The DNA de-methylating agent 5-azacytidine does not restore CYP1A induction in PCB resistant Newark Bay killifish (Fundulus heteroclitus)

Newark Bay (NB) killifish (Fundulus heteroclitus) have been chronically exposed to environmental contaminants that activate the aryl hydrocarbon receptor (AHR) and are tolerant to toxic effects and CYP1A induction provoked by AHR ligands. Resistance to CYP1A induction could be due to an epigenetic mechanism such as DNA methylation. We measured in-ovo CYP1A catalytic activity (ethoxyresorufin-O-deethylase, EROD) in NB and reference site killifish embryos aqueously exposed to various concentrations of the de-methylating agent 5-azacytidine, 5-AC (5, 50 and 500 micro(micro)M) with or without 0.2 micro(micro)g/l of the CYP1A inducer 3,3',4,4',5 pentachlorobiphenyl (IUPAC PCB126). Neither PCB126 alone, nor PCB126 plus 5-AC, induced EROD above levels in vehicle treated Newark Bay fish. In reference site fish, the same PCB126 dose provoked a 7.4-fold EROD induction relative to controls. We conclude that Newark Bay killifish are resistant to CYP1A induction by co-planar PCBs during early embryological development and our data suggests that DNA methylation does not play a critical role in resistance to CYP1A induction in this model.     

Oxidative stress in two populations of killifish (Fundulus heteroclitus) with differing contaminant exposure histories

A population of killifish (Fundulus heteroclitus) inhabiting a creosote-polluted inlet of the Elizabeth River demonstrates tolerance to the acute toxic effects exerted by contaminated sediments on reference site killifish. Previous data have suggested that upregulated antioxidant defenses contribute to short-term tolerance in killifish exposed to Elizabeth River sediments. This study investigated population differences in antioxidant defenses from wild caught Elizabeth River and reference population killifish in different seasons, and after being held in the laboratory. Parameters measured in the killifish were total glutathione concentrations (GSH(T)), activities of glutathione reductase (GR), glutathione peroxidase (GPx), and lipid peroxidation (LPO), all in adult hepatic tissues. The Elizabeth River population exhibited greater GSH(T), higher GPx activities, and increased LPO as compared to the reference population. Sex specific population differences were also observed in GSH(T) and GPx. Both populations displayed decreased GSH(T) and increased GR from early to late summer, as well as after being held in the laboratory. This study indicates that there are many factors that may contribute to differences in levels of antioxidant defenses in addition to exposure to contaminants, including reproductive status and environmental conditions.     

Assessing neuroprotective P-glycoprotein activity at the blood-brain barrier in killifish (Fundulus heteroclitus) using behavioural profiles

The neuroprotective role of P-glycoprotein, a multixenobiotic resistance transporter (ABCB1/MDR1), in the blood-brain barrier in fish was examined using behavioural toxicological assays. P-glycoprotein acts as cellular efflux pump to prevent substrates from accumulating in the brain, including environmental contaminants such as ivermectin, a common aquaculture pesticide and mammalian anti-parasitic drug. The behavioural toxicological assays were developed to determine the neuropathological effect of ivermectin in killifish (Fundulus heteroclitus). P-glycoprotein function and thus blood-brain barrier integrity can be compromised by chemosensitizers that inhibit transport activity. Fish treated with ivermectin and the P-glycoprotein inhibitor cyclosporin A were significantly more sensitive and succumbed more rapidly to tilting, lethargy, slowing of pectoral-fin movement and loss of haptic-reactivity compared to fish treated with ivermectin-only. P-glycoprotein inhibition is associated with significantly earlier onset and increased mortality in ivermectin-exposed fish. Our results suggest that P-glycoprotein confers resistance against ivermectin-induced behavioural neuropathology and mortality in fish. This assay provides us with a non-invasive tool to study P-glycoprotein function in the blood-brain barrier and evaluate the behavioural effects of potential environmental neurotoxins.     

