The peroxidase-dependent activation of butylated hydroxyanisole and butylated hydroxytoluene (BHT) to reactive intermediates. Formation of BHT-quinone methide via a chemical-chemical interaction

The food antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are shown to be metabolized to covalent binding intermediates and various other metabolites by prostaglandin H synthase and horseradish peroxidase. BHA was extensively metabolized by horseradish peroxidase (80% conversion of parent BHA into metabolites) resulting in the formation of three dimeric products. Only two of these dimers were observed in prostaglandin H synthase-catalyzed reactions. In contrast to BHA, BHT proved to be a relatively poor substrate for prostaglandin synthase and horseradish peroxidase, resulting in the formation of a small amount of polar and aqueous metabolites (23% conversion of parent BHT into metabolites). With arachidonic acid as the substrate, prostaglandin H synthase catalyzed the covalent binding of [14C]BHA and [14C]BHT to microsomal protein which was significantly inhibited by indomethacin and glutathione. The covalent binding of BHA and its metabolism to dimeric products were also inhibited by BHT. In contrast, the addition of BHA enhanced the covalent binding of BHT by 400%. Moreover, in the presence of BHA, the formation of the polar and aqueous metabolites of BHT was increased and two additional metabolites, BHT-quinone methide and stilbenequinone, were detected. The increased peroxidase-dependent oxidation of BHT in the presence of BHA is proposed to occur via the direct chemical interaction of BHA phenoxyl radical with BHT or BHT phenoxyl radical. These results suggest a potential role for phenoxyl radicals in the activation of xenobiotic chemicals to toxic metabolites.     

Studies on the mechanism of inhibition of 2-acetylaminofluorene toxicity by butylated hydroxytoluene.

Male rats were placed on a diet containing 0.05% (w/w) of the hepatic carcinogen 2-acetylaminofluorene (AAF). They ceased to gain weight. However, when the carcinogenic diet was supplemented with butylated hydroxytoluene (BHT) (0.5% w/w), an antioxidant, the animals gained weight at approximately one-half of the normal rate. This observation led to a series of experiments aimed at elucidating the mechanism(s) by which BHT reduced the toxicity of AAF. These initial studies were directed towards the effect of BHT on the extent and duration of the covalent binding of AAF with DNA. BHT feeding was shown to reduce the binding of carcinogen to hepatic DNA. Studies employing cells in culture demonstrated that BHT does not influence either excision repair or post-replication repair of DNA. These data indicate that a potential mechanism of action of BHT is at the anti-initiation level of carcinogen-induced DNA damage.     

Investigations on DNA binding in rat liver and in Salmonella and on mutagenicity in the Ames test by emodin, a natural anthraquinone

Emodin (1,6,8-trihydroxy-3-methylanthraquinone), an important aglycone found in natural anthraquinone glycosides frequently used in laxative drugs, was mutagenic in the Salmonella/mammalian microsome assay (Ames test) with a specificity for strain TA1537. The mutagenic activity was activation-dependent with an optimal amount of S9 from Aroclor 1254-treated male Sprague-Dawley rats of 20% in the S9 mix (v/v) for 10 micrograms emodin per plate. Heat inactivation of the S9 for 30 min at 60 degrees C prevented mutagenicity. The addition of the cytochrome P-448 inhibitor 7,8-benzoflavone (18.5 nmoles per plate) reduced the mutagenic activity of 5.0 micrograms emodin per plate to about one third, whereas the P-450 inhibitor metyrapone (up to 1850 nmoles per plate) was without effect. To test whether a metabolite binds covalently to Salmonella DNA, [10-(14)C]emodin was radiosynthesized, large batches of bacteria were incubated with [10-(14)C]emodin and DNA was isolated. [G-3H]Aflatoxin B1 (AFB1) was used as a positive control mutagen known to act via DNA binding. DNA obtained after aflatoxin treatment could be purified to constant specific activity. With emodin, the specific activity of DNA did not remain constant after repeated precipitations so that it is unlikely that the mutagenicity of emodin is due to covalent interaction of a metabolite with DNA. The antioxidants vitamin C and E or glutathione did not reduce the mutagenicity. Emodin was also negative with strain TA102. Thus, oxygen radicals are probably not involved. When emodin was incubated with S9 alone for up to 50 h before heat-inactivation of the enzymes and addition of bacteria, the mutagenic activity did not decrease. It is concluded that the mutagenicity of emodin is due to a chemically stable, oxidized metabolite forming physico-chemical associations with DNA, possibly of the intercalative type. In order to check whether an intact mammalian organism might be able to activate emodin to a DNA-binding metabolite, radiolabelled emodin was administered by oral gavage to male SD rats and liver DNA was isolated after 72 h. Very little radioactivity was associated with the DNA. Considering that DNA radioactivity could also be due to sources other than covalent interactions, an upper limit for the covalent binding index, CBI = (mumoles chemical bound per moles DNA nucleotides)/(mmoles chemical administered per kg body weight) of 0.5 is deduced. This is 10(4) times below the CBI of AFB1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhancement of the peroxidase-mediated oxidation of butylated hydroxytoluene to a quinone methide by phenolic and amine compounds

