The cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in leatherback sea turtle lung cells

Abstract

Hexavalent chromium [Cr(VI)] is a marine pollution of concern as recent studies show it has a global distribution, with some regions showing high Cr concentrations in marine animal tissue, and it is extensively used. Leatherback sea turtles (Dermochelys coriacea) are an endangered marine species that may experience prolonged exposures to environmental contaminants including Cr(VI). Human activities have led to global Cr(VI) contamination of the marine environment. While Cr(VI) has been identified as a known human carcinogen, the health effects in marine species are poorly understood. In this study, we assessed the cytotoxic and genotoxic effects of particulate and soluble Cr(VI) in leatherback sea turtle lung cells. Both particulate and soluble Cr(VI) induced a concentration-dependent increase in cytotoxicity. Next, using a chromosome aberration assay, we assessed the genotoxic effects of Cr(VI) in leatherback sea turtle lung cells. Particulate and soluble Cr(VI) induced a concentration-dependent increase in clastogenicity in leatherback sea turtle lung cells. These data indicate that Cr(VI) may be a health concern for leatherback sea turtles and other long-lived marine species. Additionally, these data provide foundational support to use leatherback sea turtles as a valuable model species for monitoring the health effects of Cr(VI) in the environment and possibly as an indicator species to assess environmental human exposures and effects.

Keywords: Chromate; Cytotoxicity; Genotoxicity; Hexavalent chromium; Leatherback sea turtle; Marine pollution.

Figure 1. Image of PGDC9-1LU Cells

This figure shows a representative image of the leatherback sea turtle lung cells used in this study.

Figure 2. Intracellular Chromium Ion Concentrations Increase with Increasing Particulate and Soluble Chromate Treatment

This figure shows that with increasing particulate and soluble chromate treatments, intracellular Cr ion concentrations increase in a concentration-dependent manner. Data represent an average of at least three independent experiments ± standard error of the mean. *Statistically significant compared to control (p < 0.05). (A) Lead chromate. (B) Sodium chromate.

Figure 3. Particulate and Soluble Chromate are Cytotoxic to Leatherback Sea Turtle Lung Cells

This figure shows a 24h exposure to particulate or soluble chromate reduced relative survival in a concentration-dependent manner. Data represent an average of at least three independent experiments ± standard error of the mean. *Statistically significant compared to control (p < 0.05). (A) Lead chromate. (B) Sodium chromate. C) Regression analysis was used to determine r² and LC₅₀ values for lead chromate (p = 0.018). (D) Regression analysis was used to determine r² and LC₅₀ values for sodium chromate (p = 0.016).

Figure 3. Particulate and Soluble Chromate are Cytotoxic to Leatherback Sea Turtle Lung Cells

This figure shows a 24h exposure to particulate or soluble chromate reduced relative survival in a concentration-dependent manner. Data represent an average of at least three independent experiments ± standard error of the mean. *Statistically significant compared to control (p < 0.05). (A) Lead chromate. (B) Sodium chromate. C) Regression analysis was used to determine r² and LC₅₀ values for lead chromate (p = 0.018). (D) Regression analysis was used to determine r² and LC₅₀ values for sodium chromate (p = 0.016).

Figure 4. Particulate and Soluble Chromate Induce Similar Levels of Cytotoxicity in Leatherback Sea Turtle Lung Cells

This figure shows when comparing intracellular concentrations of Cr ions, particulate and soluble chromate induce similar levels of cytotoxicity Data represent an average of at least three independent experiments ± standard error of the mean. (A). Scatter plot showing particulate and soluble chromate induce similar levels of cytotoxicity at similar intracellular Cr ion concetrations. (B) Regression analysis determined r² values for lead chromate and sodium chromate to be significant predictors of relative survival, p = 0.001 and p = 0.003, respectively.

Figure 5. Particulate and Soluble Chromate are Genotoxic to Leatherback Sea Turtle Lung Cells

This figure shows after a 24 h exposure particulate and soluble chromate induces a concentration-dependent increase in chromosome damage. No metaphases were observed at the highest concentration tested for particulate chromate (10 ug/cm²) or soluble chromate (10 uM). Data represent an average of three independent experiments ± standard error of the mean. *Statistically significant compared to control (p < 0.05). (A) Lead chromate. (B) Sodium chromate. (C). Regression analysis was used to determine r² and TC₂₀ values for the percent of metaphases with damage (p = 0.052) and total damage in 100 metaphases (p = 0.021) for lead chromate (D) Regression analysis was used to determine r² and TC₂₀ values for the percent of metaphases with damage (p = 0.031) and total damage in 100 metaphases (p = 0.017) for sodium chromate were also calculated based on regression analysis.

Figure 5. Particulate and Soluble Chromate are Genotoxic to Leatherback Sea Turtle Lung Cells

This figure shows after a 24 h exposure particulate and soluble chromate induces a concentration-dependent increase in chromosome damage. No metaphases were observed at the highest concentration tested for particulate chromate (10 ug/cm²) or soluble chromate (10 uM). Data represent an average of three independent experiments ± standard error of the mean. *Statistically significant compared to control (p < 0.05). (A) Lead chromate. (B) Sodium chromate. (C). Regression analysis was used to determine r² and TC₂₀ values for the percent of metaphases with damage (p = 0.052) and total damage in 100 metaphases (p = 0.021) for lead chromate (D) Regression analysis was used to determine r² and TC₂₀ values for the percent of metaphases with damage (p = 0.031) and total damage in 100 metaphases (p = 0.017) for sodium chromate were also calculated based on regression analysis.

Figure 6. Particulate and Soluble Chromium Induce Similar Levels of Genotoxicity

This figure shows that at similar levels of intracellular Cr ion concentrations particulate and soluble chromate induce a similar frequency of cells with damage and similar levels of total chromosome damage. Data represent an average of at least three experiments ± standard error of the mean. (A) Percent of metaphases with damage. (B) Total aberrations in 100 metaphases. (C) Regression analysis determined r² values for lead chromate and sodium chromate to be significant predictors of the percent of metaphases with damage, p = 0.002 and p = 0.032, respectively (D). Regression analysis determined r² values for lead chromate and sodium chromate to be significant predictors of total damage in 100 metaphases, p = 0.001 and p = 0.019, respectively.

Figure 6. Particulate and Soluble Chromium Induce Similar Levels of Genotoxicity

This figure shows that at similar levels of intracellular Cr ion concentrations particulate and soluble chromate induce a similar frequency of cells with damage and similar levels of total chromosome damage. Data represent an average of at least three experiments ± standard error of the mean. (A) Percent of metaphases with damage. (B) Total aberrations in 100 metaphases. (C) Regression analysis determined r² values for lead chromate and sodium chromate to be significant predictors of the percent of metaphases with damage, p = 0.002 and p = 0.032, respectively (D). Regression analysis determined r² values for lead chromate and sodium chromate to be significant predictors of total damage in 100 metaphases, p = 0.001 and p = 0.019, respectively.