Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice

A physiological examination of mice harboring a null allele at the aryl hydrocarbon (Ah) locus revealed that the encoded aryl hydrocarbon receptor plays a role in the resolution of fetal vascular structures during development. Although the aryl hydrocarbon receptor is more commonly studied for its role in regulating xenobiotic metabolism and dioxin toxicity, a developmental role of this protein is supported by the observation that Ah null mice display smaller livers, reduced fecundity, and decreased body weights. Upon investigating the liver phenotype, we found that the decrease in liver size is directly related to a reduction in hepatocyte size. We also found that smaller hepatocyte size is the result of massive portosystemic shunting in null animals. Colloidal carbon uptake and microsphere perfusion studies indicated that 56% of portal blood flow bypasses the liver sinusoids. Latex corrosion casts and angiography demonstrated that shunting is consistent with the existence of a patent ductus venosus in adult animals. Importantly, fetal vascular structures were also observed at other sites. Intravital microscopy demonstrated an immature sinusoidal architecture in the liver and persistent hyaloid arteries in the eyes of adult Ah null mice, whereas corrosion casting experiments described aberrations in kidney vascular patterns.

A multidrug resistance transporter from human MCF-7 breast cancer cells

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 663-aa member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.

An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids

Hepatic hydroxylation is an essential step in the metabolism and excretion of bile acids and is necessary to avoid pathologic conditions such as cholestasis and liver damage. In this report, we demonstrate that the human xenobiotic receptor SXR (steroid and xenobiotic receptor) and its rodent homolog PXR (pregnane X receptor) serve as functional bile acid receptors in both cultured cells and animals. In particular, the secondary bile acid derivative lithocholic acid (LCA) is highly hepatotoxic and, as we show here, a metabolic substrate for CYP3A hydroxylation. By using combinations of knockout and transgenic animals, we show that activation of SXR/PXR is necessary and sufficient to both induce CYP3A enzymes and confer resistance to toxicity by LCA, as well as other xenotoxicants such as tribromoethanol and zoxazolamine. Therefore, we establish SXR and PXR as bile acid receptors and a role for the xenobiotic response in the detoxification of bile acids.

The Chemically Inducible Plant Cytochrome P450 CYP76B1 Actively Metabolizes Phenylureas and Other Xenobiotics1

Cytochrome P450s (P450s) constitute one of the major classes of enzymes that are responsible for detoxification of exogenous molecules both in animals and plants. On the basis of its inducibility by exogenous chemicals, we recently isolated a new plant P450, CYP76B1, from Jerusalem artichoke (Helianthus tuberosus) and showed that it was capable of dealkylating a model xenobiotic compound, 7-ethoxycoumarin. In the present paper we show that CYP76B1 is more strongly induced by foreign compounds than other P450s isolated from the same plant, and metabolizes with high efficiency a wide range of xenobiotics, including alkoxycoumarins, alkoxyresorufins, and several herbicides of the class of phenylureas. CYP76B1 catalyzes the double N-dealkylation of phenylureas with turnover rates comparable to those reported for physiological substrates and produces nonphytotoxic compounds. Potential uses for CYP76B1 thus include control of herbicide tolerance and selectivity, as well as soil and groundwater bioremediation.

Conserved promoter elements in the CYP6B gene family suggest common ancestry for cytochrome P450 monooxygenases mediating furanocoumarin detoxification.

Despite the fact that Papilio glaucus and Papilio polyxenes share no single hostplant species, both species feed to varying extents on hostplants that contain furanocoumarins. P. glaucus contains two nearly identical genes, CYP6B4v2 and CYP6B5v1, and P. polyxenes contains two related genes, CYP6B1v3 and CYP6B3v2. Except for CYP6B3v2, the substrate specificity of which has not yet been defined, each of the encoded cytochrome P450 monooxygenases (P450s) metabolizes an array of linear furanocoumarins. All four genes are transcriptionally induced in larvae by exposure to the furanocoumarin xanthotoxin; several are also induced by other furanocoumarins. Comparisons of the organizational structures of these genes indicate that all have the same intron/exon arrangement. Sequences in the promoter regions of the P. glaucus CYP6B4v2/CYP6B5v1 genes and the P. polyxenes CYP6B3v2 gene are similar but not identical to the -146 to -97 region of CYP6B1v3 gene, which contains a xanthotoxin-responsive element (XRE-xan) important for basal and xanthotoxin-inducible transcription of CYP6B1v3. Complements of the xenobiotic-responsive element (XRE-AhR) in the dioxin-inducible human and rat CYP1A1 genes also exist in all four promoters, suggesting that these genes may be regulated by dioxin. Antioxidant-responsive elements (AREs) in mouse and rat glutathione S-transferase genes and the Barbie box element (Bar) in the bacterial CYP102 gene exist in the CYP6B1v3, CYP6B4v2, and CYP6B5v1 promoters. Similarities in the protein sequences, intron positions, and xanthotoxin- and xenobiotic-responsive promoter elements indicate that these insect CYP6B genes are derived from a common ancestral gene. Evolutionary comparisons between these P450 genes are the first available for a group of insect genes transcriptionally regulated by hostplant allelochemicals and provide insights into the process by which insects evolve specialized feeding habits.

