Biotransformation of carbamazepine and nicotine in juvenile American alligator (Alligator mississippiensis) in vitro hepatic S9 vs. in situ perfused liver.

American alligators (Alligator mississippiensis) are apex predators and sentinel species in the coastal wetland ecosystem along the Gulf of Mexico. There is concern for alligator exposure and susceptibility to chemical contaminants due to their high trophic level and lower metabolic capability. At present, their hepatic biotransformation capacity to metabolize or detoxify contaminants has not been comprehensively determined. In this study, the hepatic biotransformation capability of juvenile American alligators to metabolize two commonly found environmental pharmaceuticals: carbamazepine (CBZ) or nicotine (NCT) was evaluated. The formation of their respective primary metabolites, i.e., carbamazepine-10,11-epoxide (CBZ-E) and cotinine (CTN), was evaluated at 10 µM (within the human therapeutic range). The in vitro S9 and a novel in situ liver perfusion assays were used to characterize and compare metabolic ability in isolated hepatic enzymes vs. whole organ (liver). For CBZ, the perfused livers exhibited only 30% of intrinsic formation clearance (CLf,int) relative to the S9 assay. The metabolism of NCT was not detectable in the S9 assay and was only observed in the perfused liver assay. Compared to the corresponding rat models (S9 or perfused livers),alligators' CLf,int was 2060% for CBZ and 50% for NCT of rats. Additionally, NCT exposure increased lactate levels in perfused livers indicating metabolic stress. This study provides insight into the hepatic capability of alligators to metabolize CBZ and NCT using an established in vitro (S9) system and a newly developed in situ liver perfusion system.

Optimization of an in situ liver perfusion method to evaluate hepatic function of juvenile American alligators (Alligator mississippiensis).

American alligators (Alligator mississippiensis) are a sentinel species whose health is representative of environmental quality. However, their susceptibility to various natural or anthropogenic stressors is yet to be comprehensively studied. Understanding hepatic function in such assessments is essential as the liver is the central organ in the metabolic physiology of an organism, and therefore influences its adaptive capability. In this study, a novel liver perfusion system was developed to study the hepatic physiology of juvenile alligators. First, a cannulation procedure was developed for an in situ liver perfusion preparation. Second, an optimal flow rate of 0.5 ml/min/g liver was determined based on the oxygen content in the effluent perfusate. Third, the efficacy of the liver preparation was tested by perfusing the liver with normoxic or hypoxic Tyrode's buffer while various biomarkers of hepatic function were monitored in the effluent perfusate. Our results showed that in the normoxic perfusion, the aspartate transferase (AST) and lactate/pyruvate ratio in the perfusate remained stable and within an acceptable physiological range for 6 h. In contrast, hypoxia exposure significantly increased the lactate/pyruvate ratio in the perfusate after 2 h, indicating an induction of anaerobic metabolism. These results suggest that the perfused liver remained viable during the perfusion period and exhibited the expected physiological response under hypoxia exposure. The liver perfusion system developed in this study provides an experimental framework with which to study the basic hepatic physiology of alligators and elucidate the effects of environmental or anthropogenic stressors on the metabolic physiology of this sentinel species.

Evaluation of fatty acids and carnitine as biomarkers of PFOS exposure in biota (fish and dolphin) from Galveston Bay and the northwestern Gulf of Mexico.

