Using bioanalytical tools to detect and track organic micropollutants in the Ganga River near two major cities.

Major rivers in India are subject to ongoing impacts from urban drain discharges, most of which contain high levels of domestic and industrial wastewater and stormwater. The aim of the present study was to determine the levels of bioactive organic micropollutants at the discharge points of major urban drains in comparison to upstream and downstream sites. To achieve this, we employed a panel of in vitro bioanalytical tools to quantify estrogenic, androgenic, progestogenic, glucocorticoid and peroxisome proliferator-like activity in water extracts collected from two Indian cities in the Ganga Basin. Cytotoxicity of the water extracts in a human-derived cell line and the potential to cause oxidative stress in a fish cell line were also investigated. We found high levels of activity for all endpoints in samples directly receiving urban drain discharge and low levels at sites upstream from drain discharges. Estrogenicity was detected at levels equivalent to 10 ng/L 17ß-estradiol, representing a high likelihood of biomarker effects in fish. Sites located downstream from drain discharges exhibited low to intermediate activity in all assays. This study demonstrates the importance of managing urban drain discharges and the utility of applying bioanalytical tools to assess water quality.

The Toxicogenome of Hyalella azteca: A Model for Sediment Ecotoxicology and Evolutionary Toxicology.

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.

In vitro nuclear receptor inhibition and cytotoxicity of hydraulic fracturing chemicals and their binary mixtures.

The widespread use of hydraulic fracturing (HF) in oil and gas extraction operations has led to concern over environmental risks posed by chemicals used in HF fluids. Here we employed a suite of stable luciferase reporter gene assays to investigate the potential for selected HF chemicals or geogenics to activate or antagonise nuclear receptor signalling. We screened three biocides (bronopol [BP], glutaraldehyde [GA], and tetrakis(hydroxymethyl)phosphonium sulfate [THPS]), a surfactant (2-butoxyethanol), a friction reducer (polyacrylamide), and a coal seam geogenic (o-cresol) for their potential to act as agonists or antagonists of the estrogen receptor, androgen receptor, progesterone receptor (PR), glucocorticoid receptor or peroxisome proliferator-activated receptor gamma (PPARγ). None of the chemicals induced luciferase activity in any of assays used in the study. In antagonistic mode, BP, GA and THPS caused reductions in luciferase activity in the reporter assays at higher concentrations (50-100 µM), while at low concentrations (2-10 µM) GA and THPS enhanced luciferase activity in some assays relative to controls. None of the other tested chemicals exhibited antagonism in the selected assays. In most cases, altered receptor signalling only occurred at concentrations exhibiting cytotoxicity. However, PPARγ activity, and to a lesser extent PR activity, were inhibited by THPS at sub-cytotoxic concentrations. The majority of binary combinations tested exhibited significantly less-than-additive cytotoxicity, and none of the combinations exhibited synergistic cytotoxicity. In summary, the results of the present study indicate that the selected chemicals are not likely to function as direct agonists of the nuclear receptors tested, and only one chemical, THPS was an apparent partial antagonist of two nuclear receptors.

Environmental pollution affects molecular and biochemical responses during gonadal maturation of Astyanax fasciatus (Teleostei: Characiformes: Characidae).

Endocrine disrupting compounds (EDCs) have the potential to alter fish reproduction at various levels of organization. The aim of this study was to assess the impact of a natural environment with heavily anthropogenic influence on the physiological processes involved in reproduction in the freshwater fish lambari (Astyanax fasciatus) using different biomarkers. Adult males and females were collected in different seasons from two distinct sites in the same watershed: Ponte Nova Reservoir (PN) considered a pristine or small anthropogenic influence reference point; and Billings Reservoir (Bil), subjected to a large anthropogenic impact. Biological indices, such as hepatosomatic index and gonadosomatic index (GSI), gonadal histomorphology, fecundity, and biomarkers such as plasma levels of estradiol (E2) as well as hepatic gene expression of its alfa nuclear receptor (ERα), were analyzed. Hepatic vitellogenin (VTG) gene expression was evaluated in both sexes, as an indicator of xenoestrogen exposure. Females collected at PN presented a typical annual variation reflected in GSI, whereas for those sampled at Bil the index did not change through the seasons. The higher concentration of E2 in males collected at Bil during spring/2013, together with the detection of VTG gene expression, suggest the presence of EDCs in the water. These EDCs may have also influenced fecundity of females from Bil, which was higher during winter and spring/2013. Gene expression of ERα and ovarian morphology did not differ between fish from both sites. Water conditions from Bil reservoir impacted by anthropic activity clearly interfered mainly with biomarkers of biological effect such as plasma E2 levels and absolute and relative fecundity, but also altered biomarkers of exposure as VTG gene expression. These facts support the notion that waterborne EDCs are capable of causing estrogenic activity in A. fasciatus.

