azardous waste is a mixed bag. Many chemicals present at hazardous waste sites may not be very hazardous to human health. As a result, the cancer-causing potential of hazardous waste can vary from one site to the next. Determining the precise health risk at different waste sites is the first step in identifying the particularly dangerous sites that should be cleaned up first. It can also identify the sites that do not require immediate cleanup, potentially saving millions of dollars.
Using the techniques of molecular biology, ORNL researchers are developing new ways to evaluate the health risks of potentially hazardous chemicals. Examples of such chemicals are benzene in gasoline and perchloroethylene, a degreasing agent that ends up in landfills. The new methods potentially can be used to identify cancer-causing chemicals and to stop cancers by restoring normal growth patterns to cancer cells.
The tumor-suppressor gene, called wildtype p53, is responsible for production of the p53 protein, which is present in trace amounts in normal human cells. The p53 protein has the ability to stop cells from growing. Mutation or deletion of the p53 tumor-suppressor gene is now recognized as an almost universal step in the abnormal cellular growth process called cancer. Mounting evidence indicates that tumors caused by certain viruses result from inactivation by the virus of the antigrowth properties of the p53 protein.
Loss of tumor-suppressor genes by inheritance is thought to make some individuals predisposed to development of cancer. For example, individuals who have lost both copies of the tumor-suppressing retinoblastoma gene develop cancer of the eye. Similarly, it is thought that some women who lack or have a damaged p53 tumor-suppressor gene are predisposed to breast cancer. It is now possible to test breast cells to determine if copies of the p53 gene are lost or mutated. Such a screening test can help physicians identify those women at increased risk of developing breast cancer.
Exposure to certain toxic chemicals or radiation can result in the eventual appearance of a mutated p53 gene in cells. Detection of mutant protein or a loss of p53 function in suppressing cell growth may serve as a good biomarker indicating that a person or animal is at greater risk of developing cancer. However, it is likely that cells that contain a mutated p53 gene are rare events hidden in a background of a large number of normal cells. Therefore, detection of these cells requires the new molecular biology techniques that have the highest level of sensitivity. Even more sensitive and specific techniques may have to be designed to detect genetic events that are responsible for cancer before disease begins.
Craig Dees, a molecular virologist in ORNL's Health Sciences Research Division (HSRD), and Curtis C. Travis, director of the Laboratory's Center for Risk Management, are trying to design new methods for analyzing the health risks to humans using molecular biology techniques. Because of the central and near-universal role of the p53 gene in the induction of cancer, their research has focused on determining whether suppressor genes are damaged by exposure to chemicals or radiation produced through energy use or production. By better understanding the genetic events responsible for cancer, they hope to identify new biomarkers to predict human health risks.
These ORNL researchers would especially like to clarify the human health risks, if any, of exposure to cancer-causing chemicals or radiation at very low levels. "This topic is a very controversial subject in the scientific and lay community at this time," Dees says. "Bruce Ames, one of the most prominent designers of a test to detect chemical carcinogens, recently published an article in Science magazine in which he argues that our current testing methods are identifying chemicals as hazardous to humans even though they are carcinogens only in laboratory rodents. In addressing the low-level exposure issue, Ames essentially stated that it is not realistic to worry about low-level chemical exposures that are below the background level of naturally occurring sources like foods. I side with Ames, but other investigators think differently."
"In addition to the problems of determining human health risks based on exposure of laboratory animals," Travis says, "a large number of chemicals suspected to be carcinogens, like perchloroethylene, have not been shown to directly damage genetic material. It is important to determine how these chemicals cause cancer."
"Do they really pose any real health risk to humans?" Dees asks. "Maybe they put at risk only certain individuals with inherited genetic defects in growth-stimulating genes or growth-suppressing genes like p53? The answer to these suppositions lies in a better understanding of the molecular alterations that are responsible for causing a cell to suddenly grow uncontrollably. This knowledge may help us rationally design new molecular biology-based assays to identify these individuals before problems start."
For example, the normal p53 protein is known to have phosphate chemically bound to it. The processes of adding or removing phosphate from proteins is known to be a mechanism through which cells regulate protein function; these processes are called phosphorylation and dephosphorylation. Dees and Travis hypothesize that some chemicals found in hazardous waste may cause cancer not by inflicting damage directly on genetic material but rather by altering the function of proteins that control cell growth.
"One way this might work," says Dees, "would be to adversely affect the phosphorylation of key regulatory proteins like p53 that are involved in regulating growth."
For many years it has been known that some chemicals activate a specific enzyme called protein kinase C, which puts phosphate on proteins. However, no one has been able to fully explain how this effect of chemicals on protein kinase C caused loss of the genetic control of cell growth.
At a 1992 information meeting of HSRD, Dees and Travis presented the first evidence that chemicals such as benzene and perchloroethylene change the phosphorylation levels of two tumor suppressor gene products (p53 and rb105), possibly causing them to stimulate rather than suppress cell proliferation. Several other laboratories have recently confirmed the original observations of Dees and Travis.
"Presumably," Dees says, "chemical alteration of tumor-suppressor gene products contributes to the process by which these chemicals cause cells to grow at abnormally high rates. It may now be possible to screen chemical carcinogens by examining their effects on the tumor-suppressor gene products."
The almost universal occurrence of damage to p53 genes in cancer cells now provides a target for analyzing the effects of chemical carcinogens.
"We hope to be able to answer some of the pressing questions on the health effects of low-level exposure to chemicals and clarify the human health risks," says Dees. "The ultimate goal is to be able to identify specific individuals who are at increased risk and perhaps to genetically reengineer them with normal tumor-suppressor genes to ensure that they have the proper growth controls to inhibit the formation of cancer cells."
Observing the effects of various chemicals on tumor-suppressor genes could be a sensitive way of measuring the ability of these substances to cause cancer. It could help regulators make better sense of the mixed bag of hazardous waste.