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Published in final edited form as: Comp Biochem Physiol C Toxicol Pharmacol. 2015 Oct 9;178:145–155. doi: 10.1016/j.cbpc.2015.09.013

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

Jamie L Young 1,2,3,4, Sandra S Wise 1,2,3,4, Hong Xie 1,2,3,5, Cairong Zhu 6, Tomokazu Fukuda 7, John Pierce Wise Sr 1,2,3,4,*
PMCID: PMC4669981  NIHMSID: NIHMS730912  PMID: 26440299

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 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=271 uM) that human cells (LC50=471 uM), but its clastogenicity was similar between the two species. Thus, adjusting for differences in uptake indicated the explanation for the difference in potency was mostly due to uptake rather than differently affected mechanisms. Overall these data indicate sea turtles may be a useful sentinel for human health responses to marine pollution.

Keywords: chromium, chromate, sea turtle, hawksbill, genotoxicity, marine pollution, skin

1. Introduction

Ocean pollution is now a global problem. For example, we have recently documented metal pollution in all of the world’s oceans (Holmes et al., 2008a; Martino et al., 2013; Savery et al., 2013a, 2013b, 2014a, 2014b; Wise et al., 2009, 2011a). Aquatic reptiles are key species in the ocean and inhabit both coastal and pelagic ecosystems. They can be long-lived and consequently can bioaccumulate environmental pollutants. Some, such as the American alligator (Alligator mississippiensis) have been classified as endangered and recovered, while others such as the hawksbill sea turtle (Eretmochelys imbricata) remain critically endangered and at risk of extinction (IUCN, 2015). Some human impacts, like hunting and loss of egg-laying habitat, have been well-identified as important factors on the health of aquatic reptiles (e.g. see Meylan and Donnelly, 1999). The impact of environmental pollution is poorly understood. In fact, reptile toxicology, particularly genetic toxicology, is one of the most underdeveloped areas of study despite the importance of these species.

Aquatic reptiles have been used very effectively as environmental sentinels for human exposure and human disease. The most developed model species is the American alligator, which brought attention to the problem of pesticide pollution in Florida lakes and launched the field of endocrine disruption with the discovery of reduced penis length in males as a consequence of pesticide exposure in Lake Woodruff (Delany et al., 1988; Guillette et al., 1994, 1995a, 1995b, 1999; Guillette 1998). Applications of other aquatic reptiles as sentinels and models for human disease have lagged behind particularly for genetic toxicology.

We have been developing sea turtles as an aquatic animal model for metal pollution and impacts for both ecosystem and human health focusing on hexavalent chromium. We chose chromium because our recent data in whales show elevated chromium levels around the globe with some locations having levels so high they fall in the range of those found in chromate workers who died of lung cancer (Wise et al., 2009). Given the high exposures and its known ability to damage DNA and cause reproductive effects (Agency for Toxic Substances and Disease Registry, “ATSDR”, 2012), chromium must be considered as a major marine health concern (Neff, 2002). While we do use whales as model species, it is important to consider other species, particularly from different taxa such as aquatic reptiles.

Data show metal pollutants accumulate in several sea turtle species (Storelli et al., 1998, 2008; Anan et al., 2002; Franzellitti et al., 2004; Maffucci et al., 2005; Gardner et al., 2006; Frias-Expericuet et al., 2006; Andreani et al., 2008; Garcia-Fernandez et al., 2009; Jerez et al., 2010). However, chromium levels were only reported in two of these studies. Suzuki et al (2012) found low plasma Cr levels and did not consider other tissues. The other study reported lung, liver, kidney and muscle levels with lung showing the highest level of 2.29 ppm (mg/kg) (Storelli et al., 1998). While of limited insight, the data do indicate that like whales, sea turtles are being exposed to chromium. Studies of chromium’s impacts on turtles are also limited to two reports. One study found chromium was the most cytotoxic of 4 metals considered (Tan et al., 2010). The other was our report that chromium was cytotoxic and genotoxic to cultured hawksbill turtle cells (Wise et al., 2014). No studies have compared the response of sea turtle and human model systems.

Accordingly, to further develop sea turtles as a model system for human health, we compared the cytotoxic impacts of chromium in cultured human and sea turtle cells. We focused on hexavalent chromium [Cr(VI)] because the chemistry of the marine environment favors the hexavalent form and the data in whales strongly implicate Cr(VI) as the source of exposure in the marine environment (Geisler and Schmidt, 1991; Pettine and Millero, 1990; Li Chen et al., 2009a; Wise et al., 2009, 2011a). We considered both particulate and soluble Cr(VI) because the data in whales and the limited data in sea turtles indicate inhalation is a major route of exposure (Li Chen et al., 2009a; Wise et al., 2011a) and human and rodent studies show particulate Cr(VI) is more potent than soluble Cr(VI) (IARC, 1990; Holmes et al., 2008b; Wise et al., 2008a, 2008b).

2. Methods

2.1 Chemicals and Reagents

RPMI was purchased from Mediatech (Manassas, VA). Penicillin/streptomycin, Gurr’s buffer, sodium, L-glutamine and trypsin/EDTA were purchased from Invitrogen Corporation (Grand Island, NY). Dulbecco’s minimal essential medium and Ham’s F-12 (DMEM/F12) 50:50 mixture was purchased from Mediatech Inc. (Herndon, VA). Cosmic calf serum (CCS) was purchased from Hyclone (Logan, UT). Fetal Bovine Serum (FBS) was purchased from Gibco Life Technologies (Grand Island, NY).Crystal violet, acetic acid, and methanol were purchased from J.T. Baker (Phillipsburg, NJ). Tissue culture dishes, flasks, and plasticware were purchased from BD (Franklin Lakes, NJ). Lead chromate (PbCrO4), sodium chromate (NaCrO4), potassium chloride (KCl), and demecolchicine were purchased from Sigma/Aldrich (St. Louis, MO) Giemsa stain was purchased from Biomedical Specialties Inc. (Santa Monica, CA). Cytoseal 60 slide mounting medium was purchased from VWR (Bridgeport, NJ). MycoAlert detection kits were purchased from Lonza Rockland, Inc (Rockland, ME).

2.2 Cells and Cell Culture

We used hawksbill sea turtle fibroblast cells that were established from a skin biopsy of a healthy, juvenile hawksbill sea turtle (Fukuda et al., 2012) and primary human skin cells (BJ cells) previously described in Vaziri and Benchimol (1998). All cells were cultured as monolayers of adherent cells and allowed to grow to near confluence before they were subcultured or used for experiments. All cultures were fed at least two times a week and subcultured at least once a week. Hawksbill sea turtle cells were grown in RPMI supplemented with 10% FBS and maintained in a humidified incubator with 5% CO2 set at 26°C. BJ cells were grown in DMEM/F-12 50:50 mixture, supplemented with 15% CCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 ug/ml streptomycin, and 0.1 mM sodium pyruvate. The BJ cells were maintained in a humidified incubator with 5% CO2 set at 37°C. Cells were tested routinely for mycoplasma contamination. All experiments were tested on logarithmically growing cells.

