R.T. Di Giulio

Developmental exposure to a complex PAH mixture causes persistent behavioral effects in naive Fundulus heteroclitus (killifish) but not in a population of PAH-adapted killifish.

Acute exposures to some individual polycyclic aromatic hydrocarbons (PAHs) and complex PAH mixtures are known to cause cardiac malformations and edema in the developing fish embryo. However, the heart is not the only organ impacted by developmental PAH exposure. The developing brain is also affected, resulting in lasting behavioral dysfunction. While acute exposures to some PAHs are teratogenically lethal in fish, little is known about the later life consequences of early life, lower dose subteratogenic PAH exposures. We sought to determine and characterize the long-term behavioral consequences of subteratogenic developmental PAH mixture exposure in both naive killifish and PAH-adapted killifish using sediment pore water derived from the Atlantic Wood Industries Superfund Site. Killifish offspring were embryonically treated with two low-level PAH mixture dilutions of Elizabeth River sediment extract (ERSE) (TPAH 5.04 μg/L and 50.4 μg/L) at 24h post fertilization. Following exposure, killifish were raised to larval, juvenile, and adult life stages and subjected to a series of behavioral tests including: a locomotor activity test (4 days post-hatch), a sensorimotor response tap/habituation test (3 months post hatch), and a novel tank diving and exploration test (3months post hatch). Killifish were also monitored for survival at 1, 2, and 5 months over 5-month rearing period. Developmental PAH exposure caused short-term as well as persistent behavioral impairments in naive killifish. In contrast, the PAH-adapted killifish did not show behavioral alterations following PAH exposure. PAH mixture exposure caused increased mortality in reference killifish over time; yet, the PAH-adapted killifish, while demonstrating long-term rearing mortality, had no significant changes in mortality associated with ERSE exposure. This study demonstrated that early embryonic exposure to PAH-contaminated sediment pore water caused long-term locomotor and behavioral alterations in killifish, and that locomotor alterations could be observed in early larval stages. Additionally, our study highlights the resistance to behavioral alterations caused by low-level PAH mixture exposure in the adapted killifish population. Furthermore, this is the first longitudinal behavioral study to use killifish, an environmentally important estuarine teleost fish, and this testing framework can be used for future contaminant assessment.

Resistance to teratogenesis by F1 and F2 embryos of PAH-adapted Fundulus heteroclitus is strongly inherited despite reduced recalcitrance of the AHR pathway.

Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood Superfund site on the Elizabeth River (Portsmouth, VA, USA) are exposed to a complex mixture of polycyclic aromatic hydrocarbons (PAHs) from former creosote operations, but are resistant to the acute toxicity and cardiac teratogenesis caused by PAHs. The resistance is associated with a dramatic recalcitrance to induction of cytochrome P450 (CYP1) metabolism enzymes following exposure to aryl hydrocarbon receptor (AHR) agonists, along with an elevated antioxidant response and increased expression of several other xenobiotic metabolism and excretion enzymes. However, the heritability of the resistance in the absence of chemical stressors has been inconsistently demonstrated. Understanding the heritability of this resistance will help clarify the nature of population-level responses to chronic exposure to PAH mixtures and aid in identifying the important mechanistic components of resistance to aryl hydrocarbons. We compared the response of Atlantic Wood F1 and F2 embryos to benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126), and a mixture of BkF and fluoranthene (Fl) to that of F1 embryos of reference site killifish. Resistance to cardiac teratogenesis and induction of CYP mRNA expression and CYP activity was determined. We found that both Atlantic Wood F1 and F2 embryos were highly resistance to cardiac teratogenesis. However, the resistance by Atlantic Wood F2 embryos to induction of CYP mRNA expression and enzyme activity was intermediate between that of Atlantic Wood F1 embryos and reference embryos. These results suggest that resistance to cardiac teratogenesis in Atlantic Wood fish is conferred by multiple factors, not all of which appear to be fully genetically heritable.

Vitellogenin-associated maternal transfer of exogenous and endogenous ligands in the estuarine fish, Fundulus heteroclitus

Abstract Sequestration of large quantities of vitellogenin (VTG) is critical for proper oocyte development in most oviparous vertebrates. While previous studies have shown a general correlation between oocyte growth and the accumulation of various exogenous and endogenous ligands, few studies have attempted to elucidate the role VTG plays in this maternal transfer. In the present study, we have demonstrated that oocytic accumulation of [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (3H-TCDD) and [14C]-benzo(a)pyrene (14C-BaP) by gravid Fundulus heteroclitus is directly correlated with oocyte maturational status. A positive correlation was observed between oocyte maturational state (size) and both total quantity (total pmoles) as well as concentration (pmoles/g tissue) of TCDD and BaP. Further, the bi-phasic accumulation of both TCDD and BaP mirrors that previously observed for protein sequestration by cultured Fundulus oocytes. Additionally, both TCDD and BaP were associated with VTG in vivo. HPLC analysis of serum extracts has shown that VTG associates with both parent BaP and BaP metabolites. Studies with 125I-T4 (thyroxine) also suggest that this critical hormone also associates with VTG in vivo. Ongoing studies are examining the VTG-associated transport and oocytic sequestration of 125I-T4 in gravid Fundulus.