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.

Heat shock protein hsp90 regulates dioxin receptor function in vivo.

The dioxin (aryl hydrocarbon) receptor is a ligand-dependent basic helix-loop-helix (bHLH) factor that binds to xenobiotic response elements of target promoters upon heterodimerization with the bHLH partner factor Arnt. Here we have replaced the bHLH motif of the dioxin receptor with a heterologous DNA-binding domain to create fusion proteins that mediate ligand-dependent transcriptional enhancement in yeast (Saccharomyces cerevisiae). Previously, our experiments indicated that the ligand-free dioxin receptor is stably associated with the 90-kDa heat shock protein, hsp90. To investigate the role of hsp90 in dioxin signaling we have studied receptor function in a yeast strain where hsp90 expression can be down-regulated to about 5% relative to wild-type levels. At low levels of hsp90, ligand-dependent activation of the chimeric dioxin receptor construct was almost completely inhibited, whereas the activity of a similar chimeric construct containing the structurally related Arnt factor was not affected. Moreover, a chimeric dioxin receptor construct lacking the central ligand- and hsp90-binding region of the receptor showed constitutive transcriptional activity in yeast that was not impaired upon down-regulation of hsp90 expression levels. Thus, these data suggest that hsp90 is a critical determinant of conditional regulation of dioxin receptor function in vivo via the ligand-binding domain.

Interazione di nanoparticelle metalliche con menbrane biologiche: risultati di studi in vitro su cute, mucosa del cavo orale e meningi ed implicazioni per la salute

L’utilizzo di nanomateriali, ovvero una nuova classe di sostanze composte da particelle ultrafini con dimensioni comprese fra 1 e 100 nm (American Society for Testing Materials - ASTM), è in costante aumento a livello globale. La particolarità di tali sostanze è rappresentata da un alto rapporto tra la superficie e il volume delle particelle, che determina caratteristiche chimico-fisiche completamente differenti rispetto alle omologhe macrosostanze di riferimento. Tali caratteristiche sono tali da imporre una loro classificazione come nuovi agenti chimici (Royal Society & Royal Academy of Engineering report 2004). Gli impieghi attuali dei nanomateriali risultano in continua evoluzione, spaziando in diversi ambiti, dall’industria farmaceutica e cosmetica, all’industria tessile, elettronica, aerospaziale ed informatica. Diversi sono anche gli impieghi in campo biomedico; tra questi la diagnostica e la farmacoterapia. È quindi prevedibile che in futuro una quota sempre maggiore di lavoratori e consumatori risulteranno esposti a tali sostanze. Allo stato attuale non vi è una completa conoscenza degli effetti tossicologici ed ambientali di queste sostanze, pertanto, al fine di un loro utilizzo in totale sicurezza, risulta necessario capirne meglio l’impatto sulla salute, le vie di penetrazione nel corpo umano e il rischio per i lavoratori conseguente al loro utilizzo o lavorazione. La cute rappresenta la prima barriera nei confronti delle sostanze tossiche che possono entrare in contatto con l’organismo umano. Successivamente agli anni ‘60, quando si riteneva che la cute rappresentasse una barriera totalmente impermeabile, è stato dimostrato come essa presenti differenti gradi di permeabilità nei confronti di alcuni xenobiotici, dipendente dalle caratteristiche delle sostanze in esame, dal sito anatomico di penetrazione, dal grado di integrità della barriera stessa e dall’eventuale presenza di patologie della cute. La mucosa del cavo orale funge da primo filtro nei confronti delle sostanze che entrano in contatto con il tratto digestivo e può venir coinvolta in contaminazioni di superficie determinate da esposizioni occupazionali e/o ambientali. È noto che, rispetto alla cute, presenti una permeabilità all’acqua quattro volte maggiore, e, per tale motivo, è stata studiata come via di somministrazione di farmaci, ma, ad oggi, pochi sono gli studi che ne hanno valutato le caratteristiche di permeazione nei confronti delle nanoparticelle (NPs). Una terza importante barriera biologica è quella che ricopre il sistema nervoso centrale, essa è rappresentata da tre foglietti di tessuto connettivo, che assieme costituiscono le meningi. Questi tre foglietti rivestono completamente l’encefalo permettendone un isolamento, tradizionalmente ritenuto completo, nei confronti degli xenobiotici. L’unica via di assorbimento diretto, in questo contesto, è rappresentata dalla via intranasale. Essa permette un passaggio diretto di sostanze dall’epitelio olfattivo all’encefalo, eludendo la selettiva barriera emato-encefalica. Negli ultimi anni la letteratura scientifica si è arricchita di studi che hanno indagato le caratteristiche di assorbimento di farmaci attraverso questa via, ma pochissimi sono gli studi che hanno indagato la possibile penetrazione di nanoparticelle attraverso questa via, e nessuno, in particolar modo, ha indagato le caratteristiche di permeazione delle meningi. L’attività di ricerca svolta nell’ambito del presente dottorato ha avuto per finalità l’indagine delle caratteristiche di permeabilità e di assorbimento della cute, della mucosa del cavo orale e delle meningi nei confronti di alcune nanoparticelle, scelte fra quelle più rappresentative in relazione alla diffusione d’utilizzo a livello globale. I risultati degli esperimenti condotti hanno dimostrato, in vitro, che l’esposizione cutanea a Pt, Rh, Co3O4 e Ni NPs determinano permeazione in tracce dei medesimi metalli attraverso la cute, mentre per le TiO2 NPs tale permeazione non è stata dimostrata. È stato riscontrato, inoltre, che la mucosa del cavo orale e le meningi sono permeabili nei confronti dell’Ag in forma nanoparticellare.