Teratogenesis in Fundulus heteroclitus embryos exposed to a creosote-contaminated sediment extract and CYP1A inhibitors

The goal of these experiments was to explore the relationship between cytochrome P4501A (CYP1A) induction and the teratogenicity of sediments from the Atlantic Wood Industries Superfund site (Elizabeth River, VA) in Fundulus heteroclitus embryos. In these experiments we used embryos spawned from reference site adults to assess CYP1A activity and teratogenicity induced by aqueous Elizabeth River sediment extracts (ERSE). Embryo exposures to ERSE induced CYP1A activity and caused deformities, including pericardial edema, heart elongation and tail shortening. Co-exposures with various CYP1A inhibitors significantly decreased CYP1A activity and increased the teratogenicity of the sediment extract. Potential mechanisms for this increased toxicity are discussed herein.     

A rapid change in methylmercury tolerance in a population of killifish,Fundulus heteroclitus,from a golf course pond

Embryos of killifish (Fundulus heteroclitus) from Bullhead Bay, Southampton, New York, showed considerable variation in tolerance to methylmercury (meHg) in 1980, 1981 and 1983, in that some females produced eggs that were tolerant to the teratological effects of 0·05 ppm, while other females produced susceptible eggs. However, in 1982, over 40% of the females produced clutches of non-viable eggs (which could not be tested for meHg tolerance) and those fish which produced viable eggs generally produced tolerant ones. After hatching, the larvae were also more tolerant. Accompanying the shift in meHg tolerance was a trend of increasing fin ray count of the females, a parameter which has previously been correlated with the production of more tolerant embryos. In 1982, a nearby population exhibited a distribution of embryonic and larval tolerance and fin ray count comparable with that of the first population in the other years.We hypothesize that the unusually high rainfall in June, 1982 caused an inflow of pesticides and heavy metals from a golf course adjacent to the first site, and that the contaminants were responsible for the striking changes in reproductive success and meHg tolerance. Residues of chlorinated hydrocarbon insecticides were found in fish of the first, but not of the second, population. The variability in the original population may have permitted a segment of it to withstand the inflow of pollutants, while the susceptible individuals produced non-viable eggs. This may have been the cause of the rapid shift of overall tolerance in the population.     

Endocrine disruption: thyroid dysfunction in mummichogs (Fundulus heteroclitus) from a polluted habitat.

Mummichogs (Fundulus heteroclitus) from Piles Creeks (PC), New Jersey (a polluted site), are sluggish and show poorer prey capture and predator avoidance than reference fish from Tuckerton (TK). The behavioral dysfunction of the PC fish may be associated with thyroid impairment due to endocrine disruption. In this study, we compared thyroid histology and thyroid hormones in the two populations and determined experimentally whether the polluted environment could alter thyroid hormone levels. PC fish had larger thyroid follicles, greater follicle cell heights, and contained higher plasma thyroxine (T4) levels than TK fish. However, there were no significant differences in either plasma or tissue triiodothyronine (T3). TK fish held in simulated PC environments had higher plasma T4 and lower plasma T3 than field-sampled fish. PC fish held in clean water had lower plasma T4 and T3 than field-sampled fish. In either case, there was no significant difference in tissue T3 content. The contaminants in PC alter thyroid structure and function, which may relate to the behavioral differences between fish from the polluted and reference sites.     

Effects of salinity on sublethal toxicity of atrazine to mummichog (Fundulus heteroclitus) larvae

In coastal marshes, fish larvae may be exposed simultaneously to extreme salinities and to atrazine, a widely used herbicide. To assess the effects of salinity on the toxicity of atrazine, newly-hatched mummichog (Fundulus heteroclitus) were exposed to atrazine (0, 5, 50 and 500 microg/L) at three salinities (3, 15 and 35PSU). Whole body cortisol was measured after 24 h. Body length, condition factor, whole body proteins, lipids, residual masses and water contents were assessed after 96 h. Significants effects were found for both condition factor and water content. Condition factors were lower at salinity 3 and 35 compared to near isoosmotic salinity, 15PSU. In addition, atrazine decreased condition factor at 500 microg/L. Reduction in condition was likely due to retardation in axial growth since body length, percentages of proteins or lipids were not affected. In the absence of atrazine, salinity had no effect on the prevalence of dehydrated (81% water) or hyperhydrated (85% water) larvae. In larvae exposed to atrazine, the prevalence of hyperhydrated larvae increased at 3PSU and 5 microg/L atrazine and that of dehydrated larvae increased at 15 and 35PSU and 5 microg/L atrazine. Severity of dehydratation increased with atrazine concentration at salinity 35PSU. Thus, a short-term exposure to environmentally realistic concentrations of atrazine affects osmotic control in mummichog larvae with possible effects on buoyancy, survival and recruitment.     

