Structural characterization by mass spectrometry of hemoglobin adducts formed after in vivo exposure to methyl bromide

A mass spectrometric procedure is described for the structural study of the adducts formed in human hemoglobin by in vitro exposure of erythrocytes to the alkylating agent methyl bromide using different protein to reagent ratios. Peptide mapping by HPLC and tandem mass spectrometry allowed location of methylated amino acids within the protein sequence. A prominent reactivity of several nucleophilic side chains in human hemoglobin subunits was observed, which was modulated by the concentration of the alkylating agent. Cysteine residues, the main reactive sites, were fully methylated in hemoglobin exposed to a 10-fold excess of methyl bromide, differently from other residues, including histidines, showing a heterogeneous pattern of methylation that was largely directed by their environment. No evidence of methylation was found at the heme proximal histidines beta92 and alpha87. A more selective methylation was obtained when the ratio methyl bromide: hemoglobin was lowered to about 1:1. In this last case only specific residues were reactive. Among them, the N-terminal amino group of both alpha- and beta-globins, cysteine 104 in the alpha-chain and cysteine 93 (not cysteine 112) in the beta-chain, indicating a different accessibility to reaction of the sulfhydryl groups on the protein chain. Thus hemoglobin side chains are selectively modified and the degree of modification at each site is a function of the position of the single amino acid residue within the protein quaternary structure, raising the possibility that alterations of structure and functional properties of human hemoglobin following exposure to alkylating agents may be mediated through such covalent protein modifications. The results obtained demonstrate the usefulness of the analytical approach for the characterization of hemoglobin adducts with methyl bromide or similar compounds, which can constitute the basis for biomonitoring of human exposure.     

Characterization of amino acid and glutathione adducts of cis-2-butene-1,4-dial, a reactive metabolite of furan

Metabolic activation of the hepatocarcinogen furan yields metabolites that react covalently with proteins. cis-2-Butene-1,4-dial is a microsomal metabolite of furan. This reactive aldehyde is thought to be the toxic metabolite that is responsible for the carcinogenic activity of furan. In order to characterize the chemistry by which this unsaturated dialdehyde could alkylate proteins, the products formed upon reaction of cis-2-butene-1,4-dial with model nucleophiles in pH 7.4 buffer were investigated. N(alpha)-Acetyl-L-lysine (AcLys) reacts with cis-2-butene-1,4-dial to form N-substituted pyrrolin-2-one adducts. N-Acetyl-L-cysteine (AcCys) reacts rapidly with cis-2-butene-1,4-dial to form multiple uncharacterized products. The inclusion of AcLys in this reaction mixture yielded an N-substituted 3-(S-acetylcysteinyl)pyrrole adduct which links the two amino acid residues. Related compounds were isolated when cis-2-butene-1,4-dial and glutathione (GSH) were combined. In this case, cis-2-butene-1,4-dial cross-linked two molecules of GSH resulting in either cyclic or acyclic adducts depending on the relative GSH concentration. Incubation of furan with rat liver microsomes in the presence of [glycine-2-3H]GSH led to the formation of radioactive peaks that coeluted with synthetic standards for the bisgluthathione conjugates. These studies demonstrate that the reactive cis-2-butene-1,4-dial formed during the microsomal oxidation of furan reacts rapidly and completely with amino acid residues to form pyrrole and pyrrolin-2-one derivatives. Therefore, this metabolite is a likely candidate for the activated furan derivative that binds to proteins. The ease with which cis-2-butene-1,4-dial cross-links amino acids suggests that pyrrole-thiol cross-links may be involved in the toxicity observed following furan exposure.     

