Cysteine Adducts Research Articles

Modification of Peptide and Protein Cysteine Thiol Groups by Conjugation with a Degradation Product of Ascorbate

Ascorbate is an important water-soluble antioxidant, which when oxidized by reactive oxygen species is converted into dehydroascorbate (DHA). If not rapidly reduced back to ascorbate, DHA decomposes to a reactive 5-carbon compound (DHA*, +130 Da) that can modify reduced cysteinyl residues in peptides and proteins in vitro. The formation of cysteine adducts by DHA* was characterized by mass spectrometry using reduced insulin B-chain, α-lactalbumin, and hemoglobin. Mass spectrometry of DHA* modified insulin B-chain revealed the presence of one and two DHA* adducts. Enzymatic cleavage and tandem mass spectrometry of modified peptides allowed unambiguous localization of DHA* to the two cysteine residues in positions 7 and 19 of the insulin B-chain. Incubations of DHA with α-lactalbumin revealed that approximately 25% of the protein population was in a reduced state and could be modified by DHA*. The adduct was assigned to the N-terminally located cysteinyl residue in position 6. Incubation of hemoglobin with DHA followed by pepsin digestion and electrospray ionization tandem mass spectrometry (ESI-MSMS) of the peptide mixture allowed for the identification of three modified peptides. Tandem mass spectrometry of the modified peptides, two from the hemoglobin A-chain with identical mass and one from the hemoglobin B-chain, gave a complete series of y-type fragment ions, which were assigned to the cysteine containing peptides (100)LLSHCL(105) (A-chain), (101)LSHCLL(106) (A-chain), and (111)VCVLAHHFGKE(121) (B-chain). Although the DHA* adduct was lost from the peptides derived from α-lactalbumin and hemoglobin before fragmentation of the peptide bond, carbamidomethylation of the proteins prior to incubation with DHA abolished the formation of DHA*-protein adducts and confirmed that the target was indeed the cysteine thiol group. Future studies are focused on the modification of proteins by DHA* in cells and in vivo.

Formation of 8-S-l-cysteinylguanosine from 8-bromoguanosine and cysteine

When 8-bromoguanosine was incubated with cysteine at pH 7.4 and 37°C, a previously unidentified product was formed as a major product in addition to guanosine. The product was identified as a cysteine substitution derivative of guanosine at the 8 position, 8-S-l-cysteinylguanosine. The reaction was accelerated under mildly basic conditions. The cysteine adduct of guanosine was fairly stable and decomposed with a half-life of 193h at pH 7.4 and 37°C. Similar results were observed for incubation of 8-bromo-2′-deoxyguanosine with cysteine. The results suggest that 8-bromoguanine in nucleosides, nucleotides, RNA, and DNA can react with thiols resulting in stable adducts.

Development of New Neuromuscular Blocking Agents

Increased understanding of structure–activity relationships has allowed the development of neuromuscular blocking compounds and reversal agents that are unique and offer pharmacokinetic and dynamic improvements over their predecessors. Compounds have been developed that have a short duration of effect, a rapid clearance and a decrease in adverse effects. The fumarates comprise one of the series of compounds that have resulted from this research. These compounds are inactivated through a mechanism that is completely new for neuromuscular blocking agents—that of cysteine adduction. The work has also led to the development of alternative ways to enhance recovery of neuromuscular function. Sugammadex encapsulates steroidal neuromuscular blocking compounds making them unable to interact with the acetylcholine receptor and reversing their effect within minutes. Recovery from gantacurium- or CW002-induced neuromuscular block can be shortened by administration of l-cysteine. These new neuromuscular blocking and reversal agents allow for maintenance of an adequate depth of neuromuscular block intraoperatively and almost immediate recovery of neuromuscular function, a sensible goal as the risks of residual neuromuscular block are increasingly appreciated.

Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle

Cyanobacteriochromes (CBCRs) are cyanobacterial members of the phytochrome superfamily of photosensors. Like phytochromes, CBCRs convert between two photostates by photoisomerization of a covalently bound linear tetrapyrrole (bilin) chromophore. Although phytochromes are red/far-red sensors, CBCRs exhibit diverse photocycles spanning the visible spectrum and the near-UV (330-680 nm). Two CBCR subfamilies detect near-UV to blue light (330-450 nm) via a "two-Cys photocycle" that couples bilin 15Z/15E photoisomerization with formation or elimination of a second bilin-cysteine adduct. On the other hand, mechanisms for tuning the absorption between the green and red regions of the spectrum have not been elucidated as of yet. CcaS and RcaE are members of a CBCR subfamily that regulates complementary chromatic acclimation, in which cyanobacteria optimize light-harvesting antennae in response to green or red ambient light. CcaS has been shown to undergo a green/red photocycle: reversible photoconversion between a green-absorbing 15Z state ((15Z)P(g)) and a red-absorbing 15E state ((15E)P(r)). We demonstrate that RcaE from Fremyella diplosiphon undergoes the same photocycle and exhibits light-regulated kinase activity. In both RcaE and CcaS, the bilin chromophore is deprotonated as (15Z)P(g) but protonated as (15E)P(r). This change of bilin protonation state is modulated by three key residues that are conserved in green/red CBCRs. We therefore designate the photocycle of green/red CBCRs a "protochromic photocycle," in which the dramatic change from green to red absorption is not induced by initial bilin photoisomerization but by a subsequent change in bilin protonation state.

First Evidence of the Production of Odorant Polyfunctional Thiols by Bottle Refermentation

Bottle refermentation, which confers effervescence and resistance against infection and oxidation to beers, has also long been known to affect the fruity character imparted by esters. Yet it is recognized to improve the flavor perception, first by reducing stale aldehydes (trans-2-nonenal, 3-methylthiopropionaldehyde, etc.) to low-odorant alcohols, and also by bringing new pleasant odors. In this work, the polyfunctional thiol contents of a beer subjected or not to bottle refermentation were compared. A trained panel detected a strong organoleptic impact of bottle refermentation. Specific pHMB thiol extraction was applied and the extracts were analyzed by GC-MS, GC-PFPD, and GC-olfactometry (AEDA). Many sulfanylalkylalcohols, sulfanylalkylacetates, and sulfanylalkylcarbonyls were shown to be produced during the refermentation process, especially after 3 weeks. Among them, the hoppy 1-sulfanyl-3-methyl-2-butene was still perceived at the sniffing port after diluting the extract by a factor of 32,768. The major thiol, 2-sulfanylethyl acetate, reached 10 μg/L. As shown by spiking deuterated cysteine before bottle refermentation, the Ehrlich pathway revealed still efficient in the bottle. Most of the other identified polyfunctional thiols shared a common beta-sulfanyl structure, which lead us to suspect that hop cysteine adducts might be hydrolyzed by yeast-derived lyases. The spiking of 5 and 10 mg/L of S-3-(1-hydroxyhexyl)cysteine confirmed the ability of yeast to release free thiols through bottle refermentation. Therefore, a better control of the refermentation process requires both an excellent control of yeast (Ehrlich pathway and β-lyase activity) and strict selection of the hop variety (level of cysteine adducts).

Facile dimethylarsenic exchange and pyramidal inversion in its cysteine and glutathione adducts

Rapid thiolate exchange of dimethylarsonium, Me2As(+), is observed between two different thiolate species in solution. NMR is used to characterize the equilibrium constants for interthiol transfer as well the rapid intra molecular conformational dynamics which leads to the coalescence of diastereotopic methyl resonances. These rapid exchange kinetics have important consequences of arsenic's toxicity and pharmacology.

Quantitative LC/MS/MS analysis of acetaminophen–cysteine adducts (APAP–CYS) and its application in acetaminophen overdose patients

A more sensitive approach by liquid chromatography–tandem mass spectrometry (LC/MS/MS) method using the AB Sciex QTRAP 5500 was developed and validated for the detection and quantification of acetaminophen–cysteine adducts (APAP–CYS) in human plasma. Chromatographic separation was achieved using a Protecol P C18 column (2.1 mm i.d. × 100 mm, 3 microns). The mobile phase consists of A: 2 mM ammonium formate in water (0.2% formic acid) and B: 2 mM ammonium formate in acetonitrile (0.2% formic acid). The analysis was performed using positive and negative ion electrospray ionization (ESI) in multiple reaction monitoring mode (MRM) with a total run time of 7 min per assay. The MS/MS ion transitions monitored were m/z 271 → 140 for APAP–CYS (positive mode) and m/z 154 → 111 for acetaminophen-D4 (negative mode). The newly developed method showed good linearity in the range of 1.0 to 100 ng mL−1. Inter and intra batch precision and accuracy of the method ranged from 0.28 to 5.30% and 87.0 to 113% respectively. LOD was 0.5 ng mL−1 and LLOQ was 1.0 ng mL−1. Analytes were extracted from plasma samples by simple protein precipitation using acetonitrile. The method was successfully applied in a preliminary clinical study to determine APAP–CYS concentration from 46 acetaminophen overdose patients treated at outpatient clinic in the University Malaya Medical Centre.