Perfluorooctane sulfonate (PFOS) is a ubiquitous pollutant that elicits a wide range of toxic effects in exposed biota. Coastal zones in highly urbanized or industrial areas are particularly vulnerable to PFOS pollution. At present, information is lacking on biomarkers to assess PFOS effects on aquatic wildlife. This study investigated the efficacy of l-carnitine (or carnitine) and fatty acids as biomarkers of PFOS exposure in aquatic biota. The levels of PFOS, total and free carnitine, and 24 fatty acids (measured as fatty acid methyl esters or FAMEs) were measured in the liver, and muscle or blubber, of fish and dolphins sampled from Galveston Bay and the northern Gulf of Mexico (nGoM). Overall, bottlenose dolphins (Tursiops truncatus) had the highest hepatic PFOS levels. Galveston Bay fish, gafftopsail catfish (Bagre marinus), red drum (Sciaenops ocellatus), and spotted seatrout (Cynoscion nebulosus), had hepatic PFOS levels ∼8-13× higher than nGoM pelagic fish species, red snapper (Lutjanus campechanus) and yellowfin tuna (Thunnus albacares). The multivariate analysis of PFOS liver body-burdens and biomarkers found carnitine to be a more modal biomarker of PFOS exposure than FAMEs. Significant positive correlation of hepatic PFOS levels with total carnitine was evident for biota from Galveston Bay (fish only), and a significant correlation between PFOS and total and free carnitine was evident for biota from the nGoM (fish and dolphins). Given the essential role of carnitine in mediating fatty acid ß-oxidation, our results suggest carnitine to be a likely candidate biomarker of environmental PFOS exposure and indicative of potential dyslipidemia effects.

The use of in silico extreme pathway (ExPa) analysis to identify conserved reproductive transcriptional-regulatory networks in humans, mice, and zebrafish.

Vertebrate sex determination and differentiation are coordinated by the activations and maintenance of reproductive transcriptional-regulatory networks (TRNs). There is considerable interest in studying the conserved design principles and functions of reproductive TRNs given that their intricate regulation is susceptible to disruption by gene mutations or exposures to exogenous endocrine disrupting chemicals (or EDCs). In this manuscript, the Boolean rules describing reproductive TRNs in humans, mice, and zebrafish, were represented as a pseudo-stoichiometric matrix model. This model mathematically described the interactions of 35 transcription factors with 21 sex determination and differentiation genes across the three species. The in silico approach of Extreme Pathway (ExPa) analysis was used to predict the extent of TRN gene activations subject to the species-specific transcriptomics data, from across various developmental life-stages. A goal of this work was to identify conserved and functional reproductive TRNs across the three species. ExPa analyses predicted the sex differentiation genes, DHH, DMRT1, and AR, to be highly active in male humans, mice, and zebrafish. Whereas FOXL2 was the most active gene in female humans and mice; and CYP19A1A in female zebrafish. These results agree with the expectation that regardless of a lack of sex determination genes in zebrafish, the TRNs responsible for canalizing male vs. female sexual differentiation are conserved with mammalian taxa. ExPa analysis therefore provides a framework with which to study the TRNs that influence the development of sexual phenotypes. And the in silico predicted conservation of sex differentiation TRNs between mammals and zebrafish identifies the piscine species as an effective in vivo model to study mammalian reproductive systems under normal or perturbed pathologies.

Core metabolism plasticity in phytoplankton: Response of Dunaliella tertiolecta to oil exposure.

Human alterations to the marine environment such as an oil spill can induce oxidative stress in phytoplankton. Exposure to oil has been shown to be lethal to most phytoplankton species, but some are able to survive and grow at unaffected or reduced growth rates, which appears to be independent of the class and phylum of the phytoplankton and their ability to consume components of oil heterotrophically. The goal of this article is to test the role of core metabolism plasticity in the oil-resisting ability of phytoplankton. Experiments were performed on the oil- resistant chlorophyte, Dunaliella tertiolecta, in control and water accommodated fractions of oil, with and without metabolic inhibitors targeting the core metabolic pathways. We observed that inhibiting pathways such as photosynthetic electron transport (PET) and pentose-phosphate pathway were lethal; however, inhibition of pathways such as mitochondrial electron transport and cyclic electron transport caused growth to be arrested. Pathways such as photorespiration and Kreb's cycle appear to play a critical role in the oil-tolerating ability of D. tertiolecta. Analysis of photo-physiology revealed reduced PET under inhibition of photorespiration but not Kreb's cycle. Further studies showed enhanced flux through Kreb's cycle suggesting increased energy production and photorespiration counteract oxidative stress. Lastly, reduced extracellular carbohydrate secretion under oil exposure indicated carbon and energy conservation, which together with enhanced flux through Kreb's cycle played a major role in the survival of D. tertiolecta under oil exposure by meeting the additional energy demands. Overall, we present data that suggest the role of phenotypic plasticity of multiple core metabolic pathways in accounting for the oxidative stress tolerating ability of certain phytoplankton species.