De novo assembly and analysis of changes in the protein-coding transcriptome of the freshwater shrimp Paratya australiensis (Decapoda: Atyidae) in response to acid sulfate drainage water.

BACKGROUND: The atyid shrimp Paratya australiensis occurs in surface freshwater habitats throughout eastern Australia and has been used to study the ecotoxicology of contaminants such as pesticides and metals. The acidification of surface water that can occur after acid sulfate material in soils and sediments is oxidised and subsequently re-wetted is a serious environmental issue in coastal regions and inland riverine floodplains worldwide. Solubilisation of soil-associated minerals can result in high waterborne concentrations of mineral salts and dissolved metals, which together with low pH represent a potential threat to aquatic ecosystems in affected regions. The aims of the present study were to gain insight into stress responses induced by exposure to acid drainage water (ADW) in P. australiensis by determining changes in the abundance of protein-coding transcripts and to generate a comprehensive transcriptomic resource to facilitate further research into gene regulation or protein structure and function in this species. Adult P. australiensis were exposed for 24 h to undiluted ADW, 50 % ADW diluted in river water, or to river water as control, and high-throughput mRNA sequencing (RNA-Seq) conducted on whole-body tissues. A reference transcriptome was generated using de novo assembly and putative protein-coding regions were identified and annotated. Changes in transcript abundance in response to ADW exposure were determined by aligning reads to the reference transcriptome and quantifying coverage. RESULTS: A high proportion of arthropod benchmarking universal single-copy orthologues were present in the reference transcriptome. Functions associated with cuticle biosynthesis and oxidative stress were significantly enriched in the lists of transcripts exhibiting differential abundance in either direction after exposure to 50 % or 100 % ADW. Transcripts involved in osmoregulation exhibited decreased abundance following exposure to ADW. The transcriptome contained full-length coding sequences for numerous proteins known to be involved in environmental response pathways, including two putative metallothioneins, four glutathione peroxidases and 19 nuclear receptors. CONCLUSIONS: The results of the present study provide insight into stress response pathways induced in crustaceans by short-term exposure to multiple stressors present in ADW such as low pH, high salinity and dissolved metals, and represent a resource for future toxicogenomics and protein functional studies in P. australiensis.

In vitro nuclear receptor activity and in vivo gene expression analysis in Murray-Darling rainbowfish (Melanotaenia fluviatilis) after short-term exposure to fluoxetine.

Fluoxetine (FLX) is one of numerous pharmaceuticals found in treated municipal wastewater discharged to the environment. In the present study, we investigated the effects of short-term (96h) waterborne FLX exposure (1µg/L or 100µg/L) on the expression of selected genes in brain, liver, and gonads of female Murray-Darling rainbowfish (Melanotaenia fluviatilis), a small-bodied teleost of ecotoxicological relevance in the Australasia region. Plasma 17ß-estradiol (E2) levels were also determined. In the brain, no significant changes in mRNA levels were observed for the selected genes. In ovaries, 100µg/L FLX caused a 10-fold downregulation of aromatase A (cyp19a1a) mRNA and a 4-fold upregulation of estrogen receptor α (esr1) mRNA levels. In liver, mRNA levels for vitellogenin A (vtga) and choriogenin L (chgl) were downregulated by 50-fold and 18-fold compared with controls, respectively, in response to 100µg/L FLX. Concentrations of E2 in plasma were significantly lower than controls in response to 100µg/L FLX. This could be attributable to a decrease in estrogen biosynthesis as a result of the observed downregulation of cyp19a1a mRNA. To establish whether the observed changes in gene expression could be explained by the modulation of selected nuclear receptors by FLX, we employed panel of reporter gene assays in agonistic and antagonistic modes. Apart from minor activation of ERα after exposure to high concentrations (5µM), FLX did not activate or inhibit the nuclear receptors tested. Further study is required to determine whether the observed downregulation of ovarian aromatase expression and liver estrogen-regulated genes also occurs at environmentally relevant FLX concentrations over longer exposure periods.