Molecular mechanism of glyceraldehyde-3-phosphate dehydrogenase inactivation by α,β-unsaturated carbonyl derivatives.

α,β-Unsaturated carbonyls make up an important class of chemicals involved in environmental toxicity and disease processes. Whereas adduction of cysteine residues on proteins is a well-documented reaction of these chemicals, such a generic effect cannot explain the molecular mechanism of cytotoxicity. Instead, more detailed information is needed regarding the possible specificity and kinetics of cysteine targeting and the quantitative relationship between adduct burden and protein dysfunction. To address these data gaps, we incubated purified human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with acrylamide (ACR), acrolein, or methylvinyl ketone (MVK). Results show that these α,β-unsaturated carbonyl toxicants inhibited GAPDH activity in a concentration- and time-dependent manner. The rank order of enzyme inhibition (K(I)) (i.e., ACR ≪ MVK < acrolein) was related to the calculated electrophilic reactivity of each compound and to the corresponding kinetics of cysteine adduct formation. Tandem mass spectrometry revealed that adduct formation was selective at lower concentrations; i.e., ACR preferentially formed adducts with Cys152 (residues 146-162). At higher concentrations, ACR also formed adducts with Cys156 and Cys247 (residues 235-248). Adduct formation at Cys152 was correlated to enzyme inhibition, which is consistent with the regulatory role of this residue in enzyme function and its location within the GAPDH active site. Further analyses indicated that Cys152 was present in a pK(a)-lowering microenvironment (pK(a) = 6.03), and at physiological pH, the corresponding sulfhydryl group exists in the highly reactive nucleophilic thiolate state. These data suggest a general cytotoxic mechanism in which electrophilic α,β-unsaturated carbonyls selectively form adducts with reactive nucleophilic cysteine residues specifically associated with the active sites of proteins. These specialized cysteine residues are toxicologically relevant molecular targets, because chemical derivatization causes loss of protein function.

A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins

Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30picograms and the lowest amount of quantifiable cysteamine adduction is 60picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19nmol adduct/nmol protein to 0.03nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24nmol adduct/nmol protein to 0.14nmol adduct/nmol protein.

Detection of acetaminophen–cysteine adducts in cases of indeterminate liver failure is not diagnostic of intentional overdose

Detection of acetaminophen–cysteine adducts in cases of indeterminate liver failure is not diagnostic of intentional overdose

A new neuromuscular blocker AV002 and its antagonist

Background AV002 is a novel muscle relaxant with a duration dependent on the rate of cysteine adduction to the molecule,which has excellent clinical application perspective.Objective Compare AV002 with other muscle relaxants,and discuss its advantages and disadvantages.Content Animal experiments show that AV002 is a nondepolarizing neuromuscular blocking agent with fast acting and moderate duration. Its neuromuscular blocking effect will be inhibited by endogenous L-cysteine Inactivated molecules. It has minimal circulatory effects. Trend The characteristics of fast onset,reliable recovery,and an moderate duration together with rapid cysteine reversal suggest that AV002 is worthy of further study and evaluation. Key words: Nondepolarizing neuromuscular blocker; AV002; Cysteine

Spectroscopic and Computational Characterization of Substrate-Bound Mouse Cysteine Dioxygenase: Nature of the Ferrous and Ferric Cysteine Adducts and Mechanistic Implications

Cysteine dioxygenase (CDO) is a mononuclear non-heme Fe-dependent dioxygenase that catalyzes the initial step of oxidative cysteine catabolism. Its active site consists of an Fe(II) ion ligated by three histidine residues from the protein, an interesting variation on the more common 2-His-1-carboxylate motif found in many other non-heme Fe(II)-dependent enzymes. Multiple structural and kinetic studies of CDO have been carried out recently, resulting in a variety of proposed catalytic mechanisms; however, many open questions remain regarding the structure/function relationships of this vital enzyme. In this study, resting and substrate-bound forms of CDO in the Fe(II) and Fe(III) states, both of which are proposed to have important roles in this enzyme's catalytic mechanism, were characterized by utilizing various spectroscopic methods. The nature of the substrate/active site interactions was also explored using the cysteine analogue selenocysteine (Sec). Our electronic absorption, magnetic circular dichroism, and resonance Raman data exhibit features characteristic of direct S (or Se) ligation to both the high-spin Fe(II) and Fe(III) active site ions. The resulting Cys- (or Sec-) bound species were modeled and further characterized using density functional theory computations to generate experimentally validated geometric and electronic structure descriptions. Collectively, our results yield a more complete description of several catalytically relevant species and provide support for a reaction mechanism similar to that established for many structurally related 2-His-1-carboxylate Fe(II)-dependent dioxygenases.