Autocatalytic oxidation of hemoglobin by nitrite: a possible mechanism

Oxidation of oxyhemoglobin by nitrite ions to produce methemoglobin is one of the more employed procedures to oxidize the hemoprotein. The process takes place readily after a clear induction time. This behaviour is usually explained in terms of an autocatalytic reaction mechanism. However, the generally accepted mechanism is not autocatalytic and cannot explain the main features of the process. In the present work it is proposed that the characteristics of the process require the occurrence of a fast reaction between oxyhemoglobin and nitrogen dioxide. This process acts as a branching step, leading to the observed autocatalysis.     

In vitro and in vivo protective effect of honokiol on rat liver from peroxidative injury

Honokiol, a compound extracted from the Chinese medicinal herb Magnolia officinalis, has a strong antioxidant effect on the inhibition of lipid peroxidation in rat heart mitochondria. To investigate the protective effect of honokiol on hepatocytes from peroxidative injury, oxygen consumption and malondialdehyde formation for in vitro iron-induced lipid peroxidation were assayed, and the mitochondrial respiratory function for in vivo ischemia-reperfusion injury were evaluated in rat liver, respectively. The inhibitory effect of honokiol on oxygen consumption and malondialdehyde formation during iron-induced lipid peroxidation in liver mitochondria showed obvious dose-dependent responses with a concentration of 50% inhibition being 2.3 x 10(-7) M and 4.96 x 10(-7) M, respectively, that is, 550 times and 680 times more potent than alpha-tocopherol, respectively. When rat livers were introduced with ischemia 60 min followed by reperfusion for 60 min, and then pretreated with honokiol (10 micrograms/kg BW), the mitochondrial respiratory control ratio (the quotient of the respiration rate of State 3 to that of State 4) and ADP/O ratio from the honokiol-treated livers were significantly higher than those of non-treated livers during reperfusion. The dose-dependent protective effect of honokiol on ischemia-reperfusion injury was 10 microgram-100 micrograms/Kg body weight. We conclude that honokiol is a strong antioxidant and shed insight into clinical implications for protection of hepatocytes from ischemia-reperfusion injury.     

Monophosphate 32P-postlabeling assay of DNA adducts from 1,2:3,4-diepoxybutane, the most genotoxic metabolite of 1,3-butadiene: in vitro methodological studies and in vivo dosimetry

Among the main DNA-reactive metabolites of 1,3-butadiene (BD), both 1,2:3,4-butadiene diepoxide (BDE) and 1,2-epoxy-3-butene (BME) have been reported in mice and rats exposed to BD, but blood and tissue levels of these metabolites are much higher in mice than in rats under similar exposure conditions. BDE, being more reactive and genotoxic than BME, is thought to be responsible for the greater susceptibility of mice to BD carcinogenicity. While BDE is a DNA-alkylating agent and some BDE adducts have been characterized, no sufficiently sensitive method has been reported for studying BDE-DNA binding in vivo. In the present investigation, a modified dinucleotide/monophosphate version of the 32P-postlabeling assay was applied to detect BDE-DNA adducts, which were prepared by reacting BDE with calf thymus DNA or deoxyribooligonucleotides [(AC)10, (AG)10, (CCT)7 and (GGT)7] in vitro or with skin DNA of mice in vivo upon topical treatment. Optimal resolution by 2-D PEI-cellulose TLC of the highly polar 5'-monophosphate adducts was achieved at +4 degrees C using 0.3 M LiCI (DI) and 0.4 M NaCl, 0.04 M H3BO3, pH 7.6 (D2). The profiles of the 32P-postlabeled adducts were similar for calf thymus and skin DNA, with 3 major spots being detected. Adducts obtained in in vitro and in vivo experiments were compared by re- and cochromatography in 4 or 5 different solvents, and these experiments provided evidence that corresponding BDE adducts, for the most part, were identical and represented adenine derivatives. Guanine adducts were not detected by this method although literature data indicate their formation. Quantitatively, the assay responded linearly to adduct concentration, as shown in an experiment where BDE-modified skin DNA was serially diluted up to 81-fold with control DNA. The limit of detection was approximately 1 adduct in 10(8) normal nucleotides. Further, in an in vivo dosimetry study, skin DNA from groups of 8 individual mice treated with different doses of BDE (1.9, 5.7, 17, 51 and 153 mumol/mouse) for 3 days exhibited a linear relationship (r > or = 0.992) between adduct levels and dose. The results suggest that the 32P-postlabeling assay described herein will have utility in mechanistic studies and biomonitoring of DNA adduct formation from BDE and possibly other polar epoxides.     