PFASs pollution in Galveston Bay surface waters and biota (shellfish and fish) following AFFFs use during the ITC fire at Deer Park (March 17th-20th 2019), Houston, TX.

The use of aqueous film forming foams (AFFFs) as fire retardants is an critical point-source for per- and polyfluoroalkyl substances (PFASs) pollution into the aquatic environment. This study investigated PFASs pollution in the surface waters and biota (shellfish and fish) of Galveston Bay, following AFFFs use to extinguish a petrochemical fire (March 17th to 20th, 2019) of oil storage tanks at the International Terminals Company (ITC) in Deer Park (Houston, TX). The levels of up to twelve EPA priority PFASs were measured in surface waters and biota from March-November 2019. PFASs levels in surface waters showed mean total levels in March and April 2019 to be from 4× to ~300× higher than those measured in the following months. PFOS (perfluorooctanesulfonic acid) was the most abundant homolog measured at ≥66% of total PFASs. Maximal PFOS levels exceeded the State of Texas' water regulatory limit of 0.6 µg L-1 in 3% of the samples analyzed in March and April 2019. PFOS was also the most prominent homolog (≥66% of total PFASs) measured in eastern oysters (Crassostrea virginica), red drum (Sciaenops ocellatus), gafftopsail catfish (Bagre marinus), and spotted seatrout (Cynoscion nebulosus). A statistically significant elevation of PFOS body-burdens was measured in oysters and spotted seatrout in April and May 2019, respectively. A Hazard Ratio calculation for seafood safety suggests an advisory of 1-2 meals per week for gafftopsail catfish and red drum, and 2 meals per week for spotted seatrout to be protective for human exposure to PFOS. The levels in oysters indicated no immediate concerns for the dietary exposure of humans. Our results highlight a need for continual monitoring to assess the long-term fate and seafood advisories for PFASs.

Overcompensation of CoA Trapping by Di(2-ethylhexyl) Phthalate (DEHP) Metabolites in Livers of Wistar Rats.

Di(2-ethylhexyl) phthalate (DEHP) is commonly used as a plasticizer in various industrial and household plastic products, ensuring widespread human exposures. Its routine detection in human bio-fluids and the propensity of its monoester metabolite to activate peroxisome proliferator activated receptor-α (PPARα) and perturb lipid metabolism implicate it as a metabolic disrupter. In this study we evaluated the effects of DEHP exposure on hepatic levels of free CoA and various CoA esters, while also confirming the metabolic activation to CoA esters and partial ß-oxidation of a DEHP metabolite (2-ethyhexanol). Male Wistar rats were exposed via diet to 2% (w/w) DEHP for fourteen-days, following which hepatic levels of free CoA and various CoA esters were identified using liquid chromatography-mass spectrometry. DEHP exposed rats showed significantly elevated free CoA and increased levels of physiological, DEHP-derived and unidentified CoA esters. The physiological CoA ester of malonyl-CoA and DEHP-derived CoA ester of 3-keto-2-ethylhexanoyl-CoA were the most highly elevated, at eighteen- and ninety eight-times respectively. We also detected sixteen unidentified CoA esters which may be derivative of DEHP metabolism or induction of other intermediary metabolism metabolites. Our results demonstrate that DEHP is a metabolic disrupter which affects production and sequestration of CoA, an essential cofactor of oxidative and biosynthetic reactions.

Molecular mechanism of oil induced growth inhibition in diatoms using Thalassiosira pseudonana as the model species.

