DNA and Protein Adducts

Vesicants, nerve agents, and phosgene are reactive electrophiles that react covalently with nucleophilic sites on macro molecules. Reactive nucleophilic sites exist on the bases and phosphate groups of DNA molecules. An advantage of DNA as a substrate is that it is present in all tissues of the body. A disadvantage is that repair mechanisms tend to excise the alkylated moiety, resulting in a much shorter lifetime compared to alkylated proteins (for a recent review of mass spectrometry for quantitation of DNA adducts, see Koc and Swen-berg 2 ). 

The group of Ludlum developed a high-performance liquid chromatography (HPLC) method for the analysis of N7-HETE-Gua (9) and also a 32P-postlabeling method for N7-HETE-deoxyguanosine 5 -phosphate (10). 

An enzyme-linked immunosorbent assay (ELISA) was developed by Van der Schans and colleagues for detection of the mustard adduct within DNA, using monoclonal antibodies raised against N7-HETE-guanosine-5 -phosphate coupled to keyhole limpet hemocyanin (n). The ELISA was successfully applied in toxicokinetic studies in which levels of adducted DNA were followed in conjunction with measurement of intact sulfur mustard (12). 

The ELISA for detection of the DNA adduct was successfully applied to blood samples from two casualties of the Iraq-Iran conflict. These samples were collected 22 and 26 days following the alleged exposure to sulfur mustard (13). Concentrations found in lymphocytes and granulocytes were equivalent to similar levels found in human blood after treatment in vitro with 0.015-0.43 xM sulfur mustard. 

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An increased incidence of chromosome aberrations and micronuclei in peripheral lymphocytes have been reported in occupationally exposed workers. Additional studies have found a slight increase in the incidence of sister chromatid exchanges, whereas no increase has been found in several other studies. Sister chromatid exchange and chromosomal aberrations were induced in vivo in rodents and in vitro in human lymphocytes. Both DNA and protein adducts are formed in humans after styrene exposure. 

Recommendation 7. The Army should keep abreast of, and adopt where appropriate, developments in medical diagnostic techniques for detecting and quantifying low-level exposures to toxic substances, including research related to the use of DNA and protein adducts as measures of toxicologically relevant metabolites. 

Turteltaub, K.W. Livermore National Laboratory and University of California at San Francisco San Francisco, CA In vivo metabolic studies characterization of DNA and protein adducts in bone marrow Health Effects Institute... 

A number of adducts to amino acid residues have been identified by Noort and colleagues (1996) and Black et al. (1997a, b). Six different histidine residues, three glutamic acid residues, and both of the N-terminal valines were found. Alkylated cysteine, aspartic acid, lysine, and tryptophan were also detected. While the N1 and N3 histidine adducts were found to be most abundant, it was the alkylated N-terminal valine adducts that were most useful for subsequent quantification. See Detection of DNA and protein adducts of vesicants, below, for analytical details. 

Once incorporated, unbound lewisite is quickly hydrolyzed. Its predominant metabolite is 2-chlorovinylarsonous acid, CVAA (Figure 50.8). Analytical methods to confinn lewisite exposure have, at least in the past, focused on the detection and quantification of CVAA. However, Noort et al. (2002) also pointed out that due to the high affinity of arsenic towards sulfhydryl groups, adducts of lewisite/ CVAA and cysteine residues of proteins are formed. In an in vitro study, incubating " C-labeled lewisite with human blood samples, 90% of lewisite was found in erythrocytes, whereas 25 to 50% of arsenic was bound to globin. From these protein adducts, CVAA can be released to form an adduct with the antidote British Anti-Lewisite (BAL) (Fidder et al, 2000). The authors were also able to identify a specific protein adduct of lewisite formed with the cysteine residues 93 and 112 of P-globin. See Detection of DNA and protein adducts of vesicants, below, for analytical... 

A second example of sulfate bioactivation derives from the observed carcinogenicity of aromatic amineS/ such as those derived from coal tar (44). The polycyclic aromatic amines are N-hydroxylated by CYPs and then sulfated to form unstable N-O-sulfates that decompose and produce reactive nitrenium ion intermediates/ which form DNA and protein adducts. One environmental/genetic hypothesis of colon cancer etiology involves the interaction between dietary aromatic amines and the polymorphic expression of the appropriate STs for their activation to procarcinogenic reactive intermediates (44/ 45). 