We have recently demonstrated that butylated hydroxyanisole (BHA) markedly stimulates the peroxidase-dependent oxidation of butylated hydroxytoluene (BHT) to the potentially toxic BHT-quinone methide. Using both horseradish peroxidase and prostaglandin H synthase we now report the ability of a wide variety of compounds to stimulate peroxidase-dependent activation of BHT. These compounds include several phenolic compounds commonly present in pharmacologic preparations or occurring naturally in foods. The ability of a given compound to stimulate BHT oxidation was found to depend on the type of radical it forms upon peroxidase oxidation. Compounds which have been shown to form phenoxy radicals or nitrogen-centered cation radicals were observed to enhance BHT oxidation. Conversely, compounds which are known to form peroxy radicals or semiquinone radicals either inhibited or had no effect on BHT oxidation. Compounds which enhanced BHT oxidation (monitored by covalent binding of [14C]BHT to protein) were also observed to stimulate the formation of BHT-quinone methide and stilbenequinone. This suggested a common mechanism of interaction of these compounds with BHT. The stimulation of BHT covalent binding by BHA was also seen in various human and animal tissues using either arachidonic acid or hydrogen peroxide as substrate. The possible toxicologic implications of the enhancement of peroxidase-catalyzed BHT oxidation to BHT-quinone methide are discussed.     

Development of new structural alerts suitable for chemical category formation for assigning covalent and non-covalent mechanisms relevant to DNA binding

The need to assess the ability of a chemical to act as a mutagen is one of the primary requirements in regulatory toxicology. Several pieces of legislation have led to an increased interest in the use of in silico methods, specifically the formation of chemical categories and read-across for the assessment of toxicological endpoints. One of the key steps in the development of chemical categories for mutagenicity is defining the mechanistic organic chemistry associated with the formation of a covalent bond between DNA and an exogenous chemical. To this end this study has analysed, by use of a large set of mutagenicity data (Ames test), the mechanistic coverage of a recently published set of in silico structural alerts developed for category formation. The results show that the majority of chemicals with a positive result in the Ames test were assigned at least one covalent binding mechanism related to the formation of a DNA adduct. The remaining chemicals with positive data in the Ames assay were subjected to a detailed mechanistic analysis from which 26 new structural alerts relating to covalent binding mechanisms were developed. In addition, structural alerts for radical and non-covalent intercalation mechanisms were also defined. The structural alerts outlined in this study are not intended to predict mutagenicity but rather to identify mechanisms associated with covalent and non-covalent DNA binding. This mechanistic profiling information can then be used to form chemical categories suitable for filling data gaps via read-across. A strategy for chemical category formation for mutagenicity is also presented.     

