Lack of CYP1A responsiveness in species inhabiting chronically contaminated habitats: two varieties of resistance?

Organisms chronically exposed to organic pollutants such as polychlorinated biphenyls (PCBs) can develop resistance to these chemicals, a condition associated with reduced inducibility of the biomarker enzyme cytochrome P450 1A (CYP1A). This study addresses the CYP1A response of members of the families Ictaluridae and Centrarchidae, two fish families found throughout much of the United States. We measured CYP1A expression, PCB body burdens, and conducted CYP1A challenge experiments in species from these families residing in the Town Branch/Mud River system (Logan County, KY, USA), a stream system historically contaminated with high levels of PCBs. Despite PCB concentrations in muscle tissue typically associated with elevated CYP1A (16.7 to 75.2µgPCB/g wet edible flesh), resident fish in the contaminated Town Branch/Mud River sites (yellow bullhead [Ameiurus natalis], green sunfish [Lepomis cyanellus], and spotted bass [Micropterus punctulatus]) had hepatic CYP1A activity levels similar to, rather than higher than, those in reference fish, suggesting reduced sensitivity to CYP1A induction. Lack of CYP1A expression following direct contaminant exposure has often been associated with resistance to those contaminants. To determine if CYP1A in resident populations was resistant to induction by PCBs, we exposed resident fish to a single, intraperitoneal injection with a potent CYP1A inducer, 3,4,3',4'-tetrachlorobiphenyl (PCB 77). PCB 77 treatment significantly induced hepatic CYP1A activity and protein in yellow bullhead from reference, but not contaminated, sites and had no effect on CYP1A in green sunfish from either site. The low CYP1A expression levels in resident fish with elevated PCB body burdens, together with the failure of PCB injection to induce CYP1A in certain populations, indicate an acclimatory CYP1A response in yellow bullheads and likely an inherently resistant CYP1A in green sunfish. This work demonstrates for the first time acclimation of CYP1A to PCBs in a species within the family Ictaluridae and provides further support for our previous work indicating an apparent inherent lack of CYP1A sensitivity to chlorinated inducers in Centrarchids. These traits may explain, at least in part, the common association of these families with degraded habitats and indicate Lepomis members are likely to be excellent candidates for exploring the mechanistic basis of 'inherent' CYP1A resistance. This study also underlines to the need for thorough characterization of the CYP1A responsivity of a population and/or species prior to using CYP1A as a reliable biomonitoring tool.

Differential sensitivity of CYP1A to 3,3',4',4-tetrachlorobiphenyl and benzo(a)pyrene in two Lepomis species.

Although Lepomis species are abundant in a wide variety of habitats throughout North America and could serve as potentially valuable biomonitoring tools, few studies have examined the induction of pollutant biomarkers in this genus. We hypothesized that the induction of cytochrome P-450 1A (CYP1A), a sensitive and widely used indicator of response to aquatic contaminants, would serve as an effective biomarker of organic pollutant exposure in Lepomis species. We examined the response of CYP1A and two of the major pollutant-responsive phase II enzymes, glutathione S-transferase (GST), and uridine diphosphate glucuronyltransferase (UDPGT), in Lepomis exposed to organic pollutants under laboratory and field conditions. Two Lepomis species (longear sunfish, Lepomis megalottis and bluegill, Lepomis macrochirus) were exposed in the laboratory via intraperitoneal injection to corn oil (vehicle), benzo(a)pyrene (BaP) (10 and 50mg/kg), a polynuclear aromatic hydrocarbon (PAH) or 3,4,3',4'-tetrachlorobiphenyl (PCB 77) (0.1 and 1.0mg/kg), a dioxin-like planar halogenated aromatic hydrocarbon (HAH), and sacrificed 2 (BaP) or 7 (corn oil, PCB77) days later. Lepomis hepatic CYP1A exhibited differential sensitivity to these two classes of environmental contaminants. CYP1A activity was weakly induced in bluegill exposed to 1.0mg/kg PCB 77 (3 fold induction over controls) but strongly induced in both bluegill and longear sunfish exposed to 50mg/kg BaP (37 and 15 fold induction over controls, respectively). In contrast, hepatic GST activity in both species remained unchanged following the treatment with either compound and hepatic UDPGT activity, which was assessed only in BaP-treated longear sunfish, was unaffected by that chemical, indicating these phase II enzymes may not be sensitive pollutant biomarkers in this genus. Further, longear sunfish collected from a PCB contaminated site displayed relatively low levels of CYP1A activity despite PCB body burdens associated with strong induction of CYP1A activity in other fish species. The strong induction of CYP1A by BaP with much weaker CYP1A response to PCB indicates that CYP1A in Lepomis sp. could be an excellent biomarker for PAH pollution, but may not be a reliable indicator of site contamination by halogenated hydrocarbons. We conclude that Lepomis species provide a useful model for examining the regulation and potential consequences of differential pollutant sensitivity, but that CYP1A in these species should be used with caution as an indicator of halogenated contaminants.

