Organ-specific biotransformation in salmonids: Insight into intrinsic enzyme activity and biotransformation of three micropollutants.

Aquatic ecosystems continue to be threatened by chemical pollution. To what extent organisms are able to cope with chemical exposure depends on their ability to display mechanisms of defense across different organs. Among these mechanisms, biotransformation processes represent key physiological responses that facilitate detoxification and reduce the bioaccumulation potential of chemicals. Biotransformation does not only depend on the ability of different organs to display biotransformation enzymes but also on the affinity of chemicals towards these enzymes. In the present study, we explored the ability of different organs and of two freshwater fish to support biotransformation processes through the determination of in vitro phase I and II biotransformation enzyme activity, and their role in supporting intrinsic clearance and the formation of biotransformation products. Three environmentally relevant pollutants were evaluated: the polycyclic aromatic hydrocarbon (PAH) pyrene (as recommended by the OECD 319b test guideline), the fungicide azoxystrobin, and the pharmaceutical propranolol. Comparative studies using S9 sub-cellular fractions derived from the liver, intestine, gills, and brain of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) revealed significant phase I and II enzyme activity in all organs. However, organ- and species-specific differences were found. In brown trout, significant extrahepatic biotransformation was observed for pyrene but not for azoxystrobin and propranolol. In rainbow trout, the brain appeared to biotransform azoxystrobin. In this same species, propranolol appeared to be biotransformed by the intestine and gills. Biotransformation products could be detected only from hepatic biotransformation, and their profiles and formation rates displayed species-specific patterns and occurred at different magnitudes. Altogether, our findings further contribute to the current understanding of organ-specific biotransformation capacity, beyond the expression and activity of enzymes, and its dependence on specific enzyme-chemical interactions to support mechanisms of defense against exposure.

Comparative cytotoxicity induced by parabens and their halogenated byproducts in human and fish cell lines.

Parabens are a group of para-hydroxybenzoic acid (p-HBA) esters widely used in pharmaceutical industries. Their safety is well documented in mammalian models, but little is known about their toxicity in non-mammal species. In addition, chlorinated and brominated parabens resulting from wastewater treatment have been identified in effluents. In the present study, we explored the cytotoxic effects (EC50) of five parabens: methylparaben (MP), ethylparaben (EP), propylparaben (PP), butylparaben (BuP), and benzylparaben (BeP); the primary metabolite, 4-hydroxybenzoic acid (4-HBA), and three of the wastewater chlorinated/brominated byproducts on fish and human cell lines. In general, higher cytotoxicity was observed with increased paraben chain length. The tested compounds induced toxicity in the order of 4-HBA < MP < EP < PP < BuP < BeP. The halogenated byproducts led to higher toxicity with the addition of second chlorine. The longer chain-parabens (BuP and BeP) caused a concentration-dependent decrease in cell viability in fish cell lines. Intriguingly, the main paraben metabolite, 4-HBA, proved to be more toxic to fish hepatocytes than human hepatocytes by 100-fold. Our study demonstrated that the cytotoxicity of some of these compounds appears to be tissue-dependent. These observations provide valuable information for early cellular responses in human and non-mammalian models upon exposure to paraben congeners.

Effects of perfluoroalkyl substances (PFASs) and benzo[a]pyrene (BaP) co-exposure on phase I biotransformation in rainbow trout (Oncorhynchus mykiss).

The presence of perfluoroalkyl substances (PFASs) in the environment, especially in aquatic ecosystems, continues to be a significant concern for human and environmental health. Previous studies have suggested that several PFASs do not undergo biotransformation due to their chemical stability, yet perfluorooctanesulfonic acid (PFOS)- and perfluorooctanoic acid (PFOA)-exposed organisms have presented altered activity of important biotransformation pathways. Given the fundamental role of biotransformation in biological organisms and the significant distribution of PFAS in aquatic environments, the present study investigated the influence of PFOA and PFOS on phase I biotransformation enzymes in vitro using the rainbow trout liver RTL-W1 cell line and in vivo using juvenile rainbow trout. Cells and fish were exposed and co-exposed to environmentally relevant concentrations of PFOA, PFOS, and benzo[a]pyrene (BaP), for 72 h and 10 days, respectively, prior to measurements of cytotoxicity and biotransformation ability through measurements of CYP1A1-, CYP1A2-, and CYP3A4-like activities. Our results indicate that exposure to PFAS-BaP binary mixtures altered CYP1A-like activity in vivo; however, those alterations were not observed in vitro. Similarly, while BaP did not significantly induce CYP3A4 in vivo, exposure to the PFAS led to significantly lower enzymatic activity relative to basal levels. These observations may have implications for organisms simultaneously exposed to PFASs and other environmental pollutants for which biotransformation is necessary, especially in detoxification mechanisms. Furthermore, the interference with biotransformation pathways could potentially predispose exposed organisms to a compromised physiology, which may increase their vulnerability to other stressors and erode their survival fitness.