Mechanisms of butylated hydroxytoluene-mediated modulation of 2-acetylaminofluorene mutagenicity in rat and human hepatocyte/Salmonella assays

Salmonella typhimurium (TA98) mutagenesis assays were used to study the influence of the antioxidant butylated hydroxytoluene (BHT) on 2-acetylaminofluorene (2-AAF) mutagenesis, in search of the mechanism of the anticarcinogenic effects of BHT. Rats pre-treated with BHT in the diet (0.5% w/w for 10 days) provided hepatocytes and hepatocyte S9 which were more efficient in the activation of 2-AAF than were similar preparations from control rats. The increased release of mutagens from hepatocytes might explain the reported increase in the incidence of bladder tumours in BHT-treated rats. In contrast, the mutagenic activity of 2-AAF was inhibited by the in vitro addition of BHT into incubations where human or rat liver S9 and intact hepatocytes were used for metabolic activation. Both competitive and un-competitive inhibition by BHT of 7-ethoxycoumarin O-deethylation was observed in hepatocytes which suggested that the antimutagenic activity may be mediated by one or more mechanisms of cytochrome P-450 inhibition. BHT inhibition of the mutagenicity of N-OH 2-AAF and of rat urinary metabolites of 2-AAF indicated that effects other than those mediated by cytochrome P-450 also occur e.g. scavenging of reactive metabolites. It was concluded that BHT-modulation of 2-AAF metabolic activation and mutagenesis (which may relate to BHT-protection against hepatocarcinogenicity) involves multiple mechanisms.     

Investigation of absorption, metabolism kinetics and DNA-binding of intratracheally administered benzo[a]pyrene in the isolated, perfused rat lung: a comparative study between microcrystalline and particulate adsorbed benzo[a]pyrene

Benzo[a]pyrene (B[a]P) adsorbed onto urban air particles (UAP) or in microcrystalline form (MCr) was administered intratracheally to the isolated perfused lung in doses of 100 and 1.5 micrograms. The appearance rate constant calculated for B[a]P release to the perfusate buffer was significantly lower for B[a]P administered adsorbed onto UAP (0.007 +/- 0.002 min-1) compared to the microcrystalline preparation (0.051 +/- 0.030 min-1). A classical two-compartmental model fitted well to the elimination of B[a]P from the perfusate buffer, after administration in solution to the buffer reservoir; C = 24 e-0.05t + 14 e-0.01t (pmol/ml). The concentration of polar metabolites in the perfusion buffer, at the end of experiments was approx. 9-fold higher for lungs administered the microcrystalline preparation compared to UAP at 1.5 microgram doses. At the 100 microgram dose level, the difference between preparations was only 2-fold, the data indicating that enzyme saturation might be important at the high dose level. With regard to the metabolite pattern, adsorption of B[a]P onto urban air particles caused a relative increase in the formation of B[a]P-9,10-dihydrodiol, whereas the relative formation rate for phenols was decreased. The absolute levels of B[a]P metabolites covalently bound to DNA was significantly higher in lungs given the MCr preparation compared to the UAP. When calculated as the amount metabolites bound, in relation to the total amount polar metabolites at the end of perfusion, however, the UAP preparation was significantly more efficient to enhance the production of DNA binding metabolites; 2.62 +/- 0.59 X 10(-5) vs. 1.33 +/- 0.21 X 10(-5) (pmol covalently DNA-bound metabolites/mg DNA/pmol metabolites formed). The results indicate that urban air particles may exert a cocarcinogenic effect with polynuclear aromatic hydrocarbons by increasing the pulmonary residence time for the carcinogenic hydrocarbon and/or alter the metabolite pattern in a way that enhances the covalent binding of metabolites to DNA.     

The DNA binding of benzo[alpha]pyrene metabolites catalysed by rat lung microsomes in vitro and in isolated perfused rat lung.

When [³H]benzo[a]pyrene is incubated in vitro together with DNA, NADPH and rat lung microsomes, covalent binding of benzo[a]pyrene (BP) metabolites to DNA occurs. These metabolite-nucleoside complexes can be resolved into several distinct peaks by elution of a Sephadex LH-20 column with a water-methanol gradient. 3-Methylcholanthrene (MC) pretreatment of animals induces the total covalent binding in vitro several-fold and increases the amounts of at least five metabolite-nucleoside complexes associated with the 7,8-diol-9,10-epoxidcs, the 7,8-oxide or quinones oxygenated further, the 4,5-oxide and phenols oxygenated further. These increases correspond well with the increases in the production of both non-K-region and K-region metabolites of BP by lung microsomes, as determined by highpressure liquid chromatography (HPLC). On the other hand, when [³H]BP is metabolized in isolated perfused rat lung, only the peak representing the 7,8-diol-9,10-epoxide bound to nucleoside(s) is readily detectable and then only in lungs from MC-treated animals. The extent of binding of BP metabolites to lung DNA is very low, about 0.0004% of the total dose applied to the perfusion medium; more than 60% of this can be accounted for by the binding of the 7,8-diol-9,10-epoxides to nucleoside(s). It is suggested that the further metabolism leading to metabolites not available to covalent binding, (e.g. conjugation) of primary BP metabolites in the intact tissue is responsible for the differences in the metabolite-nucleoside patterns observed in vivo, as compared with microsomal metabolism in vitro.     