Resistance to polycyclic aromatic hydrocarbon toxicity and associated bioenergetic consequences in a population of Fundulus heteroclitus

Several locations in the Elizabeth River, VA, USA are highly contaminated with polycyclic aromatic hydrocarbons (PAHs) due to the release of creosote mixtures from wood treatment facilities. Interestingly, some populations of Atlantic killifish (Fundulus heteroclitus) inhabiting the Elizabeth River (ER) are resistant to PAH-induced teratogenesis. However, evolutionary resistance to PAHs due to chronic PAH exposure is associated with reduced fitness and increased susceptibility to other environmental stressors in at least one PAH-resistant ER killifish population. More specifically, wild-caught and first generation PAH-resistant juvenile killifish have altered metabolic demands when compared to non-resistant fish. Herein, we investigated this association further by examining a previously under-studied population captured from the creosote-contaminated site Republic Creosoting (Rep). We assessed PAH toxicity and effects on energy metabolism in Rep killifish in comparison with killifish from the reference site Kings Creek (KC). Following exposures to simple and complex PAH mixtures, Rep killifish exhibited several phenotypes associated with PAH resistance including decreased incidences of developmental cardiovascular deformities and recalcitrant cytochrome P450 1A (CYP1A) activity. We evaluated bioenergetics in killifish embryos throughout development and found elevated basal oxygen consumption rates in Rep embryos relative to KC embryos. Furthermore, juvenile F1 Rep fish had significantly lower maximal metabolic rates and aerobic scopes than KC juveniles. These results suggest that populations of killifish that have adapted or evolved to withstand the toxicity associated with PAHs consequently have altered energetic metabolism or demands. Such consequences could result in an enhanced vulnerability to other environmental and anthropogenic stressors in PAH-resistant killifish.

Embryonic cardiotoxicity of weak aryl hydrocarbon receptor agonists and CYP1A inhibitor fluoranthene in the Atlantic killifish (Fundulus heteroclitus).

High affinity aryl hydrocarbon receptor (AHR) ligands, such as certain polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause severe cardiac teratogenesis in fish embryos. Moderately strong AHR agonists, for example benzo[a]pyrene and β-naphthoflavone, are capable of causing similar cardiotoxic effects, particularly when coupled with cytochrome P450 1A (CYP1A) inhibitors (e.g., fluoranthene (FL). Additionally, some weaker AHR agonists (carbaryl, 2-methylindole, 3-methylindole, and phenanthrene) are known to also cause cardiotoxicity in zebrafish (Danio rerio) embryos when coupled with FL; however, the cardiotoxic effects were not mediated specifically by AHR stimulation. This study was performed to determine if binary exposure to weak AHR agonists and FL were also capable of causing cardiotoxicity in Atlantic killifish Fundulus heteroclitus embryos. Binary exposures were performed in both naïve and PAH-adapted killifish embryos to examine resistance to weak agonists and FL binary exposures. Weak agonists used in this study included the following: carbaryl, phenanthrene, 2-methylindole, 3-methylindole, indigo, and indirubin. Carbaryl, indigo, and indirubin induced the highest CYP1 activity levels in naïve killifish embryos, but no significant CYP1 induction was observed in the PAH-adapted killifish. Embryos were coexposed to subteratogenic levels of each agonist and 500μg/L FL to assess if binary administration could cause cardiotoxicity. Indigo and indirubin coupled with FL caused cardiac teratogenesis in naïve killifish, but coexposures did not produce cardiac chamber abnormalities in the PAH-adapted population. Knockdown of AHR2 in naïve killifish embryos did not prevent cardiac teratogenesis. The data suggest a unique mechanism of cardiotoxicity that is not driven by AHR2 activation.

Comparative time-course of benzo[a]pyrene-dna adduct formation, and its relationship to CYP1A activity in two species of catfish

Abstract Studies have demonstrated differences between the brown bullhead (Ameriurus nebulosus) (BB) and a closely related species of catfish, the channel catfish (Ictalurus punctatus) (CC) in activity of important liver enzymes that may affect their ability to activate or detoxify certain procarcinogens. We have measured the formation and persistence of benzof[a]pyrene-DNA adducts in the liver of these two species using the 32P-postlabeling (PPL) method, along with the effect of prior induction by β-naphthoflavone (βNF). In this time-course study, a total of 80 fish from each species were used. Half were induced with βNF (i.p. injections of 10 mgkg−1 at day 3 and 2) followed by a single benzo[a]pyrene (BaP) i.p. injection (20 mg kg−1). Liver DNA and microsomes were sampled on day 1, 3, 7, 14 and 45 after the BaP dosage—adducts and ethoxyresorufin-O-deethylase (EROD) activity were analyzed. EROD activity in CC was significantly higher at most time points than in BB. In contrast, the BaP-DNA adduct profile revealed a significantly higher level of adducts in the BB than the CC. Adduct levels at most time points were significantly higher in the BB than in the CC at p

