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Assessing the effects of contaminants in marine wildlife using cell-based assays

CURATOR
A pinboard by
Kimberly Finlayson

PhD candidate, Griffith University

PINBOARD SUMMARY

Using primary cell cultures, species specific effects of contaminants can be elucidated.

My research focuses on using primary cell cultures established from the species of interest to identify the effects of contaminants. Specifically, skin and organ cell cultures were established from several species of marine turtles (green, loggerhead, hawksbill). These cultures have then been used to test contaminants that we know accumulate in turtles against a number of endpoints, such as cell viability, genotoxicity, oxidative stress, and metabolism. In doing so, effect values can be compared to already known accumulation values to better identify populations that may be at risk.

The battery of assays can then be applied to biological samples, such as blood. Blood has been collected from turtles from different foraging grounds, and thus exposed to different types and mixtures of contaminants. The contaminants can then be extracted from the blood, and the extracts tested in the battery of assays. This will further help to identify foraging grounds at risk and exactly what those risks might be. As marine turtles show high foraging site fidelity, they can be used as an indicator of habitat health. Populations that are found to be at risk indicate an unhealthy marine environment. Results from such a study may better guide coastal management and conservation initiatives by identifying areas of high priority or concern.

This project may be used as a model for studying wildlife that can typically be difficult, such as other marine animals or endangered animals. The establishment of species specific cell cultures can be applied to virtually any other species. These cell cultures can then be used not just for toxicity testing, but a wide variety of research applications such as physiology, virology and aetiology. Ultimately these types of cell based, or in vitro, assessments can replace live animal testing.

42 ITEMS PINNED

Atrazine Alters Expression of Reproductive and Stress Genes in the Developing Hypothalamus of the Snapping Turtle, Chelydra serpentina.

Abstract: Atrazine is an herbicide used to control broadleaf grasses and a suspected endocrine disrupting chemical. Snapping turtles lay eggs between late May and early June, which could lead to atrazine exposure via field runoff. Our goal was to determine whether a single exposure to 2ppb or 40ppb atrazine during embryogenesis could induce short- and long-term changes in gene expression within the hypothalamus of snapping turtles. We treated eggs with atrazine following sex determination and measured gene expression within the hypothalamus. We selected genes a priori for their role in the hypothalamus-pituitary-gonad or the hypothalamus-pituitary-adrenal axes of the endocrine system. We did not identify any changes in gene expression 24-hours after treatment. However, at hatching AR, Kiss1R, and POMC expression was upregulated in both sexes, while expression of CYP19A1 and PDYN was increased in females. Six months after hatching, CYP19A1 and PRLH expression was increased in animals treated with 2ppb atrazine. Our study shows persistent changes in hypothalamic gene expression due to low-dose embryonic exposure to the herbicide atrazine with significant effects in both the HPG and HPA axes. Effects reported here appear to be conserved among vertebrates.

Pub.: 09 Aug '16, Pinned: 06 Oct '17

Genotoxic effects of polychlorinated biphenyls (PCB 153, 138, 101, 118) in a fish cell line (RTG-2).

Abstract: Polychlorinated biphenyls (PCBs) are persistent pollutants in aquatic environments, often causing the decline or disappearance of wild populations. The primary aim of this study was to investigate the genotoxic effects of some PCBs (PCB153 (2,2',4,4',5,5'-hexachlorobiphenyl) and 138 (2,2',3,4,4',5'-hexachloro-biphenyl), both non-dioxin-like compounds, and the pentachlorobiphenyls PCB118 (2,3',4,4',5-) and 101 (2,2',4',5,5'-), the former an ortho-substituted, low-affinity dioxin-like compound and the latter a non-coplanar congener classified as non-dioxin-like) in fish cells (RTG-2). These congeners are mostly present in surface waters and in edible aquatic organisms and the loss of DNA integrity in vitro serves as a sensitive biomarker of cytogenetic alterations and is considered as an initial step for the identification of genotoxic effects. The alkaline comet assay and the micronucleus test show clear genotoxic damage after short and longer exposure (2 and 24h) to maximum soluble, non-cytotoxic doses, evident sooner with PCBs 101 and 118. Oxidative stress situations involving ROS release, reduction in total GSH, lipid peroxidation and alteration to superoxide dismutase, seen after exposure with all the congeners, though with different kinetics, seem the most likely explanation for the genotoxic damage. This appears to be confirmed by the modified comet assay (pH 10) for detection of oxidized bases using endonuclease III. The increased generation of intracellular ROS might explain the apoptosis seen after treatment with the single PCBs and evaluated on the basis of the rise in 3-7 caspase activity. Therefore both the non-coplanar, non-dioxin-like PCBs (153, 138, 101) and the low-affinity dioxin-like compound PCB118 cause evident genotoxic damage, probably as a consequence of oxidative stress.

Pub.: 21 Apr '11, Pinned: 06 Oct '17

Effects of organochlorine compounds on cytochrome P450 aromatase activity in an immortal sea turtle cell line.

