Phosphylated tyrosine in albumin as a biomarker of exposure to organophosphorus nerve agents

The organophosphorus nerve agents sarin, soman, cyclosarin and tabun phosphylate a tyrosine residue on albumin in human blood. These adducts may offer relatively long-lived biological markers of nerve agent exposure that do not ‘age’ rapidly, and which are not degraded by therapy with oximes. Sensitive methods for the detection of these adducts have been developed using liquid chromatography-tandem mass spectrometry. Adducts of all four nerve agents were detected in the blood of exposed guinea pigs being used in studies to improve medical countermeasures. The tyrosine adducts with soman and tabun were detected in guinea pigs receiving therapy 7 days following subcutaneous administration of five times the LD₅₀ dose of the respective nerve agent. VX also forms a tyrosine adduct in human blood in vitro but only at high concentrations.     

Biomarkers of organophosphorus nerve agent exposure: comparison of phosphylated butyrylcholinesterase and phosphylated albumin after oxime therapy

Organophosphorus nerve agents inhibit the activity of cholinesterases by phosphylation of the active site serine. In addition, sarin, cyclosarin, soman and tabun have been shown to phosphylate a tyrosine residue in albumin. Therapies against nerve agent poisoning include the use of oximes to reactivate inhibited cholinesterases by displacement of the phosphyl moiety and hence detectable levels of adducts with cholinesterases may be reduced. Adducts with tyrosine have been shown to be persistent in the guinea pig in the presence of oxime therapy. Plasma samples obtained from an animal study aimed at improving therapy against nerve agent poisoning were used to compare the suitability of tyrosine and butyrylcholinesterase (BuChE) adducts as biomarkers of nerve agent exposure after treatment with therapeutic oximes. Under the terms of the project licence, these samples could be collected only on death of the animal, which occurred within hours of exposure or when culled at 23 or 24 days. Tyrosine adducts were detected in all samples collected following intra-muscular administration of twice the LD₅₀ dose of the respective nerve agent. Aged BuChE adducts were detected in samples collected within a few hours after administration of soman and tabun, but not after 23 or 24 days. No BuChE adducts were detected in animals exposed to sarin and cyclosarin where samples were collected only after 23 or 24 days.     

An in vitro and in vivo evaluation of the efficacy of recombinant human liver prolidase as a catalytic bioscavenger of chemical warfare nerve agents

In this study, we determined the ability of recombinant human liver prolidase to hydrolyze nerve agents in vitro and its ability to afford protection in vivo in mice. Using adenovirus containing the human liver prolidase gene, the enzyme was over expressed by 200- to 300-fold in mouse liver and purified to homogeneity by affinity and gel filtration chromatography. The purified enzyme hydrolyzed sarin, cyclosarin and soman with varying rates of hydrolysis. The most efficient hydrolysis was with sarin, followed by soman and by cyclosarin {apparent k_cat/K_m [(1.9?±?0.3), (1.7?±?0.2), and (0.45?±?0.04)]?×?10^5?M^?1?min^?1, respectively}; VX and tabun were not hydrolyzed by the recombinant enzyme. The enzyme hydrolyzed P (+) isomers faster than the P (?) isomers. The ability of recombinant human liver prolidase to afford 24 hour survival against a cumulative dose of 2?×?LD₅₀ of each nerve agent was investigated in mice. Compared to mice injected with a control virus, mice injected with the prolidase expressing virus contained (29?±?7)-fold higher levels of the enzyme in their blood on day 5. Challenging these mice with two consecutive 1?×?LD₅₀ doses of sarin, cyclosarin, and soman resulted in the death of all animals within 5 to 8?min from nerve agent toxicity. In contrast, mice injected with the adenovirus expressing mouse butyrylcholinesterase, an enzyme which is known to afford protection in vivo, survived multiple 1?×?LD₅₀ challenges of these nerve agents and displayed no signs of toxicity. These results suggest that, while prolidase can hydrolyze certain G-type nerve agents in vitro, the enzyme does not offer 24 hour protection against a cumulative dose of 2?×?LD₅₀ of G-agents in mice in vivo.     