CYP1A expression in caged rainbow trout discriminates among sites with various degrees of polychlorinated biphenyl contamination.

It has become increasingly apparent that resident fish can develop resistance to chemicals in their environment, thus compromising their usefulness as sentinels of site-specific pollution. By using a stream system whose resident fish appear to have developed pollutant resistance (Brammell et al., Mar Environ Res 58:251-255, 2005), we tested the hypothesis that the pollutant-inducible biomarker, cytochrome P4501A (CYP1A), as measured in field-caged juvenile rainbow trout (Oncorhynchus mykiss), would reflect relative pollution differences between reference and polychlorinated biphenyl (PCB)-contaminated sites. Trout were caged in the Town Branch/Mud River system (Logan County, KY), a stream system undergoing remediation for PCBs. Fish were held in remediated (Town Branch), unremeditated (Mud River), and reference sites for 2 weeks during spring 2002. At the end of this period, gill and hepatic CYP1A expression were measured. To evaluate the relative PCB exposure of caged trout and provide a reference point against which to calibrate CYP1A response, PCB levels were quantified in sediments from each site. Hepatic CYP1A expression in caged trout clearly detected the presence of PCBs in the Town Branch/Mud River stream system. Sediment PCB levels and hepatic CYP1A expression in caged trout produced identical pollution rankings for the study sites. Gill CYP1A expression, although suggestive of site differences, was not statistically different among sites. Unlike resident fish, which failed to show site differences in hepatic CYP1A expression in this waterway (Brammell et al. 2005), caged fish proved to be a sensitive discriminator of relative PCB contamination in this system. In summary, we determined that CYP1A expression in caged fish reflected relative in situ pollutant exposure. The exposure paradigm confirmed that 2 weeks was a sufficient caging period for evaluating CYP1A response in this species at these temperatures (13-19 degrees C). In addition, these studies demonstrate that tissue-specific CYP1A expression can provide insights into likely routes of exposure. We conclude that CYP1A expression in caged trout is a reliable and inexpensive first-pass determination of relative environmental pollutant exposure and bioavailability in aqueous systems.

Apparent lack of CYP1A response to high PCB body burdens in fish from a chronically contaminated PCB site.

Chronic exposure to organic contaminants such as polychlorinated biphenyls (PCBs) can lead to the development of resistance to these chemicals, a condition associated with reduced response of CYP1A1, a pollutant-inducible biomarker. We measured CYP1A activity (ethoxyresorufin o-deethylase, EROD) and PCB concentrations in feral fish from the Town Branch/Mud River system (Logan County, KY), a stream historically contaminated with PCBs and partially remediated. As a first step in evaluating the possible development of resistant populations in this system, we measured CYP1A expression and PCB body burdens in resident fish from sites we previously characterized as containing biologically significant levels of CYP1A inducing compounds. Mean PCB concentrations in edible flesh ranged from 75.2 to 16.7 microg/g in fish collected from Town Branch remediated sites and were relatively low (1.23 microg/g) in Town Branch reference site fish. However, hepatic CYP1A activity was similar among individuals of most species collected from reference and contaminated/remediated sites. The absence of elevated CYP1A levels in resident fish species despite the presence of significant PCB body burdens may indicate these fish have developed reduced sensitivity to CYP1A induction, a condition associated with acquired resistance to toxicants.