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.     

Differential gene expression in mummichogs (Fundulus heteroclitus) following treatment with pyrene: comparison to a creosote contaminated site

Mummichogs, Fundulus heteroclitus, an estuarine fish with a relatively small home range found along the eastern coast of the United States are well-suited to monitoring contaminant effects, including those of polycyclic aromatic hydrocarbons (PAHs). One of the common PAHs in estuaries is pyrene. We report here on efforts to develop multiple biomarkers of pyrene exposure in this species. Adult male mummichogs were exposed in the laboratory to the weak aryl hydrocarbon receptor (AhR) agonist pyrene at 0, 30, or 50 microg/L in 7-day static renewal exposures. The RNA was extracted from livers and alterations in mRNA expression were assessed by subtractive hybridization and differential display in order to produce multiple biomarkers of pyrene exposure. Genes demonstrating differential expression were confirmed by quantitative-PCR (Q-PCR) and include cytochrome P-450 1A (CYP1A), a putative hepatocyte growth factor activator, a X-ray inducible retrotransposon, and several expressed sequenced tags (ESTs). Some of these genes represent new biomarkers of pyrene exposure and potential biomarkers of PAH exposure. Therefore, similar changes were investigated at a Superfund site in Charleston, SC. Mummichogs from a creosote contaminated site and from a reference site (North Inlet National Estuarine Research Reserve near Georgetown, SC) were trapped, RNA extracted from the livers, and Q-PCR performed. Many of the genes differentially expressed following pyrene exposure were not altered at the creosote contaminated site in comparison to the reference site. However, CYP1A and an EST were induced. CYP1A induction at Diesel Creek indicates that this population of fish does not demonstrate refractory CYP1A phenotypes observed at several sites with high levels of AhR agonists. Ultimately, we anticipate that the use of multiple biomarkers of PAH exposure will provide useful information on the potential effects of toxicants.     

Interactive effects of cadmium and benzo[a]pyrene on metallothionein induction in mummichog (Fundulus heteroclitus).

Previous experiments demonstrated that exposure of mummichog to cadmium (Cd) in combination with benzo[a]pyrene (BaP) caused a higher mortality than would be expected from simple additive effects. Experiments are described here that investigated whether BaP exposure inhibits the induction of metallothionein (MT), a major detoxifying protein for Cd, or if reactive BaP metabolites compete with Cd for binding sites on MT. Fish were injected with or without BaP (18 mg/kg) in combination with a low (1 mg/kg) or high (3.2 mg/kg) dose of Cd, and in one treatment BP was dosed 4 days after Cd. The results showed a rapid induction of MT to 1.5 mg/g wet weight liver, 1 day after injecting the low Cd dose. Simultaneous BaP exposure significantly delayed the induction of MT, for both low and high Cd doses, and BaP temporarily lowered the induced MT concentration when dosed 4 days after induction by Cd. To test if binding of BaP metabolites to MT reduces the detoxification potential for Cd, microsomes of CYP1A-induced fish were incubated with MT and radiolabeled BaP. Active metabolism of BaP was observed by high-performance liquid chromatography analysis, but no association of BaP metabolites with MT was found. Neither could this be demonstrated in vivo, in liver MT isolated from mummichog dosed with 3H-BaP and Cd. These results suggest that increased toxicity of Cd in combination with BaP exposure is likely to be caused by inhibited MT synthesis, rather than by interference of BaP metabolites with Cd binding on MT.     

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.     

苯并(a)芘和芘对梭鱼肝脏DNA损伤的研究

用苯并(a)芘、芘以及它们的等量混和物,分别在浓度为0.1,1,10,20,50μg/dm3浓度下对梭鱼暴污,5d后取梭鱼肝脏和鳃用碱解旋法分别测定其DNA的损伤,结果随着污染物浓度的增加,肝脏DNA损伤程度增加;在相同浓度下,苯并(a)芘和芘的联合毒性大于苯并(a)芘和芘分别作用时的毒性之和。所以苯并(a)芘和芘对DNA损伤的联合作用应为加强作用。     

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.     

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.     