Dietary cadmium and benzo(a)pyrene increased intestinal metallothionein expression in the fish Fundulus heteroclitus

Fish were individually fed food pellets containing cadmium, benzo(a)pyrene, or a combination of the two, then analyzed for metallothionein mRNA expression in the intestine, liver, and gill using real-time RT-qPCR. An initial experiment using only cadmium showed that ingestion of pellets varied in individual fish, and estimates of cadmium dose from the numbers of ingested pellets indicated considerable individual variability in cadmium dose. Induction of intestinal metallothionein mRNA was apparent, however, and a linear dose–response relationship was observed for metallothionein expression and cadmium dose in the intestine, but not the other organs, which showed no induction. In a second experiment, the entire daily cadmium dose was provided in a single contaminated pellet that was consumed by all treated fish, effectively eliminating the effect of variable ingestion rates on dose, and the interaction between cadmium and benzo(a)pyrene was also investigated. The intestine was again the primary organ for metallothionein induction by cadmium. When benzo(a)pyrene was administered together with cadmium, induction of metallothionein was potentiated by the presence of benzo(a)pyrene, with the main effect seen in the intestine, where already high levels of induction by cadmium alone increased by 1.74-fold when benzo(a)pyrene was present.     

Impact of benzyl butyl phthalate on shoaling behavior in Fundulus heteroclitus (mummichog) populations

Fundulus heteroclitus preference for association with familiar conspecifics of similar body length was impacted by benzyl butyl phthalate (BBP); this was found to be a statically significant result with a p < 0.0001. When presented with equally sized shoals consisting of either large or small fish, the majority of unexposed (84%) and acetone exposed control (82%) fish selected the shoal of large fish. A small number of control fish chose either the shoal of small fish (6% and 10%) or the neutral zone (10% and 8%) where they were clear morphological outliers. Fish exposed to 0.1 mg/L BBP exposure daily for four weeks selected the shoal of small fish more often than unexposed or acetone controls (7.5- and 4.5-fold respectively). They also remained in the neutral zone and displayed agitation at levels more than twice that of control. Agitation and shoal choice disruption are quantifiable behavioral responses that support the use of F. heteroclitus as a model for detecting sub-lethal BBP exposure.     

Patterns of heritability of decreased EROD activity and resistance to PCB 126-induced teratogenesis in laboratory-reared offspring of killifish (Fundulus heteroclitus) from a creosote-contaminated site in the Elizabeth River,VA, USA

Killifish (Fundulus heteroclitus) from a highly contaminated site on the Elizabeth River are resistant to the acute toxicity and the cytochrome P4501A (CYP1A)-inducing activity of both the sediments from the site and chemically pure polycyclic aromatic hydrocarbons (PAHs). These effects are highly heritable for one generation, but heritable to a lesser degree by subsequent generations, in clean conditions in the laboratory. We show that offspring of this population of Elizabeth River killifish are also resistant to the teratogenicity and P4501A-inducing activity of PCB congener 126, a prototypical coplanar halogenated aromatic hydrocarbon (HAH). Furthermore, the pattern of greater resistance to acute toxicity and P4501A-inducing activity in the first generation and less in subsequent generations is also observed upon exposure to PCB-126.     

Immune function, hepatic CYP1A, and reproductive biomarker responses in the gulf killifish, Fundulus grandis, during dietary exposures to endocrine disrupters.

