Alkaline Permethylation Research Articles

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.

Covalent protein adducts of hydroquinone in tissues from rats: identification and quantitation of sulfhydryl-bound forms.

The Michael-type addition of sulfhydryl groups to benzoquinone (BQ) or substituted benzoquinones is proposed as the primary mechanism by which these electrophilic intermediates react with either cellular glutathione or protein sulfhydryls. This reaction constitutes a reductive alkylation with a substituted hydroquinone (HQ) derivative resulting from the addition. In the case of HQ, oxidative conversion of the parent material to BQ followed by conjugation with glutathione leads to metabolic activation, producing intermediates which are nephrotoxic as well as having other proposed biological activities. Chemically, BQ may react with more than 1 equiv of glutathione (or other sulfhydryl reagents) to produce HQ derivatives substituted with up to four sulfhydryl groups. Similarly, multiply substituted protein-S adducts of HQ were anticipated to occur in vivo following administration of this material. In the current studies, sulfhydryl-bound HQ protein adducts were detected and quantitated in protein isolated from rats using a modification of the alkaline permethylation procedure of Slaughter and Hanzlik [(1993) Anal. Biochem. 208, 288-295]. In particular, total protein-S adducts to HQ in kidney or blood reached a level of 420 or 80 pmol/mg of protein, respectively, 6 h following a single gavage dose of 100 mg/kg HQ. Measured half-lives of protein-S adducts in kidney and blood were 23.9 and 36.0 h, respectively. The applicability of protein-S adducts as a tissue dosimeter for HQ is discussed.

Covalent protein adducts of hydroquinone in tissues from rats: quantitation of sulfhydryl-bound forms following single gavage or intraperitoneal administration or repetitive gavage administration.

The current studies were conducted to investigate the degree and type of protein binding of hydroquinone (HQ) in the rat following single oral or intraperitoneal (ip) or repeated oral administrations. Male or female F-344 rats or male SD rats received a single dose of HQ at 0, 25, 50, or 100 mg/kg by either gavage or ip injection (SD rats only). In addition, male or female F-344 or male SD rats received HQ by gavage for 6 weeks (5 days/week) at 0, 25, or 50 mg/kg/day. Sulfhydryl-bound HQ was quantitated in protein from blood, kidneys, livers, or spleens 24 h after treatment using an alkaline permethylation procedure. The amount of total protein-S adducts increased with increasing dose in all the tissues that were assayed. Female rats had higher levels of adducts in blood, livers, and kidneys than did male rats when they were treated orally. Male F-344 rats treated orally had elevated levels of adducts in these same tissues compared to SD rats treated orally. For all genders and strains of rats and for all treatment regimens, mono-adducts predominated in livers (>72% of total). In the kidneys, tri- and tetrasubstituted adducts predominated with the summation accounting for >60% of the total. Ip administration of HQ resulted in significantly elevated levels of adducts in all the tissues that were examined, with the greatest increases seen for protein from blood and spleens. Levels of protein-S adducts of HQ in rat kidney following a single gavage administration correlated well with previously published differences in acute HQ nephrotoxicity in rats (female F-344 rat > male F-344 rat > male SD rat). Elevated levels of HQ protein-S adducts following repeated gavage administration did not correlate to measurable clinical signs of nephrotoxicity. Evidence is presented suggesting a possible role for the prostaglandin H synthase complex in the metabolic activation of HQ. In addition, protein arylation alone cannot account for the greater sensitivity of male F-344 rats toward chronic administration of HQ. The sensitivity of male F-344 rats to HQ is likely due to other factors, including the incidence and severity of chronic progressive nephropathy.

Cytotoxicity of 1,2-epoxynaphthalene is correlated with protein binding and in situ glutathione depletion in cytochrome P4501A1 expressing Sf-21 cells.