All four known cyclic adducts formed in DNA by the vinyl chloride metabolite chloroacetaldehyde are released by a human DNA glycosylase

We have previously reported that human cells and tissues contain a 1,N6-ethenoadenine (epsilon A) binding protein, which, through glycosylase activity, releases both 3-methyladenine (m3A) and epsilon A from DNA treated with methylating agents or the vinyl chloride metabolite chloroacetaldehyde, respectively. We now find that both the partially purified human epsilon A-binding protein and cell-free extracts containing the cloned human m3A-DNA glycosylase release all four cyclic etheno adducts--namely epsilon A, 3,N4-ethenocytosine (epsilon C), N2,3-ethenoguanine (N2,3-epsilon G), and 1,N2-ethenoguanine (1,N2-epsilon G). Base release was both time and protein concentration dependent. Both epsilon A and epsilon C were excised at similar rates, while 1,N2-epsilon G and N2,3-epsilon G were released much more slowly under identical conditions. The cleavage of glycosyl bonds of several heterocyclic adducts as well as those of simple methylated adducts by the same human glycosylase appears unusual in enzymology. This raises the question of how such a multiple, divergent activity evolved in humans and what may be its primary substrate.     

Characterization and evaluation by 32P-postlabelling of psoralen-type DNA adducts in HeLa cells

Samples of DNA irradiated at 405 and/or 365 nm in the presence of 8-methoxypsoralen (8-MOP) were analysed via a modified postlabelling assay using three hydrolysis enzymes other than those employed previously. These enzymes (deoxyribonucleaseI, venom phosphodiesterase and alkaline phosphatase) liberated 3'-adducted dinucleotide monophosphate instead of the 5'-modified dinucleotide monophosphate normally obtained. The first separation chromatography (D1) of samples irradiated in the presence of 8-MOP showed a single spot above the origin, and the next separation (D2) resolved this spot into two components (spots I and II). Double irradiation experiments in which samples of DNA were first irradiated at 405 nm before being irradiated at 365 nm showed that spot II could be transformed into spot I. The use of 6,4,4'-trimethylangelicin, which induced only photomonoadducts under UVA irradiation, gave only spot II. These two results indicated that spots I and II were respectively due to interstrand cross-links and monoadducts. Dose-effect experiments showed that spots I and II were dose dependent, and low-dose irradiations permitted us to measure one interstrand cross-link and two monoadducts per 10(8) base pairs.     

Inhibition of rotavirus infection in vitro and in vivo by a synthetic peptide from VP4

A synthetic peptide corresponding to bovine rotavirus C486 (BRV) VP4 amino acid sequence 232-255 (VP4-peptide) was studied with the objective of defining the origin of the protective immune response reported previously by Ijaz et al. (J. Virol. 1991, 65, 3106-3113). Pretreatment of MA-104 cells with the VP4-peptide before infection with rotavirus prevented both the attachment of 35S-labelled virus and plaque formation in vitro. In vivo studies using a murine rotavirus model demonstrated that intragastric administration of VP4-peptide protected subjects from challenge with virulent rotavirus. These results clearly indicate the importance of this epitope in virus-cell interactions and their potential as a rotavirus vaccine candidate.     