Molecular dynamics simulation studies for DNA sequence recognition by reactive metabolites of anticancer compounds

The discovery of novel anticancer molecules 5F‐203 (NSC703786) and 5‐aminoflavone (5‐AMF, NSC686288) has addressed the issues of toxicity and reduced efficacy by targeting over expressed Cytochrome P450 1A1 (CYP1A1) in cancer cells. CYP1A1 metabolizes these compounds into their reactive metabolites, which are proven to mediate their anticancer effect through DNA adduct formation. However, the drug metabolite–DNA binding has not been explored so far. Hence, understanding the binding characteristics and molecular recognition for drug metabolites with DNA is of practical and fundamental interest. The present study is aimed to model binding preference shown by reactive metabolites of 5F‐203 and 5‐AMF with DNA in forming DNA adducts. To perform this, three different DNA crystal structures covering sequence diversity were selected, and 12 DNA‐reactive metabolite complexes were generated. Molecular dynamics simulations for all complexes were performed using AMBER 11 software after development of protocol for DNA‐reactive metabolite system. Furthermore, the MM‐PBSA/GBSA energy calculation, per‐nucleotide energy decomposition, and Molecular Electrostatic Surface Potential analysis were performed. The results obtained from present study clearly indicate that minor groove in DNA is preferable for binding of reactive metabolites of anticancer compounds. The binding preferences shown by reactive metabolites were also governed by specific nucleotide sequence and distribution of electrostatic charges in major and minor groove of DNA structure. Overall, our study provides useful insights into the initial step of mechanism of reactive metabolite binding to the DNA and the guidelines for designing of sequence specific DNA interacting anticancer agents. Copyright © 2014 John Wiley & Sons, Ltd.     

2-Hydroxyemodin, an active metabolite of emodin in the hepatic microsomes of rats

The hepatic microsomes derived from rats transformed emodin (1,3,8-trihydroxy-6-methyl-anthraquinone), an anthraquinone present in fungal metabolites and constituent of rhubarb, into at least 10 anthraquinoid metabolites. Metabolite d proved to be mutagenic to Salmonella typhimurium TA1537 in the absence of activation system. MS, NMR, UV and mutagenicity test analysis revealed that metabolite d was 2-hydroxyemodin (1,2,3,8-tetrahydroxy-6-methyl-anthraquinone) and exhibited mutagenicity in doses of 2-20 micrograms/plate. In addition to this active metabolite, TLC analysis revealed the formation of 4-hydroxyemodin (metabolite a), 5-hydroxyemodin (metabolite b), 7-hydroxyemodin (metabolite d') and others. No mutagenicity of these monohydroxyemodins was demonstrated in the absence of activation system.     

Mitigation of reactive metabolite formation for a series of 3-amino-2-pyridone inhibitors of Bruton’s tyrosine kinase (BTK)

Reactive metabolites have been putatively linked to many adverse drug reactions including idiosyncratic toxicities for a number of drugs with black box warnings or withdrawn from the market. Therefore, it is desirable to minimize the risk of reactive metabolite formation for lead molecules in optimization, in particular for non-life threatening chronic disease, to maximize benefit to risk ratio. This article describes our effort in addressing reactive metabolite issues for a series of 3-amino-2-pyridone inhibitors of BTK, e.g. compound 1 has a value of 459 pmol/mg protein in the microsomal covalent binding assay. Parallel approaches were taken to successfully resolve the issues: establishment of a predictive screening assay with correlation association of covalent binding assay, identification of the origin of reactive metabolite formation using MS/MS analysis of HLM as well as isolation and characterization of GSH adducts. This ultimately led to the discovery of compound 7 (RN941) with significantly reduced covalent binding of 26 pmol/mg protein.     

