Nanoparticles can damage DNA even in cells that are not directly exposed to them, according to an in vitro study published online today (November 5) in Nature Nanotechnology -- raising further questions about the safety of nanomaterials used in clinical therapies.

Image: Wikimedia commons, Jerome Walker, Dennis Myts

"DNA damage due to nanoparticles has been described for many types of nanoparticles, but that's done in a primary or direct sense," said Andre Nel, chief of NanoMedicine at the University of California, Los Angeles, who was not involved in the study. "Indirect DNA damage to hazardous nanoparticles is not something that I have seen described before."

Scientists are using nanotechnology to develop delivery systems for drugs and imaging agents, but some studies have suggested these particles may be toxic. Researchers have linked inhalation of nanoparticles or nanotubes to cardiorespiratory disease, for example. Additionally, nanoparticle debris from implants, such as cobalt-chromium (CoCr) alloy particles which can be released from metal-on-metal orthopedic joint replacements, are known to damage human cells in culture.

To further explore the toxicity of CoCr nanoparticles, Charles Patrick Case of the University of Bristol, UK, and his colleagues examined human fibroblast cells that were exposed either directly to the particles or through a cellular barrier. (In a living organism, such a barrier might be the placenta or the lining of the lungs.) They constructed the barrier by growing a thick layer of BeWo cells -- a human cell line often used as a model barrier -- in a porous plastic insert, which they placed above a fibroblast culture. After 24 hours of exposure, the researchers measured the amount of DNA damage in the fibroblasts and found that all cells -- those protected by the BeWo barrier and those directly exposed to the CoCr -- had sustained a significant amount of damage. Parallel experiments with micron-sized particles showed a similar effect.

"When we did this experiment, we imagined the [BeWo] barrier would be an effective barrier. We didn't imagine we would see anything," Case said in a press conference in London this morning. "To our great surprise, not only did we see damage on the other side of the barrier, but we saw as much damage as if we had no barrier at all."

The team found no increase in CoCr in the media below the plastic insert, suggesting that the BeWo barrier, which was up to four cells thick, had successfully kept out the CoCr particles. When they indirectly exposed the fibroblast cultures to the micron-sized particles in the absence of the BeWo barrier -- which could not pass through the pores in the plastic insert -- the cells suffered less damage than they had when the BeWo barrier was present, suggesting that the barrier somehow mediated or intensified the damage.

"Plainly the cell barrier is doing something we didn't expect," Case said. "There is something strange going on." The researchers speculated that the particle exposure caused some type of change in the barrier's top cell layer, which was in turn somehow translated into an effect on the cells below, causing the DNA damage they observed.

When they blocked cellular gap junctions in the fibroblasts with a variety of chemical compounds to prevent intercellular communication, they saw a decrease in DNA damage, suggesting that signaling between the cells transmits the damaging effects to cells not directly exposed to the CoCr particles. Further testing suggested this process involved the release of ATP, which can act as an extracellular signaling molecule.

"Up until now, except for immune responses, which can act indirectly through cytokines and second messengers, this is the first indirect mechanism that I've seen," said toxicologist Stephan Stern of the Nanotechnology Characterization Lab of SAIC-Frederick, Inc. at the National Cancer Institute in Frederick, MD, who was not involved in the research. "Potentially, these particles can act indirectly through second messenger systems to affect target cells without directly acting on those target cells."

Case cautioned that their vitro system differs greatly from the human body. First, the researchers used extremely high concentrations of CoCr -- higher than what might be expected in vivo. Also, the BeWo barrier is not necessarily equivalent to natural cell barriers, such as the placenta. Thus, while this study proposes a possible mechanism by which nanoparticles may indirectly affect cells, there is no evidence that this will occur in the body, Stern said. Some nanoparticles, for instance, such as titanium dioxide, have "been shown to cause DNA damage [in vitro, but] in vivo studies have actually shown protection against carcinogenesis," Stern said.

Additionally, the researchers only investigated one type of nanoparticle and also showed that larger micron-size particles cause the same effect. Based on these findings alone, "we cannot generalize this to all nanoparticles or an exclusive nanoscale property," Nel said.

Still, the results of this study raise the possibility that nanoparticles may have indirect effects that need to be considered when developing nano-based technologies, Ashley Blom, an orthopedic surgeon at the University of Bristol who was not involved in the study, said at the press conference. "This work has raised some really interesting questions, and it's given us insight [into] how barriers in the body might work," he said. "This opens up a whole new field of research we need to look into."


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