Identification of Putative Nuclear Receptors and Steroidogenic Enzymes in Murray-Darling Rainbowfish (Melanotaenia fluviatilis) Using RNA-Seq and De Novo Transcriptome Assembly.

Murray-Darling rainbowfish (Melanotaenia fluviatilis [Castelnau, 1878]; Atheriniformes: Melanotaeniidae) is a small-bodied teleost currently under development in Australasia as a test species for aquatic toxicological studies. To date, efforts towards the development of molecular biomarkers of contaminant exposure have been hindered by the lack of available sequence data. To address this, we sequenced messenger RNA from brain, liver and gonads of mature male and female fish and generated a high-quality draft transcriptome using a de novo assembly approach. 149,742 clusters of putative transcripts were obtained, encompassing 43,841 non-redundant protein-coding regions. Deduced amino acid sequences were annotated by functional inference based on similarity with sequences from manually curated protein sequence databases. The draft assembly contained protein-coding regions homologous to 95.7% of the complete cohort of predicted proteins from the taxonomically related species, Oryzias latipes (Japanese medaka). The mean length of rainbowfish protein-coding sequences relative to their medaka homologues was 92.1%, indicating that despite the limited number of tissues sampled a large proportion of the total expected number of protein-coding genes was captured in the study. Because of our interest in the effects of environmental contaminants on endocrine pathways, we manually curated subsets of coding regions for putative nuclear receptors and steroidogenic enzymes in the rainbowfish transcriptome, revealing 61 candidate nuclear receptors encompassing all known subfamilies, and 41 putative steroidogenic enzymes representing all major steroidogenic enzymes occurring in teleosts. The transcriptome presented here will be a valuable resource for researchers interested in biomarker development, protein structure and function, and contaminant-response genomics in Murray-Darling rainbowfish.

Tracking multiple modes of endocrine activity in Australia's largest inland sewage treatment plant and effluent- receiving environment using a panel of in vitro bioassays.

Estrogenicity of sewage effluents, and related ecotoxicological effects in effluent-receiving environments, have been widely reported over the last 2 decades. However, relatively little attention has been given to other endocrine pathways that may be similarly disrupted by a growing list of contaminants of concern. Furthermore, the Australian evidence base is limited compared with those of Europe and North America. During a low dilution period in summer, the authors investigated multiple endocrine potencies in Australia's largest inland sewage treatment plant (STP) and the Lower Molonglo/Upper Murrumbidgee effluent-receiving environment. This STP receives 900 L/s of mostly domestic wastewater from a population of 350 000, and contributes a high proportion of total flow in the lower catchment during dry periods. A panel of in vitro receptor-driven transactivation assays were used to detect (anti)estrogenic, (anti) androgenic, (anti)progestagenic, glucocorticoid, and peroxisome-proliferator activity at various stages of the sewage treatment process. Total estrogenic and (anti)androgenic potency was removed after primary and/or secondary treatment; however, total removal efficiency for glucocorticoid potency was poorer (53-66%), and progestagenic potency was found to increase along the treatment train. Estrogenicity was detected in surface waters and bed sediments upstream and downstream of the effluent outfall, at maximum levels 10 times lower than low-hazard thresholds. Glucocorticoid and progestagenic activity were found to persist to 4 km downstream of the effluent outfall, suggesting that future research is needed on these endocrine-disrupting chemical categories in effluent-receiving systems.

Nortestosterone-derived synthetic progestogens do not activate the progestogen receptor of Murray-Darling rainbowfish (Melanotaenia fluviatilis) but are potent agonists of androgen receptors alpha and beta.

