Patricia McClellan-Green

Associations between organochlorine contaminant concentrations and clinical health parameters in loggerhead sea turtles from North Carolina, USA

Widespread and persistent organochlorine (OC) contaminants, such as polychlorinated biphenyls (PCBs) and pesticides, are known to have broad-ranging toxicities in wildlife. In this study we investigated, for the first time, their possible health effects on loggerhead sea turtles (Caretta caretta). Nonlethal fat biopsies and blood samples were collected from live turtles for OC contaminant analysis, and concentrations were compared with clinical health assessment data, including hematology, plasma chemistry, and body condition. Concentrations of total PCBs (∑PCBs), ∑DDTs, ∑chlordanes, dieldrin, and mirex were determined in 44 fat biopsies and 48 blood samples. Blood concentrations of ∑chlordanes were negatively correlated with red blood cell counts, hemoglobin, and hematocrit, indicative of anemia. Positive correlations were observed between most classes of OC contaminants and white blood cell counts and between mirex and ∑TCDD-like PCB concentrations and the heterophil:lymphocyte ratio, suggesting modulation of the immune system. All classes of OCs in the blood except dieldrin were correlated positively with aspartate aminotransferase (AST) activity, indicating possible hepatocellular damage. Mirex and ∑TCDD-like PCB blood concentrations were negatively correlated with alkaline phosphatase (ALP) activity. Significant correlations to levels of certain OC contaminant classes also suggested possible alteration of protein (↑blood urea nitrogen, ↓albumin:globulin ratio), carbohydrate (↓glucose), and ion (↑sodium, ↓magnesium) regulation. These correlations suggest that OC contaminants may be affecting the health of loggerhead sea turtles even though sea turtles accumulate lower concentrations of OCs compared with other wildlife.

Effects of organochlorine contaminants on loggerhead sea turtle immunity: comparison of a correlative field study and in vitro exposure experiments

Several laboratory and field studies indicate that organochlorine contaminants (OCs), such as poly-chlorinated biphenyls (PCBs) and pesticides, modulate immune responses in rodents, wildlife, and humans. In the present study we examined the effects of OCs on immunity in free-ranging loggerhead sea turtles (Caretta caretta). Mitogen-induced lymphocyte proliferation responses, lysozyme activity, and OC concentrations were measured from blood samples. Mitogens chosen in the lymphocyte proliferation assay were phytohemagglutinin (PHA) and concanavalin A (ConA) for T-lymphocyte stimulation, and lipopolysaccharide (LPS) and phorbol 12,13-dibutyrate (PDB) for B-lymphocyte stimulation. Lysozyme activity was significantly and negatively correlated with whole-blood concentrations of 4,4′-dichlorodiphenyldichloroethylene (4,4′-DDE) and the sum of chlordanes. Lymphocyte proliferation responses stimulated by PHA, LPS, and PDB were significantly and positively correlated with concentrations of the sum of PCBs measured in whole blood. LPS- and PDB-induced proliferation were also significantly and positively correlated with 4,4′-DDE blood concentrations. These correlative observations in free-ranging turtles suggest that current, chronic exposure to OCs may suppress innate immunity and enhance certain lymphocyte functions of loggerhead sea turtles. To further test this hypothesis, lymphocyte proliferation was measured after in vitro exposure of peripheral blood leukocytes from 16 turtles to Aroclor 1254 (0–13.5 μg/mL) or 4,4′-DDE (0–13.4 μg/mL). Both contaminants increased PHA- and PDB-induced proliferation at concentrations below those that affected cell viability. Moreover, the concentrations that enhanced PDB-induced proliferation in vitro were similar to concentrations measured in turtles with the highest proliferative responses. The similarities between the in vitro experiments and the correlative field study suggest that OC exposure modulates immunity in loggerhead turtles.

