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.

Molecular characterization and sex-specific tissue expression of estrogen receptor alpha (esr1), estrogen receptor betaa (esr2a) and ovarian aromatase (cyp19a1a) in yellow perch (Perca flavescens).

Yellow perch (Perca flavescens) exhibits an estrogen-stimulated sexual size dimorphism (SSD) wherein females grow faster and larger than males. To aid in the examination of this phenomenon, the cDNA sequences encoding estrogen receptor-alpha (esr1), estrogen receptor-betaa (esr2a) and ovarian aromatase (cyp19a1a) for the teleost yellow perch were obtained. Several tissues were analyzed from both male and female adult yellow perch for sex-specific tissue expression. The full length cDNAs of yellow perch esr1, esr2a and cyp19a1a consist of 3052 bp, 2462 bp and 1859 bp with open reading frames encoding putative proteins of 576 amino acids, 555 amino acids and 518 amino acids, respectively. Esr1 and esr2a expression was highest in female ovary and liver tissues with low to moderate expression in other tissues. Esr2a showed a more global tissue expression pattern than esr1, particularly in males but also in females. Cyp19a1a expression was highest in both male and female spleen tissue and oocytes with moderate expression in male pituitary and gill tissue. Cyp19a1a expression was moderately high in female liver tissue with undetectable expression in male liver tissue, suggesting its involvement in sexually dimorphic growth. These sequences are valuable molecular tools that can be used in future studies investigating estrogen mechanisms and actions, such as SSD, in yellow perch.

Effect of nutrient availability on the uptake of PCB congener 2,2',6,6'-tetrachlorobiphenyl by a diatom (Stephanodiscus minutulus) and transfer to a zooplankton (Daphnia pulicaria).

The objective of this study was to examine the importance of nutrient status of a diatom (Stephanodiscus minutulus) to the uptake of PCB congener #54 (2,2',6,6'-tetrachlorobiphenyl) and the subsequent transfer of PCB to a pelagic grazing zooplankton (Daphnia pulicaria). The algae, which were grown under different nutrient treatments, were then fed to a zooplankton to examine the subsequent food chain transfer of PCB. Algal cultures were grown for at least 2 weeks in a steady state condition in (1) non-limiting, (2) low-Si, (3) low-N or (4) low-P media. Steady state algal cultures were dosed with 0.2 microg L(-1) PCB and were sampled for PCB uptake after 24h. D. pulicaria were allowed to graze on these same cultures for 48 h before being analyzed for PCB body burdens. Low-Si (68% or 0.135 microg L(-1) of PCB) and low-P cultures (62%) had significantly higher percentage uptake of total PCB than the non-limiting (55%) or low-N (52%) treatments. When these values were divided by biochemical or elemental parameters, PCB per lipids (microg microg(-1)) had one of the lowest coefficients of variation (CV) across the four treatments, indicating their importance in PCB uptake. When equal biovolumes of the four different treatment cultures were fed to zooplankton, both the low-N (13.9 ng PCB mg wet weight(-1)) and the low-P (9.6 ng PCB mg wet weight(-1)) grazing D. pulicaria had significantly higher PCB per wet weight than the low-Si (5.6 ng PCB mg wet weight(-1)) and non-limited (2.6 ng PCB mg wet weight(-1)) grazing D. pulicaria. There were no significant differences between algal nutrient treatments in PCB per wet weight of zooplankton grazing on clean algal food in PCB contaminated media. This study indicates that uptake of PCB by phytoplankton can be significantly altered by nutrient availability which subsequently affects transfer to zooplankton, potentially through such responses as grazing rate and lipid assimilation.

Cadmium uptake by Ceriodaphnia dubia from different exposures: relevance to body burden and toxicity.

Uptake of cadmium from water by freshwater animals has been well documented. However, little attention has been given to the importance of dietary and combined exposure as the source for uptake. In the present study, we compared cadmium uptake from waterborne (water-only), dietary (food-only), and combined (water plus food) exposures on the freshwater cladoceran Ceriodaphnia dubia. Major test end points included cadmium whole-body burdens, feeding rate, and reproduction. The C. dubia three-brood reproduction tests were conducted according to U.S. Environmental Protection Agency methods. The results indicated that cadmium was accumulated from both water and diet. Whereas the results showed that uptake from water was more rapid than uptake from diet, both uptakes occurred independently, and cadmium concentrations in C. dubia were additive from both sources. Furthermore, the present study found cadmium trophic transfer between primary producers (i.e., Pseudokirchneriella subcapitata) and primary consumers (i.e., C. dubia). Feeding rate was reduced from all avenues of exposure. Feeding reduction in combined exposure was affected additively by waterborne and dietary cadmium. Also, reductions in neonate production were observed after exposure to all three exposure types. Both body burden and reduced feeding affected C. dubia reproduction. These results indicate the importance of both waterborne and dietary cadmium exposures as the sources for metal uptake and toxicity.