Abstract: Many classes of environmental contaminants affect the reproductive function of animals through interactions with the endocrine system. The primary components affected by endocrine active compounds (EACs) are the steroid receptors and the enzymes responsible for steroidogenesis. This study sought to develop an in vitro model for assessing EAC effects in sea turtles by examining their ability to alter cytochrome P450 aromatase (CYP19) activity. Aromatase is the enzyme responsible for the conversion of testosterone to estradiol. This enzyme is critical in the sexual differentiation of reptiles which demonstrate temperature-dependent sex determination. An immortal testis cell line GST-TS from a green sea turtle was grown in culture at 30 degrees C in RPMI 1640 media. The cells were exposed to three known aromatase inducers; dexamethasone (Dex), 8Br-cyclic AMP, or human chronic gonadotropin (HCG) and one aromatase inhibitor 4-androstenol-dione (4-OHA). In addition, the GST-TS cells were exposed to 0.1-30 microM atrazine and 3-100 microM 4,4'-DDE. The inducing compounds that have been shown to increase aromatase activity in other systems failed to induce aromatase activity in the GST-TS cells, yet exposure to the inhibiting compound, 4-OHA, did result in a significant reduction. Atrazine (0.1, 1.0 and 10 microM) significantly induced aromatase activity following a 24 h exposure, and 4,4'-DDE inhibited the activity but only at cytotoxic concentrations (100 microM). Based on these results, this in vitro model can be useful in examining the endocrine effects of EACs in sea turtles.

Pub.: 05 Jun '04, Pinned: 06 Oct '17

PCB pollution continues to impact populations of orcas and other dolphins in European waters.

Abstract: Organochlorine (OC) pesticides and the more persistent polychlorinated biphenyls (PCBs) have well-established dose-dependent toxicities to birds, fish and mammals in experimental studies, but the actual impact of OC pollutants on European marine top predators remains unknown. Here we show that several cetacean species have very high mean blubber PCB concentrations likely to cause population declines and suppress population recovery. In a large pan-European meta-analysis of stranded (n = 929) or biopsied (n = 152) cetaceans, three out of four species:- striped dolphins (SDs), bottlenose dolphins (BNDs) and killer whales (KWs) had mean PCB levels that markedly exceeded all known marine mammal PCB toxicity thresholds. Some locations (e.g. western Mediterranean Sea, south-west Iberian Peninsula) are global PCB "hotspots" for marine mammals. Blubber PCB concentrations initially declined following a mid-1980s EU ban, but have since stabilised in UK harbour porpoises and SDs in the western Mediterranean Sea. Some small or declining populations of BNDs and KWs in the NE Atlantic were associated with low recruitment, consistent with PCB-induced reproductive toxicity. Despite regulations and mitigation measures to reduce PCB pollution, their biomagnification in marine food webs continues to cause severe impacts among cetacean top predators in European seas.

Pub.: 15 Jan '16, Pinned: 06 Oct '17

Toxic effects of various pollutants in 11B7501 lymphoma B cell line from harbour seal (Phoca vitulina).

Abstract: Although, heavy metals and polycyclic aromatic hydrocarbons (PAHs) have been reported at high levels in marine mammals, little is known about the toxic effects of some of these contaminants. In this study, we assessed the immunotoxic and genotoxic effects of seven heavy metals (arsenic, vanadium, selenium, iron, zinc, silver and chromium) and one PAH (benzo[a]pyrene or B[a]P) on a lymphoma B cell line from harbour seal (Phoca vitulina). A significant reduction in lymphocyte proliferation was registered following an exposure to 0.05 microM of B[a]P, 5 microM of arsenic or selenium, 50 microM of vanadium, 100 microM of silver and 200 microM of iron. On the contrary, zinc increased the lymphoproliferative response at 200 microM. Decreased phagocytosis was observed at 20 microM of arsenic, 50 microM of B[a]P or selenium, 200 microM of zinc and 500 microM of vanadium. Micronuclei induction occurred with 0.2 microM of B[a]P, 100 microM of vanadium and with 200muM of arsenic or selenium. Exposure to 50muM of arsenic decreased G(2)/M phase of the cell cycle. Chromium did not induce any effects at the concentrations tested. Concentrations of heavy metals (except silver and vanadium) and B[a]P inducing an toxic effect are within the environmental ranges reported in the blood tissue of pinnipeds. The reduction of some functional activities of the harbour seal immune system may cause a significant weakness capable of altering host resistance to disease in free-ranging pinnipeds.

Pub.: 02 Feb '10, Pinned: 06 Oct '17

Immunotoxic effects of environmental pollutants in marine mammals.

Abstract: Due to their marine ecology and life-history, marine mammals accumulate some of the highest levels of environmental contaminants of all wildlife. Given the increasing prevalence and severity of diseases in marine wildlife, it is imperative to understand how pollutants affect the immune system and consequently disease susceptibility. Advancements and adaptations of analytical techniques have facilitated marine mammal immunotoxicology research. Field studies, captive-feeding experiments and in vitro laboratory studies with marine mammals have associated exposure to environmental pollutants, most notable polychlorinated biphenyls (PCBs), organochlorine pesticides and heavy metals, to alterations of both the innate and adaptive arms of immune systems, which include aspects of cellular and humoral immunity. For marine mammals, reported immunotoxicology endpoints fell into several major categories: immune tissue histopathology, haematology/circulating immune cell populations, functional immune assays (lymphocyte proliferation, phagocytosis, respiratory burst, and natural killer cell activity), immunoglobulin production, and cytokine gene expression. Lymphocyte proliferation is by far the most commonly used immune assay, with studies using different organic pollutants and metals predominantly reporting immunosuppressive effects despite the many differences in study design and animal life history. Using combined field and laboratory data, we determined effect threshold levels for suppression of lymphocyte proliferation to be between b0.001-10 ppm for PCBs, 0.002-1.3 ppm for Hg, 0.009-0.06 for MeHg, and 0.1-2.4 for cadmium in polar bears and several pinniped and cetacean species. Similarly, thresholds for suppression of phagocytosis were 0.6-1.4 and 0.08-1.9 ppm for PCBs and mercury, respectively. Although data are lacking for many important immune endpoints and mechanisms of specific immune alterations are not well understood, this review revealed a systemic suppression of immune function in marine mammals exposed to environmental contaminants. Exposure to immunotoxic contaminants may have significant population level consequences as a contributing factor to increasing anthropogenic stress in wildlife and infectious disease outbreaks.