Photochemotherapy: identification of a metabolite of 4, 5', 8-trimethylpsoralen

A compound, 4, 8-dimethyl, 5'-carboxypsoralen (DMeCP), has been identified in mouse urine as a major metabolite of the photoactive drug, 4, 5', 8-trimethylpsoralen (TMeP). This drug is widely used in the treatment of vitiligo and psoriasis. DMeCP is fluorescent, and nonphotosensitizing when tested on guinea pig skin. DMeCP also occurs in the urine of human patients receiving TMeP orally.     

Characterization of a hemoglobin protease secreted by the pathogenic Escherichia coli strain EB1

Many pathogenic bacteria can use heme compounds as a source of iron. Pathogenic Escherichia coli strains are capable of using hemoglobin as an iron source. However, the mechanism of heme acquisition from hemoglobin is not understood for this microorganism. We present the first molecular characterization of a hemoglobin protease (Hbp) from a human pathogenic E. coli strain. The enzyme also appeared to be a heme-binding protein. Affinity purification of this bifunctional protein enabled us to identify the extracellular gene product, and to clone and analyze its gene. A purification procedure developed for Hbp allowed us to perform functional studies. The protein interacted with hemoglobin, degraded it and subsequently bound the released heme. These results suggest that the protein is involved in heme acquisition by this human pathogen. Hbp belongs to the so-called IgA1 protease-like proteins, as indicated by the kinetics of its membrane transfer and DNA sequence similarity. The gene of this protein appears to be located on the large pColV-K30 episome, that only has been isolated from human and animal pathogens. All these characteristics indicate that Hbp may be an important virulence factor that may play a significant role in the pathogenesis of E. coli infections.     

Characterization of allergoids from ovalbumin in vitro and in vivo

Several in vivo and in vitro methods for monitoring immunological properties of two allergoids obtained by formaldehyde treatment of ovalbumin (OA) were developed. The calculated molecular weight of allergoids was 80 kD (OA-F1) and 165 kD (OA-F2), respectively. The allergenic activity in vitro of allergoids in mast-cell histamine release assay was 1000 times lower than of OA. Both allergoids showed reduced ability to induce passive cutaneous anaphylaxis in the Sprague-Dawley rats or systemic anaphylaxis in Dunkin-Harley guinea-pigs. The ability of OA and allergoids to bind to the OA-specific IgE antibodies was measured in vivo by the inhibition of passive cutaneous anaphylaxis (PCA-inhibition). Allergoid binding to IgE was 51-66% lower than the native allergen. Moreover, the avidity of OA-specific IgG antibodies, measured by ELISA-inhibition, for allergoids and allergen was of the same order. Allergoids induced a different pattern of humoral immune response from that, induced by the native allergen. Thus, after immunization of BALB/c mouse, both allergoids induced a higher production of IgG and a lower production of IgE than OA, only OA-F2 induced a lower production of IgG1. The differences in the IgA response to the immunogens was not significant. Delayed hypersensitivity studies in the BALB/c mouse showed that allergoids were 5- to 12-times less effective in inducing a cell-mediated immune response than OA. The present study provides a battery of immunological methods for preclinical testing of modified allergens.     

A novel melatonin metabolite, cyclic 3-hydroxymelatonin: a biomarker of in vivo hydroxyl radical generation

In the current study, we characterized a urinary melatonin metabolite which could provide a safe and effective method to monitor generation of HO* in humans. Using mass spectrometry (MS), proton nuclear magnetic resonance (1H NMR), COSY 1H NMR analysis, and calculations on the relative thermodynamic stability, a novel melatonin metabolite was identified as cyclic 3-hydroxymelatonin (3-OHM). 3-OHM is the product of the reaction of melatonin with HO* which was generated in two different cell-free in vitro systems. Interestingly, this same metabolite, 3-OHM, was also identified in the urine of both rats and humans. A proposed reaction pathway suggests that 3-OHM is the footprint product that results when a melatonin molecule scavenges two HO*. When rats were challenged with ionizing radiation which results in HO* generation, urinary 3-OHM increased dramatically compared to that of controls. These results strongly indicate that the quantity of 3-OHM produced is associated with in vivo HO* generation. Since melatonin exists in virtually all animal species and has a wide intracellular distribution and 3-OHM is readily detected noninvasively in urine, we suggest that 3-OHM is a valuable biomarker that can be used to monitor in vivo HO* levels in humans and other species. The measurement of urinary 3-OHM as a biomarker of HO* generation could provide clinical benefits in the diagnosis and treatment of diseases.     