The 2010 Deepwater Horizon oil-spill exposed the microbes of Gulf of Mexico to unprecedented amount of oil. Conclusive evidence of the underlying molecular mechanism(s) on the negative effects of oil exposure on certain phytoplankton species such as Thalassiosira pseudonana is still lacking, curtailing our understanding of how oil spills alter community composition. We performed experiments on model diatom T. pseudonana to understand the mechanisms underpinning observed reduced growth and photosynthesis rates during oil exposure. Results show severe impairment to processes upstream of photosynthesis, such as light absorption, with proteins associated with the light harvesting complex damaged while the pigments were unaffected. Proteins associated with photosynthetic electron transport were also damaged, severely affecting photosynthetic apparatus and depriving cells of energy and carbon for growth. Negative growth effects were alleviated when an organic carbon source was provided. Further investigation through proteomics combined with pathway enrichment analysis confirmed the above findings, while highlighting other negatively affected processes such as those associated with ferroxidase complex, high-affinity iron-permease complex, and multiple transmembrane transport. We also show that oxidative stress is not the primary route of negative effects, rather secondary. Overall, this study provides a mechanistic understanding of the cellular damage that occurs during oil exposure to T. pseudonana.

Correlations between environmental salinity levels, blood biochemistry parameters, and steroid hormones in wild juvenile American alligators (Alligator mississippiensis).

American alligators (Alligator mississippiensis) inhabit freshwater wetlands that are vulnerable to salinization caused by anthropogenic alterations to freshwater flow, in addition to storm surges, sea level rise, and droughts. Salinization of coastal freshwater habitats is a growing concern in a changing climate due to increased frequency and intensity of storm surges and drought conditions. This study opportunistically sampled juvenile male and female wild alligators in various salinities each month excluding November, December, and January for one year at Rockefeller Wildlife Refuge in coastal Louisiana. Blood plasma biochemistry parameters including electrolyte levels were subsequently measured. In addition, levels of various renin-angiotensin-aldosterone system hormones, glucocorticoids, androgens, estrogens, and progestogens were analyzed using liquid chromatography and tandem mass spectrometry. Only males were sampled in hyperosmotic environments (> 10‰) during dry conditions in late summer 2018. In juvenile males, plasma Na+, Cl-, and the progestogen 17α,20ß-dihydroxypregnenone were significantly and positively correlated with environmental salinity. However, variation in glucocorticoids, androgens, and estrogens were not associated with hypersaline water while sex steroids showed significant seasonal variation. This study demonstrated significant correlation of environmental salinity with electrolyte levels and a sex steroid in wild juvenile alligators, and to our knowledge represents the first measurement of 17α,20ß-dihydroxypregnenone in alligators.

An integrated in vivo and in silico analysis of the metabolism disrupting effects of CPI-613 on embryo-larval zebrafish (Danio rerio).

CPI-613 is a mitochondrial metabolism disrupter that inhibits tricarboxylic acid (TCA) cycle activity. The consequences of TCA cycle disruption on various metabolic pathways and overall organismal physiology are not fully known. The present study integrates in vivo experimental data with an in silico stoichiometric metabolism model of zebrafish to study the metabolic pathways perturbed under CPI-613 exposure. Embryo-larval life stages of zebrafish (Danio rerio) were exposed to 1 µM CPI-613 for 20 days. Whole-organism respirometry measurements showed an initial suppression of O2 consumption at Day 5 of exposure, followed by recovery comparable to the solvent control (0.01% DMSO) by Day 20. Comparison of whole-transcriptome RNA-sequencing at Day 5 vs. 20 of exposure showed functional categories related to O2 binding and transport, antioxidant activity, FAD binding, and hemoglobin complexes, to be commonly represented. Metabolic enzyme gene expression changes and O2 consumption rate was used to parametrize two in silico stoichiometric metabolic models representative of Day 5 or 20 of exposure. Computational simulations predicted impaired ATP synthesis, α-ketoglutarate dehydrogenase (KGDH) activity, and fatty acid ß-oxidation at Day 5 vs. 20 of exposure. These results show that the targeted disruption of KGDH may also impact oxidative phosphorylation (ATP synthesis) and fatty acid metabolism (ß-oxidation), in turn influencing cellular bioenergetics and the observed reduction in whole-organism O2 consumption rate. The results of this study provide an integrated in vivo and in silico framework to study the impacts of metabolic disruption on organismal physiology.