Pub.: 23 Nov '15, Pinned: 06 Oct '17

The occurrence of chemical elements and POPs in loggerhead turtles (Caretta caretta): an overview.

Abstract: Chemical elements and persistent organic pollutants (POPs) are globally present in aquatic systems and their potential transfer to loggerhead marine turtles (Caretta caretta) has become a serious threat for their health status. The environmental fate of these xenobiotics may be traced by the analysis of turtles' tissues and blood. Generally, loggerhead turtles exhibited a higher metal load than other turtle species, this could be explained by differences in diet habits being food the main source of exposure. Literature shows that muscle, liver and kidney are most considered for the quantification of chemical elements, while, organic compounds are typically investigated in liver and fat. This paper is an overview of the international studies carried out on the quantification of chemical elements, polychlorinated biphenyls (PCBs), organochlorines (OCs) and perfluorinated compounds (PFCs), in tissues, organs and fluids of C. caretta from the Mediterranean Sea, the Atlantic and the Pacific Oceans.

Pub.: 28 Jun '11, Pinned: 06 Oct '17

Cytotoxic and endocrine-disrupting potential of atrazine, diazinon, endosulfan, and mancozeb in adrenocortical steroidogenic cells of rainbow trout exposed in vitro.

Abstract: An in vitro bioassay for detection and quantitative assessment of chemicals with the capacity to disrupt adrenal steroidogenesis has been developed and used to compare the cytotoxic and endocrine-disrupting potential of four pesticides. Enzymatically dispersed adrenocortical cells of rainbow trout (Oncorhynchus mykiss) were exposed in vitro to atrazine, diazinon, endosulfan, and mancozeb, and cortisol secretion in response to ACTH or dibutyryl-cAMP (dbcAMP) and cell viability were determined. The effective concentration, EC50 (concentration that inhibits cortisol secretion by 50%), the median lethal concentration, LC50 (concentration that kills 50% of the cells), and the LC50/EC50 ratio were established for the test pesticides. The pesticides were ranked as follows: EC50, endosulfan < diazinon < mancozeb < atrazine; LC50, diazinon < endosulfan < mancozeb < atrazine, with diazinon as the most cytotoxic. Endosulfan and mancozeb disrupted sites downstream of the cAMP-generating step of the cortisol synthetic pathway while atrazine seemed to act upstream from the cAMP step. The in vitro adrenal bioassay can be used for screening of adrenotoxicants and for mechanistic studies of adrenotoxicity.

Pub.: 24 Apr '02, Pinned: 06 Oct '17

Pansteatitis associated with high levels of polychlorinated biphenyls in a wild loggerhead sea turtle Caretta caretta.

Abstract: We describe the morphologic and toxicological findings in a case of pansteatitis in a stranded loggerhead sea turtle Caretta caretta. At necropsy, a large amount of adipose tissue in the celomic cavity showing very firm, yellow to orange irregular formations was observed. Histological lesions ranged from the infiltration of necrotic fat by scarce multinucleated giant cells and numerous macrophages containing xylene-insoluble lipopigment inclusions to the presence of several granulomas characterized by an irregular central necrotic area consisting of lipopigment surrounded by numerous multinucleated giant cells. Microbiological cultures were negative. Celomic fat was analyzed for PCBs and DDTs, resulting in very high levels of PCB 138, 153, 180 209 (3170, 2830, 980 and 1190 ng g-1, respectively). Although a nutritional cause cannot be ruled out, the high levels of PCBs detected in the celomic fat could have induced lipid peroxidation in adipocytes, resulting in cell damage, deposition of ceroid pigment and inflammatory response. This is the first report of pansteatitis in a wild sea turtle.

Pub.: 01 Mar '13, Pinned: 22 Sep '17

High levels of polychlorinated biphenyls in tissues of Atlantic turtles stranded in the Canary Islands, Spain.

Abstract: Polychlorinated biphenyls (PCBs 28, 31, 52, 101, 138, 153, 180, and 209) were measured in tissue samples (liver and fat) from 30 loggerhead turtles Caretta caretta, 1 green turtle Chelonia mydas, and 1 leatherback Dermochelys coriacea stranded on the coasts of the Canary Islands, trying to establish a possible relation between PCB concentrations and the lesions and causes of death. Tissues from these turtles contained higher levels of PCBs than those reported in turtles from other geographical regions. Sigma PCB concentrations (1980+/-5320 ng g(-1)wet wt.) in the liver of loggerheads were higher than in the adipose tissue (450+/-1700 ng g(-1)wet wt.). Concentrations of PCB 209 in the liver (1200+/-3120 ng g(-1)wet wt.) of loggerheads and in the liver (530 ng g(-1)wet wt.) and adipose tissue (500 ng g(-1)wet wt.) of the leatherback were remarkable. Frequencies of detection of PCB 209 in the liver (15.5%) and adipose tissue (31%) were also remarkable. Cachexia was detected in 7 turtles (22%) and septicemia was diagnosed in 10 turtles (31%). Statistically, a positive correlation was detected between Sigma PCBs concentration and cachexia. Poor physical condition, cachexia and/or septicaemia could explain the high levels of PCBs and tissue distribution. However, no histological lesions exclusively attributed to the acute effects of PCBs were described. The most prevalent histological lesions were ulcerative and purulent oesophagitis, purulent dermatitis, necrotizing enteritis, and granulomatous pneumonia. The bacteria most frequently isolated were Escherichia coli, Staphylococcus sp., and Aeromonas sp. Although immunosupression as a result of PCBs pollution has been described previously, other factors in this study, such as incidental fishing, nutritional status, and exposition to different micro-organisms, make it difficult to establish a clear association between PCB concentrations and causes of death.