2.3 Preparation of Chemicals

Sodium chromate (Na2CrO4) was used as a representative soluble Cr(VI) compound (CAS# 7775-11-3, ACS reagent minimum 98% purity) and was administered as a solution in water as previously described (Wise et al., 2002). Lead chromate (PbCrO4) was used as a representative particulate Cr(VI) compound (CAS# 7758-97-6, ACS reagent minimum 98% purity) and was administered as a suspension in water as previously described (Wise et al., 2002). Sodium chromate treatment concentrations are expressed as uM because sodium chromate dissolves fully in cell culture. Lead chromate treatment concentrations are expressed as weight per surface area (ug/cm2) because it is an insoluble compound that only partially dissolves in cell culture. (Holmes et al., 2005; Wise et al., 2005)

2.4 Cytotoxicity

Cytotoxicity was determined using a clonogenic assay based on our published methods (Wise et al., 2002). Briefly, logarithmically growing cells were seeded in each well of a 6-well tissue culture plate and allowed to rest for 48 hours before being treated with either sodium chromate or lead chromate for 24 hours. After treatment, cells were resuspended in fresh medium and reseeded in four 100 mm tissue culture dishes per concentration at a density of 1000 cells/dish. Once colonies formed (about two weeks) the dishes were rinsed twice with phosphate buffered saline (PBS) and fixed in methanol for 20 minutes. The dishes were then stained with crystal violet for 30 minutes and the colonies counted. All experiments were repeated at least three times and results were expressed as a percentage of the control.

2.5 Clastogenicity

Clastogenicity was measured using a chromosomal aberration assay based on our published methods (Wise et al., 2002). Briefly, logarithmically growing cells were seeded in 100 mm tissue culture dishes and allowed to rest for 48 hours before being treated with either sodium chromate or lead chromate for 24 hours. Cells were arrested in metaphase by adding 0.1 g/ml demecolcine to each 100 mm dish 1 hour prior to the end of the treatment period. At the end of the treatment period, cells were harvested and resuspended in a hypotonic solution of 0.075 M KCl and then fixed with a 3:1 methanol:acetic acid solution. The fixative was changed twice before the cells were dropped on to clean, wet microscope slides and stained with 5% Giemsa stain in Gurr’s Buffer. Slides were analyzed for chromosome aberrations in 100 metaphase per treatment concentration, as described in our published methods (Wise et al., 2002).

2.6 Determination of Intracellular Chromium Ion levels

2.6.1 Cell Preparation

Intracellular chromium ion levels were measured using the ion uptake assay, as previously described (Holmes et al., 2005). Briefly, a monolayer of logarithmically growing cells were seeded into 100 mm tissue culture dishes, allowed to rest for 48 hours to re-enter the normal cell cycle pattern, and then treated with various concentrations of sodium chromate and lead chromate for 24 h. At the completion of treatment, 3 mL of treated culture media was saved for the determination of extracellular chromium ion levels. Cells were then harvested and the number and volume of cells determined. The cells were washed twice with PBS, resuspended in hypotonic solution, followed by the addition of 2% SDS. This solution was sheered through a needle and filtered. The filtered samples were stored at −20°C until analysis.

2.6.2 Ion Level Measurement

Intracellular chromium levels were determined using an Inductively Coupled Plasma-Optical Emissions Spectrometer (ICP-OES) using previously published methods (Holmes et al., 2005). Solutions were introduced to the nebulizer using a peristaltic pump operating at 2 mL/min. Prior to analysis, samples of intracellular fluid were diluted 5x in 0.16 M aqueous HNO3. Chromium was determined using emission wavelength at 267.716 with a minimum detection limit of 2 ppb. Yttrium was used as an internal standard. The intracellular concentrations were converted from ug/L to uM by dividing the volume of the sample, the atomic weight of the chemical, the number of cells in the sample and the average cell volume. 0 h treatments were conducted, and the values subtracted from the 24 h measurements to account for the possibility of particulate Cr(VI) passing through the filter.

2.7 Statistical Analysis

We used the statistical test SCHEFFE for each dose compared to control and each dose compared to each other for each species. A probit model was used to calculate the LC50 and the TD20 because the scatter plot showed a logistic curve.

3. Results

3.1 Cytotoxicity

Soluble and particulate Cr(VI) induced a concentration-dependent decrease in cell survival in both human and hawksbill sea turtle cells. At lower concentrations, the cytotoxic response to soluble Cr(VI) was similar in both sea turtle and human cells. For example, concentrations of 0.5, and 1 sodium chromate induced 79, and 52 percent relative survival in human cells, and 69 and 46 percent relative survival in hawksbill sea turtle cells, respectively (Fig. 1A). At higher concentrations, hawksbill cells were more resistant as 2.5 and 5 uM sodium chromate induced 24, and 4 percent relative survival, respectively compared to 4, and 0 percent relative survival in human cells (Fig. 1A). Based on administered concentration, the calculated LC50 for sodium chromate in sea turtle cells was 0.9 uM (95% confidence interval: 0.9 to 1.0) and 0.9 uM (95% confidence interval: 0.8 to 1.0) in human cells, further reflecting the similar outcomes at lower concentrations.

Fig.1.

Fig.1

Fig.1

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Hawksbill sea turtle and human skin cells exhibit similar cytotoxic responses to soluble Cr(VI), but not to particulate Cr(VI). Particulate Cr(VI) is more cytotoxic to human skin cells than hawksbill sea turtle cells. This figure shows that exposure to Cr(VI) induces cytotoxicity in human and hawksbill sea turtle skin cells in a concentration-dependent manner. (A) Soluble Cr(VI) cytotoxicity in human and hawksbill sea turtle skin cells. In sea turtle cells all concentrations were significantly different from the control except for 0.5 uM (p < 0.02 for 1uM, p < 0.002 for 2.5 uM, p < 0.001 for 5 uM). 0.5 uM was statistically different from 2.5 and 5 uM (p < 0.05 and p < 0.005, respectively). In human cells all concentrations were statistically different from the control (p < 0.04 for 0.5 uM and p < 0.0001 for 1, 2.5, and 5 uM). 0.5 uM was statistically different from 1, 2.5 and 5 uM (p < 0.007, p < 0.0001 and p < 0.0001, respectively). 1 uM was statistically different from 2.5 and 5 uM (p < 0.0001 and p < 0.0001, respectively). (B) Particulate Cr(VI) cytotoxicity in human and hawksbill sea turtle skin cells. Data represents the average of three experiments ± the standard error of the mean. In sea turtle cells all concentrations were significantly different from the control except for 0.1 and 0.5 ug/cm2 (p < 0.0003 for 1 ug/cm2 and p < 0.0001 for 5 ug/cm2). 0.1 ug/cm2 was statistically different from 0.5, 1.0, and 5 ug/cm2 (p < 0.02, p < 0.0001, and p < 0.0001, respectively). 0.5 ug/cm2 was statistically different from 1 and 5 ug/cm2 (p < 0.04 and p < 0.0001, respectively). 1 ug/cm2 was statistically different from 5 ug/cm2 (p < 0.0002). In human cells all concentrations were statistically different from the control (p < 0.002 for 0.1 ug/cm2 and p < 0.0001 for 0.5, 1 and 5 ug/cm2). 0.1 ug/cm2 was statistically different from 0.5, 1 and 5 ug/cm2 (p < 0.002, p < 0.0001, p < 0.0001, respectively). 0.5 ug/cm2 was statistically different from 5 ug/cm2 (p < 0.01).

