Chem. Res. Toxicol. 1993,6, 741-747
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Forum: Frontiers in Molecular Toxicology Applications of Mass Spectrometry to Toxicology Ian A. Blair* Departments of Pharmacology and Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37232 Received September 17,1993
Introduction
Mass spectrometry (MS) continues to play an important role in toxicological research because of the requirement for sensitive and specific analytical methodology. The last few years have seen the applications of exciting new developments in instrumentation and ionization techniques to problems in toxicology together with the sophisticated application of older MS techniques (1-4). T h e atmospheric pressure ionization (MI)techniques of electrospray (ESI) and ion-spray (nebulizer assisted E S I ) have rapidly gained wide acceptance in toxicological research because of their high sensitivity and their capabilityfor the analysis of macromolecules on relatively simple instrumentation (5,6).I n the original design of Fenn and Whitehouse (6),E S I was carried out by introduction of a solution containing the analytes at flow rates in the range of 1-15 d l m i n . The solution entered the E S I chamber through a stainless steel needle that was held at ground potential. For the generation of positive ions, the chamber and the surrounding cylindrical electrode were held at between -3 and -5 kV. The field that was generated at the needle t i p caused the eluting liquid to become charged and to form a spray of charged droplets as a consequence of the Coulombic forces that occurred. Driven by the electric field in the ESIchamber, the droplets migrated toward the analyzer region of a mass spectrometer. A countercurrent flow of bath gas heated to around 100 O C was used to help evaporation of the solvent from the droplets as they drifted toward the analyzer region. The exact mechanism for ion formation in E S I is still somewhat controversial. However, it has been suggested that, during evaporation of the solvent from the droplets, the charge density on the surface increases until the droplets reach a size where the charge density is strong enough to desorb ions from the droplet (6).The ions then emerge from aglass capillary entrained in a dry bath gas that passes through a skimmer into the analyzer of the mass spectrometer. Macromolecules can form a number of charge states depending on the number of cations that become attached to them. From a simple mathematical relationship, it is then possible to calculate the molecular weight of the analyte molecule. Small molecules generally emerge singly charged, through the attachment of eithera proton or a metal cation. Negative ions can begenerated by reversal of the source potentials, although it is necessary toprevent corona discharges with a n electron scavenger such as SFs or oxygen. *Address correspondence to this author at the Department of Pharmacology, Vanderbilt University, 84 MRB, 23rd Ave. S. at Pierce Ave., Nashville, TN 37232-6602. Phone: 615-322-2094,1 (voice mail); 615-343-1268 (FAX).
In an innovative modification of the original Fenn and Whitehouse design, Chowdhury et a1 replaced the glass capillary with a heated metal capillary (7). This allowed much improved desolvation of the charged droplets so that much higher flow rates could be obtained. The source (which has now been commercialized by Finnigan) offers the capability for ESI with conventional column chromatography at flow rates up to 1 mL/min. Higher flow rates can also be obtained by the use of a heated nebulizer coupled with discharge ionization (8). This technique, known as AP chemical ionization (APCI), serves to complement ESI as it appears to be partially useful for the analysis of small molecules (9). API-based techniques have been installed primarily on quadrupole instruments rather than sector instruments because it is much easier to operate at the high potentials that are required. However, sources are available for sector instruments, and these have proved to be useful, particularly when accurate mass determinations are required (10). The increased availability of APIMS techniques has allowed much improved access to LC/MS-based methodology ( I 1-1 5). Although ESI provides greater sensitivity than ionspray, most investigators have preferred the latter technique because of its tolerance for higher flow rates (50 pL/min) and the ability to use microbore columns instead of capillary columns. Capillary LC which is required for true ESI suffers from the necessity for very low volume injection volumes (100 nL) or the requirement for long gradient runs when higher injection volumes are used. Under reversed-phase conditions, it is possible to inject up to 20 pL of solvent, but the