PAH and PCB body-burdens in epibenthic deep-sea invertebrates from the northern Gulf of Mexico.

There is a paucity of information on the levels of PAHs and PCBs in the deep-sea (≥200 m). In this study, the body-burdens of 16 PAHs and 29 PCBs were measured in: Actinaria (sea anemones), Holothuroidea (sea cucumber), Pennatulacea (sea pens), and Crinoidea (sea lilies) in the deep Gulf of Mexico. All epibenthic species were collected at depths of approximately 2000 m. The PAH and PCB congener profile displayed a similar pattern of bioaccumulation across all four taxa. The high molecular weight PAH, dibenz[a,h]anthracene, was the most abundant PAH in all organisms, ranging from 36 to 53% of sum total PAHs. PCBs 101 and 138 exhibited the highest levels at 20-25% of total congener concentrations in all taxa. The exposure to PAHs and PCBs is likely attributed to contaminated particulate organic matter that is consumed by the deposit and filter feeding epibenthic megafauna sampled in this study.

The pharmaceutical prednisone affects sheepshead minnow (Cyprinodon variegatus) metabolism and swimming performance.

High usage of the synthetic glucocorticoids (GCs) has led to significant presence of this pharmaceutical group in surface waters where it can affect non-target organisms such as fish. Assessment of a fish's metabolism and swimming performance provide reliable sub-lethal measures of effects of GCs on oxygen-requiring processes and ability to swim. In this study, we determined time-dependent (7, 14 and 21 days) effects of the synthetic GC prednisone (1 µg L-1) on sheepshead minnow (SHM) (Cyprinodon variegatus). Standard (SMR), routine (RMR) and maximum (MMR) metabolic rate, metabolic scope (MS), excess post-exercise oxygen consumption (EPOC), cost of transport (COT) and critical swimming speed (Ucrit) were determined. Twenty-one days exposure to prednisone resulted in significantly higher SMR, RMR, MMR, MS, EPOC and COT compared with 7d and 14d prednisone fish. However, Ucrit was not significantly different between prednisone and solvent control exposed fish (within 7d, 14d, 21d groups). SMR, RMR and MMR were lower in the 7d and 14d prednisone exposed fish compared with their solvent control groups. In contrast, SMR, RMR and MMR were all significantly higher in the 21d prednisone exposed fish compared with solvent control. EPOC was significantly higher in 14d prednisone exposed fish and trending higher in 21d and 7d prednisone exposed fish compared with their solvent controls. EPOC was significantly higher in 21d compared with 7d prednisone exposed fish. A significantly higher COT was seen in the 21d compared with 7d and 14d prednisone fish. Collectively, this study showed time-dependent effects of prednisone on SHM metabolism and swimming performance.

Polycyclic aromatic hydrocarbons (PAHs) and putative PAH-degrading bacteria in Galveston Bay, TX (USA), following Hurricane Harvey (2017).

Hurricane Harvey was the wettest hurricane in US history bringing record rainfall and widespread flooding in Houston, TX. The resulting storm- and floodwaters largely emptied into the Galveston Bay. Surface water was collected from 10 stations during five cruises to investigate the concentrations and sources of 16 priority polycyclic aromatic hydrocarbons (PAHs), and relative abundances of PAH-degrading bacteria. Highest PAH levels (102-167 ng/L) were detected during the first sampling event, decreasing to 36-69 ng/L within a week. Four sites had elevated concentrations of carcinogenic benzo[a]pyrene that exceeded the Texas Standard for Surface Water threshold. The highest relative abundances of known PAH-degrading bacteria Burkholderiaceae, Comamonadaceae, and Sphingomonadales were detected during the first and second sampling events. PAH origins were about 60% pyrogenic, 2% petrogenic, and the remainder of mixed sources. This study improves our understanding on the fate, source, and distributions of PAHs in Galveston Bay after an extreme flooding event.

