David O. Norris

Antidepressant Pharmaceuticals in Two U.S. Effluent-Impacted Streams: Occurrence and Fate in Water and Sediment, and Selective Uptake in Fish Neural Tissue

Antidepressant pharmaceuticals are widely prescribed in the United States; release of municipal wastewater effluent is a primary route introducing them to aquatic environments, where little is known about their distribution and fate. Water, bed sediment, and brain tissue from native white suckers (Catostomus commersoni) were collected upstream and at points progressively downstream from outfalls discharging to two effluent-impacted streams, Boulder Creek (Colorado) and Fourmile Creek (Iowa). A liquid chromatography/tandem mass spectrometry method was used to quantify antidepressants, including fluoxetine, norfluoxetine (degradate), sertraline, norsertraline (degradate), paroxetine, citalopram, fluvoxamine, duloxetine, venlafaxine, and bupropion in all three sample matrices. Antidepressants were not present above the limit of quantitation in water samples upstream from the effluent outfalls but were present at points downstream at ng/L concentrations, even at the farthest downstream sampling site 8.4 km downstream from the outfall. The antidepressants with the highest measured concentrations in both streams were venlafaxine, bupropion, and citalopram and typically were observed at concentrations of at least an order of magnitude greater than the more commonly investigated antidepressants fluoxetine and sertraline. Concentrations of antidepressants in bed sediment were measured at ng/g levels; venlafaxine and fluoxetine were the predominant chemicals observed. Fluoxetine, sertraline, and their degradates were the principal antidepressants observed in fish brain tissue, typically at low ng/g concentrations. A qualitatively different antidepressant profile was observed in brain tissue compared to streamwater samples. This study documents that wastewater effluent can be a point source of antidepressants to stream ecosystems and that the qualitative composition of antidepressants in brain tissue from exposed fish differs substantially from the compositions observed in streamwater and sediment, suggesting selective uptake.

Antidepressants at environmentally relevant concentrations affect predator avoidance behavior of larval fathead minnows (Pimephales promelas).

The effects of embryonic and larval exposure to environmentally relevant (ng/L) concentrations of common antidepressants, fluoxetine, sertraline, venlafaxine, and bupropion (singularly and in mixture) on C-start escape behavior were evaluated in fathead minnows (Pimephales promelas). Embryos (postfertilization until hatching) were exposed for 5 d and, after hatching, were allowed to grow in control well water until 12 d old. Similarly, posthatch fathead minnows were exposed for 12 d to these compounds. High-speed (1,000 frames/s) video recordings of escape behavior were collected and transferred to National Institutes of Health Image for frame-by-frame analysis of latency periods, escape velocities, and total escape response (combination of latency period and escape velocity). When tested 12 d posthatch, fluoxetine and venlafaxine adversely affected C-start performance of larvae exposed as embryos. Conversely, larvae exposed for 12 d posthatch did not exhibit altered escape responses when exposed to fluoxetine but were affected by venlafaxine and bupropion exposure. Mixtures of these four antidepressant pharmaceuticals slowed predator avoidance behaviors in larval fathead minnows regardless of the exposure window. The direct impact of reduced C-start performance on survival and, ultimately, reproductive fitness provides an avenue to assess the ecological relevance of exposure in an assay of relatively short duration.