For particulate Cr(VI), hawksbill sea turtle cells were more resistant to lead chromate-induced cytotoxicity than human cells. For example, concentrations of 0.1, 0.5, 1 and 5 ug/cm2 lead chromate induced 64, 28, 10, and 0 % relative survival, respectively in human cells. In hawksbill sea turtle cells the same concentrations of particulate Cr(VI) induced 108, 79, 54 and 7 percent relative survival, respectively (Fig. 1B). This difference is further shown by the calculated LC50 for lead chromate which was 6.7-fold higher in sea turtle cells than in human cells (1.2 ug/cm2 compared to 0.18 ug/cm2 (95% confidence intervals for each respectively: 1.1 to 1.4 and 0.15 to 0.21)).

3.2. Clastogenicity

Soluble and particulate Cr(VI) induced a concentration-dependent increase in clastogenicity. Clastogenicity was quantified through two different measurements of chromosome aberrations. One measure was the percent of metaphases, which used the cell as the unit of comparison. The other measure, total chromosome damage, used the chromosome as the unit of comparison. Consistent with the cytotoxicity data, sea turtle cells were more resistant to the clastogenic effects of both soluble and particulate Cr(VI). For example, in human cells, sodium chromate concentrations of 0.5, 1, and 2.5 uM damaged chromosomes in 16, 27, and 39 percent of metaphases and induced 18, 30, and 64 total aberrations in 100 metaphases, respectively. In hawksbill sea turtle cells, the same sodium chromate concentrations damaged chromosomes in 9, 14, and 21% of metaphases and induced 10, 16, and 26 total aberrations in 100 metaphases, respectively. At 5 uM sodium chromate, no metaphases were observed in human cells; however, in sea turtle cells, 5 uM sodium chromate induced damage in 29 % of the metaphases and induced 39 total aberrations (Fig. 2).

Fig.2.

Fig.2

Fig.2

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Hawksbill sea turtle cells are more resistant to soluble Cr(VI)-induced clastogenicity than human skin cells. This figure shows that soluble Cr(VI) induces a concentration-dependent increase in clastogenicity in both human and hawksbill sea turtle cells. (A) Soluble Cr(VI)-induced percent of metaphases with chromosome damage. In sea turtle cells all concentrations were significantly different from the control except 0.5 and 1 uM (p < 0.004 for 2.5 uM and p < 0.0007 for 5 uM). 0.5 uM was statistically different from 2.5 and 5 uM (p < 0.05 and p < 0.005, respectively). 1 uM was statistically different from 5 uM (p < 0.03). In human cells all concentrations were statistically different from the control except 0.5 uM (p < 0.02 for 1 uM and p < 0.0008 for 2.5 uM). 0.5 uM was statistically different from 2.5 uM (p < 0.009). (B) Soluble Cr(VI)-induced total chromosome damage. In sea turtle cells all concentrations were significantly different from the control except 0.5 uM (p < 0.02 for 1 uM, p < 0.0004 for 2.5 uM, and p < 0.0001 for 5 uM). 0.5 uM was statistically different from 2.5 and 5 uM (p < 0.006 and p < 0.0002, respectively). 1 uM was statistically different from 5 uM (p < 0.001). In human cells all concentrations were statistically different from the control except 0.5 uM (p < 0.03 for 1 uM and p < 0.0002 for 2.5 uM). 0.5 uM was statistically different from 2.5 uM (p < 0.0005). 1 uM was statistically different from 2.5 uM (p < 0.006). Data represents the average of three experiments (100 metaphases per experiment) ± the standard error of the mean. NM - no metaphases observed. *Only 290 metaphases were scored at 0.5 uM sodium chromate in hawksbill sea turtle cells.

We calculated a TC20 (concentration at which 20 % of cells were damaged or 20 aberrations were produced). Based on administered concentration, the calculated TC20s for the percent of metaphases with damage and total aberrations in 100 metaphases for sodium chromate in sea turtle cells were 2.1 and 1.4 uM (95% confidence intervals for each respectively: 1.6 to 2.9 and 1.1 to 1.8), respectively, compared to 0.67 and 0.59 uM, respectively (95% confidence intervals for each respectively: 0.47 to 0.84 and 0.49 to 0.68), for these same endpoints in human cells. In other words, the TC20s for sodium chromate were 2.4-3.1 times higher in sea turtle cells than in human cells showing the sea turtle cells are more resistant.

Sea turtle cells were also more resistant to particulate Cr(VI)-induced genotoxicity. In human cells, at concentrations of 0.1, 0.5, and 1 ug/cm2 lead chromate induced damage in 16, 28 and 36 percent of metaphases with damage, respectively, and 17, 35, and 46 damaged chromosomes in 100 metaphases, respectively. At 5 ug/cm2 no metaphases were detected. In sea turtle cells, the same concentrations of lead chromate damaged chromosomes in 11, 15 and 26 percent of metaphases producing a total of 11, 17, and 30 aberrations in 100 cells (Fig. 3). Consistent with these observations, the calculated TC20s for the percent of metaphases with damage and total aberrations in 100 metaphases for lead chromate in sea turtle cells were 3.7- and 3.1-fold higher than in human cells, respectively (0.67 and 0.44 ug/cm2 (95% confidence intervals for each respectively: 0.44 to 0.94 and 0.33 to 0.56) compared to 0.18 and 0.14 ug/cm2 (95% confidence intervals for each respectively: 0.10 to 0.26 and 0.09 to 0.19), respectively).

Fig.3.

Fig.3

Fig.3

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Hawksbill sea turtle cells are more resistant particulate Cr(VI)-induced clastogenicity than human skin cells. This figure shows that particulate Cr(VI) induces a concentration-dependent increase in clastogenicity in both human and hawksbill sea turtle cells. (A) Particulate Cr(VI)-induced percent of metaphases with chromosome damage. In sea turtle cells all concentrations were significantly different from the control except 0.1 and 0.5 ug/cm2 (p < 0.002 for 1 ug/cm2 and p < 0.0001 for 5 ug/cm2). 0.1 ug/cm2 was statistically different from 1 and 5 ug/cm2 (p < 0.03 and p < 0.0008, respectively). 0.5 ug/cm2 was statistically different from 5 ug/cm2 (p < 0.002). In human cells all concentrations were statistically different from the control (p < 0.006 for 0.1 ug/cm2 and p < 0.0001 for 0.5 and 1 ug/cm2). 0.1 ug/cm2 was statistically different from 0.5 and 1 ug/cm2 (p < 0.008 and p < 0.0005, respectively). (B) Particulate Cr(VI)-induced total chromosome damage. In sea turtle cells all concentrations were significantly different from the control except 0.1 and 5 ug/cm2 (p < 0.05 for 1 ug/cm2 and p < 0.006 for 5 ug/cm2). 0.1 ug/cm2 was statistically different from 5 ug/cm2 (p < 0.004). 0.5 ug/cm2 was statistically different from 5 ug/cm2 (p < 0.005). 1 ug/cm2 was statistically different from 5 ug/cm2 (p < 0.05). In human cells all concentrations were statistically different from the control except for 0.1 ug/cm2 (p < 0.0007 for 0.5 ug/cm2 and p < 0.0001 for 1 ug/cm2). 1 ug/cm2 was statistically different from 0.5 and 1 ug/cm2 (p < 0.02 and p < 0.002, respectively). Data represents the average of three experiments ± the standard error of the mean (100 metaphases per experiment). NM - no metaphases observed. *Only 254 metaphases were scored at 5 ug/cm2 lead chromate in hawksbill sea turtle cells.