Short-term exposure to 12‰ brackish water has significant effects on the endocrine physiology of juvenile American alligator (Alligator mississippiensis).

American alligators (Alligator mississippiensis) mainly inhabit freshwater habitats but can be exposed to a wide range of salinities during storm surges, droughts or from alterations in freshwater flows. Although some salinization events last weeks, others only last a few days. This study assessed changes in the endocrine function of the renin-angiotensin-aldosterone system (RAAS) and steroid hormone production (steroidogenesis) in juvenile alligators exposed to brackish water (12‰) for 7 days. We quantified plasma levels of angiotensin II and the corticosteroids (aldosterone, corticosterone and 11-deoxycortisol). Various progestogens, androgens, and estrogens were further assessed. The protein expression for the RAAS enzymes, renin and angiotensin converting enzyme (ACE), was quantified immunohistochemically in kidney and lung tissue, respectively, and histology was performed on kidney, lung and gonad tissues. Finally, blood biochemistry parameters such as electrolyte levels and diagnostic indicators for dehydration, renal, and hepatic function were measured. Corticosterone, 11-deoxycortisol, Na+, Cl-, total protein, albumin, uric acid, and cholesterol levels were all significantly elevated in alligators exposed to brackish water compared with alligators in freshwater. The levels of 17ß-estradiol and estrone were significantly lowered while histology showed alterations in gonad tissue in the brackish water exposed group. In contrast, while there were no effects of exposure on aldosterone levels, angiotensin II was significantly reduced in brackish water exposed alligators. These results correlated with significantly decreased expressions for both renin and ACE in kidney and lung tissue. Overall, this study showed that short-term exposure of alligators to 12‰ brackish water has significant endocrine effects on juvenile alligators.

Integration of multi-tissue PAH and PCB burdens with biomarker activity in three coastal shark species from the northwestern Gulf of Mexico.

Tissue-based burdens of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were integrated with ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST) enzyme activity in bull (Carcharhinus leucas), blacktip (Carcharhinus limbatus), and bonnethead (Sphyrna tiburo) sharks from Galveston Bay, TX. The potential toxicity of these burdens was evaluated by calculation of toxic equivalents (TEQs). Concentrations of total PAHs (∑PAHs) were significantly greater in blacktip and bonnethead sharks than bull sharks in liver, but did not exhibit differences in muscle among species. Hepatic concentrations of ∑PAHs in these sharks (range of means: 1560-2200 ng/g wet wt.) were greater than concentrations previously reported in oysters from Galveston Bay (range of means: 134-333 ng/g dry wt.), which suggests that trophic dilution of PAHs may not be reflected in sharks. Total PCBs (∑PCBs) were significantly greatest in bull sharks and lowest in bonnetheads, while blacktips were intermediate to these species. EROD activity was greater in bonnetheads than the other species, whereas GST activity was significantly higher in blacktips and bonnetheads than in bull sharks. Integration of hepatic burdens with biomarker activity via constrained multivariate analysis found correlations for only a small number of individual PAH/PCB congeners. Hepatic TEQ measurements suggest potential physiological effects of these burdens compared to established TEQ thresholds for other taxa, although the likelihood of similar effects in sharks requires further study and the inclusion of toxic endpoints. Our findings indicate that sharks may be prone to the accumulation of PAHs and PCBs, which may result in negative health outcomes for these cartilaginous fishes.

Extracellular polymeric substances (EPS) producing and oil degrading bacteria isolated from the northern Gulf of Mexico.