In vitro-in vivo biotransformation and phase I metabolite profiling of benzo[a]pyrene in Gulf killifish (Fundulus grandis) populations with different exposure histories.

Chronic exposure to pollution may lead populations to display evolutionary adaptations associated with cellular and physiological mechanisms of defense against xenobiotics. This could result in differences in the way individuals of the same species, but inhabiting different areas, cope with chemical exposure. In the present study, we explore two Gulf killifish (Fundulus grandis) populations with different exposure histories for potential differences in the biotransformation of benzo[a]pyrene (BaP), and conduct a comparative evaluation of in vitro and in vivo approaches to describe the applicability of new approach methodologies (NAMs) for biotransformation assessments. Pollution-adapted and non-adapted F. grandis were subjected to intraperitoneal (IP) injections of BaP in time-course exposures, prior to measurements of CYP biotransformation activity, BaP liver concentrations, and the identification and quantification of phase I metabolites. Additionally, substrate depletion bioassays using liver S9 fractions were employed for measurements of intrinsic hepatic clearance and to evaluate the production of metabolites in vitro. Pollution-adapted F. grandis presented significantly lower CYP1A activity and intrinsic clearance rates that were 3 to 4 times lower than non-adapted fish. The metabolite profiling of BaP showed the presence of 1­hydroxy-benzo[a]pyrene in both the in vitro and in vivo approaches but with no significant population differences. Contrarily, 9­hydroxy-benzo[a]pyrene and benzo[a]pyrene-4,5-dihydrodiol, only identified through the in vivo approach, presented higher concentrations in the bile of pollution-adapted fish relative to non-adapted individuals. These observations further the understanding of the evolutionary adaptation of F. grandis inhabiting heavily polluted environments in the Houston Ship Channel, TX, USA, and highlight the need to consider the evolutionary history of populations of interest during the implementation of NAMs.

Reduced biotransformation of polycyclic aromatic hydrocarbons (PAHs) in pollution-adapted Gulf killifish (Fundulus grandis).

Anthropogenic pollution represents a significant source of selection, potentially leading to the emergence of evolutionary adaptations in chronically exposed organisms. A recent example of this scenario corresponds to Gulf killifish (Fundulus grandis) populations inhabiting the Houston Ship Channel (HSC), Texas, USA, which have been documented to have adapted to this heavily contaminated environment. Although not fully elucidated, one particularly important aspect of their adaptation involves the reduced inducibility of the aryl hydrocarbon receptor (AhR) and, potentially, the alteration of major biotransformation pathways. In the present study, we employed a modified Organization for Economic Cooperation and Development (OECD) 319-B test guideline to explore population and sex-related differences in the hepatic biotransformation of six polycyclic aromatic hydrocarbons (PAHs) in F. grandis populations with different exposure histories. Pollution-adapted F. grandis showed significantly lower hepatic clearance of PAHs than non-adapted fish, especially for high molecular weight PAHs (chrysene, benzo[k]fluoranthene, and benzo[a]pyrene), with pollution-adapted females presenting the lowest clearance. The characterization of different phase I biotransformation enzymes revealed that the basal activity of CYP1A, fundamental in the biotransformation of PAHs, was significantly lower in pollution-adapted fish, especially in females, which showed the lowest activity. Contrarily, basal CYP2C9-like activity was significantly higher in pollution-adapted fish. These results demonstrate the importance of exposure and evolutionary histories in shaping organisms' responses to pollution and provide significant evidence of sex-specific biotransformation differences in F. grandis populations.

Plasma Vitellogenin Reveals Potential Seasonal Estrogenicity in Fish from On-Site Wastewater Treatment Systems in Semi-Arid Streams Influenced by Snowmelt.