Synthetic progestogens derived from 19-nortestosterone can elicit a number of adverse effects in fish including decreased fecundity, altered hormone levels, disruption of normal breeding cycles, expression in females of male-specific biomarkers, development of male secondary sexual characteristics in females, and changes in the expression of steroidogenic genes. A recent in vitro study showed that a number of representatives from this class of progestins were potent agonists of fathead minnow androgen receptor (AR) and only weak agonists of progesterone receptor (PR) from the same species. This confirms that synthetic progestogens derived from 19-nortestosterone function as AR agonists in otomorphs, which express a single AR subtype. However, numerous perciformes are known to express two AR subtypes. We have recently shown that ARα and ARß from Murray-Darling rainbowfish (Melanotaenia fluviatilis) respond differently to certain androgens and anti-androgens. The goal of the present study was to determine concentration-response profiles for selected progestins in transactivation assays driven by rainbowfish ARα, ARß and PR in order to ascertain the relative potency of progestins against these receptors. As a means of confirming the expected activity of the progestins and reference compounds used in the study against human-derived receptors, we also established concentration-response relationships using transactivation assays driven by human PR and AR. We found that all five 19-nortestosterone-derived progestins tested were highly potent agonists of rainbowfish ARα, but that only four of the five progestins were potent agonists of rainbowfish ARß, with norgestimate exhibiting only weak activity against rainbowfish ARß. The spironolactone-derived progestin, drospirenone, was not an agonist of rainbowfish ARα or ARß but was a weak agonist of rainbowfish PR. None of the 19-nortestosterone-progestins activated rainbowfish PR. These findings confirm that the majority of 19-nortestosterone-derived progestins are likely to elicit strong androgenic activity in teleosts, but that PR-mediated effects would be minimal. In species that express two AR subtypes similar to rainbowfish ARα and ARß, biological processes mediated by a specific subtype may be affected differently by progestins such as norgestimate.

Differential ligand selectivity of androgen receptors α and ß from Murray-Darling rainbowfish (Melanotaenia fluviatilis).

Androgen receptors (ARs) mediate the physiological effects of androgens in vertebrates. In fishes, AR-mediated pathways can be modulated by aquatic contaminants, resulting in the masculinisation of female fish or diminished secondary sex characteristics in males. The Murray-Darling rainbowfish (Melanotaenia fluviatilis) is a small-bodied freshwater teleost used in Australia as a test species for environmental toxicology research. We determined concentration-response profiles for selected agonists and antagonists of rainbowfish ARα and ARß using transient transactivation assays. For both ARα and ARß, the order of potency of natural agonists was 11-ketotestosterone (11-KT)>5α-dihydrotestosterone>testosterone>androstenedione. Methyltestosterone was a highly potent agonist of both receptors relative to 11-KT. The relative potency of the veterinary growth-promoting androgen, 17ß-trenbolone, varied by more than a factor of 5 between ARα and ARß. The non-steroidal anti-androgen bicalutamide exhibited high inhibitory potency relative to the structurally related model anti-androgen, flutamide. The inhibitory potency of the agricultural fungicide, vinclozolin, was approximately 1.7-fold relative to flutamide for ARα, but over 20-fold in the case of ARß. Fluorescent protein tagging of ARs showed that the rainbowfish ARα subtype is constitutively localised to the nucleus, while ARß is cytoplasmic in the absence of ligand, an observation which agrees with the reported subcellular localisation of AR subtypes from other teleost species. Collectively, these data suggest that M. fluviatilis ARα and ARß respond differently to environmental AR modulators and that in vivo sensitivity to contaminants may depend on the tissue distribution of the AR subtypes at the time of exposure.

Assessment of multiple hormonal activities in wastewater at different stages of treatment.

Changes in the endocrine potency of municipal wastewater at 3 wastewater treatment plants (WWTPs) in Australia were investigated using a panel of in vitro receptor-driven transactivation assays. The assays were based on human estrogen receptor α, androgen receptor, progesterone receptor, glucocorticoid receptor, and peroxisome proliferator-activated receptor γ2. Total removal efficiencies for estrogenic activity in the dissolved phase were 79.8% to 99.4%. Chemical analysis of 17ß-estradiol, estrone, and 17α-ethinylestradiol levels showed that they accounted for the majority of the observed in vitro estrogenic activity in the final effluents but only 18% to 70% of estrogenic activity in the influents. Removal efficiency for androgenic activity was 97.5% to 100%. Endocrine activity levels were low in the final effluent of the WWTP with the lowest catchment population, with only estrogenic activity detected. In the final effluent of the WWTP with an intermediate catchment population, estrogenic, glucocorticoid, and peroxisome proliferator activities were detected. Estrogenic, antiandrogenic, progestagenic, glucocorticoid, and peroxisome proliferator activities were detected in the final effluent of the WWTP with the highest catchment population. The present study confirms the efficacy of secondary and tertiary treatment in reducing the concentrations of endocrine-active compounds in municipal wastewater. Further work is required to determine the possible health risks to aquatic biota posed by multiple hormonal activities present at low levels.

