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1.
The purpose of these guidelines is to provide concise guidance on the planning, performing and interpretation of studies to monitor groups or individuals exposed to genotoxic agents. Most human carcinogens are genotoxic but not all genotoxic agents have been shown to be carcinogenic in humans. Although the main interest in these studies is due to the association of genotoxicity with carcinogenicity, there is also an inherent interest in monitoring human genotoxicity independently of cancer as an endpoint.The most often studied genotoxicity endpoints have been selected for inclusion in this document and they are structural and numerical chromosomal aberrations assessed using cytogenetic methods (classical chromosomal aberration analysis (CA), fluorescence in situ hybridisation (FISH), micronuclei (MN)); DNA damage (adducts, strand breaks, crosslinking, alkali-labile sites) assessed using bio-chemical/electrophoretic assays or sister chromatid exchanges (SCE); protein adducts; and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations. The document does not consider germ cells or gene mutation assays other than HPRT or markers of oxidative stress, which have been applied on a more limited scale. 相似文献
2.
This review presents with selected examples the versatility of nuclear magnetic resonance (NMR) spectroscopy in the analysis of toxic organophosphorus (OP) compounds, i.e. OP pesticides and chemical warfare agents (CWAs). Several NMR applications of biological importance, like studies on inhibition mechanism, metabolism, and exposure determination, are presented. The review also concerns with the environmental analysis of OP compounds by NMR spectroscopy. Residue analysis of environment and food samples as well as characterization of degradation in environment is discussed. Some of the NMR studies that have been done to support the Chemical Weapons Convention, i.e. the development of suitable CWA detoxification means and the method development of verification analysis for CWAs and their degradation products, are outlined. 相似文献
3.
The analysis of proteins by RPLC commonly involves the use of TFA as an ion‐pairing agent, even though it forms adducts and suppresses sensitivity. The presence of adducts can complicate protein molecular weight assignment especially when protein isoforms coelute as in the case of histones. To mitigate the complicating effects of TFA adducts in protein LC‐MS, we have optimized TFA‐free methods for protein separation. Protein standards and histones were used to evaluate TFA‐free separations using capillary (0.3 mm id) and nanoscale (0.1 mm id) C 8 columns with the ion‐pairing agents, formic acid or acetic acid. The optimized method was then used to examine the applicability of the approach for histone characterization in human cancer cell lines and primary tumor cells from chronic lymphocytic leukemia patients. 相似文献
4.
Hemoglobin adducts are useful for the identification and quantification of electrophilic agents in vivo. A modified Edman degradation method has been extensively used for monitoring exposure to ethylene oxide through gas chromatographic-mass spectrometric measurements of hydroxyethyl adducts to the N-terminal valines in hemoglobin. In a ring test, four laboratories using different versions of the method analyzed eight human globin samples with low adduct levels from ethylene oxide. Measurements of the same adduct by a radioimmunoassay were also included. Strong correlation between the measurements by the different laboratories shows that the method in principle works well. However, there were some systematic quantitative differences. 相似文献
5.
Propanil is one of the major herbicides used on rice-paddies and is thought to produce adverse health effects through the action of its metabolite 3,4-dichloroaniline (3,4-DCA). T he feasibility of monitoring human exposure to propanil on the basis of 3,4-DCA adducts to haemoglobin (Hb) was investigated. We developed a method based on gas chromatography negative ion chemical ionization-mass spectrometry (NICI-GC-MS) to quantify 3,4-DCA released from human Hb after alkaline hydrolysis of the protein. 3,4-DCA-Hb adducts were identified in agricultural workers exposed to propanil and were detectable even 4 months after the last herbicide application. Urine samples collected at the same time had no measurable level of 3,4-DCA. 3,4-DCA-Hb adducts might be useful for monitoring human exposure to 3,4-DCA from agricultural sources. 相似文献
6.