Toxicity of cadmium to six species in two genera of crayfish and the effect of cadmium on molting success.

Nine acute (96-h) toxicity tests were conducted on six species of crayfish (Cambaridae). Six tests focused on adults, and three tests examined juveniles. Lethal concentration to 50% of a population (LC50) and lethal concentration to 10% of a population (LC10) values, respectively, for the adults of individual test species were as follows: Orconectes juvenilis, 2.44 and 0.623 mg Cd/L; Orconectes placidus, 0.487 and 0.092 mg Cd/L; Orconectes virilis, 3.30 and 0.947 mg Cd/L; Procambarus acutus, 0.368 and 0.048 mg Cd/L; Procambarus alleni, 3.07 and 0.386 mg Cd/L; and Procambarus clarkii, 2.66 and 0.486 mg Cd/L. The Orconectes LC50 genus mean acute value (GMAV) was 1.57 mg Cd/L, whereas the LC50 GMAV for Procambarus was 1.44 mg Cd/L. The LC10 GMAVs were 0.379 and 0.208 mg Cd/L, respectively. Family mean acute values (FMAVs) also were calculated for the Cambaridae using all species data (LC50, 1.51 mg Cd/L; LC10, 0.281 mg Cd/L). For tests with juvenile crayfish, the LC50 and LC10 values, respectively, were as follows: O. juvenilis, 0.060 and 0.014 mg Cd/L; O. placidus, 0.037 and 0.002 mg Cd/L; and P. clarkii, 0.624 and 0.283 mg Cd/L. The GMAVs were calculated for juvenile Orconectes (LC50, 0.047 mg Cd/L; LC10, 0.005 mg Cd/L). Additionally, FMAVs were calculated for juvenile crayfish (LC50, 0.111 mg Cd/L; LC10, 0.020 mg Cd/L). Crayfish sensitivity to Cd varied by a factor of nine among species tested as adults and by a factor of 17 among species tested as juveniles. Molting was a sensitive life stage for crayfish. Most individuals that molted shortly before or during exposure to Cd died, whereas all controls that molted in the adult assays survived. Because molting is a sensitive, recurring life-cycle event, molting individuals should be included in toxicological analysis despite some contrary recommendations.

Effects of aqueous, dietary and combined exposures of cadmium to Ceriodaphnia dubia.

While effects of water-borne metal exposures on freshwater animals have been well documented, the effect of dietary metal exposure is less understood but is gaining importance. However, little attention has been given to the importance of combining both exposure pathways. In this study, we compared effects of aqueous ('water only'), dietary ('food only') and combined ('water+food') exposures of cadmium to the freshwater cladocerans, Ceriodaphnia dubia. Major test endpoints included survival, feeding rate and reproduction. The C. dubia three-brood reproduction tests were conducted according to the United States Environmental Protection Agency (U.S. EPA) methods. Three exposure scenarios were used: aqueous, dietary, and combined aqueous and dietary exposures. Results showed that all three exposures affected survival, feeding rate and reproduction of C. dubia. Interestingly, combined exposure showed contribution effects of aqueous and dietary exposures. Lower cadmium concentrations were needed in combined exposure to produce effects as compared to higher concentrations in aqueous or dietary exposure alone. These results demonstrated the potential importance of dietary and combined exposures for consideration of cadmium regulation and risk assessment of metals.

Use of sunfish and stoneroller minnows as real-time in situ biomonitors of PCB contamination in freshwater streams.

