Dosimetry Cancer researchers are using molecular dosimetry to measure the reactivity of carcinogens with target DNA molecules and to determine whether there are threshold levels of exposure.
W
arning: The Surgeon General Has Determined That Smokine May Be Dangerous To Your Health.’ But how dangerous? And how much tobacco smoke is enough to cause the variety of cancers, heart disease, and birth defects that well-established epidemiological and animal exposure studies have shown it causes? For the more than 3000 identified compounds in tobacco smoke, as well as for a myriad of environmental and food-related contaminants and toxins, policy makers and public health officials have the unenviable job of trying to figure out, on the basis of these types of studies, the biological risk to h u m a n s exposed to these compounds. Many compounds, including known toxins a n d carcinogens, produce a wide range of reactions in individuals, and lesions or tumors may not appear until years after an exposure.
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Some carcinogens interact with each other, some initiate tumors, some promote tumors, and some must be metabolized to produce an actively genotoxic form. These complications make it difficult to quantitate the carcinogenicity of a compound by using gross measures of external or internal exposure. “Total body burden,” or the concentration of toxin taken u p by the body, is used as a measure of internal exposure. But because of physiological and metabolic differences among individuals a n d among species, t h e amount of toxin present does not necessarily reflect how much damage it will cause. Molecular dosimetry is one way cancer researchers are beginning to quantify the potency of carcinogens and to determine whether there are threshold levels of exposure to them. This technique measures the chemical reactivity of a potential or known carcinogen, mutagen, or other genotoxin with a host “target molecule”-
generally DNA. Most models of carcinogenicity a s s u m e t h a t t h e genotoxin acts by mutating or damaging DNA, but in molecular dosimetry some proteins are also examined a s easily obtainable indicators with similar reactivity. Many genotoxic agents are electrophilic species that form covalent adducts with bases along the DNA strand and with certain amino acid residues in proteins. Steven Tannenhaum, of t h e Department of Chemistry and the Division of Toxicology a t the Massachusetts Institute of Technology, says, “Molecular dosimetry a t MIT really started about 30 years ago with Gerald Wogan’s first studies of aflatoxin adduction to DNA.” Researchers took what had been discovered in animal experiments-the observation that the carcinogen formed adducts with DNA and proteins-and tried to use quantification of t h e adducts a s a measure of the effective dose, or the amount of genotoxin that actually reaches and affects the DNA. “The key to doing molecular dosimetry [effectively] is the use of new analytical techniques that can measure adduct concentrations a t the femtomole levels that might be found in people,” says Tannenbaum. One advantage of measuring the reactivity of genotoxic agents with DNA is that although physiology and metabolism vary widely among species a n d among individuals, t h e mechanisms of DNA regulation, transcription, and translation are fairly consistent among vertebrates. Because t h e chemistry of purified DNA is even more uniform, it may be more accurate to extrapolate rates of DNA-adduct formation among species than it is to extrapolate rates of tumor formation. Another advantage of molecular dosimetly is that it can be performed on h u m a n urine or blood samples in vitro rather than requiring harmful in vivo exposure to potential carcinogens. Tracking down the adducts
Tannenbaum’s group uses GCIMS with negative ion chemical ioniza-