Mechanisms of imposex induction in the mud snail, Ilyanassa obsoleta: TBT as a neurotoxin and aromatase inhibitor

The occurrence of imposex, imposition of male sex characteristics on female snails, has been extensively documented throughout the world. Tributyltin (TBT) and other organotins have been causally linked to imposex induction at levels as low as 2 ng/l. There are several proposed mechanisms of action. First, TBT has been shown to be neurotoxic and to accumulate in snail ganglia. Peptide hormones control sexual differentiation in gastropods, and one hypothesis is that TBT acts as a neurotoxin to abnormally release the peptide hormone Penis Morphogenic Factor (PMF). However, PMF has not been characterized to date. The neuropeptide APGWamide significantly induces imposex in the mud snail, Ilyanassa obsoleta, at 10(-16) moles sub-cutaneous (SQ) injection over 2 weeks, and could be the PMF in this species. A second hypothesis is that TBT inhibits aromatase activity leading to increased testosterone levels and decreased estradiol. In vitro studies with snail digestive gland microsomes showed that TBT-dosed snails not exhibiting imposex had a 52% reduction in aromatase activity. Although the role of vertebrate sex steroids is not known in gastropods, it is possible that the combination of changes in peptide and steroid hormones may lead to imposex induction at extremely low doses of TBT.

Organochlorine contaminants in sea turtles: Correlations between whole blood and fat

Monitoring toxic organochlorine (OC) compounds is an important aspect in wildlife studies, especially in protected species such as sea turtles. The goal of this study was to determine whether blood OC concentrations can predict those in adipose tissue of sea turtles. Blood offers many benefits for monitoring OCs. It can be collected nondestructively from live turtles and can be sampled repeatedly for continuous monitoring. Organochlorine concentrations in blood may better represent the exposure levels of target tissues, but blood concentrations may fluctuate more than those in fatty tissues following recent dietary exposure or lipid mobilization. Paired fat and blood samples were collected from 44 live, juvenile loggerhead sea turtles and 10 juvenile Kemps ridley sea turtle carcasses. Organochlorines were analyzed using gas chromatography with electron capture detection and mass spectrometry. Lipid-normalized OC concentrations measured in the blood significantly correlated to levels found in the fat samples of both species. This result suggests that sea turtle blood is a suitable alternative to fatty tissues for measuring OCs because blood concentrations reasonably represent those observed in the paired fat samples. However, blood OC concentrations calculated on a wet-mass basis were significantly and inversely correlated to lipid content in the fat samples. Therefore, caution should be used when monitoring spatial or temporal trends, as OC levels may increase in the blood following mobilization of fat stores, such as during long migrations, breeding, or disease events.

Induction of cytochrome P450 3A and heat shock protein by tributyltin in blue crab, Callinectes sapidus

Abstract Tributyltin (TBT), a toxic contaminant in aquatic environments, breaks down cytochrome P450 enzymes (P450s) in vitro. To determine in vivo effects of long-term TBT-exposure in aquatic invertebrates, respiration rates, heat shock protein induction, microsomal cytochrome P450 levels and metabolism of [14C]testosterone were examined in the hepatopancreas of blue crabs fed TBT. Four groups of crabs were fed fish injected with ethanol or 2, 4 or 8 μg TBT Cl dissolved in ethanol/g meat; doses of 0, 125, 250, or 500 μg kg−1 respectively. Crabs were fed TBT-treated meat every other day for 16 days. Respiration rates were significantly decreased after TBT exposure. Hepatopancreas microsomes and cytosol were prepared 24 h after the last feeding. Heat shock protein was induced at the two highest TBT exposure concentrations. Total P450 levels were similar in all samples; however, protein cross-reacting with anti-scup CYP 3A, an isozyme responsible for 6β-hydroxylation of testosterone in vertebrates, increased at the 250 μg and 500 μg TBT Cl kg−1 dose. EROD activity showed that CYP 1A was not elevated. Hydroxylation of [14C]testosterone at the 6β, 6α, 7α, and 16α positions by hepatopancreas microsomes increased significantly in crabs exposed to 250 μg TBT Cl kg−1. The lack of increase in [14C]testosterone hydroxylation at the 500 μg kg−1 dosage may result from TBT Cls disruption of P450 activity at high concentrations. Incubation of control microsomes with TBT significantly reduced spectrally quantified P450 as well as P450 activity after 2 h, showing that TBT Cl interferes with crab microsomal P450 proteins. Immunoinhibition of testosterone 6β-hydroxylation was accomplished by using scup CYP 3A antibody, showing that the antibody specifically binds to a protein with 6β-hydroxylase activity. These results indicate that blue crabs are stressed by TBT-exposure, and upregulate P450 isozymes possibly to aid in metabolism and elimination of TBT.