(Table 1) Concentrations of the main organochlorine pesticides in liver and plasma samples of ringed seals (Phoca hispida)

The present study investigates the concentrations and patterns of organochlorine pesticides (OCPs) and their metabolites in liver and plasma of two ringed seal populations (Phoca hispida): lower contaminated Svalbard population and more contaminated Baltic Sea population. Among OCPs, p,p'-DDE and sum-chlordanes were the highest in concentration. With increasing hepatic contaminant concentrations and activities of xenobiotic-metabolizing enzymes, the concentrations of 3-methylsulfonyl-p,p'-DDE and the concentration ratios of pentachlorophenol/hexachlorobenzene increased, and the toxaphene pattern shifted more towards persistent Parlar-26 and -50 and less towards more biodegradable Parlar-44. Relative concentrations of the chlordane metabolites, oxychlordane and -heptachlorepoxide, to sum-chlordanes were higher in the seals from Svalbard compared to the seals from the Baltic, while the trend was opposite for cis- and trans-nonachlor. The observed differences in the OCP patterns in the seals from the two populations are probably related to the catalytic activity of xenobiotic-metabolizing enzymes, and also to differences in dietary exposure.

Abolition of valproate-derived choleresis in the Mrp2 transporter-deficient rat

Valproic acid (VPA) is a major therapeutic agent in the treatment of epilepsy and other neurological disorders. It is metabolized in humans and rats primarily along two pathways: direct glucuronidation to yield the acyl glucuronide (VPA-G) and beta-oxidation. We have shown much earlier in the Sprague-Dawley rat that i.v. administration of sodium valproate (NaVPA) caused a marked choleresis ( mean of 3.3 times basal bile flow after doses of 150 mg/kg), ascribed to the passive osmotic flow of bile water following excretion of VPA-G across the canalicular membrane. Active biliary pumping of anionic drug conjugates across the canalicular membrane is now believed to be attributable to transporter proteins, in particular Mrp2, which is deficient in the TR- ( a mutant Wistar) rat. In the present study, normal Wistar and Mrp2-deficient TR- rats were dosed i.v. with NaVPA at 150 mg/kg. In the Wistar rats, there was a peak choleretic effect of about 3.2 times basal bile flow, occurring at about 30 to 45 min postdose ( as seen previously with Sprague-Dawley rats). In TR- rats given the same i.v. dose, there was no evidence of postdose choleresis. The choleresis was correlated with the excretion of VPA-G into bile. In Wistar rats, 62.8 +/- 7.7% of the NaVPA dose was excreted in bile as VPA-G, whereas in TR- rats, only 2.0 +/- 0.6% of the same dose was excreted as VPA-G in bile ( with partial compensatory excretion of VPA-G in urine). This study underlines the functional ( bile flow) consequences of biliary transport of xenobiotic conjugated metabolites.