Cytotoxicity of binary mixtures of human pharmaceuticals in a fish cell line: approaches for non-monotonic concentration-response relationships.

Predicting the effects of mixtures of environmental micropollutants is a priority research area. In this study, the cytotoxicity of ten pharmaceuticals to the rainbow trout cell line RTG-2 was determined using the neutral red uptake assay. Fluoxetine (FL), propranolol (PPN), and diclofenac (DCF) were selected for further study as binary mixtures. Biphasic concentration-response relationships were observed in cells exposed to FL and PPN. In the case of PPN, microscopic examination revealed lysosomal swelling indicative of direct uptake and accumulation of the compound. Three equations describing non-monotonic concentration-response relationships were evaluated and one was found to consistently provide more accurate estimates of the median and 10% effect concentrations compared with a sigmoidal concentration-response model. Predictive modeling of the effects of binary mixtures of FL, PPN, and DCF was undertaken using an implementation of the concentration addition (CA) conceptual model incorporating non-monotonic concentration-response relationships. The cytotoxicity of the all three binary combinations could be adequately predicted using CA, suggesting that the toxic mode of action in RTG-2 cells is unrelated to the therapeutic mode of action of these compounds. The approach presented here is widely applicable to the study of mixture toxicity in cases where non-monotonic concentration-response relationships are observed.

Benchmarking organic micropollutants in wastewater, recycled water and drinking water with in vitro bioassays.

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

Dietary fish oil replacement with canola oil up-regulates glutathione peroxidase 1 gene expression in yellowtail kingfish (Seriola lalandi).

The marine carnivore yellowtail kingfish (YTK, Seriola lalandi) was fed diets containing 5% residual fish oil (from the dietary fish meal) plus either 20% fish oil (FO), 20% canola oil (CO), 20% poultry oil (PO), 10% fish oil plus 10% canola oil (FO/CO) or 10% fish oil plus 10% poultry oil (FO/PO) and the effects on fish growth and hepatic expression of two glutathione peroxidase (GPx 1 and GPx 4) and two peroxiredoxin (Prx 1 and Prx 4) antioxidant genes were investigated. Partial (50%) replacement of the added dietary fish oil with poultry oil significantly improved fish growth whereas 100% replacement with canola oil significantly depressed fish growth. The fatty acid profiles of the fish fillets generally reflected those of the dietary oils except that there was apparent selective utilization of palmitic acid (16:0) and oleic acid (18:1n-9) and apparent selective retention of eicospentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). The Prx 1 and 4 genes were expressed at 10- and 100-fold the level of the GPx 4 and 1 genes, respectively, and at one-tenth the level of the highly expressed ß-actin reference gene. Dietary fish oil replacement with canola oil significantly up-regulated GPx 1 gene expression and there was a non-significant tendency towards down-regulation of Prx 1 and Prx 4. The results are discussed in terms of the effects of fish oil replacement on the peroxidation index of the diets and the resulting effects on the target antioxidant enzymes.

A glutathione peroxidase 4 (GPx4) homologue from southern bluefin tuna is a secreted protein: first report of a secreted GPx4 isoform in vertebrates.

The antioxidant enzyme glutathione peroxidase 4 (GPx4) is capable of reducing complex lipid hydroperoxides in addition to hydrogen peroxide and organic hydroperoxides. Mammals express three GPx4 isoforms that are targeted to nucleoli, mitochondria or cytosol via variable amino termini. To better understand the role of this important antioxidant enzyme in marine finfish, we determined the subcellular localisation of a GPx4 homologue from southern bluefin tuna (Thunnus maccoyii; SBT). We created constructs for the expression of the selenocysteine-to-cysteine mutant of SBT GPx4 (GPx4C) tagged with enhanced green fluorescent protein (EGFP), including or lacking a putative amino-terminal signal peptide, and expressed the fusion proteins in a fish cell line. Fluorescence microscopy revealed that the full-length GPx4C-EGFP fusion protein localised to the trans-Golgi, suggesting that tuna GPx4 may be directed to the secretory pathway. Anti-GFP immunoblotting of cell lysates and proteins from culture media showed that the secretion of SBT GPx4 into the culture medium required an amino-terminal signal peptide. According to available sequence data, the SBT GPx4 isoform studied here is representative of other piscine GPx4 isoforms, suggesting that the secretion of at least one GPx4 isoform may be common amongst teleost fish.