Propanil is one of the major herbicides used on rice-paddies and is thought to produce adverse health effects through the action of its metabolite 3,4-dichloroaniline (3,4-DCA). T he feasibility of monitoring human exposure to propanil on the basis of 3,4-DCA adducts to haemoglobin (Hb) was investigated. We developed a method based on gas chromatography negative ion chemical ionization-mass spectrometry (NICI-GC-MS) to quantify 3,4-DCA released from human Hb after alkaline hydrolysis of the protein. 3,4-DCA-Hb adducts were identified in agricultural workers exposed to propanil and were detectable even 4 months after the last herbicide application. Urine samples collected at the same time had no measurable level of 3,4-DCA. 3,4-DCA-Hb adducts might be useful for monitoring human exposure to 3,4-DCA from agricultural sources. 相似文献
7.
Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1–5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and N ε-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them – amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine – particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given. 相似文献
8.
Measurements of adducts formed with blood proteins, particularly haemoglobin, are increasingly being used to monitor human exposures to genotoxic chemicals. Information about the relationships between levels of genotoxic chemicals in the environment, e.g., concentration in the air, and levels of protein adducts in the blood is particularly important in setting safety standards and assessing risks. This paper describes the relationships between level of exposure to alkylating agents and level of haemoglobin adducts, considering the zero-order kinetics of the disappearance of these adducts. For comparison the corresponding relationship for adducts to macromolecules subjected to turnover, with first-order kinetics of disappearance, is described. For chemically stable and unstable adducts different exposure situations are considered: acute, chronic, intermittent and varying exposure levels. It is shown how an optimum solution of the problem of establishing the relationship between long-term exposure at varying levels (e.g., in work environments) and adduct level can be reached. Through mathematical derivations, which are given, expressions applicable to various exposure patterns are obtained and presented. 相似文献
9.
This paper provides a short historical overview of the development of bioanalytical methods for chemical warfare (CW) agents and their biological markers of exposure, with a more detailed overview of methods for organophosphorus nerve agents. Bioanalytical methods for unchanged CW agents are used primarily for toxicokinetic/toxicodynamic studies. An important aspect of nerve agent toxicokinetics is the different biological activity and detoxification pathways for enantiomers. CW agents have a relatively short lifetime in the human body, and are hydrolysed, metabolised, or adducted to nucleophilic sites on macromolecules such as proteins and DNA. These provide biological markers of exposure. In the past two decades, metabolites, protein adducts of nerve agents, vesicants and phosgene, and DNA adducts of sulfur and nitrogen mustards, have been identified and characterized. Sensitive analytical methods have been developed for their detection, based mainly on mass spectrometry combined with gas or liquid chromatography. Biological markers for sarin, VX and sulfur mustard have been validated in cases of accidental and deliberate human exposures. The concern for terrorist use of CW agents has stimulated the development of higher throughput analytical methods in support of homeland security. 相似文献
10.
In the past decades a large number of DNA adducts induced in the intact animal by alkylating agents have been identified. The formation and repair of these adducts are important determinants, not only of mutagenesis, tumor initiation and DNA-mediated toxicity but probably also of tumor progression. Most studies on in vivo DNA modification have been performed on isolated bulk DNA. More recently, methods have been developed to study the distribution of DNA adducts at the level of either the individual gene or the individual cell. This paper reviews immunocytochemical methods to study the formation and repair of DNA adducts and other DNA modifications at the level of the individual cell. DNA modifications induced by alkylating agents and a variety of other agents including ultraviolet radiation, aromatic amines, polycyclic aromatic hydrocarbons and platinum anti-cancer drugs will be discussed. Up to now, immunocytochemical analysis of in vivo modified DNA has largely concentrated on experimental animals. These studies have revealed striking heterogeneities with regard to formation and/or repair of DNA adducts in tissues from rat, hamster and mouse. Immunocytochemical adduct analysis can be used to identify in a convenient, fast and detailed way cell types, cell stages and sites in which biological effects of the adducts might be expressed. More recently, immunocytochemical analysis of DNA adducts also proved to be feasible on in situ exposed human samples. A number of existing and potential applications in the field of chemical carcinogenesis, experimental chemotherapy and molecular epidemiology are discussed. 相似文献
11.
