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LABORATORY PROFILE Red blood cells and toxic chemicals Toxicologists search for relationships between the health of an organism and its environment. However, the difficulties involved in identifying human diseases that result from exposure to toxic chemicals mean that such identification often comes too late for the affected individuals. Predictive information about what kinds of exposure result in diseases such as cancer would be of enormous value. In addition, the hidden connections that may occur among the huge number of potentially toxic compounds in our environment need to be understood One predictive approach is to investigate adducts between toxic chemicals and hemoglobin or DNA. Hans-Giinter Neumann, head of a research group at the Department of Toxicology of the University of Wiirzburg (Germany) is one of the pioneers in this area. Starting about 20 years ago, he and his team began searching for biomarkers of response to toxic chemicals. "What we need to know is the biologically effective dose and how or if it correlates with the external exposure undergone by an individual " says Neumann Are "long-term exposure limit" values really an accurate tool to prevent illnesses in workers? Where do the differences in individual susceptibilities of different persons come from? DNA is an obvious target for identifying exposures to genotoxic compounds; however, it undergoes numerous reactions and yields a mixture of products. Interestingly, genotoxic compounds also target hemoglobin and form stable reaction products with hemoglobin in vivo, just as they do with
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DNA. The reactions with hemoglobin, however, often yield a single product. For example, says Neumann, alkylating agents and epoxides react with the /V-terminal valine, amino and nitro arenes react with cysteine((3-93, and epoxides react with histidine. Research indicates a correlation between adduct formation in DNA and hemoglobin. Using hemoglobin as a site for biomarkers has other advantages, says Neumann. It is easily available from blood samples, where it is present in high concentration, and its lifetime is long enough (18 weeks) to allow monitoring of chronic exposure because steady-state levels are reached. And finally, the adduct levels are directly proportional to the absorbed dose within a wide linear range. At present, Neumann's group is investigating adducts that are released from hemoglobin by hydrolysis. The preferred analytical method is GC/MS, which requires derivatization of the target compound. Although derivatization is usually thought of as a disadvantage, in this case it is actually advantageous because derivatization increases the molecular mass of the analytes and separates them from the huge number of non-analyte peaks corresponding to lowmass compounds, which also appear in the mass spectrum. LC with electrochemical ot fluorescence detection is also a possible approach but LC/MS methods have not been developed for these samples Neumann's group is now applying their method to real-world problems. For example, coke oven workers are typically exposed to emissions of carcinogenic polycyclic aromatic hydrocarbons (PAHs). Neumann's group has chosen five nitroPAHs (1-nitropyrene, 2-nitrofluorene, 9-nitrophenanthrene, 3-nitrofluoranthrene, and 6-nitrochrysene) as exposure markers. In humans, the nitro group is reduced to the nitroso derivative which binds covalently as a thioamide to hemoglobin's cysteine. To track exposure of workers to these nitro-PAHs, Neumann's group collects 5-10 mL of blood enough for at least two independent sample preparations The corresponding amino-PAHs are quantitatively released from the hemoglobin by hydrolysis and derivatized to pentafluoro Dropionic acid amides before analysis by CC/MS The detection limit is in the ranee 100 ftr
According to Neumann, the main deficiency of this approach is the lack of a general sample preparation method. Overall,
Analytical Chemistry News & Features, June 1, 1997
solid-phase extraction is the preferred approach, but Neumann reports that reproducibility on C18 cartridges has been a nagging problem. Extraction efficiency and reproducibility would improve if phases had better reproducibility than what is available today, if a larger choice of phases were available, and if the pH stability were improved, he says. The vacuum rack for the disposable cartridges also needs to be modified. The initial design used by the group has analytes in contact with difficult-to-clean steel capillaries When Neumann's group analyzed their nitro-PAH results, they were surprised to find high interindividual variability in the levels of adduct formation. These variations could arise from different metabolic capacities, different toxicokinetics, different working styles, or different lifestyles (although there was no correlation with smoking behavior). Despite the variability, the difference between coke oven workers and individuals selected as controls was obvious. This was particularly relevant when the sum of the concentrations from all five nitro-PAHs was compared between the coke oven worker and the control groups Such hemoglobin adduct levels are not yet a tool to predict the cancer risk of an individual. Future research will need to look at the analysis of adducts that cannot be released by hydrolysis. In this case, it is necessary to investigate the various polypeptides, looking at which ones carry the adduct after degradation of the protein. Because GC/MS is not suitable for analysis of these large biomolecules LC/MS methods will have to be developed says Neumann Veronika R. Meyer