Diametrically opposed effects of hypoxia and oxidative stress on two viral transactivators

BackgroundMany pathogens exist in multiple physiological niches within the host. Differences between aerobic and anaerobic conditions are known to alter the expression of bacterial virulence factors, typically through the conditional activity of transactivators that modulate their expression. More recently, changes in physiological niches have been shown to affect the expression of viral genes. For many viruses, differences in oxygen tension between hypoxia and normoxia alter gene expression or function. Oxygen tension also affects many mammalian transactivators including AP-1, NFkB, and p53 by affecting the reduced state of critical cysteines in these proteins. We have recently determined that an essential cys-x-x-cys motif in the EBNA1 transactivator of Epstein-Barr virus is redox-regulated, such that transactivation is favoured under reducing conditions. The crucial Tat transactivator of human immunodeficiency virus (HIV) has an essential cysteine-rich region, and is also regulated by redox. Contrary to EBNA1, it is reported that Tat's activity is increased by oxidative stress. Here we have compared the effects of hypoxia, oxidative stress, and cellular redox modulators on EBNA1 and Tat.ResultsOur results indicate that unlike EBNA1, Tat is less active during hypoxia. Agents that generate hydroxyl and superoxide radicals reduce EBNA1's activity but increase transactivation by Tat. The cellular redox modulator, APE1/Ref-1, increases EBNA1's activity, without any effect on Tat. Conversely, thioredoxin reductase 1 (TRR1) reduces Tat's function without any effect on EBNA1.ConclusionsWe conclude that oxygen partial pressure and oxidative stress affects the functions of EBNA1 and Tat in a dramatically opposed fashion. Tat is more active during oxidative stress, whereas EBNA1's activity is compromised under these conditions. The two proteins respond to differing cellular redox modulators, suggesting that the oxidized cysteine adduct is a disulfide bond(s) in Tat, but sulfenic acid in EBNA1. The effect of oxygen partial pressure on transactivator function suggests that changes in redox may underlie differences in virus-infected cells dependent upon the physiological niches they traffic to.

Detection of protein adduction derived from styrene oxide to cysteine residues by alkaline permethylation

Styrene oxide–cysteine adduction is predominantly involved in protein covalent modification after exposure in vivo to styrene or styrene oxide. In the present study, we developed an alkaline permethylation- and GC/MS-based approach to detect styrene oxide-derived protein adduction. Permethylation of the protein adducts produced two methylthiophenylethanols, namely 2-methylthio-2-phenyl-1-ethanol and 2-methylthio-1-phenyl-1-ethanol. To improve the permethylation efficiency, reaction conditions, including temperature, time, NaOH strength, and molar ratio of CH 3I/NaOH, were explored. Under optimized conditions, the yields of the analyte formation resulting from permethylation of authentic standard α- and β-mercapturic acids, representing α and β isomers of cysteine adducts, were 35% and 28%, respectively. Permethylation of styrene oxide-modified bovine serum albumin released the two methylthiophenylethanols with an α-/β-adduction ratio of 1.5. A concentration-dependent increase in both α- and β-adduction was observed in mouse liver microsomes incubated with styrene at various concentrations. CD-1 mice were administered intraperitoneally with styrene at doses of 0, 50, and 400 mg/kg daily for 5 days. The formation of protein adducts derived from styrene oxide in whole blood in 400 mg/kg group was observed with an α/β ratio of 4.8, suggesting that the reaction of styrene oxide with cysteine residues took place more likely at the α-carbon than the β-carbon of styrene oxide.

