Hyperosmolality in the form of elevated NaCl but not urea causes DNA damage in murine kidney cells

Abstract

This study demonstrates, by using neutral comet assay and pulsed field gel electrophoresis, that hyperosmotic stress causes DNA damage in the form of double strand breaks (dsb). Different solutes increase the rate of DNA dsb to different degrees at identical strengths of hyperosmolality. Hyperosmolality in the form of elevated NaCl (HNa) is most potent in this regard, whereas hyperosmolality in the form of elevated urea (HU) does not cause DNA dsb. The amount of DNA dsb increases significantly as early as 15 min after the onset of HNa. By using neutral comet and DNA ladder assays, we show that this rapid induction of DNA damage is not attributable to apoptosis. We demonstrate that renal inner medullary cells are able to efficiently repair hyperosmotic DNA damage within 48 h after exposure to hyperosmolality. DNA repair correlates with cell survival and is repressed by 25 M LY294002, an inhibitor of DNA-activated protein kinases. These results strongly suggest that the hyperosmotic stress resistance of renal inner medullary cells is based not only on adaptations that protect cellular proteins from osmotic damage but, in addition, on adaptations that compensate DNA damage and maintain genomic integrity. C ells adapt to osmotic stress by cell volume regulation and protection of protein structure, activity, and metabolism to maintain proper cell function. Such protective responses are necessary because osmotic stress alters cell volume, the concentration and stability of proteins, and the rate of biochemical reactions (1). Proteins are protected from osmotic stress by molecular chaperones and compatible organic osmolytes (2, 3). We recently showed that mouse renal inner medullary collecting duct (mIMCD3) cells respond to hyperosmotic stress by induction of cell cycle arrest, the tumor suppressor p53, and the growth arrest and DNA damage inducible proteins GADD45 and GADD153 (4,5). These responses are known hallmarks of signaling pathways that counteract DNA damage in mammalian cells (6, 7). DNA damage is defined as an alteration of DNA structure capable of causing cellular injury and reduction of viability or reproductive fitness of the organism (8). Thus, an induction of DNA repair pathways may be required to confer the high osmotic stress tolerance that is characteristic of renal inner medullary cells. However, in contrast to the wealth of knowledge about osmotic effects on cell volume and protein stability and function, little is known about the consequences of osmotic stress on DNA integrity in mammalian cells. Studies on several mammalian cell lines, including CHO cells (9), PAP-HT25 cells (10), human peripheral lymphocytes (11), and V79 cells (12), indicate that osmotic stress can lead to chromosomal aberrations. Such aberrations may be the consequence of an increased frequency of DNA double strand breaks (dsb) or result from an inhibition of constitutive DNA-repair mechanisms. Hyperosmotic stress inhibits inducible DNA repair pathways that are activated in response to ionizing radiation in some mammalian cell...

Quantitative approaches for assessing dose–response relationships in genetic toxicology studies

“…This was originally considered suitable because mutagenic potential was thought to be a rare property of only a few chemicals to which exposure could simply be prevented. However, with accumulating experience, it has become apparent that many chemicals, both synthetic and natural, can induce genetic damage [Galloway et al, 1987;Moore and Brock, 1988;Seeberg et al, 1988;Kultz and Chakravarty, 2001;Charles et al, 2002;Claxton et al, 2010].…”

DNA damage evaluated by γH2AX foci formation by a selective group of chemical/physical stressors

“…It has also been shown that postirradiation hypertonic treatment increased the formation of chromosome aberrations and cell killing and reduced DNA synthesis, as well as an increase in γH2AX foci formation, which may result from the inhibition of DNA repair pathways activated by radiation [30,44,45]. Elevated NaCl itself also can induce DSBs in murine kidney cells, however, DSBs is not caused by hyperosmolality per se but by changes in ionic strength, cell volume, or macromolecular crowding [46]. In our experimental system, FL cells were quickly killed, however, no increase in γH2AX foci was observed.…”

Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses

“…This is in stark contrast to inorganic ions, which at high concentrations typically bind to and destabilize proteins and nucleic acids. Indeed, exposure of some cells to high NaCl medium can produce breaks in DNA (Kültz and Chakravarty, 2001).…”