Testosterone metabolism in imposex and normal Ilyanassa obsoleta: Comparison of Field and TBTA Cl-induced imposex

Abstract Tributyltin (TBT) is a marine biocide that alters activity of cytochrome P450 (P450) monooxygenases and elicits androgenization in gastropod molluscs. This study was conducted to determine whether testosterone metabolism was altered in field collected and TBT-induced imposex female mud snail, I. obsoleta . Normal and imposex snails were collected from two field sites. Imposex was induced by exposing normal snails to 2.5, 5.0, 10, 20, or 200 ng TBT l −1 seawater for 45 days at 19°C. Testosterone metabolism was studied by exposing snails to [ 14 C]-testosterone for 24 h and determining conversion by phase I (P450s and reductases) and phase II (transferases) enzymes. All snails took up similar levels of testosterone from seawater, and radioactivity partitioned primarily into gonad/digestive gland complex. Testosterone was metabolized primarily by reductases. Field collected imposex snails had decreased rates of reduced metabolites, while there was no overall change in testosterone metabolism in TBT-induced imposex snails. These findings indicate that field-collected imposex snails metabolize testosterone differently from normal snails, as well as from TBT-induced imposex snails.

Detection of gram-negative histamine-producing bacteria in fish: a comparative study.

Poisoning due to ingestion of foods with elevated levels of biogenic amines (histamine, putrescine, cadaverine, and tyramine) is well documented. Histamine fish poisoning largely is due to growth of naturally occurring bacteria associated with scombroid fish species. A rapid and reliable method is needed to screen for the presence of histamine-forming bacteria in fish. This study included a comparison of three methods for the detection of histamine-producing bacteria. A total of 152 histamine-producing and non-histamine-producing bacteria from multiple sources were screened using a modified Nivens agar method, a potentiometric method, and a PCR-based assay targeting a 709-bp fragment of the histidine decarboxylase gene. Histamine production by bacterial isolates was confirmed by high-performance liquid chromatography (HPLC). Bacterial strains were categorized as producing high amounts of histamine, low amounts of histamine, or no histamine. Of the 152 strains tested, 128 (84%) were positive with the Nivens agar method, 73 (48%) were positive with the potentiometric technique, and 74 (49%) were positive with the PCR assay. Overall, a 38% false-positive rate was observed with the modified Nivens agar method, although this method detected both low-histamine and high-histamine strains. There was a high degree of concordance (> 99%) between results of the potentiometric and PCR methods, but neither of these methods detected low-histamine bacteria. These observations support the need for a simple and straightforward yet sensitive method for detecting histamine-producing bacteria in seafood and environmental samples.