Characterization of leucine transport by toadfish liver in vivo

Kinetic analysis of L-leucine uptake by toadfish liver at 20 degrees C in vivo has been carried out after pulse injection of L-[14C]leucine into the hepatic portal vein. D-[3H]mannitol, which is taken up slowly by toadfish liver, is used as a marker for extracellular space and space accessible by simple diffusion. At normal plasma leucine concentration (0.1 mM), leucine uptake occurs rapidly (t1/2 = 0.25 min), representing a flux of 0.6 mumol/min for the liver as a whole. Analysis of the distribution of radioactive leucine among intracellular and extracellular free pools and protein-bound form at times of 30 s to 5 min after injection is consistent with operation of a concentrative or uphill transport system accounting for 40% of uptake at normal plasma concentration. Saturation of uptake occurs at increasing leucine loads; calculation of intracellular pool dilution from protein synthesis data indicates that 20-30% of liver intracellular space is occupied by incoming leucine during the first 2 min after portal injection. Maximal flux (V max) is 4.1 mumol/min per 7-g liver as a whole with Km = 0.6 mM. Competitive inhibition of leucine uptake is afforded by isoleucine and phenylalanine with lesser effects by aspartic acid, cysteine, methionine, threonine, tyrosine, and valine. No effect is observed with alanine, glycine, histidine, lysine, and proline.     

Characterization of hemoglobin variants by MALDI-TOF MS using a polyurethane membrane as the sample support

A new method for the sampling and off-site analysis of hemoglobin variants by mass spectrometry is reported. This technique uses a nonporous polyurethane membrane as the collection device and transportation medium of a blood sample for analysis. The same membrane is then used as the MALDI-TOF MS sample support for mass spectrometric analysis. Minimal invasive sample collection is afforded by collecting less than 1 microL of blood using a common lancet device. MALDI-TOF MS is performed directly on the membrane, after washing off the interfering plasma components, followed by the addition of matrix. This reduces the time of analysis and prevents sample loss. Enzymatic digestion can be performed directly on the membrane, using in this case trypsin, allowing for further characterization of the sample. The method is much less invasive compared to drawing blood with a syringe. The sample may be transported to the laboratory by regular mail, and thus the method can serve remote locations. We demonstrate the procedure by characterizing the Hb Shepherds Bush hemoglobin variant, b74-(E18)Gly-->Asp.     

Formation of 1,N6-etheno-2''-deoxyadenosine adducts by trans,trans-2, 4-Decadienal

This study was designed to investigate the effect of glucogon-like peptide-1 (GLP-1) on pancreatic beta-cell function in normal, Zucker diabetic fatty (ZDF) rats, a model for non-insulin-dependent diabetes mellitus (NIDDM or type II diabetes) and their heterozygous siblings. Pancreas perfusion and enzyme-linked immunosorbent assay (ELISA) were used to detect the changes in insulin release under fasting and hyperglycemic conditions and following stimulation with GLP-1. Animals from the ZDF/Gmi-fa rats (ZDF) were grouped according to age, sex, and phenotype (obese or lean), and compared with LA lean rats. Glucose stimulation (10 mmol/L) in obese rats showed repressed response in insulin release. Glucose plus GLP-1 stimulation caused increased insulin release in all groups. The degree of this response differed between groups: lean > obese; young > adult; female > male. The LA lean control group was most sensitive, while the ZDF overtly diabetic group had the lowest response. In addition, the pulsatile pattern of insulin secretion was suppressed in ZDF rats, especially in obese groups. These results support the hypothesis that GLP-1 can effectively stimulate insulin secretion. Insulin release was defective in ZDF obese rats and could be partially restored with GLP-1. ZDF lean rats also showed suppression of beta-cell function and there was a difference in beta-cell function related to sex in ZDF strain. This study documents the efficacy of GLP-1 to stimulate insulin release and contributes to our understanding of the pathophysiological mechanisms underlying NIDDM.     