mobile phase has to contain a t least 95 % water. For complex structural studies with limited supplies of analyte, capillary LC has offered significant advantages (16). Further studies will be required to determine whether the ability to carry out ESI at higher flow rates will obviate this advantage (7). The rapid advances that have occurred in APIMS have tended to overshadow LC/MS methodology based on thermospray (TSP)(17,181.There are, however, anumber of examples in toxicologyresearch where TSP methodology has been of importance for both structural and quantitative studies (4,19-23).It is likely that the increased sensitivity that can be attained with API-based methodology,together with the availability of robust instrumentation, will result in TSP being phased out. Tandem MS (MS/MS) used in combination with a number of different ionization techniques is assuming increasing importance in biologicalMS (24,25).Structural characterization of proteins modified by both endogenous (26,27)and exogenous substrates (28)can now be readily carried out. In fact, a major factor that has ensured the
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rapid acceptance of API-based methodology is the ability to carry out collision-induced dissociation (CID) of molecular ions. The resultant daughter ions (16),constant neutral loss spectra (29),or parent ion spectra (30)can be obtained with extremely high sensitivity. The combination of CID with a soft ionization process that produces abundant molecular species provides very powerful methodology for use in structural studies. It is also possible in some cases to obtain significant structural information from ESI without recourse to the use of a tandem mass spectrometer by carrying out in-source collisions (31). Thus, even relatively simple instrumentation can often provide rich structural information. Several exciting developments have been implemented over the last several years that also have great potential for facilitating structural determinations. Matrix-assisted laser desorption/ionization (MALDI) is rapidly establishing itself as a highly sensitive method that provides molecular weight information on macromolecules (32,331. A recent report has described the use of this technique for obtaining mass spectra of cytochrome P-450s (34). Thus, MALDI may prove to be helpful in future structurefunction studies on P-4509, a class of enzymes that play a pivotal role in xenobiotic metabolism. Progress is also being made in using laser desorption for high-sensitivity ionization, coupled with CID to induce specific fragmentation pathways. For example, Yuan et al. used laser desorption to form MH+ and then used electron ionization to induce fragmentation (35). This resulted in specific cleavages that allowed structural characterization of a glutathione conjugate of cyclophosphamide to be carried out. The use of more conventional CID techniques with MALDI in combination with MS/MS is still in its infancy. A recent report indicates that instrumental developments will soon provide this as a realistic method for structural characterization (36). For more conventional types of ionization such as fast atom bombardment (FAB) and liquid secondary ion (LSI), greatly enhanced detection has been possible by the introduction of array detectors. They allow a significant portion of the mass range to be detected without the necessity for scanning (28,37). An entire mass spectrum can then be obtained by linking together different portions of the mass range. Increased sensitivity can be obtained by the use of flow-FABor flowLSI methodology (38). With this type of sample introduction, in combination with array detection, it is possible to obtain full-scan tandem mass spectra in the low femtomole range (39). Unfortunately, few laboratories are equipped with the sophisticated instrumentation required for this methodology, and techniques based on the triple quadrupole are still by far the most popular. An interesting novel application of the flow-FAB technology involves its combination with microdialysis for on-line monitoringof xenobiotics introduced into the blood stream of animal models (40). Although a plethora of new techniques is available for toxicological studies, more traditional GC/MS methodologies continue to play an important role (41, 42). The more routine nature of these studies tends to obscure their utility. Electron capture negative chemical ionization (ECNCI) MS for both unmodified (43) and modified endogenous molecules (44) continues to provide a fertile ground for new high-sensitivity quantitative studies. The specificity that can be attained with high-performance GC places this technique at the forefront of methodology
for highly specific and sensitive trace analysis.