Pharmacodynamics and Cardiopulmonary Side Effects of CW002, a Cysteine-reversible Neuromuscular Blocking Drug in Dogs

CW002 is a novel neuromuscular blocking drug with a duration dependent on the rate of cysteine adduction to the molecule. The current study characterized the pharmacodynamics and cardiopulmonary side effects of CW002 in dogs. In eight beagles, the dose required to produce 95% neuromuscular blockade (ED95) for CW002 was first determined and cysteine reversibility was confirmed. Five to 7 days later, incrementally larger doses were injected starting with 6.25 x ED95 and doubling the dose every 15 min. Before and after injection, blood was obtained for histamine analysis. Systemic and pulmonary arterial pressures, cardiac output, and left ventricular pressure and volume were recorded along with inspiratory pressure and pulmonary compliance. Ventricular contractility and lusitropy were indexed from pressure and volume data. The ED95 for CW002 from pooled data was 0.009 mg/kg. At 3 x ED95, onset time was 2.6 +/- 0.9 min and duration was 47 +/- 9 min. The duration was shortened to 3.7 +/- 0.6 min by 50 mg/kg L-cysteine injected 1 min after CW002. At 25 x ED95, CW002 reduced mean arterial pressure with concomitant decreases in systemic vascular resistance, mean pulmonary artery pressure, cardiac output, contractility, and lusitropy, beginning at 50 x ED95. However, even at a dose of 100 x ED95, the average change in any variable was less than 20%. There were no changes in pulmonary vascular resistance or ventilation mechanics at any dose, and histamine release occurred in only two of eight animals. CW002 is a potent neuromuscular blocking drug that at doses up to 100 x ED95 produces modest hemodynamic effects that are not associated with bronchoconstriction or consistent histamine release.

Update on neuromuscular pharmacology

We review the efficacy and safety of gantacurium and AV002, two novel, investigational fumarate-based nondepolarizing neuromuscular blockers, as well as sugammadex and cysteine, two novel reversal drugs that have no acetylcholinesterase inhibition properties. Gantacurium (with a pharmacodynamic profile similar to that of succinylcholine) and AV002 (with an intermediate duration of action) have shown efficacy in animals and, for gantacurium, in humans. Animal data have shown that exogenous administration of the amino acid cysteine accelerates the natural chemical degradation of both gantacurium and AV002 via the cysteine adduction pathway. Another reversal drug, sugammadex (a modified gamma-cyclodextrin and the first selective relaxant binding agent), forms very tight complexes in a 1: 1 ratio with steroidal neuromuscular blocking agents. In a multicenter phase-2 randomized controlled study in the European Union, the efficacy and safety of gantacurium were evaluated, but results have not yet been published. Sugammadex is currently available in the European Union, but the United States Food and Drug Administration has had concerns about its safety (hypersensitivity and allergic reactions) and has asked for additional safety data. It is hoped that the widespread use of sugammadex in the European Union will provide additional information.

Eyedrops Containing SA9000 Prodrugs Result in Sustained Reductions in Intraocular Pressure in Rabbits

Poor topical bioavailability and ocular irritation have impeded the development of the diuretic, ethacrynic acid (ECA) as a clinically useful ocular hypotensive for the treatment of glaucoma. Thus, the development of analogs and prodrugs of analogs with improved ocular penetration, potency, and tolerability is required. The aim of this work is to evaluate the corneal penetration and ocular distribution of SA9000, an ECA analog. Novel SA9000 prodrugs intended to further improve ocular pharmacodynamic effect were also evaluated. SA9000 penetrated porcine corneas more effectively than ECA in corneal diffusion studies. In vivo studies in Dutch-belted (DB) rabbits indicated that topical application of a single dose (0.3%) of SA9000 could significantly reduce intraocular pressure (IOP) (approximately 25% vs. fellow untreated eye) but caused significant conjunctival hyperemia. Since this hyperemia was likely the result of its inherent thiol reactivity, SA9000 was formulated with equimolar cysteine, an exogenous thiol donor. The administration of increasing SA9000-cysteine adduct concentrations (0.3%, 0.6%, 0.9%) demonstrated that they cause less ocular irritation than unadducted SA9000 but could still significantly reduce IOP (0.3%: 8.7 +/- 2%; 0.6%: 14.4 +/- 5%; 0.9%: 23.3 +/- 4.4%) versus untreated contralateral control eyes. These data suggest that novel thiol donor adduction can improve the ocular bioavailability and tolerability of SA9000. SA9000-cysteine prodrugs may represent a new option for the topical treatment of glaucoma.

