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

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

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

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

Standardizing the determination and interpretation of Pcrit in fishes.

The critical oxygen tension (Pcrit) for fishes is the oxygen level below which the rate of oxygen consumption (MO2 ) becomes dependent upon ambient oxygen partial pressure (PO2 ). We compare multiple curve-fitting approaches to estimate Pcrit of the Gulf killifish, Fundulus grandis, during closed and intermittent-flow respirometry. Fitting two line segments of MO2  versus PO2  produced high and variable estimates of Pcrit, as did nonlinear regression using a hyperbolic (Michaelis-Menten) function. Using nonlinear regression fit to an exponential (modified Weibull) function, or linear regression of MO2 versus PO2  at low PO2 , and determining Pcrit as the PO2  when MO2 equals standard metabolic rate (SMR) yielded values that were consistent across fish and among experimental trials. The magnitude of the difference in Pcrit determined by alternative calculation methods exceeded the differences determined in closed and intermittent-flow respirometry, highlighting the need to standardize analytical as well as experimental approaches in determining Pcrit.

Polychlorinated biphenyl (PCB) contamination in Galveston Bay, Texas: Comparing concentrations and profiles in sediments, passive samplers, and fish.

The industrialized portion of the Houston Ship Channel (HSC) is heavily contaminated with anthropogenic contaminants, most prominent of which are the polychlorinated biphenyls (PCBs). This contamination has driven adaptive evolution in a keystone species for Galveston Bay, the Gulf killifish (Fundulus grandis). We investigated the geographical extent of PCB impacts by sampling 12 sites, ranging from the heavily industrialized upper portion of the HSC to Galveston Island. At each site, PCB concentrations and profiles were determined in three environmental compartments: sediment, water (polyethylene passive samplers), and fish tissue (resident Gulf killifish). We observed a steep gradient of PCB contamination, ranging from 4.00 to 100,000 ng/g organic carbon in sediment, 290-110,000 ng/g lipid in fish, and 4.5-2300 ng/g polyethylene in passive samplers. The PCB congener profiles in Gulf killifish at the most heavily contaminated sites were shifted toward the higher chlorinated PCBs and were highly similar to the sediment contamination profiles. In addition, while magnitude of total PCB concentrations in sediment and total fish contamination levels were highly correlated between sites, the relative PCB congener profiles in fish and passive samplers were more alike. This strong correlation, along with a lack of dependency of biota-sediment accumulation factors with total contamination rates, confirm the likely non-migratory nature of Gulf killifish and suggest their contamination levels are a good site-specific indicator of contamination in the Galveston Bay area. The spatial gradient of PCB contamination in Galveston Bay was evident in all three matrices studied and was observed effectively using Gulf killifish contamination as an environmentally relevant bioindicator of localized contamination in this environment.

Evolved resistance to PCB- and PAH-induced cardiac teratogenesis, and reduced CYP1A activity in Gulf killifish (Fundulus grandis) populations from the Houston Ship Channel, Texas.

The Houston Ship Channel (HSC), connecting Houston, Texas to Galveston Bay and ultimately the Gulf of Mexico, is heavily industrialized and includes several areas that have historically been identified as containing significant levels of mercury, dioxins, furans, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs). Gulf killifish, Fundulus grandis, inhabit this entire estuarine system, including the most contaminated areas. F. grandis is the sister species of the well-established estuarine model organism Fundulus heteroclitus, for which heritable resistance to both PCB and PAH toxicity has been documented in several populations. F. grandis collected from two Superfund sites on the HSC and from a reference population were used to establish breeding colonies. F1 embryos from HSC populations were approximately 1000-fold more resistant to PCB126- and 2-5-fold more resistant to coal tar-induced cardiovascular teratogenesis, relative to embryos from the reference population. Reciprocal crosses between reference and contaminated populations exhibit an intermediate level of resistance, confirming that observed protection is genetic and biparentally inherited. Ethoxyresorufin-O-deethylase (EROD) data confirm a reduction in basal and induced cytochrome P4501A (CYP1A) activity in resistant populations of F. grandis. This result is consistent with responses previously described for resistant populations of F. heteroclitus, specifically a recalcitrant aryl hydrocarbon receptor (AHR) pathway. The decreased levels of cardiovascular teratogenesis, and decrease in CYP1A inducibility in response to PCB126 and a PAH mixture, suggest that HSC F. grandis populations have adapted to chronic contaminants exposures via a mechanism similar to that previously described for F. heteroclitus. To the best of our knowledge, this is the first documentation of evolved pollution resistance in F. grandis. Additionally, the mechanistic similarities between the population adaptation observed in this study and previous work in F. heteroclitus suggest that genetic variation predating the evolutionary divergence of these two species may best explain the apparent rapid parallel evolution of pollution resistance in genetically and geographically distinct species and populations.