The increase in total aberrations compared to percent of cells with damage in human cells reflects an increased number of human cells with multiple aberrations. This effect was noticeably less in the sea turtle cells. For example, treatment with 2.5 uM sodium chromate produced an average of 10 metaphases with multiple aberrations in human cells, compared to an average of 2 in sea turtle cells. The spectrum of damage was similar in both human and sea turtle cells for both particulate and soluble Cr(VI) with chromatid breaks and gaps being the principal type of aberration (Table 1).

Table 1.

Spectrum of chromosome damage in hawksbill sea turtle and human skin cells after exposure to soluble and particulate Cr(VI).

Treatment Chromosome Aberration Type Total
aberrations
observed
Chromatid
Break		 Chromatid
Gap		 Isochromatid
Break		 Isochromatid
Gap		 Acentric
Fragment		 Double
Minute		 Dicentric Chromatid
Exchange		 Centromere
Spreading
Hawksbill sea turtle skin cells
Sodium Chromate (uM)
   0 1±0.3 2±0.6 0±0.0 0±0.0 0±0.0 0±0.0 0±0.0 0±0.0 0±0.0 3±0.3
   0.5 4±1.4 7±2.0 2±0.9 1±0.6 0±0.0 0±0.0 0±0.0 0±0.0 0±0.0 10±1.2
   1 5±3.1 6±1.2 3±1.0 2±1.2 0±0.0 0±0.0 0±0.3 0±0.0 0±0.0 16±2.9
   2.5 10±0.7 7±3.8 5±2.6 3±1.2 0±0.0 0±0.0 0±0.3 0±0.0 0±0.0 26±2.6
   5 17±0.5 15±2.0 3±1.0 4±0.0 0±0.0 0±0.0 0±0.0 0±0.0 0±0.0 39±0.5
Lead Chromate (ug/cm2)
   0 2±1.0 1±0.7 0±0.3 1±0.3 0±0.0 0±0.0 0±0.0 0±0.0 0±0.0 4±0.3
   0.1 5±0.5 5±1.0 0±0.0 1±0.0 0±0.0 0±0.0 0±0.0 0±0.0 1±0.5 11±1.0
   0.5 8±1.0 8±1.9 0±0.3 1±0.3 0±0.0 0±0.0 0±0.0 0±0.0 0±0.0 17±0.9
   1 17±6.7 7±1.9 3±0.6 2±0.6 0±0.0 0±0.0 0±0.3 0±0.0 0±0.3 30±5.7
   5 31±5.9 15±2.0 6±1.9 2±0.8 1±0.6 0±0.0 0±0.0 1±0.6 0±0.0 56±8.1
Human skin cells
Sodium Chromate (uM)
   0 0±0.3 1±0.3 1±0.7 0±0.3 0±0.0 0±0.0 0±0.0 0±0.0 0±0.0 2±1.0
   0.5 9±0.9 8±1.7 1±0.7 1±0.3 1±0.7 0±0.0 0±0.0 0±0.0 0±0.0 18±3.5
   1 16±4.3 11±1.0 1±1.0 1±0.4 1±0.4 0±0.0 0±0.0 0±0.4 0±0.0 31±6.7
   2.5 33±3.7 13±2.5 5±0.6 2±1.8 1±0.6 1±0.7 0±0.5 1±1.0 0±0.0 64±3.5
   5 NM NM NM NM NM NM NM NM NM NM
Lead Chromate (ug/cm2)
   0 1±0.3 1±0.9 0±0.0 0±0.0 0±0.0 1±0.7 0±0.0 0±0.0 0±0.0 3±1.3
   0.1 7±0.7 8±1.0 0±0.3 0±0.0 0±0.3 0±0.3 0±0.3 0±0.0 0±0.0 17±0.3
   0.5 18±2.3 12±1.2 3±1.5 1±0.3 1±0.6 0±0.0 0±0.0 1±0.3 0±0.3 35±4.5
   1 30±3.6 12±2.2 2±0.8 1±1.0 0±0.0 1±0.3 0±0.0 0±0.3 0±0.0 45±3.7
   5 NM NM NM NM NM NM NM NM NM NM
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Values represent the number of occurance of each type of chromsome abberations. NM - no metaphases.

3.3 Intracellular Chromium Concentrations

We considered the possibility that the difference between the species was due to differences in Cr uptake. To address this possibility, we measured intracellular Cr ion concentrations in human and sea turtle cells (Fig. 4). Intracellular Cr levels increased in a concentration-dependent manner in both human and sea turtle cells after both soluble and particulate Cr(VI) treatments. Human skin cells took up more Cr than sea turtle cells. After exposure to 0.5, 1, 2.5 and 5 uM sodium chromate human cells had intracellular Cr levels of 291, 476, 1,172 and 2,055 uM, respectively. Sea turtle cells treated with the same concentrations had intracellular Cr levels of 37, 265, 518 and 805 uM, respectively (Fig. 4A). After exposure to 0.1, 0.5, 1 and 5 ug/cm2 lead chromate, human cells had intracellular Cr levels of 285, 781, 1,077 and 1,581 uM, respectively. After treatment with the same concentrations of lead chromate, sea turtle cells had intracellular Cr levels of 0, 343, 507 and 1,295 uM, respectively (Fig. 4B).

Fig.4.

Fig.4

Fig.4

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Hawksbill sea turtle skin cells take up less Cr(VI) than human skin cells. This figure shows that human and hawksbill sea turtle skin cells take up Cr in a concentration-dependent manner after exposure to Cr(VI). (A) Cr uptake after soluble Cr(VI) treatment. In sea turtle cells all concentrations were significantly different from the control except 0.5 and 1 uM (p < 0.01 for 2.5 uM and p < 0.003 for 5 uM). 0.5 uM was statistically different from 2.5 and 5 uM (p < 0.04 and p < 0.002, respectively). 1 uM was statistically different from 5 uM (p < 0.02). In human cells all concentrations were statistically different from the control except 0.5 uM (p < 0.05 for 1 uM and p < 0.0001 for 2.5 and 5 uM). 0.5 uM was statistically different from 2.5 and 5 uM (p < 0.002 and p < 0.0001, respectively). 1 uM was statistically different from 2.5 and 5 uM (p < 0.005 and p < 0.0001, respectively. 2.5 uM was statistically different from 5 uM (p < 0.0009). (B) Cr uptake after particulate Cr(VI) treatment. In sea turtle cells only 5 ug/cm2 was significantly different from the control (p < 0.0003). 0.1 ug/cm2 was statistically different from 5 ug/cm2 (p < 0.0001). 0.5 ug/cm2 was statistically different from 5 ug/cm2 (p < 0.002). 1 ug/cm2 was statistically different from 5 ug/cm2 (p < 0.004). In human cells all concentrations were statistically different from the control except 0.1 and 0.5 ug/cm2 (p < 0.02 for 1 ug/cm2 and p < 0.002 for 5 ug/cm2). 0.1 ug/cm2 was statistically different from 1 and 5 ug/cm2 (p < 0.05 and p < 0.003, respectively). 0.5 ug/cm2 was statistically different from 5 ug/cm2 (p < 0.05). Data represents the average of three experiments ± the standard error of the mean.