Effluents from on-site wastewater treatment systems can influence surface water quality, particularly when infrastructure is aging, malfunctioning, and improperly installed. Municipal wastewater often contains chemical compounds that can lead to adverse biological effects, such as reproductive impairment, in organisms that are chronically exposed. A significant number of these compounds are endocrine-disrupting chemicals. Water quality influences of on-site systems are poorly studied in semi-arid regions where instream flows are seasonally dependent on snowmelt, and when instream dilution of wastewater effluents is minimal during other times of the year. Here we examined surface water estrogenicity in low order tributaries of two unique semi-arid streams with on-site wastewater treatment systems, for which seasonal instream flow fluctuations occur in Park City, UT, USA. Water samples were collected from a total of five locations along two lotic systems downstream from active on-site treatment systems. Samples were extracted for targeted chemical analyses and to perform in vivo and in vitro bioassays with juvenile rainbow trout. Estrogenic activity was measured by quantifying the concentration and expression of vitellogenin (VTG) in plasma and liver, respectively. Plasma VTG presented elevated levels in fish exposed to water samples collected at the two sites in close proximity to on-site systems and during seasons with low stream discharge, though the levels observed did not suggest severe endocrine disruption. However, long-term exposure to these surface water could compromise the fish populations. While the sensitivity of in vitro bioassays was low and targeted chemical analyses did not identify causative compounds, the use of complementary lines of evidence (e.g., in vivo biological models) was advantageous in identifying estrogenic activity in waters influenced by effluents from on-site wastewater systems.

Multi-approach assessment for the evaluation of spatio-temporal estrogenicity in fish from effluent-dominated surface waters under low instream flow.

Current practices employed by most wastewater treatment plants (WWTP) are unable to completely remove endocrine disrupting compounds (EDCs) from reclaimed waters, and consistently discharge these substances to receiving systems. Effluent-dominated and dependent surface waters, especially during low instream flows, can increase exposure and risks to aquatic organisms due to adverse biological effects associated with EDCs. Given the ecological implications that may arise from exposure to such compounds, the present a multi-approach study examined spatio-temporal estrogenic potential of wastewater effluent to fish in East Canyon Creek (ECC), Utah, USA, a unique urban river with instream flows seasonally influenced by snowmelt. Juvenile rainbow trout (Oncorhynchus mykiss) were caged at different upstream and downstream sites from an effluent discharge during the summer and fall seasons. In the summer, where approximately 50% of the streamflow was dominated by effluent, fish from the upstream and a downstream site, located 13 miles away from the effluent discharge, presented significantly elevated concentrations of plasma vitellogenin (VTG). Similarly, significantly high 17ß-estradiol to 11-ketotestosterone ratios were measured in the summer across all sites and time points, compared to the fall. In the laboratory, juvenile fish and primary hepatocytes were exposed to concentrated effluent and surface water samples. Quantification of VTG, although in significantly lower levels, resembled response patterns observed in fish from the field study. Furthermore, analytical quantification of common EDCs in wastewater revealed the presence of estriol and estrone, though these did not appear to be related to the observed biological responses, as these were more significant in sites were no EDCs were detected. These combined observations suggest potential estrogenicity for fish in ECC under continuous exposures and highlight the advantages of following weight-of-evidence (WoE) approaches for environmental monitoring, as targeted analytically-based assessments may or may not support the identification of causative contaminants for adverse biological effects.

Pharmaceutical uptake kinetics in rainbow trout: In situ bioaccumulation in an effluent-dominated river influenced by snowmelt.

Whether seasonal instream flow dynamics influence bioaccumulation of pharmaceuticals by fish is not well understood, specifically for urban lotic systems in semi-arid regions when flows are influenced by snowmelt. We examined uptake of select pharmaceuticals in rainbow trout (Oncorhynchus mykiss) caged in situ upstream and at incremental distances downstream (0.1, 1.4, 13 miles) from a municipal effluent discharge to East Canyon Creek in Park City, Utah, USA during summer and fall of 2018. Fish were sampled over 7-d to examine if uptake occurred, and to define uptake kinetics. Water and fish tissues were analyzed via isotope dilution liquid chromatography tandem mass spectrometry. Several pharmaceuticals were consistently detected in water, fish tissue and plasma, including carbamazepine, diphenhydramine, diltiazem, and fluoxetine. Pharmaceutical levels in water ranged up to 151 ng/L for carbamazepine, whereas the effluent tracer sucralose was consistently observed at low µg/L levels. During both summer and fall experiments at each of three downstream locations from effluent discharge, rainbow trout rapidly accumulated these pharmaceuticals; tissue levels reached steady state conditions within 24-96 h. Spatial and temporal differences for pharmaceutical levels in rainbow trout directly corresponded with surface water exposure concentrations, and uptake kinetics for individual pharmaceuticals did not vary among sites or seasons. Such observations are consistent with recent laboratory bioconcentration studies, which collectively indicate inhalational exposure from water governs rapid accumulation of ionizable base pharmaceuticals by fish in inland surface waters.