Pub.: 09 Dec '08, Pinned: 22 Sep '17

Mercury and selenium concentrations in leatherback sea turtles (Dermochelys coriacea): population comparisons, implications for reproductive success, hazard quotients and directions for future research.

Abstract: Leatherback sea turtles (Dermochelys coriacea) are long-distance migrants that travel thousands of km from foraging grounds to breeding and nesting grounds. These extensive journeys are fueled by ingestion of an estimated 300-400 kg of prey/d and likely result in exposure to high concentrations of environmental toxicants (e.g., mercury compounds). Increased bodily concentrations of mercury and its compounds in nesting female turtles may have detrimental effects on reproductive success. Leatherbacks have relatively low reproductive success compared with other sea turtles (global average hatching success ~50-60%). To assess toxicants and necessary nutrients as factors affecting leatherback turtle reproductive success at Sandy Point National Wildlife Refuge (SPNWR), St. Croix, U.S. Virgin Islands, we collected blood from nesting female leatherbacks and tissues from their hatchlings (blood from live turtles, liver and yolk sac from dead turtles). We compared the concentrations in those tissues to hatching and emergence success. We found that on SPNWR, hatching and emergence success were more closely related to seasonal factors than to total mercury and selenium concentrations in both nesting females and hatchlings. Selenium concentrations of nesting females were positively correlated with those of their hatchlings. Mercury and selenium in the liver of hatchlings were positively correlated with one another. Turtles with greater remigration intervals tended to have higher blood selenium concentrations, suggesting that selenium accumulates in leatherbacks through time. Through hazard quotients, we found evidence that selenium may be at or above concentrations that may cause physiologic harm to hatchlings. We also found evidence that population level differences exist for these trace elements. The concentrations of mercury and selenium established in this manuscript form a baseline for future toxicant studies.

Pub.: 25 Jun '13, Pinned: 22 Sep '17

Maternal health status correlates with nest success of leatherback sea turtles (Dermochelys coriacea) from Florida.

Abstract: Of the seven sea turtle species, the critically endangered leatherback sea turtle (Dermochelys coriacea) exhibits the lowest and most variable nest success (i.e., hatching success and emergence success) for reasons that remain largely unknown. In an attempt to identify or rule out causes of low reproductive success in this species, we established the largest sample size (n = 60-70 for most values) of baseline blood parameters (protein electrophoresis, hematology, plasma biochemistry) for this species to date. Hematologic, protein electrophoretic and biochemical values are important tools that can provide information regarding the physiological condition of an individual and population health as a whole. It has been proposed that the health of nesting individuals affects their reproductive output. In order to establish correlations with low reproductive success in leatherback sea turtles from Florida, we compared maternal health indices to hatching success and emergence success of their nests. As expected, hatching success (median = 57.4%) and emergence success (median = 49.1%) in Floridian leatherbacks were low during the study period (2007-2008 nesting seasons), a trend common in most nesting leatherback populations (average global hatching success = ∼50%). One protein electrophoretic value (gamma globulin protein) and one hematologic value (red blood cell count) significantly correlated with hatching success and emergence success. Several maternal biochemical parameters correlated with hatching success and/or emergence success including alkaline phosphatase activity, blood urea nitrogen, calcium, calcium:phosphorus ratio, carbon dioxide, cholesterol, creatinine, and phosphorus. Our results suggest that in leatherbacks, physiological parameters correlate with hatching success and emergence success of their nests. We conclude that long-term and comparative studies are needed to determine if certain individuals produce nests with lower hatching success and emergence success than others, and if those individuals with evidence of chronic suboptimal health have lower reproductive success.

Pub.: 24 Feb '12, Pinned: 22 Sep '17

Potential adverse effects of inorganic pollutants on clinical parameters of loggerhead sea turtles (Caretta caretta): results from a nesting colony from Cape Verde, West Africa.

Abstract: A large number of nesting loggerhead sea turtles (n = 201) were sampled to establish the blood levels of 11 elements (Cu, Mn, Pb, Zn, Cd, Ni, Cr, As, Al, Hg, and Se). Almost all of the samples showed detectable levels of these 11 elements, and Zn and Se exhibited the highest concentrations (median values as high as 6.05 and 2.28 μg/g, respectively). The median concentrations of the most toxic compounds, As, Cd, Pb, and Hg, were relatively low (0.38, 0.24, 0.06, and 0.03 μg/g, respectively). We also determined the haematological and biochemical parameters in a subsample of 50 turtles to evaluate the potential effects of these contaminants on clinical parameters and found several associations. Our study reinforces the usefulness of blood for the monitoring of the levels of contaminating elements and their adverse effects on blood parameters in sea turtles.

Pub.: 04 Sep '13, Pinned: 22 Sep '17

Potential adverse health effects of persistent organic pollutants on sea turtles: evidences from a cross-sectional study on Cape Verde loggerhead sea turtles.