The application of the fluoride reactivation process to the detection of sarin and soman nerve agent exposures in biological samples

The fluoride reactivation process was evaluated for measuring the level of sarin or soman nerve agents reactivated from substrates in plasma and tissue from in vivo exposed guinea pigs (Cava porcellus), in blood from in vivo exposed rhesus monkeys (Macaca mulatta), and in spiked human plasma and purified human albumin. Guinea pig exposures ranged from 0.05 to 44 LD50, and reactivated nerve agent levels ranged from 1.0 ng/mL in plasma obtained from 0.05 LD50 sarin-exposed guinea pigs to an average of 147 ng/g in kidney tissue obtained from two 2.0 LD50 soman-exposed guinea pigs. Positive dose-response relationships were observed in all low-level, 0.05 to 0.4 LD50, exposure studies. An average value of 2.4 ng/mL for reactivated soman was determined in plasma obtained from two rhesus monkeys three days after a 2 LD50 exposure. Of the five types of guinea pig tissue studied, plasma, heart, liver, kidney and lung, the lung and kidney tissue yielded the highest amounts of reactivated agent. In similar tissue and with similar exposure procedures, reactivated soman levels were greater than reactivated sarin levels. Levels of reactivated agents decreased rapidly with time while the guinea pig was alive, but decreased much more slowly after death. This latter chemical stability should facilitate forensic retrospective identification. The high level of reactivated agents in guinea pig samples led to the hypothesis that the principal source of reactivated agent came from the agent-carboxylesterase adduct. However, there could be contributions from adducts of the cholinesterases, albumin and fibrous tissue, as well. Quantitative analysis was performed with a GC-MS system using selected ion monitoring of the 99 and 125 ions for sarin and the 99 and 126 ions for soman. Detection levels were as low as 0.5 ng/mL. The assay was precise and easy to perform, and has potential for exposure analysis from organophosphate nerve agents and pesticides in other animal species.     

Analogues with fluorescent leaving groups for screening and selection of enzymes that efficiently hydrolyze organophosphorus nerve agents

Enzymes that efficiently hydrolyze highly toxic organophosphorus nerve agents could potentially be used as medical countermeasures. As sufficiently active enzymes are currently unknown, we synthesized twelve fluorogenic analogues of organophosphorus nerve agents with the 3-chloro-7-oxy-4-methylcoumarin leaving group as probes for high-throughput enzyme screening. This set included analogues of the pesticides paraoxon, parathion, and dimefox, and the nerve agents DFP, tabun, sarin, cyclosarin, soman, VX, and Russian-VX. Data from inhibition of acetylcholinesterase, in vivo toxicity tests of a representative analogue (cyclosarin), and kinetic studies with phosphotriesterase (PTE) from Pseudomonas diminuta, and a mammalian serum paraoxonase (PON1), confirmed that the analogues mimic the parent nerve agents effectively. They are suitable tools for high-throughput screens for the directed evolution of efficient nerve agent organophosphatases.     

Reactivating potency of obidoxime, pralidoxime, HI 6 and HLö 7 in human erythrocyte acetylcholinesterase inhibited by highly toxic organophosphorus compounds