Modified DNA Bases The role of MS in the analysis of modified DNA has been critically reviewed in an excellent article by McCloskey and Crane (4). Quantitative analyses of modified DNA bases are assuming increasing importance as markers of carcinogen exposure (45-47). In spite of the rapid advances being made with LC/MS, important contributions to this area are still being made by GC/MS (46,48). ECNCI MS has proved to particularly useful in the quantitative analysis of ethenoguanine, the N-3 adduct of guanine formed by reaction of DNA with the human carcinogen vinyl chloride. Derivatization of ethenoguanine with pentafluorobenzyl (PFB) bromide resulted in the formation of two regioisomeric PFB derivatives (49).The limit of detection for the two derivatives was 190 amol on column. When used in combination with [l3C41-heavy isotope-labeled internal standard, a limit of detection was found to be 60 fmol/pmol of guanine. It was possible to show that the livers of young rats dosed with 600 ppm vinyl chloride for 4 days had concentrations of 1.8 f 0.3 pmol of ethenoguanine/pmol of guanine. The ethenoguanine concentrations were highest in the liver and were found to persist with a half-life >30 days (50). Although the etheno adduct was formed initially in much lower concentrations than the N-7 adduct [7-(2’-oxoethyl)guanine], it was found to persist muchlonger (51). ECNCI is still unquestionably the most sensitive MS technique available for the analysis of modified bases, as highlighted by a recent report that zeptomole sensitivity can be attained (44). All that is required is the attachment of a suitable electrophore to the analyte molecule (42-44,4% 51). The quantification and identification of oxidized DNA bases from in vitro experiments still rest heavily on GC/ EIMS methodology (52). DNA is first hydrolyzed under standard conditions, and the resulting DNA bases are converted to silyl derivatives prior to GC/MS analysis. For in vitro experiments, this normally provides adequate sensitivity. In fact, it was possible to use GC/MS to quantify four modified deoxynucleotides in tumors of breast tissue of human subjects (53). Extensive cleanup of complex biological fluids (such as urine) is normally required before GC/MS analysis can be carried out (19). The availability of stable isotope-labeled modified bases will allow future quantitative studies to be carried out with high specificity (54). Immunoaffinity procedures are becoming available for the extraction of modified bases from biological fluids (55-58). This methodology provides a rapid and specific method for cleaning up samples prior to mass spectral analysis and should be particularly useful for urine samples (58). LC/MS methodologies are making a striking impact for the structural and quantitative analysis of modified bases ( 4 ) . Until recently, LC/MS analysis of base adducts was carried out primarily by methods based on TSP (18).For example, TSP MS was employed in the analysis of the malondialdehyde adduct of guanine (MlG). Using this methodology, it was possible to show that concentrations of the adduct in human urine were less than 500 fmol/mL (22). It is anticipated that API-based methodology will provide increased sensitivity both for structural confirmation and for quantification of the M1G adduct in biological fluids.
Forum: Frontiers in Molecular Toxicology
FAB in combination with MS/MS has proved to be useful for the detection and structural characterization of polyaromatic hydrocarbon-deoxynucleoside adducts (59). Positive ion spectra of guanosine adducts showed a protonated molecular ion MH+and a more intense aglycon ion resulting from loss of the deoxyribose. CID of the protonated molecular ion provided significant structural information including, in some cases, the site of covalent attachment. LSI MS was used in the characterization of DNA base adduct standards generated from styrene 7,8oxide with 2'-deoxyguanosine 3'-phosphate (60,611. The standards were then used in a 32P-postlabelingprocedure for the detection of base adducts in mononuclear cells from workers exposed to styrene oxide and from DNA treated with styrene 7,B-oxide in vitro. FAB MS/MS provided important information on the structures of phosphoramide mustard/DNA adducts (30, 6 1 , 6 2 ) . By use of parent and daughter ion scans it was possible to detect as little as 80 pmol from reaction between calf thymus DNA and phosphoramide mustard (62). It was possible to identify two isobaric alkylguanines in human urine by a novel application of deuterium exchange in combination with MSIMS (64). Confirmation of structure was provided by comparison with authentic standards.
