in whole brain or discrete brain regions (cerebrum, cerebellum, optic lobe). We assessed neurobehaviors on post hatch (PH) days 1 and 7. Despite accumulating relatively high levels of Hg in the brain, embryos and hatchlings did not consistently display neurochemical changes consistent with those seen in wild birds and laboratory mammals. Hatchlings also did not demonstrate behavioral alterations. Pathology did not indicate a difference in occurrence and types of lesions between control and dosed birds. These findings suggest that in ovo MeHg exposure alone may not be responsible for neurological impacts in bird. This work draws attention to factors, such as age and species, that may influence responses to MeHg in birds.

Postmortem stability of brain GABAergic and glutamatergic receptors and enzymes under ecological conditions.

Neurochemical biomarkers have emerged as useful tools for assessing the subclinical neurological impacts of environmental toxicants in birds and other wildlife. Careful consideration of biomarker stability is necessary before implementing their use on tissues from ecological studies, as receptors and enzymes in the brain may be affected by postmortem conditions. The goal of this study was to evaluate the postmortem stability of key GABAergic and glutamatergic receptors (N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid (GABAA-benzodiazepine)) and enzymes (glutamine synthetase (GS), glutamic acid decarboxylase (GAD)) under environmentally relevant field and storage conditions to determine their suitability as biomarkers. We exposed chicken embryo brains to postmortem environmental and storage conditions typical for ecological studies (12, 24, and 48 h at 7 °C or 25 °C; 1, 4, and 8 weeks at -80 °C or -20 °C; 1 or 2 freeze thaw cycles), and measured [3H] MK-801 binding to the NMDA receptor, [3H] flunitrazipam binding to the GABAA-benzodiazepine receptor, GS activity, and GAD activity. We found that [3H] MK-801 binding is stable under all conditions studied. GAD activity was fairly stable under each storage and environmental temperatures for all durations, but was significantly less stable when stored at -20 °C than at -80 °C. [3H] flunitrazipam binding and GS activity were both impacted by environmental and storage temperature and duration, and might best be utilized in studies of samples with similar histories. Our findings here demonstrate that caution is warranted when comparing samples with different collection and storage histories, but that some biomarkers are fairly stable under various conditions.

Multiple metals exposure and neurotoxic risk in bald eagles (Haliaeetus leucocephalus) from two Great Lakes states.

In the present study, the authors determined concentrations of several elements (As, Cd, Co, Cu, Cr, Mn, Pb, Sb, Zn) in the brains and livers of 46 bald eagles (Haliaeetus leucocephalus) from two Great Lakes states, Michigan and Minnesota. To explore whether exposures are of neurological concern, the authors assessed their associations with neurochemical receptors (N-methyl-D-aspartate [NMDA] and γ-aminobutyric acid A [GABA(A)]) and enzymes (glutamine synthetase [GS] and glutamic acid decarboxylase [GAD]) that play critical roles in vertebrate neurobehavior and reproduction. For most elements, levels in the livers and brains did not differ between region and gender. Hepatic Pb levels averaged 33.1 ppm (dry wt), 30.4% of all carcasses exceeded proposed avian Pb thresholds (>26.4 ppm), and in 30.8% of the birds examined evidence of Pb pellets or fragments was found. Significant changes in the activities of GS and GAD were related to brain concentrations of several metals (Pb, Cd, Co, Cu, Zn). No relationships were found among any of the nine elements and NMDA or GABA(A) receptor levels. When combined with the authors' previous study on these same eagles that showed Hg-associated alterations in GS, GAD, and NMDA receptor levels, the present research suggests that bald eagles are exposed to various elements, especially Pb and Hg, that are capable of causing changes in GABAergic and glutamatergic neurotransmission. The functional significance of these neurochemical changes warrants attention.

Mercury exposure and neurochemical impacts in bald eagles across several Great Lakes states.

In this study, we assessed mercury (Hg) exposure in several tissues (brain, liver, and breast and primary feathers) in bald eagles (Haliaeetus leucocephalus) collected from across five Great Lakes states (Iowa, Michigan, Minnesota, Ohio, and Wisconsin) between 2002-2010, and assessed relationships between brain Hg and neurochemical receptors (NMDA and GABA(A)) and enzymes (glutamine synthetase (GS) and glutamic acid decarboxylase (GAD)). Brain total Hg (THg) levels (dry weight basis) averaged 2.80 µg/g (range: 0.2-34.01), and levels were highest in Michigan birds. THg levels in liver (r(p) = 0.805) and breast feathers (r(p) = 0.611) significantly correlated with those in brain. Brain Hg was not associated with binding to the GABA(A) receptor. Brain THg and inorganic Hg (IHg) were significantly positively correlated with GS activity (THg r(p) = 0.190; IHg r(p) = 0.188) and negatively correlated with NMDA receptor levels (THg r(p) = -0245; IHg r(p) = -0.282), and IHg was negatively correlated with GAD activity (r(s) = -0.196). We also report upon Hg demethylation and relationships between Hg and Se in brain and liver. These results suggest that bald eagles in the Great Lakes region are exposed to Hg at levels capable of causing subclinical neurological damage, and that when tissue burdens are related to proposed avian thresholds approximately 14-27% of eagles studied here may be at risk.

Investigation of spatial trends and neurochemical impacts of mercury in herring gulls across the Laurentian Great Lakes.

Herring gulls (Larus argentatus) bioaccumulate mercury (Hg) but it is unknown whether they are exposed at levels of neurological concern. Here we studied brain tissues from gulls at five Great Lakes colonies and one non-Great Lakes colony during spring of 2001 and 2003. Total brain Hg concentrations ranged from 0.14 to 2.0 microg/g (dry weight) with a mean of 0.54 microg/g. Gulls from Scotch Bonnet Island, on the easternmost edge of the Great Lakes, had significantly higher brain Hg than other colonies. No association was found between brain Hg concentration and [3H]-ligand binding to neurochemical receptors (N-methyl-D-aspartate, muscarinic cholinergic, nicotinic cholinergic) or nicotinic receptor alpha-7 relative mRNA expression as previously documented in other wildlife. In conclusion, spatial trends in Hg contamination exist in herring gulls across the Great Lakes basin, and herring gulls accumulate brain Hg but not at levels associated with sub-clinical neurochemical alterations.