Staphylococcus epidermidis causes nosocomial infections by colonizing and forming biofilms on indwelling medical devices. This process involves specific interactions between cell wall‐anchored (CWA) proteins and host proteins adsorbed onto the biomaterial. Here, we have explored the molecular forces by which the S. epidermidis CWA protein serine‐aspartate repeat protein F (SdrF) binds to type I collagen, by means of advanced atomic force microscopy (AFM) techniques. Using single‐cell force spectroscopy, we found that SdrF mediates bacterial adhesion to collagen‐coated substrates through both weak and strong bonds. Single‐molecule force spectroscopy demonstrated that these bonds involve the A and B regions of SdrF, thus revealing that the protein is capable of dual ligand‐binding activity. Both weak and strong bonds showed high dissociation rates, indicating they are much less stable than those formed by the well‐characterized ‘dock, lock and latch’ mechanism. Collectively, our results show that CWA proteins can bind to ligands by novel mechanisms. We anticipate that AFM will greatly contribute to the identification of novel binding partners and binding mechanisms in staphylococcal CWA proteins. 相似文献
12.
Age-related changes in carbonylation of mitochondrial proteins were determined in mitochondria from the flight muscles of Drosophila melanogaster. Reactivity with antibodies against (i) adducts of dinitrophenyl hydrazone (DNP), commonly assumed to react broadly with derivatized carbonyl groups, (ii) malondialdehyde (MDA), or (iii) hydroxynonenal (HNE), was compared at five different ages of flies. MDA and HNE are carbonyl-containing products of lipid peroxidation, which can form covalent adducts with proteins. Specific objectives were to address the following inter-related issues: (1) what are the sources of adducts involved in protein carbonylation in mitochondria during aging; (2) is carbonylation by different adducts detectable solely by the DNP antibodies, as assumed widely; (3) can the adducts formed by lipid peroxidation products in vivo, be used as markers for monitoring age-associated changes in oxidative damage to proteins. The total amounts of immunoreactive proteins, detected by all three antibodies, were found to increase with age; however, the immunodensity of individual reactive bands and the magnitude of the increases were variable, and unrelated to the relative abundance of a protein. While some protein bands were strongly immunopositive for all three antibodies, others were quite selective. The amounts of high molecular weight cross-linked proteins (>200kDa) increased with age. In general, the anti-HNE antibody reacted with more protein bands compared to the anti-MDA or -DNP antibody. The results suggest that sources of the carbonyl-containing protein adducts vary and no single antibody reacts with all of them. Overall, the results indicate that HNE shows robust age-associated increases in adductation with mitochondrial proteins, and is a good marker for monitoring protein oxidative damage during aging. 相似文献
13.
An off-line mass spectrometry method that combines immuno-spin trapping and chromatographic procedures has been developed for selective detection of the nitrone spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) covalently attached to proteins, an attachment which occurs only subsequent to DMPO trapping of free radicals. In this technique, the protein-DMPO nitrone adducts are digested to peptides with proteolytic agents, peptides from the enzymatic digest are separated by HPLC, and enzyme-linked immunosorbent assays (ELISA) using polyclonal anti-DMPO nitrone antiserum are used to detect the eluted HPLC fractions that contain DMPO nitrone adducts. The fractions showing positive ELISA signals are then concentrated and characterized by tandem mass spectrometry (MS/MS). This method, which constitutes the first liquid chromatography-ELISA-mass spectrometry (LC-ELISA-MS)-based strategy for selective identification of DMPO-trapped protein residues in complex peptide mixtures, facilitates location and preparative fractionation of DMPO nitrone adducts for further structural characterization. The strategy is demonstrated for human hemoglobin, horse heart myoglobin, and sperm whale myoglobin, three globin proteins known to form DMPO-trappable protein radicals on treatment with H(2)O(2). The results demonstrate the power of the new experimental strategy to select DMPO-labeled peptides and identify sites of DMPO covalent attachments. 相似文献
14.