The microsomal metabolism of pentachlorophenol and its covalent binding to protein and DNA

The microsomal metabolism of pentachlorophenol (PCP) was investigated, with special attention to the conversion dependent covalent binding to protein and DNA. The two metabolites detected were tetrachloro-1,2- and tetrachloro-1,4-hydroquinone. Microsomes from isosafrole (ISF)-induced rats were by far the most effective in catalyzing the reaction: the rate of conversion was increased 7-fold over control microsomes. All other inducers tested (hexachlorobenzene (HCB), phenobarbital (PB) and 3-methylcholanthrene (3MC) gave 2--3-fold increases over control. There are indications that the 1,2- and 1,4-isomers are produced in different ratio's by various cytochrome P-450 isoenzymes: Microsomes from PB- and HCB-treated rats produced the tetrachloro-1,4- and tetrachloro-1,2-hydroquinone in a ratio of about 2, while microsomes from rats induced with 3 MC and ISF showed a ratio of about 1.3. When PCP was incubated with microsomes from rats treated with HCB, a mixed type inducer of P-450, the ratio between formation of the 1,4- and 1,2-isomers decreased with increasing concentration of PCP, suggesting the involvement of at least two P-450 isoenzymes with different Km-values. The overall apparent Km-value for HCB-microsomes was 13 microM both for the formation of the soluble metabolites and the covalent binding to microsomal protein, suggesting both stem from the same reaction. The covalent binding could be inhibited by ascorbic acid and this inhibition was accompanied by an increase in formation of tetrachlorohydroquinones (TCHQ). Although a large variation was observed in rates of conversion between microsomes treated with different (or no) inducers, the rate of covalent binding to microsomal protein was remarkably constant. A conversion-dependent covalent binding to DNA was observed in incubations with added DNA which was 0.2 times the amount of binding to protein (37 pmol/mg DNA).     

Effect of 2(3)-tert-butyl-4-hydroxyanisole on benzo[a]pyrene metabolism and DNA-binding of benzo[a]pyrene metabolites in isolated mouse hepatocytes

Benzo[a]pyrene (BP) metabolism and the conjugation and DNA-binding of BP metabolites, was studied using isolated hepatocytes from mice maintained on a diet containing 2(3)-tert-butyl-4-hydroxyanisole (BHA) (7.5 g/kg food) to discover the mechanisms involved in the anticarcinogenic effects of this antioxidant. The antioxidant feeding produced: (a) profound differences in the BP metabolite pattern, (b) no increase in the levels of either the glucuronic acid, the sulfate or the glutathione conjugates and (c) a marked decrease in the level of BP metabolites bound to intracellular DNA. Therefore, the inhibition of DNA-binding observed after administration of BHA, may be due to the change in BP metabolism rather than to an increase in the conjugation of reactive metabolites.     

Nucleic acid binding and mutagenicity of active metabolites of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a potent mutagen and carcinogen present in heated foodstuffs. The covalent binding of MeIQx to calf thymus DNA and calf liver RNA with microsomal activation was demonstrated. A major metabolite which exerts a direct mutagenic effect on S. typhimurium TA98 was found by HPLC analysis after incubation of MeIQx with rat liver microsomal fraction. The metabolite was identified as 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-OH-MeIQx). Synthetic N-OH-MeIQx was found to bind non-enzymatically to DNA and RNA at neutral pH even at 0 degrees C. Addition of acetic anhydride increased the binding of N-OH-MeIQx to DNA 10 times. These results suggest that MeIQx is metabolized to N-OH-MeIQx by microsomal cytochrome P-450 and further activated to an acetylated form that binds efficiently to nucleic acids in rat liver. Preferential modification of polyguanylic acid suggests that guanine residues of DNA are mainly modified with MeIQx. Synthetic N-OH-MeIQx exerted direct mutagenic activity on S. typhimurium TA98 inducing 150,000 rev/micrograms. Pentachlorophenol (PCP) caused a dose-dependent inhibition of this mutagenic effect, but 2,6-dichloro-4-nitrophenol (DCNP) did not. Thus the acetyltransferase of S. typhimurium seems to be important for the high mutagenicity of MeIQx after its microsomal activation.     