Protein and Peptide Adducts Structural characterization of endogenous proteins and peptides has been greatly simplified by the availability of modern methods of MS (16, 24-27). The same MS methodology can be employed in the characterization of proteins and peptides covalently modified by xenobiotics. A highly active area of research involves the analysis of glutathione adducts formed during xenobiotic metabolism. A standard approach has been to use a combination of positive FAB or LSI MS (where intense MH+ ions are observed) and negative FAB or LSI MS (where intense [M - HI- negative ions are observed). This approach has been employed in the analysis of GSH adducts of 1,2dibromoalkanes (65)and other dihaloalkanes (66),chlorambucil (23),methazolamide (67),butylated hydroxytoluene (68),benzene (69),eugenol (70),N-(l-methyl-3,3-diphenylpropy1)formamide (711, methyl isocyanate (721, Nmethylformamide (731, dopamine (74),butadiene monoxide (7.9, and p-aminophenol (76). It is evident from the FAB mass spectra of these compounds that there is a paucity of structural information. Further information can be readily obtained by carrying out CID on MH+ coupled with the use of daughter ion scanning in a tandem mass spectrometer. GSH adducts undergo well-defined cleavages including the loss of glutamic acid (129 Da), the loss of methyl glutamate (143 Da) from the corresponding methyl ester, and the loss of methylglycine (89 Da) from the N-(alkoxycarbonyl) methyl ester derivative (3,73,7779). These specific losses can be useful in screening studies for unknown GSH adducts during studies for xenobiotic metabolism. A major problem with static methods of analysis such as FAB and LSI is that minor interfering substances can sometimes obscure the mass spectrum. Thus, it is highly desirable that chromatography be linked to the ionization technique. LC/TSP MS methodology has had limited success with GSH adducts, but the emerging techniques based on API show real promise. Kassahun et al. demonstrated that APCI MS/MS could provide struc-
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turally informative daughter ions on the methylated GSH derivative of the reactive metabolite derived from valproic acid (80).There have also been several conference reports that API-based methods are useful for the characterization of GSH adducts (3). A variety of xenobiotic alkylating agents and xenobiotics that require metabolic activation bind covalently to hemoglobin (81). MS has been employed extensively in the quantitative and structural analysis of these adducts. In some cases it has been found that the degree of hemoglobin alkylation can be correlated with the extent of binding to DNA, and so this can provide a noninvasive method for biomonitoring (45,46,82). Methods based on G U M S include hemoglobin adducts of 4,4'-methylenedianiline (83), l-(2-chloroethyl)-l-nitrosoureas(841, 4'methylenebis(2-chloroaniline)(85),1,2-bis(2-chloroethyl)N-nitrosourea (861,styrene oxide (87),ethylene oxide (70, 88,891,and griseofulvin (90). Detailed structural analysis of hemoglobin adducts can provide insight into the nature of the reactive metabolite involved in binding. I t has been possible, in some cases, to also characterize the site of covalent attachment of the macromolecule. For example, it was demonstrated that arylamide was metabolized to glycidamide in the rat by characterization of the hemoglobin adduct (91). Hemoglobin obtained from rats that had been treated with acrylamide was subjected to acid hydrolysis. S42-Carboxy-2-hydroxyethy1)cysteinewas identified by GUMS analysis of the hydrolysate. This modified cysteine is the predicted product from acid-catalyzed hydrolysis of glycidamide attached to hemoglobin cysteine residues. The most successful approach to characterization of the actual site of covalent attachment to the hemoglobin has involved protease digestion followed by identification of the release modified peptides. An elegant study reported by Kaur et al. employed MS to identify the sites of covalent attachment of radiolabeled styrene 7,8-oxide with human hemoglobin (28). Peptide fragments were purified by HPLC after trypsin digestion of the modified hemoglobin. Peptides containing radioactivity were then subjected to LSI MS/MS using high-energy CID to induce specific peptide cleavages. Unambiguous assignment of specific residues that had been modified was then possible. The externally accessible histidines were found to be the dominant sites for alkylation. Using similar methodology, it was demonstrated that benzo[alpyrene anti-diol epoxide bound to hemoglobin a chain aspartate-47 (92). In this study, the alkylated carboxy chains were first converted to N-(2,3-dihydroxypropyl)amides,and the protein was then subjected to trypsin digestion. Peptide fragments containing N-(2,3-dihydroxypropyl)amide were identified by FAB/MS (92).
