Document nkmEo6jDky8Xw872D3GMoEQj2

:u URL 04925 Measuring mutagens and carcinogens in the body Setting limits for chemical exposure in how chemicals in the environment cause the workplace has always been a com genetic lesions, mutations and cancer," plicated and controversial procedure, says Frederick J. de Serres, associate mainly because it is difficult to extrapo director for genetics at the National In late from animal exposure studies and stitute of Environmental Health Sci airborne chemical levels to adverse hu ences (Research Triangle Park, N. C.). man health effects. To solve this prob In fact, OSHA has already made use lem, and to better study how chemicals of these new detection techniques in cause cancer, researchers have devel setting guidelines for ethylene oxide ex oped new techniques enabling them to posure. The oxide, used as a steriiant determine how much of a given chemi for surgical equipment, came under fire cal actually gets into the human body in 1983 when investigators found that it and damages key biological molecules. caused tumors in laboratory animals. This new detection technology, says a "The exposure limits we set," says the spokesman for the Occupational Health OSHA spokesman, "were based in part and Safety Administration (OSHA), "will on data from the new technology." provide the type of evidence that will The technology was designed to mea help in rule making and will put regula sure exceedingly low levels of com tions on firm scientific footing." The pounds that have become linked chemi spokesman adds that the technology cally to DNA or proteins. Some should be "one of the most promising techniques use monoclonal antibodies to find the chemically modified biological molecules, known as adducts of DNA or protein. Others combine classical chem ical techniques with highly sen sitive analytical methods, such as gas chromatography com bined with mass spectrometry or fluorescence spectrophoto metry, to gain the same end. Sensitivity. But while the techniques may differ in meth od and in the adducts they can detect, all are capable of find ing less that one adduct in 10 million unmodified molecules. Says Regina P. Santella, assis tant professor of public health at Columbia University (New York City), "We're talking about sensitivity down in the range at which the toxic ef fects of a chemical are first seen." Randerath: Careful isolation ot chemically modified DNA. Of all the chemicals in exis tence, both natural and man developments in monitoring exposure to made, the most dangerous are the very chemicals in the workplace and assess few that are able to enter a cell and ing their potential health hazard." react with the individual nucleotide While air filters and chemical-sensi bases that make up DNA. Researchers tive badges can detect surface contami studying cancer-causing processes in nation by a particular chemical, finding laboratory animals and cultured human chemically modified biological molecules cells have determined that the process is incontrovertible proof that a person of entry and reaction is a crucial step in was not only exposed to the chemical the development of cancer and muta but also that it was incorporated into tions. But extending this process from the person's system. "Now, we can be animals and human cells to live humans gin answering some key questions on has been a problem. "We've had no way of proving that such reactions occurred in humans." says Kurt Randerath, pro fessor of pharmacology at Baylor Col lege of Medicine (Houston). Thus, Ran derath notes, it has been difficult "to prove that certain substances cause can cer and birth defects." Cigarette smoke is a substance that has eluded positive linkage with cancer. Hundreds, if not thousands, of studies have documented that cigarette smok ing causes cancer in laboratory animals, and population studies indicate the same holds true in humans. The tobacco industry, however, claims steadfastly that cigarette smoke has never been do cumented to produce the biochemical damage in humans that leads to cancer. Proof? But now Santella, Randerath and other researchers have found ad ducts of DNA and benzopyrene--a ma jor suspected carcinogen in cigarette smoke--in tumors taken from human patients with lung cancer. Such adducts were absent in lung tissue taken from nonsmokers. "I would say," say's one researcher, "this is pretty good evi dence that a compound in cigarette smoke is causing these tumors." Randerath has also been studying the interaction of DNA with various chemi cals important to the chemical process industry, such as aromatic amines, azo compounds, heterocyclic drug intermedi- `Evidence to help rule making and to put regulations on firm scientific footing' ates and nitroaromatic compounds. While his data are not yet complete, Randerath says that only a few of the substances tested so far form adducts with human DNA. However, he notes, those compounds that do form adducts "will probably prove toxic." To measure DNA adducts in human samples, Randerath isolates DNA from white blood cells exposed to a suspected carcinogen, breaks the DNA into individ ual nucleotide bases, and uses the en zyme polynucleotide kinase to label both normal and modified bases (the ad ducts) with radioactive phosphate. Next, he separates the adducts by thin-layer chromatography--the adducts migrate differently on the chromatogram than unmodified bases--and uses autoradiog raphy to measure the adduces relative to normal bases. "This method is sensi tive and fairly easy to perform," he says. "But, more important, you don't need to know beforehand what com pound the DNA has reacted with." However, those assays that use mon- 24 Chemical Week/March 6. 