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tion (NICI) o r electron ionization (EI) detection to measure t h e a d duct-forming capabilities of aromatic amines and PAHs-particularly reactive metabolites of benzo[alpyrene (BaP)-with hemoglobin (Hb) or DNA. I n many studies of humans, Hb is obtainable in greater quantity than DNA and its adducts have been used as surrogates or biomarkers for DNA adduct formation a t critical sites, although actual rates of adduct formation for Hb and DNA may differ. Hb is found endosed in red blwd cells (RBCs), and its accessibility as a target is similar to that of DNA, which is enclosed within the nucleus. To measure the rate of adduct formation in Hb for known PAHs, Tannenbaum isolates RBCs from human blood, incubates them with known doses of purified active forms of the carcinogens, washes and lyses them, removes the membranes by centrifugation, a n d precipitates t h e Hb (which now presumably contains adducts with PAH residues) from the cytosol by adding HCl and acetone. The isolated Hb is washed with 1-butanol to remove free PAHs and sulfur-bonded adducts to glutathione (GSH), a coprecipitating peptide that plays an important role in scavenging toxins from the body and is also studied in molecular dosimetry. The Hb is digested to completion with a nonselective protease, and individual amino acids are extracted with ethyl acetate and separated by either C,, HPLC or GCIMS. The PAHSusii are diastereomers of B ~ P epoxides ( t h e actively genotoxic forms of BaP). When Hb from 1 mL of whole blood is mixed with 350 nmol of epoxide, the resulting amino
acid-BaP adducts, isolated in the form of diols and tetrols, can be detected in the single-femtomole range. Part of the attraction of molecular dosimetry is that it permits chemists to predict how active a potential carcinogen might be in vivo, although extrapolation of structure-function characteristics cannot be substituted for testing any particular carcinogen experimentally. According to Tannenbaum, simple chemical properties such a s electrophilicity are bett e r for predicting carcinogenic activity of small alkylating agents t h a n of l a r g e r compounds. With bulky carcinogenic agents, steric factors and chirality come into play, and it becomes harder to predict genotoxicity from t h e compounds’ chemical structures. In addition, the method of hydrolyzing the adductsacid- or base-catalyzed, or neutralcan affect whether the adducts are recovered as syn or onti forms. Optimal GC/MS detection methods vary by the type of carcinogen being tested and by the target molecule, says Tannenbaum. Most of his group’s MS methods were developed for small derivatized adducts with MW c 1000. Because Hb adduct esters with BaP analogues are somewhat labile, the adducts are derivatized before GCIMS is performed. Analysis of the adducts by GCIMS is semi-automated for aromatic amines but not, as yet, for PAHs, because t h e d e r i v a t i z a t i o n method for NICI-MS detection of PAH adducts is trickier. For most PAH-Hb adducts, NICI-MS results in much less fragmentation t h a n does EI-MS. ‘With NICI, the advantage over E1 is in the signal-to-noise ratio, not in
the sensitivity,” says Tannenbaum. HPLC with fluorescence l i n e narrowing spectroscopy (FLNS) is another adduct detection method being used by Tannenbaum’s group. Tannenbaum says, “We picked up the technique from Gerald Small’s group [at Iowa State University]. FLNS is comparable in sensitivity to GC/MS, but no one has done a sideby-side comparison yet.” (See also Jankowiak, R.; Small, G. J. Anal. Chem. 1989, 61, 1023 A-1031 A.) Tannenbaum says he is planning a comparative study of the two methods in the next year and adds that although FLNS is still primarily qualitative, his group has figured out a way to make it quantitative for Hb adducts.
