Spring 2003 — vol. 21, no. 1 logos home page Contact the logos editor Argonne home page Feature articles Ice smoothie gives heart attacks the 'Big Chill,' may save lives Cadmium causes calcium loss in bones within hours of exposure X-rays penetrate secrets of diesel engine Argonne Update Insect breathing mechanism discovered Surface disorder is more than skin deep Portable detector finds concealed nuclear weapons Gammasphere returns to Argonne

Cadmium causes calcium loss in bones within hours of exposure

by Jennifer Ann Hutt

A team of Argonne researchers has demonstrated for the first time that cadmium releases calcium from bone within hours of exposure, even at concentrations below current Occupational Safety and Health Administration (OSHA) standards. In humans, this response might lead to the bone disease osteoporosis.

CALCIUM RELEASE — Biochemist Maryka Bhattacharyya's group measured the calcium mice released just hours after exposure to cadmium.

"We believe the bone response to cadmium is happening now in some factories," senior biochemist Maryka Bhattacharyya said. "It's just that people don't know about it until they are older, and they start to break bones because they have osteoporosis."

Cadmium is a toxic metal found in industrial workplaces that smelt and refine zinc ores or produce nickel-cadmium batteries. It is also present in cigarette smoke.

Argonne scientists are the first to show that cadmium's effect on bone begins only hours after exposure, long before the onset of kidney damage that cadmium is known to cause, and at blood concentrations below the OSHA action level of 5 parts per billion.

Argonne researchers have begun to explore how cadmium interacts with bone cells during the first hours after exposure and have identified specific genes involved in the bone response. Their findings were recently published in the journal Toxicology and Applied Pharmacology.

Understanding the steps cadmium goes through to cause bone loss may lead to more appropriate regulation of cadmium exposure in industrial settings. "The government can set better standards when they know how something is working," Bhattacharyya said. The research is funded by the Department of Energy and the National Institute of Environment Health Sciences.

Knowing how cadmium interacts with bone may make it possible to screen for early steps in that process, detecting the potential for excess bone loss before much damage occurs.

A delicate balance

In healthy bone, a balance exists between the rates of bone formation and demineralization, or break down. Specialized cells build calcified bone matrix, while others degrade it. The bone-degrading cells, called osteoclasts, attach to bone surfaces and secrete acid and other substances that dissolve the bone matrix, releasing calcium. As long as bone formation and demineralization occur at the same rate, the amount of bone present remains stable.

As we age, a new equilibrium is reached that typically results in a slow, age-dependent net loss of bone mineral. For one in eight Americans over the age of 65, the excess bone loss results in osteoporosis.

Cadmium accelerates net bone loss. For example, Argonne research indicates that women who smoke cigarettes may experience increased bone loss because tobacco smoke contains cadmium. This bone loss should be even more evident after menopause when the bone cell turnover rate is highest.

Ouch, ouch!

For many years, researchers have believed that cadmium exposure was most damaging to kidneys; bone loss was secondary. So OSHA bases its exposure standards on the potential of kidney damage and the risk of cadmium-related cancer, not bone loss.

A serious bone disease found in the Jinzu River basin of Japan first hinted that cadmium might cause serious bone loss. Itai-itai disease, which means "ouch, ouch," is a painful result of chronic cadmium poisoning from mining byproducts dumped upstream. These patients have extreme bone demineralization. But such high amounts of cadmium exposure also cause other health problems including kidney damage that could also be responsible for the bone loss, so it was difficult for researchers to isolate the bone response.

In 1988, Argonne researchers developed the first mouse model for the human itai-itai disease. They demonstrated in mice that calcium is released from bone after exposure to small amounts of cadmium, independent of kidney damage. "The data suggest that cadmium causes bone loss by increasing the formation and activity of bone-dissolving cells," Bhattacharyya said.

In mice, a single oral cadmium dose resulted in increased levels of bone calcium excreted in mice feces. This bone response began within eight hours after exposure at blood concentrations of cadmium ranging from two to five parts per billion.

Gene searching

To understand how cadmium affects the bones so rapidly, the Argonne researchers turned to genes. Genes are expressed when parts of the genetic code are copied to produce protein. By looking at how bone-cell genes are expressed before and after cadmium exposure, Bhattacharyya and her team of Allison Wilson, Elizabeth Cerny, Akhila Regunathan, Tony Flores and Jesus Villareal identified those immediately affected by the metal's presence.

The team isolated mRNA from mouse bones before exposure and two or four hours after cadmium ingestion. Called messenger RNA, this genetic material serves as a coded message from the DNA instructing the cell to produce a certain protein. Proteins work together to carry out various functions in the cell. Typically, the more mRNA a gene expresses, the more corresponding protein will be produced.

After isolation, the mRNA was used as a template to produce matching complementary strands of DNA, called cDNA. The matching strands retain the genetic code embedded in the mRNA, but allow researchers to introduce fluorescent markers that identify the strands as either normal or cadmium exposed. The exposed and unexposed cDNA were then mixed together and analyzed using a microarray — a tiny grid on a slide with probes for about 8,500 mouse genes.

"The remarkable power of this approach is that we can query an organism's entire genome at once," Bhattacharyya said.

Each probe is a known piece of genetic material mounted on a microchip that binds selectively with matching strands of cDNA that are washed over the chip.

If expression was higher in the cadmium-exposed animal — more of the genetic material was being copied — more cDNA marked "cadmium-exposed" than "unexposed" would be readily available to bind to the probe. Using the fluorescent markers, researchers could detect changes in expression for each gene.

This process yielded about 20 genes for researchers to investigate further. They then used another biological tool, northern analysis, to take a closer look at these genes. Like the microarray, northern analysis uses probes to identify the presence and abundance of specific gene messages. However, northern analysis examines one gene at a time and yields more precise results.

Protection and destruction

The northern analysis research showed that cadmium activates two types of gene expression in bone cells shortly after exposure: protective processes and toxic-response processes.

Gene expression for two protective proteins increased. One, metallothionein, is the body's way of guarding against cadmium. It tightly binds to cadmium in the cell to keep it away from the bone cell's toxic response sites. Without metallothionein, the toxic effects of cadmium are doubled. The other protective protein adds iron to the cell. Bhattacharyya said she believes this protective response is triggered to counteract the anemia response to cadmium.

Most of the observed cadmium responses increased bone-demineralization gene expression. Expression increased for genes that code for proteins that help form osteoclasts, enhance the osteoclast's ability to secrete acid, build the junction between the osteoclast and bone cells, and aid signaling between osteoclasts and osteoblasts, or bone-forming cells.

The researchers also discovered a gene of unknown function that increased 18-fold within four hours of cadmium exposure. They plan to clone and study this gene to determine its role in the bone cell's response to cadmium.

Although most of the observed changes in gene expression were small — increases between two- and four-fold — they were consistent among different strains of mice, indicating to Bhattacharyya's team that cadmium is having a real effect on gene expression. However, just because genes are sending messages for protein production doesn't always mean the proteins will be produced as expected.

In addition to exploring the gene of unknown function, Bhattacharyya now plans to investigate proteins corresponding to genes identified in this study to see whether their production increases in response to cadmium. She and her team are anxious to trace the cadmium response pathway from initial exposure through gene expression and protein production to eventual bone loss. Their work may help lay the foundation for future health standards.

For more information, please contact Catherine Foster (630/252-5580 or cfoster@anl.gov) at Argonne.