Structure of SRP14 from theSchizosaccharomyces pombesignal recognition particle

The signal recognition particle (SRP) Alu domain has been implicated in translation elongation arrest in yeasts and mammals. Fission yeast SRP RNA is similar to that of mammals, but has a minimal Alu-domain RNA lacking two stem-loops. The mammalian Alu-domain proteins SRP9 and SRP14 bind their cognate Alu RNA as a heterodimer. However, in yeasts, notably Saccharomyces cerevisiae, SRP14 is thought to bind Alu RNA as a homodimer, the SRP9 protein being replaced by SRP21, the function of which is not yet clear. Structural characterization of the Schizosaccharomyces pombe Alu domain may thus help to identify the critical features required for elongation arrest. Here, the crystal structure of the SRP14 subunit of S. pombe SRP (SpSRP14) which crystallizes as a homodimer, is presented. Comparison of the SpSRP14 homodimer with the known structure of human SRP9/14 in complex with Alu RNA suggests that many of the protein-RNA contacts centred on the conserved U-turn motif are likely to be conserved in fission yeast. Initial attempts to solve the structure using traditional selenomethionine SAD labelling failed. However, two As atoms originating from the cacodylate buffer were found to make cysteine adducts and strongly contributed to the anomalous substructure. These adducts were highly radiation-sensitive and this property was exploited using the RIP (radiation-damage-induced phasing) method. The combination of SAD and RIP phases yielded an interpretable electron-density map. This example will be of general interest to crystallographers attempting de novo phasing from crystals grown in cacodylate buffer.

Protein Modification by Acrolein: Formation and Stability of Cysteine Adducts

The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to identify in vitro and in vivo. In this study, model peptides with cysteine, lysine, and histidine residues were used to examine the reactivity of acrolein. Results from these experiments show that acrolein reacts rapidly with cysteine residues through Michael addition to form M+56 Da adducts. These M+56 adducts are, however, not stable, even though spontaneous dissociation of the adduct is slow. Further studies demonstrated that when acrolein and model peptides are incubated at physiological pH and temperature, the M+56 adducts decreased gradually accompanied by the increase of M+38 adducts, which are formed from intramolecular Schiff base formation. Adduct formation with the side chains of other amino acid residues (lysine and histidine) was much slower than cysteine and required higher acrolein concentration. When cysteine residues were blocked by reaction with iodoacetamide and higher concentrations of acrolein were used, adducts of the N-terminal amino group or histidyl residues were formed, but lysine adducts were not detected. Collectively, these data demonstrate that acrolein reacts avidly with protein cysteine residues and that the apparent loss of protein-acrolein Michael adducts over time may be related to the appearance of a novel (M+38) adduct. These findings may be important in identification of in vivo adducts of acrolein with protein cysteine residues.

Human Cytochrome P450 1A2 Involvement in the Formation of Reactive Metabolites from a Species-Specific Hepatotoxic Pyrazolopyrimidine Derivative, 5-n-Butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidine

5-n-Butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidine) (OT-7100) is a pyrazolopyrimidine derivative with potential analgesic effects. Exclusively limited elevations in serum levels of aspirate- and alanine-aminotransferase were abnormally observed in a clinical study, in contrast to no toxicological potential to experimental animals. The aim of this study was to clarify the mechanism responsible for species-specific hepatotoxicity of this model compound. OT-7100 was primarily metabolized to a carboxylic acid derivative and an amino derivative (5-n-butyl-pyrazolo[1,5-a]pyrimidine, M-5) by hydrolysis in humans and rats. In human liver, pyrazolo[1,5-a]pyrimidine derivative M-5 was further metabolized to mainly M-23OH (a C-3-position hydroxyl derivative, 3-hydroxy-5-n-butyl-pyrazolo[1,5-a]pyrimidine). Studies with recombinant cytochrome P450s (P450s), correlation analysis using a panel of human liver microsomes as well as immunoinhibition with anti-P450 antibodies collectively suggested that human liver microsomal P450 1A2 preferentially metabolized M-5 to predominantly M-23OH. Human liver microsomes were capable of activating M-5 to a covalently bound metabolite faster than rat liver microsomes: reduced glutathione prevented the bindings. A cysteine adduct derivative of M-23OH at the C-6-position was structurally confirmed. On the contrary, rat liver microsomal P450 1A2 could metabolize M-5 to equally M-23OH, M-22OH (a C-6-position hydroxyl derivative, 6-hydroxy-5-n-butyl-pyrazolo[1,5-a]pyrimidine), or an unknown metabolite. These results suggest that differences in the regiospecific metabolic function of human and rat P450 1A2 would be responsible for the human-specific metabolic activation of the primary metabolite of OT-7100 to a proximate form. It is presumed that hepatotoxicity associated with OT-7100 could be likely related to the formation of a human-specific reactive metabolite from M-23OH. OT-7100 activation by inducible P450 1A2 may therefore exhibit marked individual differences.