Abstract: The Cape Verde nesting population of loggerhead sea turtles (Caretta caretta) is the third largest population of this species in the world. For conservation purposes, it is essential to determine how these reptiles respond to different types of anthropogenic contaminants. We evaluated the presence of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) in the plasma of adult nesting loggerheads from Boa Vista Island, Cape Verde, and studied the effects of the contaminants on the health status of the turtles using hematological and biochemical parameters. All turtles had detectable levels of non-dioxin like PCBs, whereas dioxin-like congeners (DL-PCBs) were detected in only 30% of the turtles. Packed cell volume decreased with higher concentrations of PCBs, which suggests that PCB exposure could result in anemia in sea turtles. In addition, a negative association between some OCPs and white blood cells (WBC) and thrombocyte estimate was noted. The DDT-metabolite, p,p'-DDE was negatively correlated with the Na/K ratio and, additionally, a number of correlations between certain PAHs and electrolyte balances were found, which suggest that exposure to these environmental contaminants could affect the kidneys and salt glands in sea turtles. Additionally, several correlations were observed between these environmental pollutants (OCPs and PAHs) and enzyme activity (GGT, ALT, ALP and amylase) and serum protein levels, pointing to the possibility that these contaminants could induce adverse metabolic effects in sea turtles. Our results indicate that anthropogenic pollutants are present in the Cape Verde loggerhead turtle nesting population and could exert negative effects on several health parameters. Because of the importance of this loggerhead nesting population, protective regulations at national and international levels as well as international action are necessary for assuring the conservation of this population.

Pub.: 15 May '13, Pinned: 22 Sep '17

Comparative study of organohalogen contamination between two populations of Eastern Atlantic loggerhead sea turtles (Caretta caretta).

Abstract: We evaluated the presence of 37 organohalogen contaminants in plasma samples from 162 juvenile and 197 adult loggerhead turtles (Caretta caretta) from the archipelagos of the Canary Islands and Cape Verde, respectively, and compared the contamination profiles found. We detected five organochlorine pesticides (OCP) and 16 polychlorinated biphenyls (PCBs). The concentrations of the two groups of contaminants were higher in turtles from the Canary Islands (OCPs, 1.04 vs. 0.37 ng/mL; PCBs, 1.92 vs. 0.08 ng/mL). We also observed a different profile of PCB contamination between the two populations. In addition, there was a negative correlation between body size and the total concentration of PCBs in the Canary Islands turtles, but not in turtles from Cape Verde. The present study presents the first data on the organochlorine contaminants (OCs) of live turtles from Canary Islands. In addition, we perform a comparison of the levels and profiles of OCs between these two different groups of loggerhead sea turtles from the Eastern Atlantic.

Pub.: 12 Oct '13, Pinned: 22 Sep '17

Metal distribution and metallothionein in loggerhead (Caretta caretta) and green (Chelonia mydas) sea turtles.

Abstract: The first aim of our study was to determine the concentrations of selected trace elements (Zn, Cu, Fe, Mn, Cd and Pb) in tissues of green turtles from Tortuguero National Park on the North Caribbean coast of Costa Rica and of loggerheads from the Mediterranean Sea. Zn, Cu, Fe, Mn and Cd were present at detectable concentrations in all samples and showed clear organotropism, whereas Pb was not always over the detection limit and did not show any particular tissue distribution. The two species presented significant differences: Cu and Cd in liver and kidney of Chelonia mydas were significantly higher with respect to the concentrations found in Caretta caretta. The second and major goal of our study was to evaluate hepatic and renal metallothionein (MT) as a biomarker of environmental metal exposure. The present paper is the first to describe and quantify MT in kidney and liver of loggerhead turtles and in kidney of green turtles. MT concentrations were higher in green than in loggerhead turtles. In addition, positive correlations were found between Cu and Cd concentrations and Cu-MT and Cd-MT in liver and kidney in both species, suggesting a pivotal role of MT in metal storage and detoxification. The quantification of metals and MT in liver and kidney may be a valid biomarker of metal exposure in the aquatic environment to assess the health of marine sea turtles as long as accurate analytical methods are adopted.

Pub.: 21 Nov '07, Pinned: 21 Sep '17

First application of comet assay in blood cells of Mediterranean loggerhead sea turtle (Caretta caretta).

Abstract: The aim of this study was to validate the comet assay in erythrocytes of Caretta caretta, a species never investigated for genotoxicity. We studied 31 loggerhead sea turtles from three Italian marine rescue centres. Peripheral blood samples were collected from all the animals and the comet assay applied. All comet cells were analysed using two methods: visual scoring and computer image analysis. The % DNA in tail mean value ± SD and Damage Index were 21.56 ± 15.41 and 134.83 ± 94.12, respectively. A strong and statistically significant statistically correlation between the two analytical methods was observed (r = 0.95; p < 0.05). These results demonstrate that the comet assay is a useful method to detect the possible effects of genotoxic agents in loggerhead sea turtle and to increase the knowledge about the ecotoxicological health status of this threatened species.

Pub.: 12 Oct '13, Pinned: 21 Sep '17

Vitellogenin (VTG) conservation in sea turtles: anti-VTG antibody in Chelonia mydas versus Caretta caretta.

Abstract: Vitellogenin (VTG) is considered as a marker of endocrine disruption. A Western blot method for VTG quantification in Caretta caretta turtle plasma was developed using anti-VTG antibody for Chelonia mydas. A screening of samples (n = 61) collected in the southern Mediterranean Sea around Lampedusa Island, Italy, was performed. The antibody showed a good cross-reactivity with C. caretta VTG, suggesting a certain conservation of the core of the protein in different sea turtle species. The optimal operative condition for Western blot analysis consists of using diluted plasma at 1:50. In field samples, a certain mismatch with morphological sexing was observed, and VTG was detected in young animals. These results suggest the possibility of a precocious activation of VTG-encoding genes before sexual maturation and/or exposure to endocrine disrupter substances.