A long-term polychlorinated biphenyl (PCB) monitoring study was conducted fortwo moderately impacted freshwater streams in Kentucky. Streamwater, sediment, and fish were analyzed for Aroclors 1248, 1254, and 1260 during 1988-2005. Only 8 of 263 water samples showed detectable PCBs. The low occurrences of PCB detections in streamwater indicated that PCBs were transitory in the water column, rapidly mobilizing into biotic and sediment compartments. One component of this study focused on species-specific patterns of PCB residues in fish, especially the green sunfish (Lepomis cyanellus), longear sunfish (L. megalotis), bluegill (L. macrochirus), and stoneroller minnow (Campostoma anomalum). Stoneroller minnows had higher PCB concentrations and increased frequency of detection when compared to sport fish. Aroclor 1248 was detected 80% of the time in stoneroller minnows from Big Bayou creek, whereas it was only detected in 25-39% of sport fish. In comparison, Aroclors 1254 and 1260 in sport fish were detected 49-69% of the time. These results indicate that higher chlorinated PCB congeners found in Aroclors 1254 and 1260 were not as readily metabolized and excreted by sport fish. No relationships were found between sunfish age and PCB concentrations, which demonstrated that sunfish exposed to low PCB contamination can effectively regulate PCBs, regardless of age. In addition, at low PCB levels (<0.50 microg/g), green sunfish body burden did not correlate with lipid content. A certain PCB threshold concentration, > or = 1.00 microg/g, must be exceeded before correlations between PCB body burden and lipid content are observed. These results indicate that, at least for species such as the sunfish, the use of the octanol-water partition coefficient (Kow) under low-level PCB exposure would appear to have little predictive value. Studies by Sanborn et al. (1975) found the green sunfish to be particularly adept at metabolizing organochlorine compounds and PCBs. This field study supports their laboratory findings. Green sunfish may have an enhanced P450 system, or due to low body lipid content, more effectively shunt PCBs into metabolic pathways that detoxify these compounds.

Metal trophic transfer from algae to cladocerans and the relative importance of dietary metal exposure.

We initially investigated effects and uptake of cadmium and copper on Pseudokirchneriella subcapitata (primary producer; formerly known as Selenastrum capricornutum). Our major efforts focused on the effects of metal-laden (i.e., Cd, Cu) P. subcapitata when used to provide dietary metal exposure to Ceriodaphnia dubia (primary consumer). Major test endpoints included feeding rate, survival, and reproduction. Metal body burden in C. dubia increased dose dependently at dietary exposure concentrations of 0.6 microg/g algal dry weight or less for cadmium and 74.7 microg/g algal dry weight or less for copper. However, it decreased sharply when dietary concentrations exceeded 0.6 microg/g algal dry weight for cadmium and 74.7 microg/g algal dry weight copper. Both dietary cadmium and copper affected all the major test endpoints. Whereas reproduction was observed to be the most sensitive endpoint, survival was observed to be the least sensitive endpoint. These results demonstrated the potential importance of dietary exposure for consideration in metal regulations or risk assessment.

Modeling the effects of chronic toxicity on fish populations: the influence of life-history strategies.

Five life-history scenarios were composed to simulate fish population adaptations for survival under various environmental conditions. The scenarios encompassed differences in stage-specific survival, fecundity and hatch success, number of spawning events, and life-span effect response to chronic toxic impacts. The strategies represent a good survivor/generalist (GS), a species exhibiting high young-of-the-year survival, a species exhibiting high adult survival, a species with no parental care or guarding behavior, and an anadromous salmonid strategy. Although the modeled scenarios were similar in population growth rate and imposed toxic effects, differences concerning the influence of various traits were found. Scenarios characterized by a short life span, short time to reproductive maturity, moderate to high survival to reproductive maturity, large number of spawning events, and parental guarding behaviors experienced less perturbation from the imposed chronic stresses. The GS scenario, modeled after the round goby (Neogobius melanostomus), exemplified these characteristics. Scenarios exhibiting little difference between reproductive effort for younger and older adults recovered quickly from stressors on fecundity and adult survival rates. Greater population decline in response to commensurate impacts was seen for life-history strategies with long life span, no parental guarding behaviors, semelparity and annual iteroparity, high adult survival rates, and moderate to low fecundity.

Population survivorship index for fish and amphibians: application to criterion development and risk assessment.

Criterion development currently focuses on physiological responses when determining permitted chemical concentrations in environmental media. Losses of biodiversity attributed to chemical pollution may be related not only to physiological sensitivity but also to inherent population characteristics that are not included in risk assessment or criterion development. In the present study, we proposed a process and a tool for summarizing population- and species-level information that contributes to long-term survivorship. We investigated the influences of life-history strategies of fish and amphibians that contribute to variations in population sensitivity or resiliency. A life-history survivorship index for application in criterion development and risk assessment was developed to help identify which species in a study system possess life-history strategies inherently sensitive to chemical stressors. It includes life-history characteristics of fecundity, number of spawning events, life stages, parental care, attrition rates, time to reproductive maturity, and other factors. Survivorship index values may be used as multipliers to permitting criteria, resulting in toxicological-ecological guidelines that incorporate toxicological information with regional ecological and population characteristics for local or regional application. We intended the procedure to serve as a step toward incorporating population and ecological information into regulatory procedures with the goal of protecting biodiversity from chronic toxic impacts.

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.