Sinking marine oil snow was found to be a major mechanism in the transport of spilled oil from the surface to the deep sea following the Deepwater Horizon (DwH) oil spill. Marine snow formation is primarily facilitated by extracellular polymeric substances (EPS), which are mainly composed of proteins and carbohydrates secreted by microorganisms. While numerous bacteria have been identified to degrade oil, there is a paucity of knowledge on bacteria that produce EPS in response to oil and Corexit exposure in the northern Gulf of Mexico (nGoM). In this study, we isolated bacteria from surface water of the nGoM that grow on oil or Corexit dispersant. Among the 100 strains isolated, nine were identified to produce remarkable amounts of EPS. 16S rRNA gene analysis revealed that six isolates (strains C1, C5, W10, W11, W14, W20) belong to the genus Alteromonas; the others were related to Thalassospira (C8), Aestuariibacter (C12), and Escherichia (W13a). The isolates preferably degraded alkanes (17-77%), over polycyclic aromatic hydrocarbons (0.90-23%). The EPS production was determined in the presence of a water accommodated fraction (WAF) of oil, a chemical enhanced WAF (CEWAF), Corexit, and control. The highest production of visible aggregates was found in Corexit followed by CEWAF, WAF, and control; indicating that Corexit generally enhanced EPS production. The addition of WAF and Corexit did not affect the carbohydrate content, but significantly increased the protein content of the EPS. On the average, WAF and CEWAF treatments had nine to ten times more proteins, and Corexit had five times higher than the control. Our results reveal that Alteromonas and Thalassospira, among the commonly reported bacteria following the DwH spill, produce protein rich EPS that could have crucial roles in oil degradation and marine snow formation. This study highlights the link between EPS production and bacterial oil-degrading capacity that should not be overlooked during spilled oil clearance.

In Silico analysis of perturbed steroidogenesis and gonad growth in fathead minnows (P. promelas) exposed to 17α-ethynylestradiol.

The multi-factorial nature of adverse reproductive effects mediated by endocrine disrupting compounds (or EDCs) makes understanding the mechanistic basis of reproductive dysfunction a highly pertinent area of research. As a consequence, a main motivator for continued research is to integrate 'multi-leveled' complexity (i.e., from genes to phenotype) using mathematical methods capable of encapsulating properties of physiological relevance. In this study, an in silico stoichiometric model of piscine steroidogenesis was augmented with a 'biomass' reaction associating the underlying stoichiometry of steroidogenesis with a reaction representative of gonad growth. The ability of the in silico model to predict perturbed steroidogenesis and subsequent effects on gonad growth was tested by exposing reproductively active male and female fathead minnows (Pimephales promelas) to 88 ng/L of the synthetic estrogen, 17α-ethynylestradiol (EE2). The in silico model was parameterized (or constrained) with experimentally quantified concentrations of selected steroid hormones (using mass spectrometry) and fold changes in gene expression (using RT-qPCR) for selected steroidogenic enzyme genes, in gonads of male and female fish. Once constrained, the optimization framework of flux balance analysis (FBA) was used to calculate an optimal flux through the biomass reaction (analogous to gonad growth) and associated steroidogenic flux distributions required to generate biomass. FBA successfully predicted effects of EE2 exposure on fathead minnow gonad growth (%gonadosomatic index or %GSI) and perturbed production of steroid hormones. Specifically, FBA accurately predicted no effects of exposure on male %GSI and a significant reduction for female %GSI. Furthermore, in silico simulations accurately identified disrupted reaction fluxes catalyzing productions of androgens (in male fish) and progestogens (in female fish), an observation which agreed with in vivo experimentation. The analyses presented is the first-ever to successfully associate underlying flux properties of the steroidogenic network with gonad growth in fish, an approach which can incorporate in silico predictions with toxicological risk assessments.

Effects of progesterone and norethindrone on female fathead minnow (Pimephales promelas) steroidogenesis.