Pub.: 26 Nov '09, Pinned: 21 Sep '17

Oxidative stress indicators and chemical contaminants in East Pacific green turtles (Chelonia mydas) inhabiting two foraging coastal lagoons in the Baja California peninsula.

Abstract: In order to determine the potential effects of contaminants in juveniles of East Pacific green turtle, Chelonia mydas, captured alive, circulating trace metal and organochlorine pesticide concentrations were correlated with body condition, antioxidant enzyme activities and lipid peroxidation levels. Turtles were sampled in Punta Abreojos (PAO) and Bahía Magdalena (BMA). Turtles from PAO showed higher silicon and cadmium concentrations, but lower α-hexachlorocyclohexane, γ-hexachlorocyclohexane, hexachlorobenzene and aldrin concentrations than individuals from BMA. In BMA cadmium concentration decreased as the standard carapace length of the turtles increased. In PAO concentrations of α-hexachlorocyclohexane, heptachlor and hexachlorobenzene were positively correlated with the weight of the individuals. Lipid peroxidation levels were positively correlated with cadmium concentrations. In turtles captured in PAO, enzymatic antioxidant activities correlated mostly with pesticide concentrations, while in individuals from BMA enzyme activities were correlated with trace element concentrations. Correlations between antioxidant enzyme activities and concentration of xenobiotics suggest physiological sensitivity of East Pacific green turtles to chemicals. Regional differences found could be influenced by habitat conditions such as currents, upwellings (PAO) and agricultural activities (BMA). We suggest that, combined, circulating contaminant concentrations, lipid peroxidation levels and antioxidant enzyme activities in sea turtles could be used as biomarkers of the habitat conditions.

Pub.: 08 Mar '11, Pinned: 21 Sep '17

Polychlorinated biphenyls and biotransformation enzymes in three species of sea turtles from the Baja California peninsula of Mexico.

Abstract: Concentrations of polychlorinated biphenyls (PCBs) as well as the expression patterns of cytochrome P450 (CYP) enzymes and glutathione-S-transferase (GST) activities were measured in livers of loggerhead (Caretta caretta), green (Chelonia mydas), and olive ridley (Lepidocheyls olivacea) sea turtles from the Baja California peninsula of Mexico. The mean concentrations of total PCBs were 18.1, 10.5, and 15.2 ng/g wet weight (ww) respectively for the three species and PCB 153 was the dominant congener in all samples. Total PCB concentrations were dominated by penta- and hexa-chlorinated biphenyls. The mean estimated TEQs were 42.8, 22.9, and 10.4 pg/g (ww) for loggerhead, green, and olive ridley, respectively, and more than 70% was accounted for by non-ortho PCBs. Western blots revealed the presence of hepatic microsomal proteins that cross-reacted with anti-CYP2K1 and anti-CYP3A27 antibodies but not with anti-CYP1A antibody. There were no significant differences in GST activities between species. Grouping congeners based on structure-activity relationships for CYP isoenzymes suggested limited activity of CYP1A contribution to PCB biotransformation in sea turtles. These results suggest potential accumulation of PCBs that are CYP1A substrates and provide evidence for biotransformation capacity, which differs from known animal models, highlighting the need for further studies in reptiles, particularly those threatened with extinction.

Pub.: 23 Jul '09, Pinned: 21 Sep '17

The characterization of cytosolic glutathione transferase from four species of sea turtles: loggerhead (Caretta caretta), green (Chelonia mydas), olive ridley (Lepidochelys olivacea), and hawksbill (Eretmochelys imbricata).

Abstract: Glutathione s-transferases (GST) play a critical role in the detoxification of exogenous and endogenous electrophiles, as well as the products of oxidative stress. As compared to mammals, GST activity has not been extensively characterized in reptiles. Throughout the globe, most sea turtle populations face the risk of extinction. Of the natural and anthropogenic threats to sea turtles, the effects of environmental chemicals and related biochemical mechanisms, such as GST catalyzed detoxification, are probably the least understood. In the present study, GST activity was characterized in four species of sea turtles with varied life histories and feeding strategies: loggerhead (Caretta caretta), green (Chelonia mydas), olive ridley (Lepidochelys olivacea), and hawksbill (Eretmochelys imbricata). Although similar GST kinetics was observed between species, rates of catalytic activities using class-specific substrates show inter- and intra-species variation. GST from the spongivorous hawksbill sea turtle shows 3-4.5 fold higher activity with the substrate 4-nitrobenzylchloride than the other 3 species. GST from the herbivorous green sea turtle shows 3 fold higher activity with the substrate ethacrynic acid than the carnivorous olive ridley sea turtle. The results of this study may provide insight into differences in biotransformation potential in the four species of sea turtles and the possible health impacts of contaminant biotransformation by sea turtles.

Pub.: 23 May '09, Pinned: 21 Sep '17

Basic oxidative stress metabolites in eastern Pacific green turtles (Chelonia mydas agassizii).