Synthesis of adducts formed by iodine oxidation of aromatic hydrocarbons in the presence of deoxyribonucleosides and nucleobases

Polycyclic aromatic hydrocarbons (PAH) undergo two main pathways of metabolic activation related to the initiation of tumors: one-electron oxidation to give radical cations and monooxygenation to yield bay-region diol epoxides. Synthesis of standard adducts is essential for identifying biologically formed adducts. Until recently, radical cation adducts were synthesized by oxidation of the PAH in an electrochemical apparatus, not readily available in many organic chemistry laboratories. We have developed a convenient and efficient method for synthesizing PAH-nucleoside adducts by using I2 as the oxidant. Adducts of benzo[a]pyrene (BP), dibenzo[a, l]pyrene (DB[a,l]P), and 7,12-dimethylbenz[a]anthracene were synthesized with deoxyguanosine (dG), deoxyadenosine, guanine (Gua), or adenine in either Me2SO or dimethylformamide (DMF) with or without AgClO4. When, for example, the potent carcinogen BP was dissolved in DMF in the presence of 3 equiv of I2, 5 equiv of dG, and 1 equiv of AgClO4, 45% of the BP was converted to BP-6-N7Gua. When BP was placed under the same reaction conditions in the absence of AgClO4, the extent of formation of BP-6-N7Gua decreased to 30%. When the potent carcinogen DB[a,l]P was dissolved in DMF in the presence of 3 equiv of I2, 5 equiv of dG, and 1 equiv of AgClO4, 43% of the DB[a,l]P was converted to DB[a,l]P-10-N7Gua. In the more polar solvent Me2SO under the same reaction conditions, however, the yield of DB[a,l]P-10-N7Gua was only 20%. Synthesis of adducts with the oxidant I2 is more convenient and, in some cases, more efficient than synthesis by electrochemical oxidation. This method simplifies the synthesis of PAH-nucleoside and nucleobase adducts that are essential for studying biologically formed PAH-DNA adducts.     

MPP(+)-like neurotoxicity of a pyridinium metabolite derived from haloperidol: in vivo microdialysis and in vitro mitochondrial studies

Intracerebral (intrastriatal, intranigral and intracortical) microdialysis studies were conducted in conscious rats to investigate the comparative dopaminergic and serotonergic neurotoxic potential of the pyridinium metabolite 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]pyridinium (HPP+), derived from the extensively used neuroleptic agent haloperidol and 1-methyl-4-phenylpyridinium (MPP+), the pyridinium metabolite derived from the parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Although HPP+ was less potent than MPP+ on the dopaminergic system, the two compounds displayed comparable toxic effects on the serotonergic system. HPP+ also proved to be a weaker inhibitor of mitochondrial respiration than MPP+ in vivo as measured by increases in extracellular lactate levels. On the other hand, HPP+ was a more potent inhibitor of mitochondrial respiration in vitro than MPP+, with IC50 values of 12 microM (HPP+) and 160 microM (MPP+). Quantitative estimations established that the concentrations of the more hydrophobic HPP+ in the brain tissues surrounding the microdialysis probe were less than those of MPP+ after comparable perfusions. Consequently, the inherent toxicity of HPP+ relative to MPP+ may be greater than suggested by the results observed in the microdialysis experiments. These data support previous speculations that HPP+ may contribute to some of the persistent extrapyramidal side effects associated with chronic haloperidol treatment.