As knowledge of contaminants capable of adversely modulating endocrine functions increases, attention is focused on the effects of synthetic progestins as environmental endocrine disrupters. In the present study, effects of exposure to a synthetic progestin (norethindrone, 168 ± 7.5 ng/L) and endogenous progestogen (progesterone, 34 ± 4.1 ng/L) on steroidogenesis in adult female fathead minnows were examined. In vivo exposure to either compound lowered expression (nonsignificant) of luteinizing hormone (LHß) levels in the brain along with significantly down-regulating the beta isoform of membrane progesterone receptor (mPRß) in ovary tissue. The correspondence between lowered LHß levels in the brain and mPRß in the ovary is suggestive of a possible functional association as positive correlations between LHß and mPR levels have been demonstrated in other fish species. In vitro exposure of ovary tissue to progesterone resulted in significantly elevated progestogen (pregnenolone, 17α-hydroxyprogesterone, and 17α,20ß-dihydroxypregnenone) and androgen (testosterone) production. Whereas in vitro exposure to norethindrone did not significantly impact steroid hormone production but showed decreased testosterone production relative to solvent control (however this was not significant). Overall, this study showed that exposure to a natural progestogen (progesterone) and synthetic progestin (norethindrone), was capable of modulating LHß (in brain) and mPRß expression (in ovary).

Levonorgestrel exposure to fathead minnows (Pimephales promelas) alters survival, growth, steroidogenic gene expression and hormone production.

Human pharmaceuticals are commonly detected in the environment. Concern over these compounds in the environment center around the potential for pharmaceuticals to interfere with the endocrine system of aquatic organisms. The main focus of endocrine disruption research has centered on how estrogenic and androgenic compounds interact with the endocrine system to elicit reproductive effects. Other classes of compounds, such as progestins, have been overlooked. Recently, studies have investigated the potential for synthetic progestins to impair reproduction and growth in aquatic organisms. The present study utilizes the OECD 210 Early-life Stage (ELS) study to investigate the impacts levonorgestrel (LNG), a synthetic progestin, on fathead minnow (FHM) survival and growth. After 28 days post-hatch, survival of larval FHM was impacted at 462 ng/L, while growth was significantly reduced at 86.9 ng/L. Further analysis was conducted by measuring specific endocrine related mRNA transcript profiles in FHM larvae following the 28 day ELS exposure to LNG. Transcripts of 3ß-HSD, 20ß-HSD, CYP17, AR, ERα, and FSH were significantly down-regulated following 28d exposure to 16.3 ng/L LNG, while exposure to 86.9 ng/L significantly down-regulated 3ß-HSD, 20ß-HSD, CYP19A, and FSH. At 2,392 ng/L of LNG, a significant down-regulation occurred with CYP19A and ERß transcripts, while mPRα and mPRß profiles were significantly induced. No significant changes occurred in 11ß-HSD, CYP11A, StAR, LHß, and VTG mRNA expression following LNG exposure. An ex vivo steroidogenesis assay was conducted with sexually mature female FHM following a 7 day exposure 100 ng/L LNG with significant reductions observed in pregnenolone, 17α,20ß-dihydroxy-4-pregnen-3-one (17,20-DHP), testosterone, and 11-ketotestosterone. Together these data suggest LNG can negatively impact FHM larval survival and growth, with significant alterations in endocrine related responses.

A community-driven global reconstruction of human metabolism.

Multiple models of human metabolism have been reconstructed, but each represents only a subset of our knowledge. Here we describe Recon 2, a community-driven, consensus 'metabolic reconstruction', which is the most comprehensive representation of human metabolism that is applicable to computational modeling. Compared with its predecessors, the reconstruction has improved topological and functional features, including ∼2× more reactions and ∼1.7× more unique metabolites. Using Recon 2 we predicted changes in metabolite biomarkers for 49 inborn errors of metabolism with 77% accuracy when compared to experimental data. Mapping metabolomic data and drug information onto Recon 2 demonstrates its potential for integrating and analyzing diverse data types. Using protein expression data, we automatically generated a compendium of 65 cell type-specific models, providing a basis for manual curation or investigation of cell-specific metabolic properties. Recon 2 will facilitate many future biomedical studies and is freely available at http://humanmetabolism.org/.