The treatment of poisoning by highly toxic organophosphorus compounds (nerve agents) is unsatisfactory. Until now, the efficacy of new potential antidotes has primarily been evaluated in animals. However, the extrapolation of these results to humans is hampered by species differences. Since oximes are believed to act primarily through reactivation of inhibited acetylcholinesterase (AChE) and erythrocyte AChE is regarded to be a good marker for the synaptic enzyme, the reactivating potency can be investigated with human erythro‐cyte AChE in vitro. The present study was undertaken to evaluate the ability of various oximes at concentrations therapeutically relevant in humans to reactivate human erythrocyte AChE inhibited by different nerve agents. Isolated human erythrocyte AChE was inhibited with soman, sarin, cyclosarin, tabun or VX for 30?min and reactivated in the absence of inhibitory activity over 5–60?min by obidoxime, pralidoxime, HI 6 or HLö 7 (10 and 30?μM). The AChE activity was determined photometrically. The reactivation of human AChE by oximes was dependent on the organophosphate used. After soman, sarin, cyclosarin, or VX the reactivating potency decreased in the order HLö 7 > HI 6 > obidoxime > pralidoxime. Obidoxime and pralidoxime were weak reactivators of cyclosarin-inhibited AChE. Only obidoxime and HLö 7 reactivated tabun-inhibited AChE partially (20%), while pralidoxime and HI 6 were almost ineffective (5%). Therefore, HLö 7 may serve as a broad-spectrum reactivator in nerve agent poisoning at doses therapeutically relevant in humans.     

Crystal structures of human group-VIIA phospholipase A2 inhibited by organophosphorus nerve agents exhibit non-aged complexes

The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P_R and P_S stereoisomers at the P-chiral center. The tabun complex displayed only the P_R stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.     

Selective N1-alkylation of 2'-deoxyguanosine with a quinolinyl quinone methide

Nucleobase modification by quinone methides (QMs) has been extensively studied in the past decade, and multiple QM adducts were observed. For 2'-deoxyguanosine (dG), the N (2)-dG alkylation adduct was favored under aqueous buffered conditions over other N1-dG, N7-dG, and N7-guanine adducts. We report in this communication that the N1-dG adduct was selectively formed with a quinolinyl QM in 30% aqueous DMF and 10 mM phosphate buffer (pH 7.0) as a favored dG alkylation product. The quinolinyl QM was formed through the fluoride-induced desilylation and elimination of acetate, and the structure of the N1-dG adduct was fully established by one- and two-dimensional NMR analyses. In addition, the concentration of salt played a significant role in N1-dG adduct formation. Further HPLC analysis indicated that the addition of salt decreased the rate of QM formation from the acetate intermediate, although an in-depth mechanistic study is needed.     

Binding and hydrolysis of soman by human serum albumin

Human plasma and fatty acid free human albumin were incubated with soman at pH 8.0 and 25 degrees C. Four methods were used to monitor the reaction of albumin with soman: progressive inhibition of the aryl acylamidase activity of albumin, the release of fluoride ion from soman, 31P NMR, and mass spectrometry. Inhibition (phosphonylation) was slow with a bimolecular rate constant of 15 +/- 3 M(-1) min (-1). MALDI-TOF and tandem mass spectrometry of the soman-albumin adduct showed that albumin was phosphonylated on tyrosine 411. No secondary dealkylation of the adduct (aging) occurred. Covalent docking simulations and 31P NMR experiments showed that albumin has no enantiomeric preference for the four stereoisomers of soman. Spontaneous reactivation at pH 8.0 and 25 degrees C, measured as regaining of aryl acylamidase activity and decrease of covalent adduct (pinacolyl methylphosphonylated albumin) by NMR, occurred at a rate of 0.0044 h (-1), indicating that the adduct is quite stable ( t1/2 = 6.5 days). At pH 7.4 and 22 degrees C, the covalent soman-albumin adduct, measured by MALDI-TOF mass spectrometry, was more stable ( t1/2 = 20 days). Though the concentration of albumin in plasma is very high (about 0.6 mM), its reactivity with soman (phosphonylation and phosphotriesterase activity) is too slow to play a major role in detoxification of the highly toxic organophosphorus compound soman. Increasing the bimolecular rate constant of albumin for organophosphates is a protein engineering challenge that could lead to a new class of bioscavengers to be used against poisoning by nerve agents. Soman-albumin adducts detected by mass spectrometry could be useful for the diagnosis of soman exposure.     