Toxicity of aqueous fullerene in adult and larval Fundulus heteroclitus

Aqueous suspensions of fullerene aggregates (aqua-nC60) were used to investigate the movement of carbon-based nanomaterials in a marine water column and to determine their effects on different life stages of a marine teleost. Fullerene aggregates formed precipitates as a result of mixing in natural seawater, and levels of aqua-nC60 were significantly increased in bottom waters after 24 h. Exposure of Fundulus heteroclitus embryos, larvae, and adults to increasing concentrations of aqua-nC60 resulted in very little mortality, and no median lethal concentrations could be calculated at < or = 10 mg/L. Aggregates of aqua-nC60 did adhere to the chorion but did not affect development of the embryos or their hatching success. Movements of aqua-nC60 through the chorion and into the embryo tended to increase with higher exposure levels; however, the concentrations were extremely low and did not differ significantly. Larvae exposed to increasing concentrations of aqua-nC60 exhibited a significant dose-dependent increase in total glutathione (GSH). This was accompanied by a decreasing trend in lipid peroxidation (LPO), but LPO was not statistically different between treatments. Adult F. heteroclitus exposed to increasing concentrations of aqua-nC60 exhibited an increase in total GSH in liver tissue but not in the gill. No significant effects on LPO were observed in either tissue. Thus, we conclude that aqua-nC60 affects the oxidative stress response of F. heteroclitus and that increased antioxidant defenses provide some physiological tolerance for these materials. Environmental factors influencing uptake, metabolism, and physiological response following exposure, however, need further investigation.

Development of molecular-based methods for determination of high histamine producing bacteria in fish.

Histamine (or scombroid) fish poisoning is a significant cause of food borne disease in the United States. In this study, we describe the development of a molecular-based technique which uses digoxigenin (DIG) labeled DNA probes for the detection of gram negative bacteria producing high amounts of histamine (>1000 ppm). A cocktail of PCR amplification fragments corresponding to a 709 bp fragment of the histidine decarboxylase (hdc) gene of four high producing bacteria (Morganella morganii, Enterobacter aerogenes, Raoultella planticola and Photobacterium damselae) was DIG-labeled and screened against a strain bank of 152 gram negative bacteria isolated from scrombroid fish and their harvest environment. The probe cocktail reacted specifically (100%) with the high histamine producing strains but failed to react with low histamine producers and non-producers. To further evaluate the feasibility of the approach, fish homogenate inoculated with known concentrations of four high histamine producing bacterial strains was plated on modified Nivens medium (culture method) and trypticase soy agar supplemented with 2% NaCl (for colony lift hybridization). The colony lift hybridization counts did not differ significantly from the level of the initial inoculum (p>0.05), while the modified Nivens counts were significantly lower (p<0.05) than either inoculum or colony lift counts. The use of digoxigenin (DIG) labeled DNA probes with colony lift hybridization shows promise for accurate and specific enumeration of histamine producing bacteria in scombroid fish.

Dietary CdSe/ZnS quantum dot exposure in estuarine fish: Bioavailability, oxidative stress responses, reproduction, and maternal transfer

Continued development, use, and disposal of quantum dots (QDs) ensure their entrance into aquatic environments where they could pose a risk to biological organisms as whole nanoparticles or as degraded metal constituents. Reproductive Fundulus heteroclitus were fed a control diet with lecithin, diets containing 1 or 10 μg of lecithin-encapsulated CdSe/ZnS QD/day, or a diet containing 5.9 μg CdCl2/day for 85 days. Cadmium concentrations in liver, intestine, and eggs were quantified with inductively coupled plasma mass spectrometry. In fish fed 10 μg QD/day, QDs or their degradation products traversed the intestinal epithelia and accumulated in the liver. Less than 0.01% of the QDs cadmium was retained in the liver or intestinal tissues. This compares to 0.9% and 0.5% of the cadmium in the intestine and liver, respectively of fish fed a CdCl2 diet. Cadmium was also detected in the eggs from parents fed 10 μg QD/day. No significant changes in hepatic total glutathione, lipid peroxidation, or expression of genes involved in metal metabolism or oxidative stress were observed. While QDs in the diet are minimally bioavailable, unusual levels of vitellogenin transcription in male fish as well as declining fecundity require further investigation to determine if endocrine disruption is of environmental concern.