Metabolomic-based assessment reveals dysregulation of lipid profiles in human liver cells exposed to environmental obesogens.

Significant attention has been given to the potential of environmental chemicals to disrupt lipid homeostasis at the cellular level. These chemicals, classified as obesogens, are abundantly used in a wide variety of consumer products. However, there is a significant lack of information regarding the mechanisms by which environmental exposure can contribute to the onset of obesity and non-alcoholic fatty liver disease (NAFLD). Several studies have described the interaction of potential obesogens with lipid-related peroxisome proliferator-activated receptors (PPAR). However, no studies have quantified the degree of modification to lipidomic profiles in relevant human models, making it difficult to directly link PPAR agonists to the onset of lipid-related diseases. A quantitative metabolomic approach was used to examine the dysregulation of lipid metabolism in human liver cells upon exposure to potential obesogenic compounds. The chemicals rosiglitazone, perfluorooctanoic acid, di-2-ethylexylphthalate, and tributyltin significantly increased total lipids in liver cells, being diglycerides, triglycerides and phosphatidylcholines the most prominent. Contrarily, perfluorooctane sulfonic acid and the pharmaceutical fenofibrate appeared to lower total lipid concentrations, especially those belonging to the acylcarnitine, ceramide, triglyceride, and phosphatidylcholine groups. Fluorescence microscopy analysis for cellular neutral lipids revealed significant lipid bioaccumulation upon exposure to obesogens at environmentally relevant concentrations. This integrated omics analysis provides unique mechanistic insight into the potential of these environmental pollutants to promote diseases like obesity and NAFLD. Furthermore, this study provides a significant contribution to advance the understanding of molecular signatures related to obesogenic chemicals and to the development of alternatives to in vivo experimentation.

Altered expression and activity of phase I and II biotransformation enzymes in human liver cells by perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS).

Human exposure assessments for perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) have been mostly limited to the quantification of these chemicals in different environmental matrices, but only a few studies have addressed toxicological aspects associated with them. It has been suggested that both PFOA and PFOS are highly stable chemicals that are not metabolized, yet previous reports have described abnormal activity of important biotransformation pathways. Therefore, the goal of the present study was to investigate the effects of PFOA and PFOS on phase I and II biotransformation enzymes at the gene expression and activity levels, and by using the well-established human liver HepaRG cell line. Cells were exposed to a wide range of PFOA and PFOS concentrations for 24 or 48 h, prior to cytotoxicity measurements, and quantification of expression and activity of three cytochrome P450 enzymes (CYP1A2, CYP2C19 and CYP3A4) and two conjugation enzymes (glutathione-S-transferase (GST-M1) and UDP-glucuronosyltransferase (UGT-1A1)). Expression of all CYP enzymes was significantly reduced from exposure to both PFOA and PFOS after 48 h and from concentrations as low as 40-50 ng/L, with CYP3A4 also presenting the lowest activity. Among the conjugation enzymes, the expression of UGT was significantly reduced only by PFOA after 48 h of exposure, yet no significant alterations in its activity were observed. While the specific chemico-biological interactions of these compounds with gene expression and biotransformation pathways is not clear, the results from this study suggest that the interference of PFOA and PFOS with phase I and II biotransformation enzymes could potentially lead to adverse outcomes resulting from the inability of biotransformation pathways to function as needed.

Oxidative Potential of Chemical Mixtures Extracted from Contaminated Galveston Bay, TX Seafood Using a Human Cell Co-culture Model.