Demasculinization of male fish by wastewater treatment plant effluent

Adult male fathead minnows (Pimephales promelas) were exposed to effluent from the City of Boulder, Colorado wastewater treatment plant (WWTP) under controlled conditions in the field to determine if the effluent induced reproductive disruption in fish. Gonadal intersex and other evidence of reproductive disruption were previously identified in white suckers (Catostomus commersoni) in Boulder Creek downstream from this WWTP effluent outfall. Fish were exposed within a mobile flow-through exposure laboratory in July 2005 and August 2006 to WWTP effluent (EFF), Boulder Creek water (REF), or mixtures of EFF and REF for up to 28 days. Primary (sperm abundance) and secondary (nuptial tubercles and dorsal fat pads) sex characteristics were demasculinized within 14 days of exposure to 50% and 100% EFF. Vitellogenin was maximally elevated in both 50% and 100% EFF treatments within 7 days and significantly elevated by 25% EFF within 14 days. The steroidal estrogens 17β-estradiol, estrone, estriol, and 17α-ethynylestradiol, as well as estrogenic alkylphenols and bisphenol A were identified within the EFF treatments and not in the REF treatment. These results support the hypothesis that the reproductive disruption observed in this watershed is due to endocrine-active chemicals in the WWTP effluent.

Genome-wide meta-analysis in alopecia areata resolves HLA associations and reveals two new susceptibility loci

Alopecia areata (AA) is a prevalent autoimmune disease with ten known susceptibility loci. Here we perform the first meta-analysis in AA by combining data from two genome-wide association studies (GWAS), and replication with supplemented ImmunoChip data for a total of 3,253 cases and 7,543 controls. The strongest region of association is the MHC, where we fine-map 4 independent effects, all implicating HLA-DR as a key etiologic driver. Outside the MHC, we identify two novel loci that exceed statistical significance, containing ACOXL/BCL2L11(BIM) (2q13); GARP (LRRC32) (11q13.5), as well as a third nominally significant region SH2B3(LNK)/ATXN2 (12q24.12). Candidate susceptibility gene expression analysis in these regions demonstrates expression in relevant immune cells and the hair follicle. We integrate our results with data from seven other autoimmune diseases and provide insight into the alignment of AA within these disorders. Our findings uncover new molecular pathways disrupted in AA, including autophagy/apoptosis, TGFß/Tregs and JAK kinase signaling, and support the causal role of aberrant immune processes in AA.

Predator avoidance performance of larval fathead minnows (Pimephales promelas) following short-term exposure to estrogen mixtures

Aquatic organisms exposed to endocrine disrupting compounds (EDCs) at early life-stages may have reduced reproductive fitness via disruption of reproductive and non-reproductive behavioral and physiological pathways. Survival to reproductive age relies upon optimal non-reproductive trait expression, such as adequate predator avoidance responses, which may be impacted through EDC exposure. During a predator-prey confrontation, larval fish use an innate C-start escape behavior to rapidly move away from an approaching threat. We tested the hypotheses that (1) larval fathead minnows exposed to estrogens, a primary class of EDCs, singularly or in mixture, suffer a reduced ability to perform an innate C-start behavior when faced with a threat stimulus; (2) additive effects will cause greater reductions in C-start behavior; and (3) effects will differ among developmental stages. In this study, embryos (post-fertilization until hatching) were exposed for 5 days to environmentally relevant concentrations of estrone (E1), 17beta-estradiol (E2), and 17alpha-ethinylestradiol (EE2) singularly and in mixture. Exposed embryos were allowed to hatch and grow in control well water until 12 days old. Similarly, post-hatch fathead minnows were exposed for 12 days to these compounds. High-speed (1000frames/s) video recordings of escape behavior were collected and transferred to National Institutes of Health Image for frame-by-frame analysis of latency period, escape velocity, and total escape response (combination of latency period and escape velocity). When tested 12 days post-hatch, only E1 adversely affected C-start performance of larvae exposed as embryos. Conversely, larvae exposed for 12 days post-hatch did not exhibit altered escape responses when exposed to E1, while adverse responses were seen in E2 and the estrogen mixture. Ethinylestradiol exposure did not elicit changes in escape behaviors at either developmental stage. The direct impact of reduced C-start performance on survival, and ultimately, reproductive fitness provides an avenue to assess the ecological relevance of exposure in an assay of relatively short duration.