Abstract: Analysis of hematological and biochemical parameters, including oxidative stress indicators, is an invaluable tool in wildlife health assessment, particularly for threatened or endangered species. This study was aimed at obtaining baseline information of oxidative stress indicators in eastern Pacific green turtles (Chelonia mydas agassizii) from a relatively undisturbed habitat at Bahía Magdalena, Baja California Sur, Mexico. Tissues were analyzed for superoxide radical (O(2)(*-) production, lipid peroxidation (measured as thiobarbituric acid reactive substances, TBARS), and antioxidant enzyme activity (superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST)). Overall levels for all variables were within ranges reported for other reptile species. Results suggest differences in oxidative metabolism among tissues (p< or =0.05). Liver, lung and muscle had the highest levels of O(2)(*-) production. Liver revealed the highest TBARS levels. Liver and muscle showed the highest SOD activity, while liver and kidney had the highest CAT and GST activities. These data provide baseline values of the oxidative stress indicators in tissues from eastern Pacific green turtles. Development of a biomarker system to assess the health of wildlife species, especially one that could detect early exposure to environmental pollutants or emerging diseases, would provide a useful tool in the long-term conservation of the species.

Pub.: 26 Aug '06, Pinned: 21 Sep '17

Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata).

Abstract: The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n = 56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.

Pub.: 15 Nov '12, Pinned: 21 Sep '17

Preparation, cryopreservation, and growth of cells prepared from the green turtle (Chelonia mydas)

Abstract: Techniques are described for preparing, preserving, and growing cell cultures from 30 to 40-day old green turtle embryos (2.0–3.0 cm length) including cells derived from skeletal muscle, liver, heart, kidney, eye, lung, and brain. Acceptable growth of all cells occurred in all standard cell culture media tested, with optimum growth temperature near 30 °C. These cell cultures will be used in the study of sea turtle viral diseases including fibropapillomatosis, which is currently epidemic in some green turtle populations.

Pub.: 01 Sep '97, Pinned: 21 Sep '17

Establishment and characterization of 13 cell lines from a green turtle (Chelonia mydas) with fibropapillomas.

Abstract: Thirteen cell lines were established and characterized from brain, kidney, lung, spleen, heart, liver, gall bladder, urinary bladder, pancreas, testis, skin, and periorbital and tumor tissues of an immature male green turtle (Chelonia mydas) with fibropapillomas. Cell lines were optimally maintained at 30 degrees C in RPMI 1640 medium supplemented with 10% fetal bovine serum. Propagation of the turtle cell lines was serum dependent, and plating efficiencies ranged from 13 to 37%. The cell lines, which have been subcultivated more than 20 times, had a doubling time of approximately 30 to 36 h. When tested for their sensitivity to several fish viruses, most of the cell lines were susceptible to a rhabdovirus, spring viremia carp virus, but refractory to channel catfish virus (a herpesvirus), infectious pancreatic necrosis virus (a birnavirus), and two other fish rhabdoviruses, infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus. During in vitro subcultivation, tumor-like cell aggregates appeared in cell lines derived from lungs, testis, and periorbital and tumor tissues, and small, naked intranuclear virus particles were detected by thin-section electron microscopy. These cell lines are currently being used in attempts to isolate the putative etiologic virus of green turtle fibropapilloma.

Pub.: 26 Aug '99, Pinned: 21 Sep '17

Correlation among thermosensitive period, estradiol response, and gonad differentiation in the sea turtle Lepidochelys olivacea.

Abstract: Reptile embryos with temperature sex determination have a thermosensitive period (TSP). The finding that exogenous estradiol (E2) overcomes the effect of male-promoting temperature led to the idea that temperature may regulate estrogen concentration in the gonad during TSP. Since interspecific variations in TSP and in the effect of exogenous E2 exist, we undertook a study in the olive ridley Lepidochelys olivacea. Four parameters were correlated: the TSP (time dimension), the thermosensitive stages (rate of development), gonad development (histological aspect), and the estradiol response. Two kinds of experiments were performed: (1) Eggs were shifted once, at different stages of development, from a male-promoting temperature to a female-temperature (or vice versa) for the remainder of development. (2) Eggs at male-promoting temperature were treated once with 6 or 12 microg of estradiol (E2) at various times of incubation. Sex ratio was established around hatching in each experimental series. We found that the temporal dimension of the TSP was around 7 days (Days 20-27 of incubation) at a male-promoting or a female-promoting temperature. The rate of development of the whole embryo and gonadal growth was faster at female-promoting temperature than at male-promoting temperature. Formation of the genital ridge began at stage 21-22 and histological differentiation of the gonads occurred around stage 26-27. Although these stages coincided with the TSP, at male-promoting temperature the thermosensitive stages occurred earlier (from stages 20-21 to stages 23-24) than at female-promoting temperature (from stages 23-24 to stages 26-27). Thus, at male promoting-temperature, sex was determined in embryos with incipient or undifferentiated gonads. In contrast, E2 treatment continued to feminize the gonads of embryos at a male-promoting temperature beyond the TSP up to stage 25-26, but the E2-induced ovaries were significantly smaller than temperature-induced ovaries. It is suggested that the doses of E2 used were higher than the concentration of endogenous E2 required for normal sex determination. The lack of correlation between sex determination and gonad differentiation suggests that irreversible molecular processes underlying sex determination occur earlier at male- than at female-promoting temperature. Results suggest that the male sex may be the default state and that the female condition must be imposed upon it.

Pub.: 01 Sep '97, Pinned: 21 Sep '17

The effect of organochlorines and heavy metals on sex steroid-binding proteins in vitro in the plasma of nesting green turtles, Chelonia mydas.