Use of fluorogenic probes to differentiate between hydrophilic and lipophilic antioxidant activity in a fish cell line.

In finfish aquaculture, dietary antioxidants have been shown to improve indicators of general fish health and to inhibit the oxidative deterioration of polyunsaturated fatty acids. To facilitate the characterization of novel antioxidants or antioxidant mixtures, we developed assays for antioxidant activity in a fish cell line. We used 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) to determine the protective effects of a panel of representative antioxidant compounds against the formation of reactive oxygen species (ROS) under conditions that promote oxidative stress, whereas protective effects against lipid peroxidation were measured using the thiobarbituric acid reactive substances (TBARS) assay and a novel implementation of 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C(11)-BODIPY(581/591)). We found that the highly hydrophilic antioxidant, sodium ascorbate, inhibited H(2)DCFDA oxidation but had no effect on lipid peroxidation, whereas the highly hydrophobic antioxidant, α-tocopherol, potently inhibited lipid peroxidation but did not prevent H(2)DCFDA oxidation. The data suggest that a single assay is not sufficient for estimating antioxidant activity in cultured fish cells.

Development of a fish cell culture model to investigate the impact of fish oil replacement on lipid peroxidation.

Fish oils are rich in omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), predominantly 20:5n-3 and 22:6n-3, whereas vegetable oils contain abundant C(18)-PUFA, predominantly 18:3n-3 or 18:2n-6. We hypothesized that replacement of fish oils with vegetable oils would increase the oxidative stability of fish lipids. Here we have used the long established and easily cultivated FHM cell line derived from the freshwater fish species fathead minnow (Pimephales promelas) to test this hypothesis. The FHM cells were readily able to synthesize 20:5n-3 and 24:6n-3 from 18:3n-3 but 22:6n-3 synthesis was negligible. Also, they were readily able to synthesize 20:3n-6 from 18:2n-6 but 20:4n-6 synthesis was negligible. Mitochondrial ß-oxidation was greatest for 18:3n-3 and 20:5n-3 and the rates for 16:0, 18:2n-6, 22:6n-3 and 18:1n-9 were significantly lower. Fatty acid incorporation was predominantly into phospholipids (79-97%) with very little incorporation into neutral lipids. Increasing the fatty acid concentration in the growth medium substantially increased the concentrations of 18:3n-3 and 18:2n-6 in the cell phospholipids but this was not the case for 20:5n-3 or 22:6n-3. When they were subjected to oxidative stress, the FHM cells supplemented with either 20:5n-3 or 22:6n-3 (as compared with 18:3n-3 or saturated fatty acids) exhibited significantly higher levels of thiobarbituric reactive substances (TBARS) indicating higher levels of lipid peroxidation. The results are discussed in relation to the effects of fatty acid unsaturation on the oxidative stability of cellular lipids and the implications for sustainable aquaculture.

Naturally occurring DNA transfer system associated with membrane vesicles in cellulolytic Ruminococcus spp. of ruminal origin.

A genetic transformation system with similarities to those reported for gram-negative bacteria was found to be associated with membrane vesicles of the ruminal cellulolytic genus Ruminococcus. Double-stranded DNA was recovered from the subcellular particulate fraction of all the cellulolytic ruminococci examined. Electron microscopy revealed that the only particles present resembled membrane vesicles. The likelihood that the DNA was associated with membrane vesicles (also known to contain cellulosomes) was further supported by the adherence of the particles associated with the subcellular DNA to cellulose powder added to culture filtrates. The particle-associated DNA comprised a population of linear molecules ranging in size from <20 kb to 49 kb (Ruminococcus sp. strain YE73) and from 23 kb to 90 kb (Ruminococcus albus AR67). Particle-associated DNA from R. albus AR67 represented DNA derived from genomic DNA of the host bacterium having an almost identical HindIII digestion pattern and an identical 16S rRNA gene. Paradoxically, particle-associated DNA was refractory to digestion with EcoRI, while the genomic DNA was susceptible to extensive digestion, suggesting that there is differential restriction modification of genomic DNA and DNA exported from the cell. Transformation using the vesicle-containing fraction of culture supernatant of Ruminococcus sp. strain YE71 was able to restore the ability to degrade crystalline cellulose to two mutants that were otherwise unable to do so. The ability was heritable and transferred to subsequent generations. It appears that membrane-associated transformation plays a role in lateral gene transfer in complex microbial ecosystems, such as the rumen.