There has been significant recent progress toward the development of human carcinogen—DNA adduct biomonitoring methods. 32P-Postlabelling is a technique which has found wide application in human studies. 32P-Postlabelling involves enzymatic preparation and labelling of DNA samples, followed by chromatographic separation of carcinogen—nucleotide adducts from unadducted nucleotides. Thin-layer ion-exchange and high-performance liquid chromatography (HPLC) have been utilized. This paper critically reviews 32P-postlabelling methods for analysis of bulky, polyaromatic carcinogen—DNA adducts and details a strategy to optimize this technique for monitoring human samples. Development of a human carcinogen biomonitoring method requires that the biomarker meet certain criteria: that the biomarker be responsive to exposures known to increase human cancer risk, to reductions in those exposures, and to the influence of metabolic differences. In addition, reliable samples must be available by non-invasive means. The ability of 32P-postlabelling to meet these criteria is traced in the literature and discussed. Identification of specific carcinogen—DNA adducts is a difficult task due to the low (femtomole) levels in human target tissues. Because co-chromatography in thin-layer chromatography (TLC) is generally not considered to be proof of chemical identity, both synchronous fluorescence and HPLC in conjunction with 32P-postlabelling and TLC are used to confirm the identity of specific carcinogen-DNA adducts in human samples. Mass spectrometry is a highly specific method, the sensitivity of which has been improved to the point which may allow its use to confirm the identity of carcinogen—DNA adducts isolated by 32P-postlabelling and other methods. The literature relating to the use of mass spectral techniques in carcinogen—DNA adduct analysis is reviewed. 相似文献
15.
Acetylcholinesterases (AChEs) form conjugates with certain highly toxic organophosphorus (OP) agents that become gradually resistant to reactivation. This phenomenon termed "aging" is a major factor limiting the effectiveness of therapy in certain cases of OP poisoning. While AChE adducts with phosphonates and phosphates are known to age through scission of the alkoxy C-O bond, the aging path for adducts with phosphoroamidates (P-N agents) like the nerve agent N,N-dimethylphosphonocyanoamidate (tabun) is not clear. Here we report that conjugates of tabun and of its butyl analogue (butyl-tabun) with the E202Q and F338A human AChEs (HuAChEs) age at similar rates to that of the wild-type enzyme. This is in marked contrast to the large effect of these substitutions on the aging of corresponding adducts with phosphates and phosphonates, suggesting that a different aging mechanism may be involved. Both tabun and butyl-tabun appear to be similarly accommodated in the active center, as suggested by molecular modeling and by kinetic studies of phosphylation and aging with a series of HuAChE mutants (E202Q, F338A, F295A, F297A, and F295L/F297V). Mass spectrometric analysis shows that HuAChE adduct formation with tabun and butyl-tabun occurs through loss of cyanide and that during the aging process both of these adducts show a mass decrease of 28 +/- 4 Da. Due to the nature of the alkoxy substituent, such mass decrease can be unequivocally assigned to loss of the dimethylamino group, at least for the butyl-tabun conjugate. This is the first demonstration that AChE adducts with toxic P-N agents can undergo aging through scission of the P-N bond. 相似文献
16.
A number of oxidative stress agents cause DNA and protein damage, which may compromise genomic integrity. Whereas oxidant-induced DNA damage has been extensively studied, much less is known concerning the occurrence and fate of nuclear protein damage, particularly of proteins involved in the regulation and maintenance of chromatin structure. Protein damage may be caused by the formation of reactive carbonyl species such as glyoxal, which forms after lipid peroxide degradation. It may also result from degradation of early protein glycation adducts and from methylglyoxal, formed in the process of glycolytic intermediate degradation. Major adducts indicative of protein damage include the advanced glycation end product (AGE) carboxymethyllysine (CML) and argpyrimidine protein adducts. Thus, the formation of CML and argpyrimidine protein adducts represents potential biomarkers for nuclear protein damage deriving from a variety of sources. The purpose of this study was to identify and quantify AGE adducts formed in vivo in a nuclear protein, specifically histone H1, using CML and argpyrimidine as biomarkers. Histone H1 was isolated from calf thymus collected immediately after slaughter under conditions designed to minimize AGE formation before isolation. Using antibodies directed against oxidative protein adducts, we identified CML, argpyrimidine, and protein crosslinks present in the freshly isolated histone H1. Detailed mass spectroscopy analysis of histone H1 revealed the presence of two specific lysine residues modified by CML adducts. Our results strongly suggest that glycation of important nuclear protein targets such as histone H1 occurs in vivo and that these oxidative changes may alter chromatin structure, ultimately contributing to chronic changes associated with aging and diseases such as diabetes. 相似文献
17.