1965 0 URL 04926 oelonal antibodies to detect DNA ad ducts work only if the adduct is known beforehand; then an antibody specific to that adduct can be prepared. This limits use of these assays, even though they are just as sensitive and are easier to use than those that rely on chromato graphic separations. Santella, for exam ple, prepared synthetic adducts by re acting benzopyrene with DNA. "We knew the structure of the adduct and knew how to make it," she explains. "Without that type of information, anti body methods aren't practical." One way to increase the practicality of antibody assays, though, would be to use chromatographic techniques to iso late adducts. Once isolated, the struc ture of the adduct could be deter mined--and thus the identity of the chemical that attacked the DNA--and a synthetic adduct could be prepared. This adduct could then be used to make an antibody. Many investigators are planning to use this production method, Randerath says, because monoclonal as says would be "more suitable for rou tine monitoring." Another approach to such routine monitoring is to bypass the DNA com pletely and, instead, look at blood pro teins. "We know that many carcinogens react with proteins, too," says Santella, "and it's a lot easier and more practical to get hemoglobin from red blood cells than DNA from white blood cells." Adduct stability. Moreover, protein monitoring would alleviate one concern that researchers have about the stabil ity of DNA adducts. Such adducts are repaired by the body to some extent, although the degree of repair depends on the particular adduct, where on the DNA molecule it is located, and on a person's individual metabolism. Proteins in red blood cells, on the other hand, are not repaired by the body, but exist un touched for the lifetime of the blood cell, about four months. Says Santella: "Looking at hemoglobin adducts could give us an accurate picture of total ex posure over several months time." The techniques for detecting very small numbers of adducts are well es tablished. What remains before these techniques become standard in monitor ing and regulatory activities is for re searchers to demonstrate their utility on a large number of common work place chemicals. This, in the estimate of those working in the field, is beginning; within a year or two, a sufficient data base should be accumulated to judge the technique's relative merits. JOSEPH ALPER in Washington, with B. J. Spalding Close to FDA's finish line: Five AIDS-linked tests Less than 10 months after National Cancer 15 for hepatitis B tests, | and because its AIDS Institute scientist Rob || test is nearly the same ert Gallo discovered a virus, human T-cell lymphotrophic virus I* for lab technicians to I run, Abbott will immeI diately grab 60% of the type III (HTLV-III), be I U. S. market, says ana- lieved to cause acquired ! lyst Peter F. Drake o; immune deficiency syn drome (AIDS), five com k' C' ' I Kidder Peabody & Co. j (New York City). "It's panies have unveiled tests that detect anti bodies to the virus in human blood. Food and I vI id Hacklan No. i HHS health priority. going to be a very easy training" for those used to Abbott's hepati tis tests, says David A. Drug Administration (FDA) approval of Thompson, president of Abbott's Diag one or more of the tests, delayed for nostics Div., who expects his firm to several weeks, is imminent. take about 50% of the market. When approval comes, the race will At least one major market segment be on among five key contenders for an may already be sealed. The American estimated $175 million worldwide mar Red Cross (ARC) will need an estimated ket--$70 million in the U. S.--for blood 6 million tests/year for its blood banks. and plasma screening. The outcome ARC has clinically tested all five kits should rest on several factors: the accu and picked one based on its own data, racy of the firms' tests; the firms' start says Dr. S. Gerald Sandler, ARC asso ing position in the blood banks, plasma ciate vice-president for medical opera houses and clinics that make up the tions. Although each of the companies market for the tests; and the approval procured its own samples for clinical date for each test Awaiting the start tests, ARC used uniform samples. ing signal are Abbott Laboratories Pending FDA approval, ARC won't (North Chicago), Electro-Nucleonics (Co say which test it has chosen. But other lumbia, Md.) and three joint ventures: blood banks and the plasma industry Litton Bionetics (Kensington, Md.) with are "up for grabs," says Kathryne Johnson & Johnson's Ortho Diagnostics Carr, assistant to the scientific director (Raritan, N. J.); Genentech (San Francis at Genetic Systems. She thinks Abbott co) with Baxter-Travenol (Deerfield, "will get a good run for its money" in 111.); and Biotech Research Laboratories both areas. Apart from ARC, members (Rockville, Md.) with Du Pont (Wilming of the American Assn, of Blood Banks ton, Del.). will use 6 million tests/year, while the Two more. Lagging behind these plasma industry, represented by the companies by at least several months American Blood Resources Assn. are two other firms that are developing (ABRA), will use 10 million tests/year. tests based on independently discovered Not far behind. ABRA Executive Di AIDS-associated viruses: Genetic Sys rector Robert W. Reilly says Litton/Or tems (Seattle), which has a marketing tho has wide blood bank contacts, while agreement with American Hospital Sup Travenol/Genentech and Electro-Nucle ply (Evanston, 111.); and a joint venture onics are both plugged into the plasma of Chiron Corp. (Emeryville, Calif.) and market. Nobody has the edge with free Pandex Laboratories (Mundelein, 111.). standing clinical labs, he says. Du Pont The five leading tests are enzyme- claims an advantage with plasma linked immunosorbent assays (ELISA), houses, to whom it sells centrifuges. "I in which either beads or microtiter plate believe Abbott will probably be No. 1, wells coated with inactivated parts of but I firmly believe that we probably the Gallo virus will cause antibodies in won't be very far behind," says David the blood being tested to link to an en D. Mooberry, group vicepresident of zyme that causes a color change. This is Du Pont's Biomedical Products Dept. not unlike tests already used to detect Kidder Peabody's Drake, though, pre hepatitis B antigen in a market already dicts that because of the accuracy of its dominated by Abbott. test, Electro-Nucleonics will take second Because of its position in the market place (table, p.26). That firm, he says, March 6, 1985/Chemical Week 25