The gulf killifish, Fundulus grandis, was used to determine the influence of biological rhythms on three biomarker responses. We first developed monoclonal antibodies against the model's immunoglobulins and vitellogenin in order to measure antibody responses and vitellogenesis, respectively. We then treated adults with 10, 1, 1, and 10 ppm of Aroclor 1254, tribuyltin, 3-methylcholanthrene, and nonyl-phenol, respectively, in mixtures over a 16-week period. The study followed Vibrio anguillarum-specific antibody responses, hepatic CYP1A, and plasma vitellogenin levels in the morning and again in the evening at 2-week intervals. The contaminated diet suppressed secondary antibody responses, but only in the morning. The contaminated diet also altered CYP1A, but not vitellogenesis. In addition, fish in the control group exhibited daily and seasonal differences in specific antibody levels and CYP1A induction. Moreover, circulating vitellogenin levels in control males sampled in the morning increased throughout the exposure, but remained below those of females. This study underscores the need to consider normal physiological rhythms when employing biomarkers in toxicology.     

Cloning a new cytochrome P450 isoform (CYP356A1) from oyster Crassostrea gigas

We have cloned the full-length cDNA of the first member of a new cytochrome P450 (CYP) family from the Pacific oyster Crassostrea gigas. This new CYP gene was obtained based on an initial 331bp fragment previously identified among the list of the differentially expressed genes in oysters exposed to untreated domestic sewage. The full-length CYP has an open reading frame of 1500bp and based on its deduced amino acid sequence was classified as a member of a new subfamily, CYP356A1. A phylogenetic analysis showed that CYP356A1 is closely related to members of the CYP17 and CYP1 subfamilies. Semi-quantitative RT-PCR was performed to analyze the CYP356A1 expression in different tissues of the oyster (digestive gland, gill, mantle and adductor muscle). Results showed slightly higher CYP356A1 expression in digestive gland and mantle, than the other tissues, indicating a possible role of the CYP356A1 in xenobiotic biotransformation and/or steroid metabolism.     

Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.     

Benzo(a)pyrene induced glycine N-methyltransferase messenger RNA expression in Fundulus heteroclitus embryos

Glycine N-methyltransferase (GNMT) is a mediator in the methionine and folate cycles, and is responsible for the transfer of a methyl group from S-adenosylmethionine (SAM) to glycine forming S-adenosylhomocysteine (SAH) and sarcosine. All the known DNA methyltransferases use SAM as a methyl donor thus, GNMT is critically involved in regulation of DNA methylation. Altered GNMT activities have been associated with liver pathologies including hepatocellular carcinoma. The homotetramer form of GNMT is enzymatically active, but the homodimeric form has been suggested as the 4S PAH-binding protein which may mediate CYP1A expression. To further understand the role of GNMT in benzo(a)pyrene (BaP)-related toxicity, full length Fundulus heteroclitus GNMT cDNA was cloned from adult liver. The open reading frame (ORF) of GNMT is 888 base pairs long and encodes a deduced protein of 295 amino acids which has 74% identity with human GNMT. Expression of GNMT mRNA was determined by quantitative RT-PCR. In unfertilized, 2 days postfertilization (dpf), and 3 dpf embryos GNMT was constitutively higher than in 4, 7, 10 or 14 dpf embryos. Embryos were also exposed to waterborne BaP at 10 and 100 μg L⁻¹, and by 10 dpf the higher BaP dose caused increased expression of GNMT mRNA. These results suggest that PAH exposure may alter expression of an important physiological methylation mediator. Future work will be necessary to determine enzyme level effects of BaP exposure as well.     

Evaluating prey capture by larval mummichogs (Fundulus heteroclitus) as a potential biomarker for contaminants

We evaluated larval prey capture as a "behavioral biomarker" of contamination by examining feeding behavior of larval mummichogs (Fundulus heteroclitus) from many different sites, including a severely contaminated "Superfund" site, moderately contaminated sites, and reference areas. Prey capture ability was related to sediment contaminant levels. The levels of contaminants at a site were highly correlated with each other, so that the impact of individual contaminants was confounded. The number of captures of brine shrimp by mummichog larvae from all sites was highly variable, but significant negative correlations of prey capture were seen with mercury, lead, zinc, cadmium, and PCBs. As observed previously with adults, polyaromatic hydrocarbons (PAHs) did not appear to impair prey capture ability. The only site in which prey capture rates of 8-day old larvae were severely affected was the most highly contaminated Superfund site, Berry's Creek, NJ. This implies that larval prey capture is not as sensitive a behavioral biomarker for contamination as adult behavior studied previously.     