These Cr uptake data reveal that the explanation for the differences in cytotoxicity and genotoxicity were due to differential Cr uptake and the sea turtle cells were most likely resistant to the administered concentration because they took up less Cr. Correcting for this difference by considering the cytotoxicity and genotoxicity data based on intracellular concentration revealed a very different pattern. Specifically, soluble Cr(VI) was more cytotoxic to sea turtle cells than human cells, but its clastogenicity was similar between the two species (Fig. 5A, Fig. 6A, Fig. 6B). For example, based on intracellular concentration, the calculated LC50 for sodium chromate in sea turtle cells was almost 2-fold lower than in human cells (271 uM compared to 471 uM (95% confidence intervals for each respectively: 253 to 289 and 444 to 499)). By contrast, the calculated TC20s, based on intracellular concentration, were essentially the same. Specifically 1.3-fold higher in sea turtle cells for the percent of metaphases with damage (434 uM compared to 342 uM (95% confidence intervals for each respectively: 326 to 586 and 234 to 431)) and 1.1 lower for sea turtle cells for the total aberrations in 100 metaphases (298 uM compared to 323 uM (95% confidence intervals for each respectively: 217 to 373 and 272 to 269)).

Fig.5.

Fig.5

Fig.5

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Difference in Cr uptake indicate that hawksbill sea turtle cells are more sensitive to soluble Cr(VI)-induced cytotoxicity, but not to particulate Cr(VI)-induced cytotoxicity. This figure shows Cr(VI)-induced cytotoxicity in human and hawksbill sea turtle skin cells as a function of intracellular Cr ion levels. (A) Soluble Cr(VI)-induced cytotoxicity in human and hawksbill sea turtle cells as a function of intracellular Cr ion levels. (B) Particulate Cr(VI)-induced cytotoxicity in human and hawksbill sea turtle cells as a function of intracellular Cr ion levels. Data represents the average of three experiments ± the standard error of the mean.

Fig.6.

Fig.6

Fig.6

Fig.6

Fig.6

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Difference in Cr uptake can explain the observed difference between human and hawksbill sea turtle skin cells to Cr(VI)-induced clastogenicity. This figure shows that exposure to Cr(VI) induces a concentration-dependent increase in clastogenicity in human and hawksbill sea turtle cells as a function of intracellular chromium concentration. (A) Soluble Cr(VI)-induced percent metaphases with chromosome damage as a function of intercellular Cr(VI) concentration. (B) Soluble Cr(VI)-induced total chromosome damage as a function of intercellular Cr(VI) concentration. (C) Particulate Cr(VI)-induced percent metaphases with chromosome damage as a function of intercellular Cr(VI) concentration. (D) Particulate Cr(VI)-induced total chromosome damage as a function of intercellular Cr(VI) concentration.

By contrast, particulate Cr(VI) induced similar amount of cytotoxicity and genotoxicity in human and sea turtle cells after correcting for difference in intracellular uptake (Fig 5B, Fig 6C, Fig 6D). Based on administered dose, discussed above, hawksbill sea turtle cells were more resistant to lead chromate-induced cytotoxicity than human cells with a calculated LC50 6.7-fold higher in sea turtle cells. However, based on intracellular concentration, the calculated LC50 for lead chromate in sea turtle cells was only 1.6-fold higher in sea turtle cells than in human cells ((558 uM compared to 351 uM (95% confidence intervals for each respectively: 512 to 610 and 312 to 388)). Similarly, the calculated TC20s dropped from 3.7- and 3.1-fold higher for the percent of metaphases with damage and total aberrations, respectively in sea turtle cells to essentially the same level in both species (353 and 304 uM, in sea turtles respectively (95% confidence intervals for each respectively: 211 to 471 and 236 to 365), compared to 368 and 318 uM, respectively, for these same endpoints in human cells (95% confidence intervals for each respectively: 238 to 473 and 233 to 390)).

4. Discussion

Aquatic reptiles are an underutilized model for environmental health, despite studies showing they can be effective monitors for pollution and models for human health. We have begun developing sea turtles as a possible model system to consider genotoxic outcomes. Using Cr(VI) as a representative environmental concern, we directly compared the cytotoxic and clastogenic effects of particulate and soluble Cr(VI) in primary human and sea turtle skin fibroblasts. Our data show that Cr(VI) is cytotoxic and clastogenic to skin cells from both species; however, sea turtles cells are more resistant to Cr(VI)-induced cytotoxicity and genotoxicity than human cells. Much of this difference results from sea turtle cells taking up less Cr than human cells. However, correcting for differential uptake revealed interesting differences – soluble Cr(VI) induced similar amounts of chromosome damage in the two species, but induced less cell death in sea turtle cells. By contrast, for particulate Cr(VI) the amount of cytotoxicity and genotoxicity in human and sea turtle cells was similar.

This study is the first to compare cytotoxicity in marine reptiles and human cells. Previous studies compared the cytotoxic effects of particulate and soluble Cr(VI) in North Atlantic right whale (Eubalaena glacialis) and sperm whale (Physeter macrocephalus) cells to human cells. Like the outcome in sea turtle cells, they found Cr(VI) was cytotoxic to whale cells (Li Chen et al., 2009b, 2012). However, they found conflicting results with right whale cells behaving opposite of the sea turtle cells and being similar to humans for soluble Cr(VI) and more sensitive to particulate Cr(VI) (Li Chen et al., 2009b). Sperm whale cells, however, were similar to human cells for particulate Cr(VI), but were resistant to the cytotoxic effects of soluble Cr(VI) after correcting for uptake (Li Chen et al., 2012).

The cytotoxic results in sea turtle cells are particularly intriguing when considered with its genotoxic effects. Sea turtle cells incurred similar amounts of genotoxic damage, but less cell death after soluble Cr(VI) exposure, after correcting for difference in Cr uptake. These data suggest more damaged cells would survive in sea turtle cells creating a greater opportunity for a carcinogenic outcome. The explanation for this difference is uncertain as cell death pathways in sea turtle cells have not been studied. However, these data imply the cell death pathways in sea turtle cells may be less robust than in human cells. Alternatively, these outcomes may reflect differences in crosstalk between DNA damage and cell death pathways in the two species. Further research is needed to distinguish between these possibilities.