Abstract: In this study on green turtles, Chelonia mydas, from Peninsular Malaysia, the effect of selected environmental toxicants was examined in vitro. Emphasis was placed on purported hormone-mimicking chemicals such as dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene, dieldrin, lead, zinc and copper. Five concentrations were used: high (1 mg/L), medium (10(-1) mg/L), low (10(-2) mg/L), very low (10(-6) mg/L) and control (diluted carrier solvent but no toxicants). The results suggest that environmental pesticides and heavy metals may significantly alter the binding of steroids [i.e. testosterone (T) and oestradiol] to the plasma proteins in vitro. Competition studies showed that only Cu competed for binding sites with testosterone in the plasma collected from nesting C. mydas. Dieldrin and all heavy metals competed with oestradiol for binding sites. Furthermore, testosterone binding affinity was affected at various DDT concentrations and was hypothesised that DDT in vivo may act to inhibit steroid-protein interactions in nesting C. mydas. Although the precise molecular mechanism is yet to be described, DDT could have an effect upon the protein conformation thus affecting T binding (e.g. the T binding site on the steroid hormone binding protein molecule).

Pub.: 28 Feb '09, Pinned: 21 Sep '17

Mitogen-induced lymphocyte proliferation in loggerhead sea turtles: comparison of methods and effects of gender, plasma testosterone concentration, and body condition on immunity.

Abstract: A fully functioning immune system is vital to the survival of threatened and endangered sea turtles. Immunological protection against diseases in any organism can be reduced by a number of natural and anthropogenic factors, such as seasonal changes, malnutrition, disease states, and contaminant exposure. These factors are even more critical when they occur in endangered species or populations. To identify alterations in the immunological health of loggerhead sea turtles (Caretta caretta), the mitogen-induced lymphocyte proliferation (LP) assay was developed using peripheral blood leukocytes (PBLs). Collection and culture conditions were optimized for this assay using non-lethal blood samples collected from free-ranging turtles along the southeastern US coast. During the collection, two anticoagulants (sodium heparin and lithium heparin) were compared to determine effects of different ions on assay results. Optimal culture conditions were established for loggerhead PBLs while two different methods of measuring LP were compared: (1) the traditional radioactive (3)H-thymidine assay and (2) a non-radioactive, colorimetric method utilizing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium (MTT). The results indicate that the (3)H-thymidine and the non-radioactive MTT methods did not correlate with each other and that the use of heparin type did not influence the results of the LP assay. Lastly, using these optimized methods, we investigated the effect of gender, plasma testosterone concentration, and body condition on LP in loggerhead turtles and found that none of the parameters largely influenced LP.

Pub.: 29 Dec '04, Pinned: 21 Sep '17

Relationship of blood mercury levels to health parameters in the loggerhead sea turtle (Caretta caretta).

Abstract: Mercury is a pervasive environmental pollutant whose toxic effects have not been studied in sea turtles in spite of their threatened status and evidence of immunosuppression in diseased populations.In the present study we investigate mercury toxicity in loggerhead sea turtles (Caretta caretta) by examining trends between blood mercury concentrations and various health parameters.Blood was collected from free-ranging turtles, and correlations between blood mercury concentrations and plasma chemistries, complete blood counts, lysozyme, and lymphocyte proliferation were examined. Lymphocytes were also harvested from free-ranging turtles and exposed in vitro to methylmercury to assess proliferative responses.Blood mercury concentrations were positively correlated with hematocrit and creatine phosphokinase activity, and negatively correlated with lymphocyte cell counts and aspartate amino-transferase. Ex vivo negative correlations between blood mercury concentrations and B-cell proliferation were observed in 2001 and 2003 under optimal assay conditions. In vitro exposure of peripheral blood leukocytes to methylmercury resulted in suppression of proliferative responses for B cells (0.1 microg/g and 0.35 microg/g) and T cells (0.7 microg/g).The positive correlation between blood mercury concentration and hematocrit reflects the higher affinity of mercury species for erythrocytes than plasma, and demonstrates the importance of measuring hematocrit when analyzing whole blood for mercury. In vitro immunosuppression occurred at methylmercury concentrations that correspond to approximately 5% of the individuals captured in the wild. This observation and the negative correlation found ex vivo between mercury and lymphocyte numbers and mercury and B-cell proliferative responses suggests that subtle negative impacts of mercury on sea turtle immune function are possible at concentrations observed in the wild.

Pub.: 17 Oct '07, Pinned: 21 Sep '17

Comparative cytotoxicity and genotoxicity of soluble and particulate hexavalent chromium in human and hawksbill sea turtle (Eretmochelys imbricata) skin cells.

Abstract: Chromium is both a global marine pollutant and a known human health hazard. In this study, we compare the cytotoxicity and genotoxicity of both soluble and particulate chromate in human and hawksbill sea turtle (Eretmochelys imbricata) skin fibroblasts. Our data show that both soluble and particulate Cr(VI) induce concentration-dependent increases in cytotoxicity, genotoxicity, and intracellular Cr ion concentrations in both human and hawksbill sea turtle fibroblasts. Based on administered concentration, particulate and soluble Cr(VI) were more cytotoxic and clastogenic to human cells than sea turtle cells. When the analysis was based on the intracellular concentration of Cr, the data showed that the response of both species was similar. The one exception was the cytotoxicity of intracellular Cr ions from soluble Cr(VI), which caused more cytotoxicity in sea turtle cells (LC50=271μM) than that of human cells (LC50=471μM), but its clastogenicity was similar between the two species. Thus, adjusting for differences in uptake indicated that the explanation for the difference in potency was mostly due to uptake rather than differently affected mechanisms. Overall these data indicate that sea turtles may be a useful sentinel for human health responses to marine pollution.

Pub.: 07 Oct '15, Pinned: 21 Sep '17

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