In vitro kinetics of nerve agent degradation by fresh frozen plasma (FFP)

Great efforts have been undertaken in the last decades to develop new oximes to reactivate acetylcholinesterase inhibited by organophosphorus compounds (OP). So far, a broad-spectrum oxime effective against structurally diverse OP is still missing, and alternative approaches, e.g. stoichiometric and catalytic scavengers, are under investigation. Fresh frozen plasma (FFP) has been used in human OP pesticide poisoning which prompted us to investigate the in vitro kinetics of OP nerve agent degradation by FFP. Degradation was rapid and calcium-dependent with the G-type nerve agents tabun, sarin, soman and cyclosarin with half-lives from 5 to 28 min. Substantially longer and calcium-independent degradation half-lives of 23–33 h were determined with the V-type nerve agents CVX, VR and VX. However, at all the tested conditions, the degradation of V-type nerve agents was several-fold faster than spontaneous hydrolysis. Albumin did not accelerate the degradation of nerve agents. In conclusion, the fast degradation of G-type nerve agents by FFP might be a promising tool, but would require transfusion shortly after poisoning. FFP does not seem to be suitable for detoxifying relevant agent concentrations in case of human poisoning by V-type nerve agents.     

Role of carboxylesterase in soman, sarin and tabun poisoning in rats

TOCP (triorthocresyl phosphate) inhibits carboxylesterase (CarbE) activity in rat plasma and liver and significantly increases soman, sarin and tabun toxicity. Application of these agents after TOCP caused strong additional inhibition of CarbE and cholinesterases (ChE) in rat red blood cells, plasma, liver, brain, diaphragm and intercostal muscle. After phenobarbital pretreatment, which induced CarbE activity in plasma and liver by 80% and that of plasma ChE by 33%, acute toxicity of soman and tabun was greatly decreased, while that of sarin remained unaffected.     

Emergency management of chemical weapons injuries

The potential for chemical weapons to be used in terrorism is a real possibility. Classes of chemical weapons include nerve agents, vesicants (blister agents), choking agents, incapacitating agents, riot control agents, blood agents, and toxic industrial chemicals. The nerve agents work by blocking the actions of acetylcholinesterase leading to a cholinergic syndrome. Nerve agents include sarin, tabun, VX, cyclosarin, and soman. The vesicants include sulfur mustard and lewisite. The vesicants produce blisters and also damage the upper airways. Choking agents include phosgene and chlorine gas. Choking agents cause pulmonary edema. Incapacitating agents include fentanyl and its derivatives and adamsite. Riot control agents include Mace and pepper spray. Blood agents include cyanide. The mechanism of toxicity for cyanide is blocking oxidative phosphorylation. Toxic industrial chemicals include agents such as formaldehyde, hydrofluoric acid, and ammonia.     

Changes in spinal cord reflexes following subchronic exposure to soman and sarin

The organophosphorus agents soman and sarin were administered subchronically and the spinal monosynaptic (MSR) and dorsal root (DRR) reflexes were assessed in spinal cord-transected cats. Neither soman nor sarin produced behavioral signs of delayed neurotoxicity. However, both soman and sarin significantly reduced the area under the MSR and DRR with only minimal changes in the excitability of the potentials as determined using quipazine, a serotonin agonist. The changes observed in these reflexes resulting from soman or sarin exposure are discussed in relation to alterations in the central terminals of primary afferent neurons.     

Effects of water and polymer content on covalent amide-linked adduct formation in peptide-containing amorphous lyophiles