Increasing levels of pollution in Galveston Bay, TX, are of significant concern for populations that directly depend on fishing activities. Efforts to evaluate contaminant levels in commercial fish have been largely limited to the quantification of chemical mixtures in fish tissue, but little information exists about the toxicological potential of these chemicals on consumption of contaminated seafood. The present study makes use of a human cell co-culture model, mimicking the digestive system, to address the oxidative potential of chemical mixtures in seafood. Chemical extractions were performed on fillets from three fish species and oysters collected from different areas in Galveston Bay. The resulting extracts were used to expose intestinal and liver cells before the measurement of cytotoxicity and activity of antioxidant enzymes. The pesticide 4,4'-DDE was found in nearly all samples from all sites in concentrations ranging from 0.23-9.4 µg/kg. Similarly, total PCBs found in fish and oyster tissue ranged from 0.68-65.65 µg/kg, with PCB-118 being the most common congener measured. In terms of cytotoxicity, oyster extracts led to significant cell mortality, contrary to observations for fish extracts. Antioxidant enzymes, while not directly related to the presence of chemical mixtures in tissue, presented evidence of potential increases in activity from spotted trout extracts. Observations from this study suggest the need to evaluate toxicological aspects of contaminated seafood and support the use of in vitro models for the screening of accumulated chemicals.

A Mesocosm Assessment of the Effect of Bioturbation by the Ghost Shrimp (Lepidophthalmus louisianensis) on the Fate of Petroleum Hydrocarbons in the Intertidal Zone.

To fully assess the long-term impacts of oil spills like the 2010 Deepwater Horizon incident in the northern Gulf of Mexico, the potential for organisms other than microbes to affect the fate and distribution of the oil may have to be considered. This influence could be substantial for abundant bioturbating benthic animals like the ghost shrimp Lepidophthalmus louisianensis. An assessment of the influence of these ghost shrimp on petroleum hydrocarbons was conducted in laboratory micro- and mesocosms containing coastal Gulf of Mexico sediment, seawater, and oil or the polynuclear aromatic hydrocarbon (PAH) pyrene. In an experiment with pyrene added to the water column, the ghost shrimp presence lowered water-column pyrene concentrations. In an experiment with oil added to the sediment surface, the ghost shrimp presence decreased PAH concentrations in the sediment surface layer but increased these in the water column and subsurface sediment. A companion study and a mass-balance analysis indicated a net loss of PAHs through an enhancement of microbial degradation. In an experiment in which oil was added as a narrow subsurface layer in the sediment, the ghost shrimp presence appeared to broaden the oil's depth distribution. Taken together, these results demonstrate that ghost shrimp can significantly influence the biodegradation and distribution of spilled oil in coastal ecosystems. Environ Toxicol Chem 2020;39:637-647. © 2019 SETAC.

Altered Metabolomic Profile in Patients with Peripheral Artery Disease.

Peripheral artery disease (PAD) is a common atherosclerotic disease characterized by narrowed or blocked arteries in the lower extremities. Circulating serum biomarkers can provide significant insight regarding the disease progression. Here, we explore the metabolomics signatures associated with different stages of PAD and investigate potential mechanisms of the disease. We compared the serum metabolites of a cohort of 26 PAD patients presenting with claudication and 26 PAD patients presenting with critical limb ischemia (CLI) to those of 26 non-PAD controls. A difference between the metabolite profiles of PAD patients from non-PAD controls was observed for several amino acids, acylcarnitines, ceramides, and cholesteryl esters. Furthermore, our data demonstrate that patients with CLI possess an altered metabolomic signature different from that of both claudicants and non-PAD controls. These findings provide new insight into the pathophysiology of PAD and may help develop future diagnostic procedures and therapies for PAD patients.

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.

Applicability of in vitro methods in evaluating the biotransformation of polycyclic aromatic hydrocarbons (PAHs) in fish: Advances and challenges.

The biotransformation of polycyclic aromatic hydrocarbons (PAHs) and the biochemical mechanisms involved in such process continue to be intensively studied in the fields of environmental science and toxicology. The investigation of PAH biotransformation in fish is fundamental to understand how piscine species cope with PAH exposure, as these compounds are ubiquitous in aquatic ecosystems and impact different levels of biological organization. New approaches are continuously developed in the field of ecotoxicology, allowing live animal testing to be combined with and, in some cases, replaced with novel in vitro systems. Many in vitro techniques have been developed and effectively applied in the investigation of the biochemical pathways driving the biotransformation of PAH in fish. In vitro experimentation has been fundamental in the advancement of not only understanding PAH-mediated toxicity, but also in highlighting suitable cell-based models for such investigations. Therefore, the present review highlights the value and applicability of in vitro systems for PAH biotransformation studies, and provides up-to-date information on the use of in vitro fish models in the evaluation of PAH biotransformation, common biomarkers, and challenges encountered when developing and applying such systems.

Applicability of a human cell co-culture model to evaluate antioxidant responses triggered by chemical mixtures in fish and oyster homogenates.