Reactive Intermediates Insight into the mechanisms of xenobiotic toxicity can sometimes be gained by mass spectral analysis of the putative reactive metabolite. In a study of valproic acid metabolism carried out by Levy et al., GC/MS was employedto monitor the formation of 2-propyl-4-pentenoic acid, a metabolite thought to mediate the toxic side effects of the drug (93). Formation of the metabolite was found to increase during coadministration of carbamazepine or phenytoin. This suggested that the metabolite was responsible for the increased incidence of valproateinduced toxicity during coadministration of P-450 inducers
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such as phenytoin and carbamazepine. When hepatotoxic doses of acetaminophen were administered to rats, dosedependent binding to hemoglobin was observed (81).Acid hydrolysis of the hemoglobin resulted in the release of 3-cystein-S-yl-~-hydroxyacetanilide, which was identified after derivatization by GC/MS. This finding was consistent with the formation of N-acetyl-p-benzoquinone imine as the reactive metabolite derived from acetaminophen (81). A subsequent study by Lauriault et al. showed that diethyldithiocarbamate and dithiothreitol were antidotes to the cytotoxicity of N-acetyl-p-benzoquinone imine (94).FAB MS was then used to demonstrate that inhibition of toxicity was a consequence of two different mechanisms. Diethyldithiocarbamate formed an S-linked adduct to remove the reactive metabolite whereas dithiothreitol caused reduction back to acetaminophen. The solvent 1,2,3-trichloropropane causes a number of toxic effects in laboratory animals (95).Major metabolites from rats dosed with 1,2,3-trichloropropanewere identified by FAB MS and shown to result from glutathione conjugation and oxidation. Diverse toxic effects of 1,2,3trichloropropane were ascribed to bioactivation through these pathways (95).The reactive metabolite derived from a related halocarbon, bromotrichloromethane, was shown to be derived from the trichloromethyl radical (96). Reductive metabolism of bromotrichloromethane by ferrous deoxymyoglobin resulted in the formation of soluble heme-derived products that were shown by FAB MS to contain the trichloromethyl moiety (96).A protein-bound heme adduct was characterized by Edman degradation coupled with FAB MS and quadrupole Fourier transform MS (97). I t was shown to consist of a prosthetic heme group modified by CClz (derived from bromotrichloromethane) covalently bound to the proximal histidine93 (97). Covalent binding of the heme prosthetic group to the apoprotein was though to cause a change in tertiary structure which then altered its catalytic activity and enhanced its susceptibility to proteolysis. GC/MS was used in an elegant study of Slaughter and Hanzlik to show that a quinone methide was responsible for the major amount of covalent binding to hepatic proteins in bromobenzene-treated rats (98). Hepatic proteins were subjected to alkaline permethylation with potassium hydroxide and methyl iodide. Products were purified by HPLC and structural characterization of methylated thioanisoles carried out by GUMS. Further confirmation of this novel pathway was obtained by treating rats with deuterated bromobenzene and analyzing the products for deuterium content. LC MS/MS and APIbased ionization techniques have allowedthe identification of a novel, potentially toxic, pyridinium metabolite of haloperidol to be carried out (99).The metabolite was identified in the urine of patients dosed with haloperidol with human liver microsomes (99). The pyridinium metabolite was similar to that observed with the Parkinsoninducing agent MPTP which is known to selectively destroy nigrostriatal neurons in primates. Insight into the toxicity of the anticancer drug busulfan was provided by GC/MS quantification of parent drug concentrations in cerebrospinal fluid and peripheral blood plasmsa (100). There was a significant increase in the cerebrospinal fluid/plasma ratio in patients dosed with >16 mg/kg. This correlated with increase neurotoxic side effects, implicating a dose-dependent toxicity. FAB/MS
was used in a study designed to help elucidate the mechanism of 3’-azido-3’-deoxythyidine (AZT) toxicity (101).The major metabolite of AZT had previously been identified as the glucuronide conjugate. Substantial amounts of two other metabolites were subsequently identified as the reduced 3’-aminO product (AMT) and its corresponding glucuronide (101).AMT was found to be much more toxic than the parent drug in clonogenic assays for drug cytotoxicity. This suggested that AMT may be the metabolite responsible for the cytotoxic effect of AZT in patients treated with the drug.