Evidence for resistance to benzo[a]pyrene and 3,4,3'4'-tetrachlorobiphenyl in a chronically polluted Fundulus heteroclitus population

Halogenated aromatic hydrocarbons (HAHs) and polynuclear aromatic hydrocarbons (PAHs) are major environmental contaminants. Fish species that are chronically exposed to these compounds can develop resistance to their toxic effects. In all fish species studied to date, toxicant resistance has been accompanied by decreased inducibility of the xenobiotic metabolizing enzyme, cytochrome P450 1A (CYPIA). CYP1A induction is mediated through the Aryl Hydrocarbon Receptor (AHR). Although these compounds mediate their effects through this pathway, there have been resistant populations in which one chemical class cannot induce CYPIA expression (HAHs) while the other (PAHs) can. Resistance to PAHs was examined in a HAH-resistant population of Fundulus heteroclitus collected from a site contaminated with both compound classes (Newark Bay, NJ). Fish were injected intraperitoneally with the HAH 3,4,3',4'-tetrachlorobiphenyl (PCB77), benzo[a]pyrene (B[a]P, a PAH) or vehicle and sacrificed after 2 (B[a]P) or 5 days (PCB77, vehicle). We found no significant increase in CYP1A mRNA levels in resistant Newark Bay F. heteroclitus treated with either B[a]P or PCB77, while there was a 3.9 fold (PCB77) and 4.2 fold (B[a]P) increase in CYP1A mRNA in Flax fish relative to controls. AHR labeling studies revealed significantly (P < 0.05) lower levels of hepatic AHR in Newark fish (1,770 +/- 1,693.2 DPM) relative to Flax fish (6,082.5 +/- 1,709.9 DPM). Overall, these data suggest Newark F. heteroclitus are resistant to both PAHs and HAHs at the level of CYP1A mRNA, which might be mediated, in part, though lower expression of AHR. We are currently studying the promoter sequence to determine its role in chemical resistance.     

Effects of single and combined exposures to benzo(a)pyrene and 3,3'4,4'5-pentachlorobiphenyl on EROD activity and development in Fundulus heteroclitus

The goal of this study was to assess the interactive effects of a polynuclear aromatic hydrocarbon (PAH), benzo(a)pyrene (BaP), and a co-planar halogenated aromatic hydrocarbon (HAH), 3,3'4,4'5-pentachlorobiphenyl (PCB 126) on ethoxyresorufin-O-deethylase (EROD) activity and occurrence of deformities in Fundulus heteroclitus. While each compound administered alone elicited dose-dependent increases in EROD activity, the combined treatment of these two compounds generally did not elicit an additive EROD response. There was a significant correlation between deformity occurrence and EROD induction in embryos dosed with PCB alone but not for embryos dosed with BaP alone, or a combination of BaP and PCB 126.     

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.     

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.     

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.     

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.     

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.     

Altered feeding habits and strategies of a benthic forage fish (Fundulus heteroclitus) in chronically polluted tidal salt marshes

Responses in feeding ecology of a benthic forage fish, mummichogs (Fundulus heteroclitus), to altered prey resources were investigated in chronically polluted salt marshes (the Arthur Kill-AK, New York, USA). The diet niche breadth of the AK populations of mummichogs was significantly lower than that of the reference population, reflecting reduced benthic macroinfaunal species diversity. Most of the AK populations also had 2-3 times less food in their gut than the reference population. This disparity in gut fullness among the populations appeared to be partly due to ingested prey size shifts; some of the AK populations ingested fewer large prey than the reference population. Furthermore, benthic assemblages were strongly associated with sediment-associated mercury; gut fullness of the AK populations also significantly decreased with increasing mercury body burdens. These results indicate that chronic pollution may have directly (chemical bioaccumulation) and indirectly (reduced prey availability) altered the feeding ecology of mummichogs.     

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.     

Benzo(a)pyrene in the sediment of Osaka Bay and Keihin Canal and its estimated sources

Benzo(a)pyrene (BP) in the sediment of Osaka Bay were determined. High concentration was found at two stations near the mouth of Shin-Yodo and off Kobe respectively (0.98g g^–1 dry mud basis, 1.1g g^–1). The concentration decreases regularly from the mouth of the river further into the bay. The other supplemental determination was carried on the sediment and the seawater of Keihin Canal. Notable BP concentration of 89g g^–1 was observed in the sediment beneath the effluent outlet of a gasworks. The BP in the sediment near the ironworks was also considerable, while the BP was relatively less in the sediment beneath the effluent outlet of refineries. These data suggest that coke plants working in gasworks or ironworks may be the larger sources of BP than refineries. The results of Osaka Bay coincide with this hypothesis.Cadmium, lead, copper, zinc, nickel, ignition loss and fine sand content were measured from the same sediment samples of Osaka Bay. Cd, Pb, Cu and ignition loss showed the regular distributions which resemble to BP and accordingly, good correlations with BP. No significant correlations were found between BP and Ni, Zn and fine sand content which showed no regular distributions.     

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.