The accumulation of chemical compounds in fish tissue represents significant health concerns for seafood consumers, but little is known about the risks to human health associated with such substances. The identification of adverse biological responses upon exposure to contaminants has been facilitated by the development of in vitro systems resembling the human dietary pathway. The present study explores the applicability of an organotypic co-culture system, using intestinal (Caco-2) and hepatic (HepaRG) cell lines, to provide insight into the toxicity of chemical mixtures found in commercially available seafood. Chemical extractions were conducted utilizing fish and oyster standard reference material (SRM) from the U.S. National Institute of Standards and Technology (NIST). Cells were seeded in monoculture and co-culture systems and exposed to SRM extracts before measurements of cytotoxicity and antioxidant responses. Exposure to oyster extracts led to significant cell mortality in monocultures. HepaRG cells in monoculture expressed lower levels of glutathione peroxidase and superoxide dismutase than HepaRG cells in co-culture, upon exposure to both oyster and fish extracts. These observations illustrate the importance of organotypic co-culture models to explore biological responses that could be otherwise difficult to evaluate in monocultures, and the adverse effects associated with the consumption of contaminated seafood.

Xenobiotic metabolism in the fish hepatic cell lines Hepa-E1 and RTH-149, and the gill cell lines RTgill-W1 and G1B: Biomarkers of CYP450 activity and oxidative stress.

The use of fish cell cultures has proven to be an effective tool in the study of environmental and aquatic toxicology. Valuable information can be obtained from comparisons between cell lines from different species and organs. In the present study, specific chemicals were used and biomarkers (e.g. 7-Ethoxyresorufin-O-deethylase (EROD) activity and reactive oxygen species (ROS)) were measured to assess the metabolic capabilities and cytotoxicity of the fish hepatic cell lines Hepa-E1 and RTH-149, and the fish gill cell lines RTgill-W1 and G1B. These cell lines were exposed to ß-naphthoflavone (BNF) and benzo[a]pyrene (BaP), the pharmaceutical tamoxifen (TMX), and the organic peroxide tert-butylhydroperoxide (tBHP). Cytotoxicity in gill cell lines was significantly higher than in hepatic cells, with BNF and TMX being the most toxic compounds. CYP1-like associated activity, measured through EROD activity, was only detected in hepatic cells; Hepa-E1 cells showed the highest activity after exposure to both BNF and BaP. Significantly higher levels of CYP3A-like activity were also observed in Hepa-E1 cells exposed to TMX, while gill cell lines presented the lowest levels. Measurements of ROS and antioxidant enzymes indicated that peroxide levels were higher in gill cell lines in general. However, levels of superoxide were significantly higher in RTH-149 cells, where no distinctive increase of superoxide-related antioxidants was observed. The present study demonstrates the importance of selecting adequate cell lines in measuring specific metabolic parameters and provides strong evidence for the fish hepatocarcinoma Hepa-E1 cells to be an excellent alternative in assessing metabolism of xenobiotics, and in expanding the applicability of fish cell lines for in vitro studies.

Crude oil toxicity to fiddler crabs (Uca longisignalis and Uca panacea) from the northern Gulf of Mexico: Impacts on bioturbation, oxidative stress, and histology of the hepatopancreas.

The intensive drilling and extraction of fossil fuels in the Gulf of Mexico result in a considerable risk of oil spills impacting its coastal ecosystems. Impacts are more likely to be far-reaching if the oil affects ecosystem engineers like fiddler crabs, whose activities modify biogeochemical processes in the sediment. The present study investigated effects of oil on the fiddler crabs Uca longisignalis and Uca panacea, which are important as ecosystem engineers and as prey for a wide variety of species. The present study used mesocosms and microcosms to investigate the effects of crude oil on fiddler crab burrowing and to assess cellular and tissue damage by the oil. Fiddler crabs were exposed for periods of 5 or 10 d to oil concentrations up to 55 mg/cm2 on the sediment surface. Their burrowing was delayed, their burrows were smaller, and they transported less sediment in the presence of oil. The hepatopancreas had elevated levels of oxidative stress and a higher abundance of blister cells, which play a role in secretory processes. Interspecific differences were observed; most effects were strongest in U. panacea, though burrowing was more strongly affected in U. longisignalis. The present study demonstrates that crude oil is likely to impact fiddler crabs and many species that depend on them for their diet or for the ecological changes that result from their burrowing. Environ Toxicol Chem 2018;37:491-500. © 2017 SETAC.