Deamidation of asparagine-containing proteins and peptides results in the formation of hydrolysis products via a reactive succinimide intermediate. In amorphous lyophile formulations at low water content, nucleophilic amine groups in neighboring molecules can effectively compete with water for reaction with the succinimide intermediate resulting in the formation of a variety of covalent amide-linked adducts. This study examines the effects of changes in percentage of a polymeric excipient [hypromellose (HPMC)] and water content on the degradants formed from a model asparaginyl peptide (Gly-Phe-L-Asn-Gly) in amorphous solids also containing an excess of Gly-Val and carbonate buffer and stored at 40°C. Degradation of Gly-Phe-L-Asn-Gly and formation of succinimide intermediates, aspartyl peptides, and covalent amide-linked adducts were monitored by high-performance liquid chromatography. In all formulations and storage conditions, the formation kinetics of aspartyl hydrolysis products and covalent adducts could be described by a mechanism-based model that assigned a central role to the succinimide intermediate. Increasing the percentage of HPMC (i.e., reactant dilution) favored the formation of hydrolysis products over covalent amide-linked adducts, consistent with the bimolecular nature of covalent adduct formation. Increases in water content as relative humidity (RH) was varied from 33% to 75% produced orders-of-magnitude increases in the rate constants for succinimide formation and hydrolysis with both becoming nearly constant at high water contents. A bell-shaped profile for the dependence of the rate of covalent adduct formation on water content was observed, a result that may be indicative of phase separation at higher RHs.     

Partial characterization of an enzyme that hydrolyzes sarin, soman, tabun, and diisopropyl phosphorofluoridate (DFP)

The properties of a rat liver enzyme that hydrolyzes organophosphorus (OP) inhibitors of cholinesterases were studied. The rates of hydrolysis of OP inhibitors were determined by continuous titration of released hydrogen ions, using a pH stat method. Centrifugation of homogenates at 205,000 g for 30 min demonstrated that the activity was in the soluble fraction. Hydrolysis of sarin, soman, and diisopropyl phosphorofluoridate (DFP), but not of tabun, was stimulated by the addition of Mn2+ and Mg2+. Hydrolysis of sarin greater than soman greater than tabun greater than DFP. Unlike other OP hydrolases that preferentially hydrolyze the non-toxic isomers of soman, this enzyme hydrolyzed all four soman isomers at approximately the same rate. This result was obtained in vitro by gas chromatographic analysis of enzyme-catalyzed soman hydrolysis and confirmed in vivo by demonstrating reduced toxicity in mice of soman partially hydrolyzed by this enzyme. Km and Vmax were determined by fitting V vs [S] to a hyperbolic function using regression analysis. Km values ranged from 1.1 mM for soman to 8.9 mM for tabun. Vmax values ranged from 54 nmol/min/mg protein for DFP to 2694 for sarin. The enzyme was stable for at least 2 months at -90 degrees but was inactivated by heating at 100 degrees for 5 min. Elution profiles from gel filtration by high pressure liquid chromatography showed that the hydrolytic activity for the OP inhibitors eluted in a single peak, suggesting that a single enzyme was responsible for the observed hydrolysis. Further purification and characterization of this enzyme should prove useful for the development of methods for detection, detoxification, and decontamination of these cholinesterase inhibitors.     

Protective efficacy of catalytic bioscavenger, paraoxonase 1 against sarin and soman exposure in guinea pigs

Human paraoxonase 1 (PON1) has been portrayed as a catalytic bioscavenger which can hydrolyze large amounts of chemical warfare nerve agents (CWNAs) and organophosphate (OP) pesticides compared to the stoichiometric bioscavengers such as butyrylcholinesterase. We evaluated the protective efficacy of purified human and rabbit serum PON1 against nerve agents sarin and soman in guinea pigs. Catalytically active PON1 purified from human and rabbit serum was intravenously injected to guinea pigs, which were 30 min later exposed to 1.2 × LCt₅₀ sarin or soman using a microinstillation inhalation exposure technology. Pre-treatment with 5 units of purified human and rabbit serum PON1 showed mild to moderate increase in the activity of blood PON1, but significantly increased the survival rate with reduced symptoms of CWNA exposure. Although PON1 is expected to be catalytic, sarin and soman exposure resulted in a significant reduction in blood PON1 activity. However, the blood levels of PON1 in pre-treated animals after exposure to nerve agent were higher than that of untreated control animals. The activity of blood acetylcholinesterase and butyrylcholinesterase and brain acetylcholinesterase was significantly higher in PON1 pre-treated animals and were highly correlated with the survival rate. Blood O₂ saturation, pulse rate and respiratory dynamics were normalized in animals treated with PON1 compared to controls. These results demonstrate that purified human and rabbit serum PON1 significantly protect against sarin and soman exposure in guinea pigs and support the development of PON1 as a catalytic bioscavenger for protection against lethal exposure to CWNAs.     