Cortisol and Pacific Salmon: A New Look at the Role of Stress Hormones in Olfaction and Home-stream Migration

Abstract Pacific salmon (genus Oncorhynchus) exhibit an interesting and uncommon life-history pattern that combines semelparity, anadromy, and navigation (homing). During smoltification, young salmon imprint on the chemical composition of their natal stream water (the home-stream olfactory bouquet or “HSOB”); they then migrate to the ocean where they spend a few years feeding prior to migrating back to their natal freshwater stream to spawn. Upstream migration is guided by the amazing ability to discriminate between the chemical compositions of different stream waters and thus identify and travel to their home-stream. Pacific salmon demonstrate marked somatic and neural degeneration changes during home-stream migration and at the spawning grounds. The appearance of these pathologies is correlated with a marked elevation in plasma cortisol levels. While the mechanisms of salmonid homing are not completely understood, it is known that adult salmon continuously utilize two of their primary sensory systems, olfaction and vision, during homing. Olfaction is the primary sensory system involved in freshwater homing and “HSOB” recognition, and will be emphasized here. Previously, we proposed that the increase in plasma cortisol during Pacific salmon home-stream migration is adaptive because it enhances the salmons ability to recall the imprinted memory of the “HSOB” (Carruth, 1998; Carruth et al., 2000b). Elevated plasma concentrations of cortisol could prime the hippocampus or other olfactory regions of the brain to recall this memory and, therefore, aid in directing the fish to their natal stream. Thus, specific responses of salmon to stressors could enhance reproductive success.

Fish Endocrine Disruption Responses to a Major Wastewater Treatment Facility Upgrade

The urban-water cycle modifies natural stream hydrology, and domestic and commercial activities increase the burden of endocrine-disrupting chemicals, such as steroidal hormones and 4-nonylphenol, that can disrupt endocrine system function in aquatic organisms. This paper presents a series of integrated chemical and biological investigations into the occurrence, fate, and effects of endocrine-disrupting chemicals in the City of Boulder Colorados WWTF and Boulder Creek, the receiving stream. Results are presented showing the effects of a full-scale upgrade of the WWTF (that treats 0.6 m(3) s(-1) of sewage) from a trickling filter/solids contact process to an activated sludge process on the removal of endocrine-disrupting compounds and other contaminants (including nutrients, boron, bismuth, gadolinium, and ethylenediaminetetraacetic acid) through each major treatment unit. Corresponding impacts of pre- and postupgrade effluent chemistry on fish reproductive end points were evaluated using on-site, continuous-flow experiments, in which male fathead minnows (Pimephales promelas) were exposed for 28 days to upstream Boulder Creek water and WWTF effluent under controlled conditions. The upgrade of the WWTF resulted in improved removal efficiency for many endocrine-disrupting chemicals, particularly 17β-estradiol and estrone, and fish exposed to the postupgrade effluent indicated reduction in endocrine disruption relative to preupgrade conditions.

Immunohistochemical localization of corticotropin-releasing factor- and arginine vasotocin-like immunoreactivities in the brain and pituitary of the American bullfrog (Rana catesbeiana) during development and metamorphosis

Immunoperoxidase staining for corticotropin-releasing factor (CRF) in the median eminence was sparce or absent from premetamorphic tadpoles, but increased dramatically by late prometamorphosis. Quantitative photometry revealed that CRF-like immunostaining material in the median eminence was most dense in metamorphic climax tadpoles. Arginine vasotocin (AVT)-like immunostaining material was visualized in perikarya of the magnocellular nucleus, with extensive fiber staining seen in the medial basal and infundibular hypothalamus as well as in the median eminence and pars nervosa of the pituitary. AVT-like immunoreactive perikarya were virtually absent in premetamorphic tadpoles, but their number increased greatly by Taylor-Kollros stage XII and continued to increase after this stage. Quantitative photometry revealed that AVT-like immunoreactivity in the pars nervosa increased greatly at Taylor-Kollros stage XII and remained intense after this stage. AVT-like immunoreactivity did not appear in the median eminence until Taylor-Kollros stage XVI. Localization of AVT-like immunoperoxidase staining around portal vessels in the median eminence suggests an anatomical mechanism for delivery of AVT to anterior pituitary corticotropes. These results indicate that both CRF and vasotocinergic neuronal systems develop just before the activation of interrenal steroidogenesis which occurs during the later stages of metamorphosis in this species.