Rhizodeposits of Trifolium pratense and Lolium perenne: Their comparative effects on 2,4-D mineralization in two contrasting soils

Rhizosphere enhanced biodegradation of organic pollutants has been reported frequently and a stimulatory role for specific components of rhizodeposits postulated. As rhizodeposit composition is a function of plant species and soil type, we compared the effect of Lolium perenne and Trifolium pratense grown in two different soils (a sandy silt loam: pH 4, 2.8% OC, no previous 2,4-D exposure and a silt loam: pH 6.5, 4.3% OC, previous 2,4-D exposure) on the mineralization of the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid). We investigated the relationship of mineralization kinetics to dehydrogenase activity, most probable number of 2,4-D degraders (MPN2,4-D) and 2,4-D degrader composition (using sequence analysis of the gene encoding alpha-ketoglutarate/2,4-D dioxygenase (tfdA)). There were significant (P < 0.01) plant-soil interaction effects on MPN2,4-D and 2,4-D mineralization kinetics (e.g. T pratense rhizodeposits enhanced the maximum mineralization rate by 30% in the acid sandy silt loam soil, but not in the neutral silt loam soil). Differences in mineralization kinetics could not be ascribed to 2,4-D degrader composition as both soils had tfdA sequences which clustered with tfdAs representative of two distinct classes of 2,4-D degrader: canonical R. eutropha JMP134-like and oligotrophic alpha-proteobacterial-like. Other explanations for the differential rhizodeposit effect between soils and plants (e.g. nutrient competition effects) are discussed. Our findings stress that complexity of soil-plant-microbe interactions in the rhizosphere make the occurrence and extent of rhizosphere-enhanced xenobiotic degradation difficult to predict.

Rhizodeposition and the enhanced mineralization of 2,4-dichlorophenoxyacetic acid in soil from the Trifolium pratense rhizosphere

Enhanced biodegradation of organic xenobiotic compounds in the rhizosphere is frequently recorded although the specific mechanisms are poorly understood. We have shown that the mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) is enhanced in soil collected from the rhizosphere of Trifolium pratense[e.g. maximum mineralization rate = 7.9 days(-1) and time at maximum rate (t(1)) = 16.7 days for 12-day-old T. pratense soil in comparison with 4.7 days(-1) and 25.4 days, respectively, for non-planted controls). The purpose of this study was to gain a better understanding of the plant-microbe interactions involved in rhizosphere-enhanced biodegradation by narrowing down the identity of the T. pratense rhizodeposit responsible for stimulating the microbial mineralization of 2,4-D. Specifically, we investigated the distribution of the stimulatory component(s) among rhizodeposit fractions (exudates or root debris) and the influence of soil properties and plant species on its production. Production of the stimulatory rhizodeposit was dependent on soil pH (e.g. t(1) for roots grown at pH 6.5 was significantly lower than for those grown at pH 4.4) but independent of soil inorganic N concentration. Most strikingly, the stimulatory rhizodeposit was only produced by T. pratense grown in non-sterile soil and was present in both exudates and root debris. Comparison of the effect of root debris from plant species (three each) from the classes monocotyledon, dicotyledon (non-legume) and dicotyledon (legume) revealed that legumes had by far the greatest positive impact on 2,4-D mineralization kinetics. We discuss the significance of these findings with respect to legume-rhizobia interactions in the rhizosphere.

The genetics of primary congenital glaucoma

One of the objectives of the molecular biological study of glaucoma is to establish how the disease develops as a result of the production of aberrant gene products. Many of the genes associated with glaucoma code for proteins which are likely to be directly or indirectly involved in the development and/or function of cells within the trabecular meshwork. The identification of specific defects in these genes is likely to lead to a better understanding of the mechanisms involved in PCG and glaucoma in general and to the development of alternative therapies to surgery. The CYP1B1 gene in particular, which is a linked to congenital glaucoma, and is expressed in the trabecular meshwork, codes for a member of the cytochrome P450 group of proteins. These iron binding proteins constitute a family of enzymes involved in the processes of xenobiotic metabolism, growth, and development. The discovery of the CYP1B1 gene in PCG emphases the importance of abnormalities in the molecular structure of proteins expressed in cells of the trabecular network as a cause of PCG. The identification of specific genetic defects leads to the possibility of more widespread screening for PCG especially in affected families and hence, the possibility of the identification of asymptomatic carriers of the disease. Early identification of 'at risk' parents may then enable earlier detection of PCG and intervention in the infant.

