Transcriptomic analysis of cultured whale skin cells exposed to hexavalent chromium [Cr(VI)].

Hexavalent chromium Cr(VI) is known to produce cytotoxic effects in humans and is a highly toxic environmental contaminant. Interestingly, it has been shown that free ranging sperm whales (Phyester macrocephalus) may have exceedingly high levels of Cr in their skin. Also, it has been demonstrated that skin cells from whales appear more resistant to both cytotoxicity and clastogenicity upon Cr exposure compared to human cells. However, the molecular genetic mechanisms employed in whale skin cells that might lead to Cr tolerance are unknown. In an effort to understand the underlying mechanisms of Cr(VI) tolerance and to illuminate global gene expression patterns modulated by Cr, we exposed whale skin cells in culture to varying levels of Cr(VI) (i.e., 0.0, 0.5, 1.0 and 5.0 µg/cm²) followed by short read (100 bp) next generation RNA sequencing (RNA-seq). RNA-seq reads from all exposures (≈280 million reads) were pooled to generate a de novo reference transcriptome assembly. The resulting whale reference assembly had 11K contigs and an N50 of 2954 bp. Using the reads from each dose (0.0, 0.5, 1.0 and 5.0 µg/cm²) we performed RNA-seq based gene expression analysis that identified 35 up-regulated genes and 19 down-regulated genes. The experimental results suggest that low dose exposure to Cr (1.0 µg/cm²) serves to induce up-regulation of oxidative stress response genes, DNA repair genes and cell cycle regulator genes. However, at higher doses (5.0 µg/cm²) the DNA repair genes appeared down-regulated while other genes that were induced suggest the initiation of cytotoxicity. The set of genes identified that show regulatory modulation at different Cr doses provide specific candidates for further studies aimed at determination of how whales exhibit resistance to Cr toxicity and what role(s) reactive oxygen species (ROS) may play in this process.

Comparative cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human and sperm whale (Physeter macrocephalus) skin cells.

Chromium (Cr) is a global marine pollutant, present in marine mammal tissues. Hexavalent chromium [Cr(VI)] is a known human carcinogen. In this study, we compare the cytotoxic and clastogenic effects of Cr(VI) in human (Homo sapiens) and sperm whale (Physeter macrocephalus) skin fibroblasts. Our data show that increasing concentrations of both particulate and soluble Cr(VI) induce increasing amounts of cytotoxicity and clastogenicity in human and sperm whale skin cells. Furthermore, the data show that sperm whale cells are resistant to these effects exhibiting less cytotoxicity and genotoxicity than the human cells. Differences in Cr uptake accounted for some but not all of the differences in particulate and soluble Cr(VI) genotoxicity, although it did explain the differences in particulate Cr(VI) cytotoxicity. Altogether, the data indicate that Cr(VI) is a genotoxic threat to whales, but also suggest that whales have evolved cellular mechanisms to protect them against the genotoxicity of environmental agents such as Cr(VI).

The genotoxicity of particulate and soluble chromate in sperm whale (physeter macrocephalus) skin fibroblasts.

Hexavalent chromium is a marine pollutant of concern, both for the health of ocean ecosystems and for public health. Hexavalent chromium is known to induce genotoxicity in human and other terrestrial mammals. It is also known to be present in both water and air in the marine environment. However, currently there are limited data concerning both chromium levels and its toxicological effects in marine mammals. This study investigated the cytotoxic and genotoxic effects of soluble and particulate hexavalent chromium in sperm whale skin fibroblasts. Both forms of hexavalent chromium induced concentration-dependent increases in cytotoxicity and genotoxicity indicating that these compounds can be a health risk if the whales are exposed to them. These data support a hypothesis that chromium is a concern in the marine environment in general and for the health of sperm whales in particular.

The cytotoxicity and genotoxicity of hexavalent chromium in Steller sea lion lung fibroblasts compared to human lung fibroblasts.

In this study we directly compared soluble and particulate chromate cytotoxicity and genotoxicity in human (Homo sapiens) and sea lion (Eumetopias jubatus) lung fibroblasts. Our results show that hexavalent chromium induces increased cell death and chromosome damage in both human and sea lion cells with increasing intracellular chromium ion levels. The data further indicate that both sodium chromate and lead chromate are less cytotoxic and genotoxic to sea lion cells than human cells, based on an administered dose. Differences in chromium ion uptake explained some but not all of the reduced amounts of sodium chromate-induced cell death. By contrast, uptake differences could explain the differences in sodium chromate-induced chromosome damage and particulate chromate-induced toxicity. Altogether they indicate that while hexavalent chromium induces similar toxic effects in sea lion and human cells, there are different mechanisms underlying the toxic outcomes.

Particulate depleted uranium is cytotoxic and clastogenic to human lung epithelial cells.