Naphthalene is metabolized by several cytochrome P-450 (CYP) monooxygenases to 1,2-epoxynaphthalene. However, the subsequent interactions of the epoxide with macromolecules in the cells, and the significance of these interactions to cellular injury, are not well characterized. Additionally, CYP1A1, which can metabolize naphthalene to 1,2-epoxynaphthalene, may be induced by a number of xenobiotics. Yet, the in situ interaction between naphthalene and CYP1A1 alone, without the influence of other xenobiotic metabolizing enzymes, has not been examined. Using a model eukaryotic expression system capable of over-expressing recombinant CYP1A1, we found that naphthalene was toxic to cells expressing CYP1A1 in a dose- (LC50: 0.3 mM) and time-dependent (LT50: 12 h) manner. Naphthalene treatment of CYP1A1-expressing cells resulted in a 47% decrease in cellular glutathione (GSH) levels. Pretreatment with ethyl ester GSH, a GSH analog, protected CYP1A1-expressing cells such that viability was 30% greater than for cells treated with naphthalene alone. Cytotoxicity was strongly correlated (r2: 0.96) with covalent binding of cellular proteins. Alkaline permethylation techniques showed that cysteinyl-SH groups of cellular proteins are a nucleophilic target of the epoxide metabolite. These results suggest that, in the absence of other pathways, naphthalene is modified by CYP1A1 to 1,2-epoxynaphthalene, which subsequently binds cellular sulfhydryl groups on proteins and GSH.

Evidence of quinone metabolites of naphthalene covalently bound to sulfur nucleophiles of proteins of murine Clara cells after exposure to naphthalene.

Naphthalene-induced Clara cell toxicity in the mouse is associated with the covalent binding of electrophilic metabolites to cellular proteins. Epoxide and quinone metabolites of naphthalene are proposed to be the reactive metabolites responsible for covalent binding to proteins. To identify the nature of reactive metabolites bound to proteins (cysteine residues), we alkaline-permethylated proteins obtained from mouse Clara cells incubated with 0.5 mM naphthalene in vitro. Alkaline permethylation of protein adducts produced (methylthio)naphthalene derivatives detected by GC-MS. 3,4-Dimethoxy(methylthio)naphthalene was observed to be a predominant (methylthio)naphthalene derivative formed in the alkaline-permethylated protein sample obtained from Clara cells after exposure to naphthalene. This indicates that 1,2-naphthoquinone is a major metabolite covalently bound to cysteine residues of the cellular proteins. We have developed an immunoblotting approach to detect 1,2-naphthoquinone covalently bound to cysteine residues of proteins [Zheng, J., and Hammock, B. D. (1996) Chem. Res. Toxicol. 9, 904-909]. To identify 1,2-naphthoquinone covalently bound to sulfur nucleophiles of proteins, homogenates obtained from naphthalene-exposed Clara cells were separated by SDS-PAGE followed by Western blotting and immunostaining with the antibodies. Two protein bands with 24 and 25 kDa were detected by the antibodies, further supporting the view that 1,2-naphthoquinone is a reactive metabolite of naphthalene which binds to Clara cell proteins in vitro.

Interaction of p-benzoquinone and p-biphenoquinone with microtubule proteins in vitro

p-benzoquinone (BQ) and p-biphenoquinone (BPQ) are metabolites of the human myelotoxin and leukemogen benzene, which has been reported to induce aneuploidy in mammalian cells. Because a possible mechanism for the aneuploidogenic effect of benzene may be the disruption of the mitotic spindle by covalent binding of BQ and BPQ to microtubule proteins (MTP), we have studied the reaction of these quinones with MTP and its consequences for microtubule (MT) formation under cell-free conditions. Both BQ and BPQ inhibit the assembly of MTP to MT in a concentration-dependent manner. This interaction is accompanied by a spectral change of the quinones and loss of free sulfhydryl groups of MTP. With 40 μM BQ or BPQ, 50% inhibition of MT assembly was observed and associated with the loss of 1.3 thiol groups per tubulin dimer. Further analysis showed that native MTP form monoadducts, but no diadducts nor disulfide bonds with both BQ and BPQ. The formation of covalent quinone/MTP monoadducts was unequivocally demonstrated by GC/MS analysis of the respective thioanisols liberated by alkaline permethylation. Denatured MTP or glutathione or 2-mercaptoethanol gave rise to the virtually exclusive formation of monoadducts with BQ but led to a high proportion of disulfide bonds with BPQ. Therefore, BQ and BPQ react differently with thiol compounds capable of disulfide bond formation. The fact that both quinones form only monoadducts with native MTP can be explained by the assumption that the thiol groups of native tubulin are not prone to oxidative disulfide bond formation. This proposition was supported by the lack of native MTP to form disulfide bridges upon treatment with hydrogen peroxide/horseradish peroxidase under conditions leading to a complete oxidation of glutathione. The covalent binding of the benzene metabolites BQ and BPQ to critical thiol groups of tubulin inhibits MT formation under cell-free conditions and may also interfere with the formation of a functional spindle apparatus in the mitotic cell, thus leading to the abnormal chromosome segregation and aneuploidy induction reported for benzene.