It is interesting to note for particulate Cr(VI) the amount of cytotoxicity and genotoxicity in human and sea turtle cells was similar. The explanation for why the outcome is different for soluble Cr(VI), likely reflects the contribution of the cation (lead versus sodium) to cytotoxicity. Lead ions induce apoptosis in mammalian cells (Pulido and Parrish, 2003), although this outcome has not been studied for lead in reptile cells. However, our previous report showed low lead levels inside sea turtle cells after lead chromate exposure (Wise et al., 2014). It is also possible the difference reflects a cytotoxic impact of particle internalization on the cells. Such an outcome is consistent with our previous observations in human, whale and sea lion cells (Li Chen et al., 2009b, 2012; Wise et al., 2020).

This study is also the first to compare genotoxic outcome in marine reptiles and human cells. Previous studies, using whale cells, also compared the genotoxic effects in those cells to human cells (Li Chen et al., 2009b, 2012). As was the case with sea turtle cells, they found Cr(VI) was genotoxic to whale cells (Li Chen et al., 2009b, 2012). However, they found, primary cells from North Atlantic right whale (Eubalaena glacialis) and sperm whale (Physeter macrocephalus), were resistant to the genotoxic effects of particulate and soluble Cr(VI) after correcting for uptake (Li Chen et al., 2009b, 2012). The data in this study differs from those studies as the sea turtle cells had similar amounts of genotoxicity as human cells. This outcome supports our previous hypothesis that marine mammals may have evolved novel cellular mechanisms to protect them from genotoxic exposures.

The differences in Cr(VI) uptake between the sea turtle and human cells were also notable. Sea turtle cells took up less Cr than the human cells. The underlying mechanism for this outcome is uncertain. Cr(VI) uptake is mediated by anion transport proteins (Wise et al., 2008a). The most likely explanation, albeit untested, is that sea turtle cells have fewer anion transport proteins in their cellular membranes resulting in reduced Cr uptake. Of course, it is also possible that sea turtle cells evolved to use a different transport protein for Cr(VI) that has a lesser Cr(VI) affinity. It is interesting to note previous reports showed North Atlantic right whale, sperm whale and Steller sea lion cells also took up less Cr than human cells suggesting this phenotype is a common feature of cells in the marine environment (Li Chen et al., 2009b, 2012; Wise et al., 2010).

It would be interesting to understand these treatments in the context of sea turtles in the ocean. However, currently, there aren’t a good exposure profile for sea turtles as Cr(VI) levels have not been determined in their environment. A dietary exposure could be considered, though there is limited data from potential prey species, which for hawksbill turtles is mostly marine sponges. Focusing on this exposure route, data show total Cr levels from marine sponges in the Mediterranean Sea (Perez et al., 2004) ranged from 0.15 to 2.3 ug total Cr/g tissue (converted from dry weight to wet weight with a 0.25 adjustment factor (Wise et al., 2011a,b)). Given hawksbill sea turtles eat about 1,500 g prey/day NMFS, 2014), then the sea turtles daily exposure might range from 225-3,412 ug total Cr just from prey consumption [for example: 0.15 ug Cr/g tissue × 1,500 g sponge tissue/day = 225 ug total Cr/day]. Given our study treatment range of 0.52-273 ug total Cr/day, our exposure regimen generally falls below the low end of the range of what sea turtles could potentially be exposed to in their diet. Thus, Cr seems to be a significant health hazard for sea turtles as well as humans.

In summary, our study shows sea turtle cells respond similarly to human cells to the cytotoxic and genotoxic effect of Cr(VI) indicating sea turtles can be a useful model for metal genotoxicity in human systems. There are some interesting nuances between the species with sea turtle cells possibly differing in their cell death pathways or crosstalk between cell death and DNA damage pathways. The data further suggest sea turtle cells may be more sensitive to the cytotoxic effects of lead ions. We intend to investigate these differences further and expand our comparisons to other metals and other aquatic reptiles including other sea turtles.

Acknowledgments

The authors would like to acknowledge Tania Li Chen and Alexandra Smith for technical assistance and Shouping Huang and Christy Gianios, Jr. for the administrative and information technology support, respectively. We would also like to thank The Vieques Conservation and Historical Trust, TICATOVE, and the Vieques Office of the United States Fish and Wildlife Service for their support for this work. We also thank Dr. Steve Patierno for the BJ cells. Research reported in this publication was also supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Number R01ES016893 (JPW). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional funding was provided by the Maine Space Grant Consortium (JPW), The Ocean Foundation (JPW), the Henry Foundation (JPW), the Curtis and Edith Munson Foundation (JPW). Work was conducted under NMFS permit 16305-00 (J.PW.).

Footnotes

★

This paper is based on a presentation given at the 7th Aquatic Animal Models of Human Disease Conference, hosted by Texas State University (Dec 13 - Dec 19, 2014)

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Conflict of Interest

The authors declare that there are no conflicts of interest.