Studies on Low Dose Sub–acute Administration of Soman,Sarin and Tabun in the Rat

Abstract: The effects of low–dose administration of the organophosphate cholinesterase inhibitors, soman, sarin and tabun, on growth rates over 85 days were studied in rats. Acetylcholinesterase (AChE) activity was determined in the striatum and the remainder of the brain 24 hrs following the last exposure to these agents. Further, the cumulative mortality of daily administration of several doses of soman, sarin and tabun for 25 days was studied. The animals treated with 25 μg/kg of soman or sarin for 85 days demonstrated reduced growth rates which returned to control levels after 30 days. The animals which received 50 μg/kg of sarin also grew at reduced rates which returned to control levels after 35 days, while the tabun–treated (100 μg/kg) animals required 38 days to return to control growth rates. The striatal AChE activity of the soman–treated group was reduced to 36% of control while the AChE activities of the high–dose sarin–treated group were reduced to 66% of control. The striatal AChE activity of the tabun–treated group was only 13% of control. It is suggested that growth rates may be used to monitor the development of tolerance to lowdose administration of organophosphate cholinesterase inhibitors     

Studies on the reactivity of acyl glucuronides--III. Glucuronide-derived adducts of valproic acid and plasma protein and anti-adduct antibodies in humans.

The major metabolite of the anti-epileptic agent valproic acid (VPA) is its acyl glucuronide conjugate (VPA-G), which undergoes non-enzymic, pH-dependent rearrangement via acyl migration to a mixture of beta-glucuronidase-resistant forms (collectively VPA-G-R). We have compared the reactivity of VPA-G and VPA-G-R towards covalent VPA-protein adduct formation by incubation in buffer, human serum albumin (HSA) and fresh human plasma at pH 7.4 and 37 degrees. In all three media, the predominant reaction of VPA-G over 30 hr was rearrangement to VPA-G-R (ca. 24%). Hydrolysis was quite minor (ca. 2%) and covalent adduct formation negligible (when protein was present). On the other hand, both hydrolysis (ca. 27%) and adduct formation (ca. 7%) were extensive when VPA-G-R was incubated with HSA or plasma. These data do not support a transacylation mechanism for VPA-protein adduct formation, since this pathway should be much more highly favoured by VPA-G (an acyl-substituted acetal) than VPA-G-R (simple esters). VPA-protein adducts were found in the plasma of epileptic patients taking VPA chronically (mean 0.77 +/- SD 0.63 microgram VPA equivalents/mL, N = 17). An enzyme linked immunosorbent assay was developed, using HSA modified by incubation with VPA-G-R, to test the immunoreactivity of the patients' plasma. Of 57 patients tested, nine showed measurable levels of antibodies to these adducts, but the titres were very low, with no difference in response to modified and unmodified protein detectable at plasma dilutions of 1:16 or greater. These results suggest that the VPA-protein adducts have little immunogenicity, and are in agreement with clinical observations that drug hypersensitivity responses have not been associated with VPA therapy. Thus, although the in vitro data show that VPA-G is an example of a relatively unreactive acyl glucuronide, covalent VPA-plasma protein adducts and anti-adduct antibodies are nonetheless formed in vivo, at least in some patients on chronic therapy with the drug.     