Hepatic histopathology in two populations of the mummichog,Fundulus heteroclitus

The pollution-tolerant mummichog, Fundulus heteroclitus, inhabits a heavily contaminated estuary, Piles Creek (PC), in Linden, NJ. PC contaminants include PAHs, PCBs and many metals, including high Hg and Pb. PC mummichog livers and kidneys were compared with those from a pristine estuary in Tuckerton (TK), NJ, by standard histopathological methods, with quantification by computerized image analysis. PC livers had > 2 x the lipid ratios of TK livers. Cell sizes and shapes were not different between populations. TK livers had a 25% incidence of basophilic areas and an age-related 30% incidence of macrophage aggregates; PC livers had none of these lesions. PC livers had a 100% incidence of necrotic foci, compared with 55% of TK livers. Inflammation was noted in 50% of TK livers but only 27% of PC livers. Curiously, the TK livers had a 45% incidence of metacercarial cysts of a digenetic trematode, while none were found in the PC livers. The TK focal necrosis and macrophage aggregates coincided with parasite infestation. There were no observed kidney lesions in either population. Positive biomarkers in this species thus included high hepatic lipid content, possibly necrosis, but no other lesions. The lack of trematode cysts in PC livers may reflect the lack of an intermediate host in this low biodiversity estuary.     

DNA adducts in hematopoietic tissues and blood of the mummichog (Fundulus heteroclitus) from a creosote-contaminated site in the Elizabeth River, Virginia.

Hydrophobic DNA adducts were examined in liver, anterior kidney, spleen, and blood of tumor-prone mummichog (Fundulus heterclitus) from the creosote-contaminated Atlantic Wood (AW) site (Elizabeth River, Virginia). DNA adducts eluted in a diagonal radioactive zone, characteristic of polycyclic aromatic hydrocarbon exposure, in all examined tissues of AW fish. Mummichog demonstrated significantly higher levels of DNA adducts in spleen (394 +/- 109 nmol adducts/mol nucleotides) than in liver (201 +/- 77 nmol adducts/mol nucleotides) or anterior kidney (211 +/- 68 nmol adducts/mol nucleotides; P = 0.036). The levels of DNA adducts in the pooled blood (pool of four) were 142 nmol adducts/mol nucleotides. DNA adducts were not detected in the liver, anterior kidney, spleen and blood of fish collected from the reference site (< 2 nmol adducts/mol nucleotides). The high levels of DNA adducts detected in tissues of AW mummichog may be linked to the increased cancer incidence and immunosuppression in this population.     

Development of thyroid abnormalities in mummichogs,Fundulus heteroclitus,from a polluted site

Previous studies showed that adult and 6-month-old young-of-year Fundulus heteroclitus from a polluted site have enlarged thyroid follicles and (in adults) higher thyroxine levels compared to fish from an unpolluted reference site. This study's purpose was to discover when in development these differences begin to appear. For 15-20 mm fish, the average follicle size of the polluted population was significantly larger than that of the reference population, similar to the pattern found in older fish. However, for the smaller size (12-14.5 mm), the average follicle size of fish from the polluted site was significantly smaller. Although not statistically significant, radioimmunoassay measurements of thyroxine levels were consistent with follicle differences: in larger sizes (20-25 and 15-20 mm) fish from the polluted site had higher thyroxine levels but the smaller size (12-14.5 mm) had lower levels. Therefore, the thyroid abnormalities seen in adults begin when the fish are approximately 15-20 mm long. However, the follicle size reversal for the 12-14.5 mm range may also indicate abnormality at that developmental stage.     

cDNA cloning,sequencing, and differential expression of a heart-type fatty acid-binding protein in the mummichog (Fundulus heteroclitus)

A heart fatty acid-binding protein (H-FABP) cDNA was isolated from the liver of the mummichog (Fundulus heteroclitus) and the complete cDNA sequence and predicted amino acid sequence was determined. This cDNA binds to a mRNA product of 0.95 kb with the highest level of RNA expression in the male liver, gills, and gonads. Exposure of mummichogs to the polycyclic aromatic hydrocarbon (PAH) pyrene significantly down-regulated H-FABP expression at the highest concentration. This study provides evidence that environmentally relevant toxicants can modulate H-FABP expression.