References

  1. Agency for Toxic Substances and Disease Registry (ATSDR) Toxicological profile for Chromium. U.S. Department of Health and Human Services, Public Health Service; Atlanta, GA: 2012. [PubMed] [Google Scholar]
  2. Anan Y, Kunito T, Sakai H, Tanabe S. Subcellular distribution of trace elements in the liver of sea turtles. Mar. Pollut. Bull. 2002;45:224–229. doi: 10.1016/s0025-326x(02)00106-6. [DOI] [PubMed] [Google Scholar]
  3. Andreani G, Santoro M, Cottignoli S, Fabbri M, Carpene E, Isani G. Metal distribution and metallothionein in loggerhead (Caretta caretta) and green (Chelonia mydas) sea turtles. Sci. Total Environ. 2008;390:287–294. doi: 10.1016/j.scitotenv.2007.09.014. [DOI] [PubMed] [Google Scholar]
  4. Delany MF, Bell JU, Sundlof SF. Concentrations of contaminants in muscle of the American alligator in Florida. J. Wildlife Disease. 1988;24(1):62–66. doi: 10.7589/0090-3558-24.1.62. [DOI] [PubMed] [Google Scholar]
  5. Franzellitti S, Locatelli C, Gerosa G, Vallini C, Fabbri E. Heavy metal in tissues of loggerhead turtles (Caretta caretta) from the northwestern Adriatic Sea. Comp. Biochem. Physiol. C. 2004;138:187–194. doi: 10.1016/j.cca.2004.07.008. [DOI] [PubMed] [Google Scholar]
  6. Frias-Expericuet MG, Osuna-Lopez JI, Ruiz-Telles A, Quitero-Alvarez JM, Lopez-Lopez G, Izaguirre-Fierro G, Voltolina D. Heavy metals in the tissues of the sea turtle Lepidochelys olivacea from a nesting site of the northwest coast of Mexico. Bull. Environ. Contam. Toxicol. 2006;77:179–185. doi: 10.1007/s00128-006-1048-1. [DOI] [PubMed] [Google Scholar]
  7. Fukuda T, Kurita J, Saito T, Yuasa K, Kurita M, Donai K, Nitto H, Soichi M, Nishimori K, Uchida T, Isogai E, Onuma M, Sone H, Oseko N, Inoue-Murayama M. Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbracata) J. In Vitro Cell. Dev. Biol. Anim. 2012;48(10):660–665. doi: 10.1007/s11626-012-9565-1. [DOI] [PubMed] [Google Scholar]
  8. Garcia-Fernandez AJ, Gomez-Ramirez P, Matinez-Lopez E, Hernandez-Garcia A, Maria-Mojica P, Romero D, Jimenez P, Castillo JJ, Bellido JJ. Heavy metals in tissues from loggerhead turtles (Caretta caretta) from the south western Mediterranean (Spain) Ecotoxicol. Environ. Saf. 2009;72:557–563. doi: 10.1016/j.ecoenv.2008.05.003. [DOI] [PubMed] [Google Scholar]
  9. Gardner SC, Fitzgerald SL, Vargas BA, Rodriguez LM. Heavy metal accumulation in four species of sea turtles from the Baja California peninsula, Mexico. BioMetals. 2006;19:91–99. doi: 10.1007/s10534-005-8660-0. [DOI] [PubMed] [Google Scholar]
  10. Geisler CD, Schmidt D. An overview of chromium in the marine environment. Dt. Hydrogr. Z. 1991;44:185–196. [Google Scholar]
  11. Guillette LJ, Gross TS, Masson GR, Matter JM, Percival HF, Woodward AR. Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lake in Florida. Environ. Health Perspect. 1994;102:680–688. doi: 10.1289/ehp.94102680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guillette LJ, Jr., Gross TS, Gross DA, Rooney AA, Percival HF. Gonadal steroidogenesis in vitro from juvenile alligators obtained from contaminated or control lakes. Environ. Health Perspect. 1995a;103(suppl 4):31–36. doi: 10.1289/ehp.95103s431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Guillette LJ, Jr., Crain DA, Rooney AA, Pickford DB. Organization versus activation: the role of endocrine-disrupting contaminants (EDCs) during embryonic development in wildlife. Environ. Health Perspect. 1995b;107(suppl 7):157–164. doi: 10.1289/ehp.95103s7157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guillette LJ., Jr. Endocrine disruption contaminants and alligator embryos: a less from wildlife? In: Eisenbrand G, Daniel H, Dayan AD, Elias PS, Grunow W, Kemper FH, Loser E, Metzler M, Schlatter J, editors. Hormonally active agents in food. A Deutsche Forschungsgemeinschaft symposium. Wiley—VCH; Weinheim: 1998. pp. 72–88. [Google Scholar]
  15. Holmes AL, Wise SS, Xie H, Gordon N, Thompson WD, Wise JP., Sr. Lead ions do not cause human lung cells to escape chromate-induced cytotoxicity. Toxicol. Appl. Pharmol. 2005;203:167–176. doi: 10.1016/j.taap.2004.08.006. [DOI] [PubMed] [Google Scholar]
  16. Holmes AL, Wise SS, Goertz CEC, Lawrence JD, Gulland FMD, Gelatt T, Beckmen KB, Burek K, Atkinson S, Bozza M, Taylor R, Zheng T, Zhang Y, Aboueissa A-M, Wise JP., Sr. Metal tissue levels in Steller sea lion (Eumetopias jubatus) pups. Mar. Pollut. Bull. 2008a;56:1416–1421. doi: 10.1016/j.marpolbul.2008.05.007. [DOI] [PubMed] [Google Scholar]
  17. Holmes AL, Wise SS, Wise JP., Sr. Carcinogenicity of hexavalent chromium. Indian J. Med. Res. 2008b;128:353–372. [PubMed] [Google Scholar]
  18. IARC . Monographs on the Evaluation of Carcinogenic Risks to Humans: Chromium, Nickel and Welding. Vol. 49. International Agency for Research on Cancer; Lyons, France: 1990. [PMC free article] [PubMed] [Google Scholar]
  19. IUCN [Downloaded on 24 June];The IUCN Red List of Threatened Species. 2015 Version 2015.2. www.iucnredlist.org.
  20. Jerez S, Motas M, Canovas RA, Talavera J, Almela RM, del Rio AB. Accumulation and tissue distribution of heavy metals and essential elements in loggerhead turtles (Caretta caretta) from Spanish Mediterranean coastline of Murcia. Chemosphere. 2010;78:256–264. doi: 10.1016/j.chemosphere.2009.10.062. [DOI] [PubMed] [Google Scholar]
  21. Li Chen T, Wise SS, Kraus S, Shaffiey F, Grau M, Thompson WD, Zheng T, Zhang Y, Romano T, O’Hara T, Wise JP., Sr. Particulate hexavalent chromium is cytotoxic and genotoxic to the North Atlantic right whale (Eubalaena glacialis) lung and skin fibroblasts. Environ. Mol. Mutagen. 2009a;50:387–393. doi: 10.1002/em.20471. [DOI] [PubMed] [Google Scholar]
  22. Li Chen T, Wise SS, Homes A, Shaffiey F, Wise JP, Jr., Thompson WD, Kraus S, Wise JP., Sr. Cytotoxicity and gentoxicity of hexavalent chromium in human and North Atlantic right whale (Eubalaena glacialis) lung cells. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2009b;150:487–494. doi: 10.1016/j.cbpc.2009.07.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li Chen T, LaCerte C, Wise SS, Holmes A, Martino J, Wise JP, Jr., Thompson WD, Wise JP., Sr. Comparative cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human and sperm whale (Physeter macrocephalus) skin cells. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2012;155:143–150. doi: 10.1016/j.cbpc.2011.03.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maffucci F, Caurant F, Bustamante P, Bentivegna F. Trace element (Cd, Cu, Hg, Se, Zn) accumulation and tissue distribution in loggerhead turtles (Caretta caretta) from the western Mediterranean sea (southern Italy) Chemosphere. 2005;58:535–542. doi: 10.1016/j.chemosphere.2004.09.032. [DOI] [PubMed] [Google Scholar]