Boysen G and Hecht SS (2003) Analysis of DNA and protein adducts of benzo [a]pyrene in human tissues using structure-specific methods. Mutation Research 543 17-30. 

Farmer, P. B. (2004a). DNA and protein adducts as markers of genotoxicity. Toxicol Lett 149, 3-9. 

Perera (1988) reviewed the data on the shape of the dose-response curves for adducts and mutations in a number of systems. He reported linearity in a variety of systems, including epidemiology studies, for the formation of DNA and protein adducts, both for chemicals that must be metabolically activated [(e.g., polycyclic aromatic hydrocarbons (PAHs) and 4-aminobiphenyl (4-ABP)], and for direct-acting chemicals, such as ethylene oxide and propylene oxide. He also noted that in vitro and in vivo studies have found a good correlation between levels of the critical DNA adduct (i.e., the one responsible for most of the chemical-induced mutations) and the frequency of induced mutations, indicating a linear dose-response for mutations. 

Perera, F. P. (1988). The significance of DNA and protein adducts in human biomonitoring studies. Mutat Res 205, 255-269. 

The ultrahigh sensitivity of AMS enables it to follow trace amounts of radioisotope-labeled drugs in animals and humans postadministration. In addition to microdose PK studies, AMS has been utilized in many aspects of pharmaceutical research, such as absolute bioavailability determination, mass balance and metabolite profiling, and identifications of DNA and protein adducts. 

Santella RM, Perera FP, Young TL, et al. 1995. Polycyclic aromatic hydrocarbon-DNA and protein adducts in coal tar treated patients and controls and their relationship to glutathione S-transferase genotype. Mutat Res 334(2) 117-224. 

In mammalian systems, nitroaromatic compounds are further reduced to amines and/or hydroxylamines, which may subsequently form DNA and protein adducts. These stable metabolites may be formed by the reduced oxygen tension and high local single-electron transferring enzyme concentration, for example, P-450R in microsomes may favor the free radical dismutation over their reoxidation by oxygen (Equation 9.8). Subsequently, the nitroso compounds formed (Equations 9.7 and 9.8) will be reduced to hydroxylamines (ArNHOH) and/or amines (ArNHj). The stable metabolites may also be formed by the two-electron reduction of nitroaromatics by certain flavoenzymes. In fact, the enzymatic two-electron reduction may be considered as the four-electron reduction, since after the first two-electron (hydride) transfer, the reduction of an intermediate nitroso compound to hydroxylamine (ArNHOH) proceeds faster [55] ... 

Dingley KH, Curtis KD, Nowell S, Felton JS, Lang NP, Turteltaub KW. DNA and protein adduct formation in the colon and blood of humans after exposure to a dietary-relevant dose of 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine. Cancer Epidemiol Biomarkers Prev 1999 8 507-512. 

Laboratory animal experiments demonstrate that dose-response relationships exist between carcinogen exposure and adduct levels, suggesting the utility of these studies In human exposure assessments. In animals, DNA and protein adduct levels correlate with dose of PAHs (63-65). tobacco-smoke condensate (43.60.66). H-nltrosamlnes (67-70). aromatic amines (21) (Beland, F. A., unpublished data Poirier, M. C., unpublished data) and heterocyclic amines (72.73). 

Table I. Monoclonal Antibodies Recognizing Carcinogen-DNA and Protein Adducts...

Fig. 3 Mammary tumors originating in MMTV-PyMT females exhibit distinct regions of hypoxia. Two hours prior to sacrifice, a PyMT -i- female transgenic mouse bearing a late-stage mammary carcinoma was injected i.p. with hypoxyprobe-1 (NPI, Inc.), which binds to DNA and protein adducts created by hypoxic exposure[228]. Tumor sections were probed with anti-hypoxyprobe-1 antibodies (brown staining) and counterstained with hematoxylin (200 X magnification)...

From these protein adducts, CVAA can be released to form an adduct with the antidote British anti-lewisite (BAL Fidder et al., 2000). The authors were also able to identify a specific protein adduct of lewisite formed with cysteine residues 93 and 112 of p-globin. See the section "Detection of DNA and protein adducts of vesicants," later in tiiis chapter, for analytical details. Figure 56.8 summarizes the biotransformation and reversal of adduct formation by BAL. 

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