Characterization of adducts formed in the reaction of 2-chloro-4-methylthiobutanoic acid with 2'-deoxyguanosine

2-chloro-4-methylthiobutanoic acid (CMBA) is a direct-acting mutagen found in salt-nitrite-treated Sanma fish or similarly treated methionine solution. In this study, CMBA was reacted with 2'-deoxyguanosine (dG) in phosphate buffer (pH 7.4) at 37 degrees C. The HPLC-UV analysis showed that two products were mainly formed during the reaction. These were isolated, purified by semipreparative HPLC, and characterized as N7-guanine adducts: N7-(3-carboxy-3-methylthiopropyl)guanine (A1) and N7-(1-carboxy-3-methylthiopropyl)guanine (A2). Furthermore, liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analysis was employed to investigate the possible formation of minor products during the time-course of the reaction of CMBA with dG. It was found that N7-dG adducts, the precursors of A1 and A2, were formed early in the reaction and that subsequently the spontaneous depurination occurred to yield stable N7-guanine adducts A1 and A2. Stability studies in phosphate buffer (pH 7.4) at 37 degrees C showed that the amount of each N7-dG adduct decreased rapidly with a half-life of 6 h and 4 h to yield A1/A2, respectively. A regioisomer of N7-dG adducts was also observed in the LC/ESI-MS/MS analysis, but it was not characterized in detail because it was present only in trace amounts. On the basis of structural features, A1 and A2 seemed to be formed from the reaction of dG with 1-methyl-2-thietaniumcarboxylic acid, an intermediate resulting from the cyclization of CMBA. However, A2 might also have formed from the direct reaction of dG and CMBA. N7-Alkylation of the guanine residue and subsequent depurination are known to produce apurinic sites in DNA that induce point mutations and may be responsible for the observed CMBA-induced mutagenesis.     

Effects of the modulating agent WR2721 and its main metabolites on the formation and stability of cisplatin-DNA adducts in vitro in comparison to the effects of thiosulphate and diethyldithiocarbamate.

The influence of the modulating agent WR2721, its active thiol-metabolite WR1065 and the symmetrical disulphide WR33278 on the in vitro formation and stability of cis-diamminedichloroplatinum(II) (cisplatin, CDDP)-DNA adducts was investigated and compared with the effects of the highly nucleophilic modulating agents diethyldithiocarbamate (DDTC) and thiosulphate (TS). Salmon sperm DNA (0.5 mg/mL) was incubated with 25 micrograms/mL (83 microM) cisplatin for 1 hr in 50 mM phosphate buffer, pH 7.2 at 37 degrees in the absence or presence of modulating agent. DDTC and TS were potent inhibitors of the platination of the DNA (95 and 89%, respectively, with 4.2 mM of modulating agent). The WR-compounds were also remarkably active in the inhibition of DNA platination. Prevention of adduct formation in the presence of 4.2 mM WR-compound decreased in the order WR1065 (74%) greater than WR33278 (63%) greater than WR2721 (51%). The prevention of CDDP-DNA adduct formation by WR1065 was strongly concentration-dependent up to 4.2 mM but at higher concentrations this protection hardly increased at all. In the presence of the modulating agents, increased levels of CDDP monofunctionally bound to a guanine residue were observed with a simultaneous decrease in the relative abundance of bifunctional adducts. All modulators were also able to reverse part of the CDDP-DNA adducts formed. After a 2-hr incubation of already platinated salmon sperm DNA with 4.2 mM of modulating agent, the removal of Pt from DNA amounted to about 43% with DDTC, 28% with WR1065 and 13-14% with TS, WR2721 and WR33278. Even CDDP bifunctionally bound to two adjacent guanines in the same DNA strand, which is considered to be a very stable adduct, was partly reversed. Our observations suggest that WR2721, especially when administered prior to or concomitantly with CDDP, can be expected to protect those tissues from CDDP-induced damage to DNA that are able to efficiently dephosphorylate WR2721 followed by uptake of the thiol metabolite WR1065. This stresses the importance of a selective formation and uptake of WR1065 by non-tumour tissues for the successful use of WR2721 as a protective agent in combination with platinum-based cancer chemotherapy.