  25. Martino J, Wise SS, Perkin C, Sironi M, Wise JP., Sr. Metal levels in southern right whales (Eubalaena australis) from Península Valdés, Argentina. J. Environ. Anal. Toxicol. 2013;3(6):1–6. doi: 10.4172/2161-0525.1000190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Meylan A, Donnelly M. Status justification for listing the hawksbill turtle (Eretmochelys imbricata) as critically endangered on the 1996 IUCN red list of threatened animals. Chelonian Conserv. Biol. 1999;3(2):200–224. [Google Scholar]
  27. National Marine Fisheries Service [website accessed May 29, 2014];Office of Protected Resources. 2014 http://www.nmfs.noaa.gov/pr/species/turtles/hawksbill.htm.
  28. Neff JM. Chromium in the ocean. In: Neff JM, editor. Bioaccumulation in Marine Organisms: Effect of Contaminants from Oil Well Produced Water. Elsevier Ltd; 2002. pp. 131–143. [Google Scholar]
  29. Perez T, Vacelet J, Rebouillon P. In situ comparative study of severalMediterranean sponges as potential biomonitors of heavy metals. Boll. Mus. Ist. Biol. Univ. Genova. 2004;68:517–525. [Google Scholar]
  30. Pettine M, Millero FJ. Chromium speciation in seawater: the probable role of hydrogen peroxide. Limnol. Oceanogr. 1990;35:730–736. [Google Scholar]
  31. Pulido MD, Parrish AR. Metal-induced apoptosis: mechanisms. Review. Mutat. Res. 2003;533:227–241. doi: 10.1016/j.mrfmmm.2003.07.015. [DOI] [PubMed] [Google Scholar]
  32. Savery LC, Wise SS, LaCerte C, Wise J, Gianios C, Jr., Thompson WD, Perkins C, Mason MD, Payne R, Kerr I, Wise JP., Sr. Global assessment of silver pollution using sperm whales (Physeter macrocephalus) as an indicator species. J. Environ. Anal. Toxicol. 2013a;3(2):1–8. [Google Scholar]
  33. Savery LC, Evers DC, Wise SS, LaCerte C, Wise J, Gianios C, Jr., Kerr I, Payne R, Thompson WD, Perkins C, Zheng T, Zhu C, Benedict L, Wise JP., Sr. Global mercury and selenium concentrations in skin from free-ranging sperm whales (Physeter macrocephalus) Sci. Total Enviro. 2013b;450-451:59–71. doi: 10.1016/j.scitotenv.2013.01.070. [DOI] [PubMed] [Google Scholar]
  34. Savery LC, Wise J, Wise SS, LaCerte C, Gianios C, Jr., Thompson WD, Perkins C, Zheng T, Zhu C, Wise JP., Sr. Global assessment of arsenic pollution using sperm whales (Physeter macrocephalus) as an emerging aquatic model organism. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2014a;163:55–63. doi: 10.1016/j.cbpc.2014.01.003. [DOI] [PubMed] [Google Scholar]
  35. Savery LC, Wise J, Wise SS, LaCerte C, Gianios C, Jr., Thompson WD, Perkins C, Zheng T, Zhu C, Wise JP., Sr. Global assessment of oceanic lead pollution using sperm whales (Physeter macrocephalus) as an indicator species. Mar. Pollut. Bull. 2014b;79:236–244. doi: 10.1016/j.marpolbul.2013.12.007. [DOI] [PubMed] [Google Scholar]
  36. Storelli MM, Ceci E, Marcotrigiano GO. Distribution of heavy metal residues in some tissues of the Caretta carretta (Linnaeus) specimen beached along the Adriatic Sea (Italy) Bull. Environ. Contam. Toxicol. 1998;60:546–552. doi: 10.1007/s001289900660. [DOI] [PubMed] [Google Scholar]
  37. Storelli MM, Barone G, Storelli GO, Marcotrigiano GO. Total and subcellular distribution of trace elements (Cd, Cu and Zn) in the liver and kidney of green turtles (Chelonia mydas) from the Mediterranean Sea. Chemosphere. 2008;70:908–913. doi: 10.1016/j.chemosphere.2007.06.069. [DOI] [PubMed] [Google Scholar]
  38. Suzuki K, Noda J, Yanagisawa M, Kawazu I, Sera K, Fukui D, Asakawa M, Yokota H. Relationships between carapace sizes and plasma major and trace element status in captive hawksbill sea turtles (Eretmochelys imbricata) J. Vet. Med. Sci. 2012;74:167–1680. doi: 10.1292/jvms.12-0099. [DOI] [PubMed] [Google Scholar]
  39. Tan F, Wang M, Wang W, Alonso Aguirre A, Lu Y. Validation of an in vitro cytotoxicity test for four heavy metals using cell lines derived from a green sea turtle (Chelonia mydas) Cell Biol. Toxicol. 2010;26:255–263. doi: 10.1007/s10565-009-9130-1. [DOI] [PubMed] [Google Scholar]
  40. Vaziri H, Benchimol S. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr. Biol. 1998;8:279–282. doi: 10.1016/s0960-9822(98)70109-5. [DOI] [PubMed] [Google Scholar]
  41. Wise SS, Holmes AL, Moreland JA, Xie H, Sandwick SJ, Stackpole MM, Fomchenko E, Teufack S, May AJ, Jr., Katsifas SP, Wise JP., Sr. Human lung cell growth is not stimulated by lead ions after lead chromate-induced genotoxicity. Mol. Cell. Biochem. 2005;279:75–84. doi: 10.1007/s11010-005-8217-0. [DOI] [PubMed] [Google Scholar]
  42. Wise SS, Holmes AL, Wise JP., Sr. Hexavalent chromium-induced DNA damage and repair mechanisms. Rev. Environ. Health. 2008a;23(1):39–57. doi: 10.1515/reveh.2008.23.1.39. [DOI] [PubMed] [Google Scholar]
  43. Wise SS, Xie H, Fukuda T, Thompson WD, Wise JP., Sr. Hexavalent chromium is cytotoxic and genotoxic to hawksbill sea turtle cells. Toxicol. Appl. Pharmol. 2014;279:113–118. doi: 10.1016/j.taap.2014.06.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wise JP, Sr., Wise SS, Little JE. The cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human lung cells. Mutat. Res. 2002;517:221–229. doi: 10.1016/s1383-5718(02)00071-2. [DOI] [PubMed] [Google Scholar]
  45. Wise JP, Sr., Wise SS, Kraus S, Shaffiey F, Grau M, Li Chen T, Perkins C, Thompson WD, Zheng T, Zhang Y, Romano T, O’Hara T. Hexavalent chromium is cytotoxic and genotoxic to the North Atlantic right whale (Eubalaena glacialis) lung and testes fibroblasts. Mutat. Res. 2008b;650:30–38. doi: 10.1016/j.mrgentox.2007.09.007. [DOI] [PubMed] [Google Scholar]
  46. Wise JP, Sr., Payne R, Wise SS, LaCerte C, Wise J, Gianios C, Jr., Thompson WD, Perkins C, Zheng T, Zhu C, Benedict L, Kerr I. A global assessment of chromium pollution using sperm whales (Physeter macrocephalus) as an indicator species. Chemosphere. 2009;75:1461–1467. doi: 10.1016/j.chemosphere.2009.02.044. [DOI] [PubMed] [Google Scholar]
  47. Wise JP, Sr., Wise SS, Holmes AL, Lacerte C, Shaffiey F, Aboueissa A-M. The cytotoxicity and genotoxicity of hexavalent chromium in Stellar sea lion lung fibroblasts compared to human lung fibroblasts. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2010;152:91–98. doi: 10.1016/j.cbpc.2010.03.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wise JP, Wise SS, LaCerte C, Wise JP, Aboueissa A. The genotoxicity of particulate and soluble chromate in sperm whale (Physeter macrocephalus) skin fibroblasts. Environ. Mol. Mutagen. 2011a;52(1):43–49. doi: 10.1002/em.20579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wise JP, Sr., Thompson WD, Wise SS, LaCerte C, Wise J, Gianios C, Jr., Perkins C, Zheng T, Benedict L, Mason MD, Payne R, Kerr I. A global assessment of gold, titanium, strontium, and barium pollution using sperm whales (Physeter macrocephalus) as an indicator species. J. Ecosys. Ecograph. 2011b;1(1):1–8. [Google Scholar]

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