Covalent binding of benzoquinone to reduced ribonuclease. Adduct structures and stoichiometry.

As a model for the reaction of chemically reactive quinone metabolites with cellular proteins in vivo, the reactions of benzoquinone (BQ; 1-64 mol/mol of protein) with bovine pancreatic ribonuclease A (RNase), reduced RNase, S-(amidomethylated) reduced RNase, and reduced guanidinated RNase were investigated. The reaction stoichiometry and products were characterized by means of HPLC, UV-vis spectrophotometry, electrospray mass spectrometry, amino acid analysis, alkaline permethylation analysis, and measurement of the covalent binding of [14C]BQ to protein. Native RNase and S-(amidomethylated) reduced RNase show no reaction with BQ over 60 min at pH 7.4-9.6, whereas reduced RNase, which has 8 free SH groups/mol, reacts rapidly with exactly 24 molecules of BQ, of which 8 become covalently bound to protein-SH groups while 16 are reduced to hydroquinone. Half of the latter is formed via BQ oxidation of the initially formed S-(2,5-dihydroxyphenyl)cysteine moieties. Michael addition of a second protein nucleophile to each resulting S-(2,5-quinoyl)cysteine moiety, followed by reoxidation of that addition product by BQ, generates the second group of 8 molecules of HQ and results in cross-linking. Reduced guanidinated RNase, in which most of the lysines are blocked by guanidination with O-methylisourea, also reacts rapidly with BQ, but only ca. 16 equiv are consumed; of these, 8 become covalently bound to protein-SH groups while the others are reduced to HQ. Thus, even though the lysine residues in native RNase and S-(amidomethylated) reduced RNase do not react with BQ, they may react with (2,5-quinonyl)-S-protein moieties.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of Covalent Adducts to Protein Sulfur Nucleophiles by Alkaline Permethylation

We recently reported on the identification of metabolites of the hepatotoxin bromobenzene covalently bound to rat liver protein sulfur nucleophiles (D. E. Slaughter and R. P. Hanzlik, Chem. Res. Toxicol. 4, 349-359 (1991). Central to that study was our development of a method called alkaline permethylation which converts protein- S adducts of xenobiotic electrophiles to stable extractable thioanisole derivatives. We report here on substantial improvements to our original alkaline permethylation method which should greatly expand its potential utility. Specifically, we have developed significantly milder reaction conditions, eliminated side reactions, improved the amount and consistency of thioanisole yields from various mercapturic acid model compounds, and increased the overall sensitivity of the method at least 50-fold. Using the procedure described herein it is routinely possible to generate, detect, and identify by GC/MS as little as 2 pmol of a thioanisole derivative. This method is potentially quite general and should prove useful for studies in the toxicology of reactive metabolites, for industrial hygiene and biomonitoring, and for agrichemical residue analysis.

Dihydroxylated mercapturic acid metabolites of bromobenzene.