Monoamines stimulate sex reversal in the saddleback wrasse

Monoamine neurotransmitters (norepinephrine, dopamine, and serotonin) play an important role in reproduction and sexual behavior throughout the vertebrates. They are the first endogenous chemical signals in the regulation of the hypothalamo-pituitary-gonadal (HPG) axis. In teleosts with behavioral sex determination, much is known about behavioral cues that induce sex reversal. The cues are social, processed via the visual system and depend on the ratio of females to males in the population. The mechanisms by which these external behavioral cues are converted to an internal chemical regulatory process are largely unknown. The protogynous Hawaiian saddleback wrasse, Thalassoma duperrey, was used to investigate the biological pathway mediating the conversion of a social cue into neuroendocrine events regulating sex reversal. Because monoamines play an important role in the regulation of the HPG axis, they were selected as likely candidates for such a conversion. To determine if monoamines could affect sex reversal, drugs affecting monoamines were used in an attempt to either induce sex reversal under non-permissive conditions, or prevent sex reversal under permissive conditions. Increasing norepinephrine or blocking dopamine or serotonin lead to sex reversal in experimental animals under non-permissive conditions. Increasing serotonin blocked sex reversal under permissive conditions, while blocking dopamine or norepinephrine retarded the process. The results presented here demonstrate that monoamines contribute significantly to the control sex reversal. Norepinephrine stimulates initiation and completion of gonadal sex of reversal as well as color change perhaps directly via its effects on the HPG axis. Dopamine exercises inhibitory action on the initiation of sex reversal while 5-HT inhibits both initiation and completion of sex reversal. The serotonergic system appears to be an integral part of the pathway mediating the conversion of a social cue into a neuroendocrine event. The complex organization of neurochemical events controlling the psychosocial, physiological, and anatomical events that constitute reversal of sexual identity includes monoamine neurotransmitters.

Monoaminergic changes associated with socially induced sex reversal in the saddleback wrasse

The process of sex reversal in fishes is socially mediated and requires a total reorganization of the hypothalamo-pituitary-gonadal axis. When the ratio of males to females in a population of saddleback wrasse (Thalassoma dupperrey) is too low, the largest female becomes male over the course of 6 to 8 weeks. This event requires the conversion of external social cues into internal chemical cues. In an attempt to investigate the role monoamines might play in this process, two females were housed together in floating enclosures in order to induce sex reversal in the larger. Brains were sampled at various time points throughout the process of sex reversal. Monoamines were measured in the amygdala, preoptic area, ventral hypothalamus, locus coeruleus and raphe nucleus. Changes were demonstrated in monoamine metabolism for all brain regions examined. The most important changes in monoamine-system activation were seen during the first week of sex reversal. It is during this time that transitional animals undergo behavioral sex reversal. There is an increase in serotonergic activity in the amygdala which is likely related to territorial acquisition. The absence of male aggression results in a less stressful environment for the female and a reduction in serotonergic activity in the preoptic area allowing for an increase in noradrenergic activity potentially triggering the reorganization of the reproductive axis. In the ventral hypothalamus, there is a decrease in noradrenergic and increase in dopaminergic activity associated with this change from female to male. The locus coeruleus shows an increase in noradrenergic activity later in the process of sex reversal which is probably a response to more circulating androgens. In the raphe nucleus, there is a decrease in serotonergic activity at the time of behavioral sex reversal. This decrease in serotonergic activity is linked to the behavioral component of sex reversal. This study suggests that monoamines play a very important role in both behavioral and gonadal sex reversal in the saddleback wrasse, the former under the control of serotonin in the raphe and the latter mediated via serotonergic effects on norepinephrine in the preoptic area.