Specific Increase in MDR1 Mediated Drug-Efflux in Human Brain Endothelial Cells following Co-Exposure to HIV-1 and Saquinavir

Persistence of HIV-1 reservoirs within the Central Nervous System (CNS) remains a significant challenge to the efficacy of potent anti-HIV-1 drugs. The primary human Brain Microvascular Endothelial Cells (HBMVEC) constitutes the Blood Brain Barrier (BBB) which interferes with anti-HIV drug delivery into the CNS. The ATP binding cassette (ABC) transporters expressed on HBMVEC can efflux HIV-1 protease inhibitors (HPI), enabling the persistence of HIV-1 in CNS. Constitutive low level expression of several ABC-transporters, such as MDR1 (a.k.a. P-gp) and MRPs are documented in HBMVEC. Although it is recognized that inflammatory cytokines and exposure to xenobiotic drug substrates (e.g HPI) can augment the expression of these transporters, it is not known whether concomitant exposure to virus and anti-retroviral drugs can increase drug-efflux functions in HBMVEC. Our in vitro studies showed that exposure of HBMVEC to HIV-1 significantly up-regulates both MDR1 gene expression and protein levels; however, no significant increases in either MRP-1 or MRP-2 were observed. Furthermore, calcein-AM dye-efflux assays using HBMVEC showed that, compared to virus exposure alone, the MDR1 mediated drug-efflux function was significantly induced following concomitant exposure to both HIV-1 and saquinavir (SQV). This increase in MDR1 mediated drug-efflux was further substantiated via increased intracellular retention of radiolabeled [3H-] SQV. The crucial role of MDR1 in 3H-SQV efflux from HBMVEC was further confirmed by using both a MDR1 specific blocker (PSC-833) and MDR1 specific siRNAs. Therefore, MDR1 specific drug-efflux function increases in HBMVEC following co-exposure to HIV-1 and SQV which can reduce the penetration of HPIs into the infected brain reservoirs of HIV-1. A targeted suppression of MDR1 in the BBB may thus provide a novel strategy to suppress residual viral replication in the CNS, by augmenting the therapeutic efficacy of HAART drugs.

Covalent protein adduction of nitrogen mustards and related compounds

Chemical warfare agents continue to pose a global threat despite the efforts of the international community to prohibit their use in warfare. For this reason, improvement in the detection of these compounds remains of forensic interest. Protein adducts formed by the covalent modification of an electrophilic xenobiotic and a nucleophilic amino acid may provide a biomarker of exposure that is stable and specific to compounds of interest (such as chemical warfare agents), and have the capability to extend the window of detection further than the parent compound or circulating metabolites. This research investigated the formation of protein adducts of the nitrogen mustard chemical warfare agents mechlorethamine (HN-2) and tris(2-chloroethyl)amine (HN-3) to lysine and histidine residues found on the blood proteins hemoglobin and human serum albumin. Identified adducts were assessed for reproducibility and stability both in model peptide and whole protein assays. Specificity of these identified adducts was assessed using in vitro assays to metabolize common therapeutic drugs containing nitrogen mustard moieties. Results of the model peptide assays demonstrated that HN-2 and HN-3 were able to form stable adducts with lysine and histidine residues under physiological conditions. Results for whole protein assays identified three histidine adducts on hemoglobin, and three adducts (two lysine residues and one histidine residue) on human serum albumin that were previously unknown. These protein adducts were determined to be reproducible and stable at physiological conditions over a three-week analysis period. Results from the in vitro metabolic assays revealed that adducts formed by HN-2 and HN-3 are specific to these agents, as metabolized therapeutic drugs (chlorambucil, cyclophosphamide, and melphalan) did not form the same adducts on lysine or histidine residues as the previously identified adducts formed by HN-2 and HN-3. Results obtained from the model peptide and full protein work were enhanced by comparing experimental data to theoretical calculations for adduct formation, providing further confirmatory data. This project was successful in identifying and characterizing biomarkers of exposure to HN-2 and HN-3 that are specific and stable and which have the potential to be used for the forensic determination of exposure to these dangerous agents.