Bromobenzene is metabolized to electrophilic epoxides and quinones which covalently bind to protein sulfur nucleophiles, yet no quinone-derived mercapturic acid metabolites of bromobenzene have been reported. To search for them, phenobarbital-induced Sprague-Dawley rats were dosed (0.5-1.5 mmol/kg, ip) with either bromobenzene, 2-, 3-, or 4-bromophenol (BP), 3- or 4-bromocatechol (BC), or 2-bromohydroquinone (BHQ). Urine (0-24 h) was alkaline permethylated (2 N NaOH/CH3I/80 degrees C), and the resulting thioanisole derivatives were analyzed by GC/MS. Three dimethoxythioanisoles and eight bromodimethoxythioanisoles were formed by alkaline permethylation of urine of rats treated with bromobenzene or 3-BP; alkaline permethylation of urine from rats in other treatment groups afforded characteristic subsets of these derivatives. The major thioanisole from all groups except 3-BC or 4-BC was 2,5-dimethoxythioanisole, which arises from (2,5-dihydroxyphenyl)mercapturic acid. The latter was isolated from rat urine and is the first debrominated metabolite of bromobenzene reported to date. Its formation from both 4-bromophenol and BHQ requires two parallel mechanisms for bromine loss: (1) nucleophilic addition to 1,4-benzoquinone formed by hydroxylative debromination of 4-bromophenol and (2) nucleophilic displacement of bromine from 2-bromo-1,4-benzoquinone by sulfur. The yields of quinone-derived mercapturic acids formed from bromobenzene are quite low (less than 1% of dose) compared to the high yields of epoxide-derived mercapturic acids (40% of dose). Potential reasons for this are discussed.

Identification of epoxide- and quinone-derived bromobenzene adducts to protein sulfur nucleophiles

Bromobenzene (BB) hepatotoxicity is widely attributed to the alkylation of cellular proteins by chemically reactive metabolites, particularly BB-3,4-oxide. This laboratory recently reported the first conclusive evidence that BB epoxides actually do alkylate proteins; i.e., acid hydrolysates of hepatic proteins from phenobarbital-(PB-) induced BB-treated rats contain S-(o-, S-(m-, and S-(p-bromophenyl)cysteine [Weller, P.E., and Hanzlik, R.P. (1991) Chem. Res. Toxicol. 4, 17-20]. However, these three compounds account for less than 0.5% of total protein covalent binding. Bromoquinone metabolites of BB are also suspected of alkylating proteins. To search for such adducts to protein cysteinyl or methionyl residues, we heated hepatic proteins from PB-induced BB-treated rats with a two-phase mixture of 16 N KOH and CH3I ("alkaline permethylation"). Under these conditions S-alkylated residues are cleaved via elimination and the phenoxide and thiophenoxide groups on the fragments are methylated. Product analysis by 14C HPLC and GC/MS revealed o-, m-, and p-bromothioanisoles in amounts comparable to the content of S-(bromophenyl)cysteines found by acid hydrolysis (para much greater than meta, ortho). This, too, clearly implicates protein-SH alkylation by BB-2,3- and 3,4-oxides. In addition, 2,3-dimethoxy-5-bromothioanisole and another unidentified isomer were observed. However, by far the major adduct (5-6% of total covalent binding) was 2,5-dimethoxythioanisole (i.e., a debrominated adduct). When BB-d5 was administered, the latter contained mostly 3 deuterium atoms/mol. These latter results clearly show that alkylation of protein sulfur nucleophiles in vivo by quinone metabolites is 10-15 times more extensive than their alkylation by BB epoxides. After BB-d5 was administered, the bromothioanisoles and dimethoxybromothioanisoles contained 4 and 2 deuterium atoms/mol, respectively. A weighted average calculation of deuterium retention across the six major sulfur adducts agreed well with 3H/14C retention ratios determined earlier for total liver protein covalent binding of dual-labeled [3H/14C]BB, indicating that the overall pattern of BB metabolite binding to all protein nucleophiles may closely parallel that seen here specifically for protein sulfhydryl groups. The identification of a variety of specific BB-derived adducts to protein now affords the opportunity to investigate their relative contributions to the toxicity of bromobenzene.