“Real-llfe” molecular doolmetry Molecular dosimetry can also be used to monitor real-life adduct formation in people who have been exposed to environmental carcinogens. Tannenbaum’s group has assayed blood samples from smokers for BaPlike adducts by isolating Hb from RBCs, hydrolyzing it with protease, passing t h e digest solution twice over an irnmunoflmity column with a monoclonal antibody ligand raised against PAH adducts, and concentrating the eluted adducts. Originally the adducts were analyzed by synchronous fluorescence spectroscopy a t M = 34 nm, and the amount of pyrene-like fluorescent species was calculated by using a transanti[l4C1BaP-tetrol s t a n d a r d curve. Tannenbaum says h i s group h a s since abandoned that method for the more accurate GCIMS and FLNS techniques. Typical in vivo environmental concentrations a r e 0.2-10 pmollg of Hb. Although Hb adducts provide a convenient and abundant source of Table 1. Blomarkers for carcinogenicity assessment by information on genotoxicity, DNA molecular dosimetry adducts are most closely tied to the initiation of carcinogenesis and mutagenesis, and they indicate longer Acute Detoxification GSH adduct Urine exposure to genotoxins. They can be or metabolic metabolites determined in target tissues such a s activation liver, kidney, bone marrow, and Shon-term General Serum albumin Peripheral germ-line cells, but fairly invasive blood internal adducts procedures must be used to collect exposure, likely the samples. However, products of genotoxic DNA repair and excision are often Peripheral 120 days Semichronic Effectivedose, Hemoglobin excreted intact in urine and can be blood likely adducts determined by several methods. genoloxic Gerald Wogan, director of the DiEfiective dose, DNA adducts Target tissues > 1: vision of Toxicology a t MIT, has cargenotoxic ried out small-scale population studUrine Effectivedose, DNA adduct possible repair ies in China and in Gambia, West detoxifim1ioi products Africa, to determine the relationship with reparr between dietary intake of aflatoxin I and formation of adducts to DNA or
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to serum albumin, a biomarker for short-term exposure. Aflatoxin, a mycotoxin produced by Aspergillus Puuus, can contaminate cereals, peanuts, and other fwds. It has been linked to increased incidences of liver cancer in China and Africa. Wogan’s group used monoclonal antibody immnnoaffnity chromatography, followed by HPLC with UV or fluorescence detection, to detect DNA-aflatoxin adducts and aflatoxin breakdown products in urine samples. Other DNA adduct detection meth-’ ods include ”P postlabeling, a less instrumentally complex method that is nonselective and has a detection limit of less than one adduct per los or lolo nucleotide bases in samples of 1-10 g DNA. The DNA is digested with endonucleases to nucleotide-3’monophosphates and enzymatically radiolabeled with [Y-~~PI-ATP at the 5‘-position. The labeled adducts are resolved by four-directional TLC for a fingerprint of adduct nucleotides and quantified by autoradiography. Molecular dosimetry as an epidemiological tool Molecular dosimetry is not the ultimate measuring tool for carcinogenic risk, says Tannenbaum, but rather part of an overall scheme of toxic exposure assessment performed at several levels, each providing a different type of information, The chemical interaction of a purified k n o w n agent with a purified target molecule in an isolated system determines the effective dose once the agent has access to the target molecule. Uptake and adduction i n intact cells indicate some of the localized physiological and metabolic factors that influence the target molecule’s exposure to an agent. Whole-body controlledexposure studies i n a n i m a l s a r e needed to determine the toxicokinetics of exposure to an agent, and large population studies-many of them morbidity and mortality evaluations-indicate the outcomes of exposure. Because each level of exposure evaluation gives a different piece of information, the in vitro methods of molecular dosimetry are not likely to replace animal exposure studies altogether, says Tannenbaum. “Molecular dosimetry may simplify animal studies-it may even shorten them by helping direct their focus on the most likely agents a n d mechanisms-but ultimately, it’s not likely to replace them,” he remarks. Still, prospects are good that molecular dosimetry will become part of standard regulatory genotoxicity risk
assessment schemes to provide more precise information on the action of agents on human target molecules. In 1991 the International Agency for Research on Cancer assigned a group to meet in Lyon, France, to evaluate carcinogenic risks. The group concluded, “As t h e range of d a t a on mechanisms of action of carcinogens increases, so the set of possible . . . substantive types of evidence available . . . will increase. At the same time, inclusion in human studies of measures related t o mechanisms
(e.g., molecular dosimetry. . .) will increase the scoDe and sensitivitv of epidemiologicai research” (Cuncer Res. 1992.52.2357-61). Although molecular dosimetry will not replace animal testing or epidemiological outcome studies outright, it may change some of the current risk classifications by providing a refined connection between exposure and genotoxicity and by giving epidemiologists a new molecular tool for assessing carcinogenic risk. Deborah Noble
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