Increased sensitivity to oxidative stress in a creosote-adapted population of mummichog (Fundulus heteroclitus)

Abstract With increasing frequency, aquatic organisms inhabit regions where biochemical adaptations to contaminants are essential for survival. The resultant selection for individuals better adapted to a polluted environment may result in a population better able to survive certain stressors, but less fit in terms of other stressors. Research with F 1 offspring of the mummichog Fundulus heteroclitus collected from a highly creosote-contaminated site on the Elizabeth River has explored the nature of the resistance demonstrated by this population, as well as some of the costs associated with it. F 1 larvae and juveniles from the Elizabeth River were more able to survive exposure to contaminated sediments from the Elizabeth River, either in the presence or absence of UV light, than were F 1 offspring of control site (York River) Fundulus . On the other hand, the Elizabeth River F 1 population was more susceptible to phototoxicity as mediated by pure anthracene or fluoranthene, indicating a possible cost of adaptation. Furthermore, preliminary studies suggest that Elizabeth River Fundulus are more sensitive to another source of oxidative stress, H 2 O 2 . Ongoing studies are exploring the biochemical and genetic bases of these differences.

Differential phase I and phase II enzyme activities in brown bullhead and channel catfish exposed to β-naphthoflavone

Abstract This research explores biochemical mechanisms underlying the differential susceptibility of two related species of Ictaluridae (channel catfish and brown bullhead) to polycyclic aromatic hydrocarbon (PAH)-mediated liver cancer. The hypothesis is that the observed difference in the relatively insensitive catfish versus more sensitive bullhead is due to differences in hepatic phase II or epoxide hydrolase (EH) activities which could enhance excretion of PAHs. Fish of each species were treated with either corn oil or β-naphthoflavone (10 mg/kg in corn oil) at both 3 and 2 days prior to first sampling. Microsomal and cytosolic liver preparations from four fish per treatment were prepared 2, 7, and 14 days after the final dosing. EROD activities were higher in catfish as previously reported. In contrast, 1-chloro-2,4-dinitrobenzene-GST activities were significantly higher in bullhead. GST was not induced in either species in dosed versus control animals. An EH assay using benzo[a]pyrene-7,8-epoxide was optimized with catfish microsomes. Activities were 5.2±0.57 nmol/mg/min and did not show time or dose-related differences. Bullhead appear to have higher constitutive EH activities compared to catfish. Current studies are ongoing with EH inhibitors to investigate this difference.

Genome-wide scan reveals signatures of selection related to pollution adaptation in non-model estuarine Atlantic killifish (Fundulus heteroclitus)

In many human-altered ecosystems, organisms are increasingly faced with more diverse and complex environmental stressors and pollutant mixtures, to which the adaptations necessary to survive exposure are likely to be numerous and varied. Improving our understanding of the molecular mechanisms that underlie complex polygenic adaptations in natural settings requires significant toxicological, biochemical, physiological, and genomic data rarely available for non-model organisms. Here, we build upon two decades of study of adaptation to anthropogenic pollutants in a population of Atlantic killifish (Fundulus heteroclitus) that inhabits the creosote-contaminated Atlantic Wood Industries Superfund (AW) site on the Elizabeth River, Virginia in the United States. To better understand the genotypes that underlie previously characterized resistance to PCBs and PAHs, we performed Restriction site-Associated DNA sequencing (RADseq) on killifish from AW and two relatively clean reference sites (Kings Creek-KC, and Mains Creek-MC). Across the genome, we analyzed over 83,000 loci and 12,000 single nucleotide polymorphisms (SNPs). Shared across both comparisons of killifish from polluted (AW) and relatively unpolluted (KC and MC) sites, we found eight genomic regions with smoothed FST values significantly (p < 0.001) elevated above background. Using the recently published F. heteroclitus reference genome, we identified candidate genes in these significant regions involved in the AHR pathway (e.g. AIP, ARNT1c), as well as genes relating to cardiac structure and function. These genes represent both previously characterized and potentially novel molecular adaptations involved with various aspects of resistance to these environmental toxins.

Erratum to: Antioxidant Rescue of Selenomethionine-Induced Teratogenesis in Zebrafish Embryos.

The original publication incorrectly states that manganese superoxide dismutase-2 (sod2) was measured during the experiment. Copper/zinc superoxide dismutase-1 (sod1) was actually measured using the following primers for sod1 (GeneBank ID Y12236): forward primer 50-CTA GCC CGC TGA CAT TAC ATC-30, reverse primer 50-TTG CCC ACA TAG AAA TGC AC-30. The primer sequences listed in Table 1 for sod2 are incorrect.