Depleted uranium (DU) is commonly used in military applications and consequently exposure to soldiers and non-combatants is potentially frequent and widespread. DU is suspected to be a carcinogen, potentially affecting the bronchial cells of the lung. Few studies have considered DU in human bronchial cells. Accordingly, we determined the cytotoxicity and clastogenicity of particulate DU in human bronchial epithelial cells (BEP2D cells). DU-induced concentration-dependent cytotoxicity in human bronchial epithelial cells, and was not clastogenic after 24h but induced chromosomal aberrations after 48h. These data indicate that if DU is a human bronchial carcinogen, it is likely acting through a mechanism that involves DNA breaks after longer exposures.

Depleted uranium induces neoplastic transformation in human lung epithelial cells.

Depleted uranium (DU) is commonly used in military armor and munitions, and thus, exposure of soldiers and noncombatants is frequent and widespread. Previous studies have shown that DU has both chemical and radiological toxicity and that the primary route of exposure of DU to humans is through inhalation and ingestion. However, there is limited research information on the potential carcinogenicity of DU in human bronchial cells. Accordingly, we determined the neoplastic transforming ability of particulate DU to human bronchial epithelial cells (BEP2D). We observed the loss of contact inhibition and anchorage independent growth in cells exposed to DU after 24 h. We also characterized these DU-induced transformed cell lines and found that 40% of the cell lines exhibit alterations in plating efficiency and no significant changes in the cytotoxic response to DU. Cytogenetic analyses showed that 53% of the DU-transformed cell lines possess a hypodiploid phenotype. These data indicate that human bronchial cells are transformed by DU and exhibit significant chromosome instability consistent with a neoplastic phenotype.

A global assessment of chromium pollution using sperm whales (Physeter macrocephalus) as an indicator species.

Chromium (Cr) is a well-known human carcinogen and a potential reproductive toxicant, but its contribution to ocean pollution is poorly understood. The aim of this study was to provide a global baseline for Cr as a marine pollutant using the sperm whale (Physeter macrocephalus) as an indicator species. Biopsies were collected from free-ranging whales around the globe during the voyage of the research vessel The Odyssey. Total Cr levels were measured in 361 sperm whales collected from 16 regions around the globe detectable levels ranged from 0.9 to 122.6 microg Cr g tissue(-1) with a global mean of 8.8+/-0.9 microg g(-1). Two whales had undetectable levels. The highest levels were found in sperm whales sampled in the waters near the Islands of Kiribati in the Pacific (mean=44.3+/-14.4) and the Seychelles in the Indian Ocean (mean=19.5+/-5.4 microg g(-1)). The lowest mean levels were found in whales near the Canary Islands (mean=3.7+/-0.8 microg g(-1)) and off of the coast of Sri Lanka (mean=3.3+/-0.4 microg g(-1)). The global mean Cr level in whale skin was 28-times higher than mean Cr skin levels in humans without occupational exposure. The whale levels were more similar to levels only observed previously in human lung tissue from workers who died of Cr-induced lung cancer. We conclude that Cr pollution in the marine environment is significant and that further study is urgently needed.

Particulate and soluble hexavalent chromium are cytotoxic and genotoxic to Steller sea lion lung cells.

Hexavalent chromium is an environmental contaminant. Within the environment, marine waters are a common site for hexavalent chromium deposition. We have recently reported significantly high levels of chromium in skin tissue from North Atlantic right whales. These findings demonstrate that marine species are being exposed to chromium. It is possible that such exposures may be playing a role in population declines evident among certain marine mammals, such as the Steller sea lion. We developed a Steller sea lion lung cell line from Steller sea lion lung tissue. Hexavalent chromium was cytotoxic to these primary lung fibroblasts as 1, 2.5, 5, 10 and 25microM sodium chromate induced 104, 99, 92, 58 and 11% relative survival, respectively. It was also genotoxic as 0, 1, 2.5, 5 and 10microM sodium chromate damaged chromosomes in 6, 11, 21, 36, and 39% of metaphases and damaged 6, 12, 27, 49 and 57 total aberrations in 100 metaphases, respectively. We also considered the toxicity of particulate hexavalent chromium, as it is the more potent carcinogen in humans. We found that 0.1, 0.5, 1, 5 and 10microg/cm(2) particulate chromate induced 95, 88, 91, 70, and 52% relative cell survival, respectively. These concentrations were genotoxic and damaged chromosomes in 9, 13, 18, and 23% of metaphases and induced 9, 15, 20 and 30 total aberrations per 100 metaphases, respectively. These data indicate that if sufficiently exposed, chromium may adversely affect the struggling Steller sea lion population. It would be prudent to investigate the effects chromium has in other Steller sea lion organs in order to derive a better understanding of how chromium in the marine environment may be affecting the declining Steller sea lion population.