Accurate cancer biomarkers are needed for early detection, disease classification, prediction of therapeutic response and monitoring treatment. While there appears to be no shortage of candidate biomarker proteins, a major bottleneck in the biomarker pipeline continues to be their verification by enzyme linked immunosorbent assays. Multiple reaction monitoring (MRM), also known as selected reaction monitoring, is a targeted mass spectrometry approach to protein quantitation and is emerging to bridge the gap between biomarker discovery and clinical validation. Highly multiplexed MRM assays are readily configured and enable simultaneous verification of large numbers of candidates facilitating the development of biomarker panels which can increase specificity. This review focuses on recent applications of MRM to the analysis of plasma and serum from cancer patients for biomarker verification. The current status of this approach is discussed along with future directions for targeted mass spectrometry in clinical biomarker validation. 相似文献
18.
Irreversible inactivation of human acetylcholinesterase (hAChE) by organophosphorous pesticides (OPs) and chemical weapon agents (CWA) has severe morbidity and mortality consequences. We present data from quantum mechanics/molecular mechanics (QM/MM) and 80 classical molecular dynamics (MD) simulations of the apo and soman-adducted forms of hAChE to investigate the effects on the dynamics and protein structure when the catalytic Serine 203 is phosphonylated. We find that the soman phosphonylation of the active site Ser203 follows a water assisted addition-elimination mechanism with the elimination of the fluoride ion being the highest energy barrier at 6.5 kcal/mole. We observe soman-dependent changes in backbone and sidechain motions compared to the apo form of the protein. These alterations restrict the soman-adducted hAChE to a structural state that is primed for the soman adduct to be cleaved and removed from the active site. The altered motions and resulting structures provide alternative pathways into and out of the hAChE active site. In the soman-adducted protein both side and back door pathways are viable for soman adduct access. Correlation analysis of the apo and soman adducted MD trajectories shows that the correlation of gorge entrance and back door motion is disrupted when hAChE is adducted. This supports the hypothesis that substrate and product can use two different pathways as entry and exit sites in the apo form of the protein. These alternative pathways have important implications for the rational design of medical countermeasures. 相似文献
19.
The inducible repair enzyme O 6-methylguanine-DNA methyltransferase (MGMT) eliminates O 6-methylguanine adducts in DNA and protects the cells from damaging effects of alkylating agents. We have found that anti-MGMT antibodies recognize both the MGMT protein with a mol. weight?~?24 kDa and a protein with a mol. weight?~?48 kDa, which was named MARP (anti-methyltransferase antibody recognizable protein). A number of growth factors and cytokines were shown to regulate the expression of MGMT and MARP proteins. The ranges of concentrations of several growth factors and cytokines that caused increasing or decreasing protein amounts in human cell cultures were determined. The results of special biological experiments have allowed us to assume a possible role of MARP in the repair of alkyl adducts in human cells. 相似文献
20.
Mitomycin C (MC), an anti-cancer drug, and its analog, decarbamoylmitomycin C (DMC), are DNA-alkylating agents. MC is currently used in the clinics and its cytotoxicity is mainly due to its ability to form Interstrand Crosslinks (ICLs) which impede DNA replication and, thereby, block cancer cells proliferation. However, both MC and DMC are also able to generate monoadducts with DNA. In particular, we recently discovered that DMC, like MC, can form deoxyadenosine (dA) monoadducts with DNA. The biological role played by these monoadducts is worthy of investigation. To probe the role of these adducts and to detect them in enzymatic digests of DNA extracted from culture cells treated by both drugs, we need access to reference compounds i.e. MC and DMC dA-mononucleoside adducts. Previous biomimetic methods used to generate MC and DMC mononucleoside adducts are cumbersome and very low yielding. Here, we describe the diastereospecific chemical synthesis of both C-1 epimers of MC and DMC deoxyadenosine adducts. The key step of the synthesis involves an aromatic substitution reaction between a 6-fluoropurine 2′-deoxyribonucleoside and appropriately protected stereoisomeric triaminomitosenes to form protected-MC-dA adducts with either an S or R stereochemical configuration at the adenine-mitosene linkage. Fluoride-based deprotection methods generated the final four reference compounds: the two stereoisomeric MC-dA adducts and the two stereoisomeric DMC-dA adducts. The MC and DMC-dA adducts synthesized here will serve as standards for the detection and identification of such adducts formed in the DNA of culture cells treated with both drugs. 相似文献
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