Summary and Future Trends Metabolism studies continue to be of great importance in toxicology research. Of particular utility is the isotope cluster technique that allows the recognition of xenobiotic metabolites in complex biological matrices such as urine and plasma (102-104). Several methods have been developed for the deconvolution of isotope clusters from stable isotope analogs so that mass spectral identification is facilitated (105-106).API-based ionization techniques, exemplified in a number recently published studies, are having a tremendous impact on both structural and quantitative studies of xenobiotics (5,7,9,11-13,15,16, 99,107-113). As these techniques continue to proliferate, the extensive use of GC/MS methodology (41,42) will probably be curtailed. However, LC/MS methods are unable to compete with the extremely high sensitivity that can be attained with ECNCI MS, and so this technique will probably continue to be the method of choice when compounds are present in the femtomole to attomole range (42-44,49-51,114).MS/MS has had a tremendous impact both for structural studies and for rapid and specific quantitative analyses (16,24-26).Methods based on this highly specific technique will undoubtedly continue to proliferate in the future. MALDI/MS is starting to have an impact on toxicological research, particularly for macromolecular determinations (4,32,33, 115),and as MS/MS methodology becomes available (36),the technique will probably make a significant contribution to the structural analysis of small molecules. Ion trap methodology continues to spark interest because of the low cost and minimal space requirements associated with this technique (3,116,119). In spite of these advantages, commercialization of sophisticated ion trap methodology is proceeding at arather slow pace. MS methods based on triple quadrupole (16), tandem four sector (24,28), and hybrid instruments (79) continue to dominate the published literature. The high cost of tandem sector instruments has limited their use to only a few laboratories. However, exciting developments in array detectors (37) coupled with high-sensitivity introduction systems based on ESI (10)and flow FAB/ LSI (39) provide unique capabilities that cannot be attained with quadrupole and hybrid instrumentation. This suggeststhat tandem sector instruments will continue to provide important structural information, particularly where high-energy CID and unit mass resolution are required. An interesting innovative approach to metabolism studies that involves the use of stable isotopes has been developed by Abramson and his co-workers (120,122). They have introduced the chemical reaction interface (CRI) mass spectrometer for quantitative metabolism studies. In this technique, organic molecules containing
Forum: Frontiers in Molecular Toxicology
a stable isotope are subjected to chromatography. As they elute from the chromatography column, they are converted to individual atoms in a microwave furnace. The atoms are then allowed to recombine in the presence of sulfur dioxide as an oxidizinggas, and the resulting carbon dioxide and nitric oxide are then analyzed in a conventional quadrupole mass spectrometer. A stable isotope-enriched chromatogram is then generated where the relative amount of each metabolite is independent of ita structure but proportional to the number of stable isotopes present. I t is anticipated that CRI methodology will ultimately provide an alternative to the use of radioisotopes for in uiuo metabolism studies. There is a real need for this type of methodology in view of the increasing costs involved in the disposal of radioactive waste, the questionable ethics in dosing normal subjects with radioactivity, and the fact that it is almost impossible to justify dosing women and children with radioactivity. In summary, it is anticipated the API MS and MS/MS will assume a major role in both quantitative and structural studies in the future. High-sensitivity quantitative analyses will continue to rely on capillary GC/ECNCI MS. Ready access to these techniques will be important for investigators working on molecular dosimetry as well as those engaged in more fundamental mechanistic and structural aspects of toxicology.
Acknowledgment. The preparation of this review was greatly facilitated by stimulating discussions with Dr. Ajai Chaudhary and the editorial assistance of Lamar Dixon. Support of NIH Grants ES00267 and GM31304 and the Hancock Laboratory for Cancer Research are gratefully acknowledged.
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