Inhibition of tubulin assembly and covalent binding to microtubular protein by valproic acid glucuronide in vitro

Acyl glucuronides are reactive metabolites of carboxylate drugs, able to undergo a number of reactions in vitro and in vivo, including isomerization via intramolecular rearrangement and covalent adduct formation with proteins. The intrinsic reactivity of a particular acyl glucuronide depends upon the chemical makeup of the drug moiety. The least reactive acyl glucuronide yet reported is valproic acid acyl glucuronide (VPA-G), which is the major metabolite of the antiepileptic agent valproic acid (VPA). In this study, we showed that both VPA-G and its rearrangement isomers (iso-VPA-G) interacted with bovine brain microtubular protein (MTP, comprised of 85% tubulin and 15% microtubule associated proteins {MAPs}). MTP was incubated with VPA, VPA-G and iso-VPA-G for 2 h at room temperature and pH 7.5 at various concentrations up to 4 mM. VPA-G and iso-VPA-G caused dose-dependent inhibition of assembly of MTP into microtubules, with 50% inhibition (IC₅₀) values of 1.0 and 0.2 mM respectively, suggesting that iso-VPA-G has five times more inhibitory potential than VPA-G. VPA itself did not inhibit microtubule formation except at very high concentrations (≥2 mM). Dialysis to remove unbound VPA-G and iso-VPA-G (prior to the assembly assay) diminished inhibition while not removing it. Comparison of covalent binding of VPA-G and iso-VPA-G (using [¹⁴C]-labelled species) showed that adduct formation was much greater for iso-VPA-G. When [¹⁴C]-iso-VPA-G was reacted with MTP in the presence of sodium cyanide (to stabilize glycation adducts), subsequent separation into tubulin and MAPs fractions by ion exchange chromatography revealed that 78 and 22% of the covalent binding occurred with the MAPs and tubulin fractions respectively. These experiments support the notion of both covalent and reversible binding playing parts in the inhibition of microtubule formation from MTP (though the acyl glucuronide of VPA is less important than its rearrangement isomers in this regard), and that both tubulin and (perhaps more importantly) MAPs form adducts with acyl glucuronides.     

Inhibition by butylated hydroxytoluene of excision repair synthesis and semiconservative DNA synthesis

Butylated hydroxyutoluene (BHT), a commonly used food antioxidant, inhibited excision repair synthesis in normal human peripheral lymphocytes damaged by uv light. Inhibition increased with increasing drug concentration to give 50% inhibition at approximately 20 μM. The acid, aldehyde, and alcohol derivatives at the 4-methyl position did not inhibit significantly DNA repair synthesis while semiconservative DNA synthesis was inhibited by both BHT and the metabolites. The significance of these findings to the reported biological effects of the antioxidant is unknown.     

Studies on the covalent binding of an intermediate(s) in prostaglandin biosynthesis to tissue macromolecules.

We investigated the covalent binding of intermediates in prostaglandin biosynthesis to tissue macromolecules. Following incubation of [1-14C]arachidonic acid with the microsomal fraction from guinea pig lung, ram or bovine seminal vesicle, human platelets, rabbit kidney, or rat stomach fundus, the amount of covalent binding of arachidonic acid metabolites expressed as percentage of total arachidonic acid metabolized varied from tissue to tissue ranging from 3% in human platelets to 18.2% in ram seminal vesicles. In general, the thromboxane synthesizing tissues had less covalently bound metabolites than the other tissues. The amount of covalently bound metabolites was increased in the guinea pig lung microsomes when the thromboxane synthetase inhibitor, N-0164, was added to the incubation mixture. The covalent binding of arachidonic acid metabolite(s) was greatly reduced by the addition of glutathione to the incubation mixture. In addition to the covalently bound metabolites, water-soluble metabolites derived from arachidonic acid metabolism were also observed. The amount of water-soluble metabolites was small in each tissue except for the rat stomach fundus. In the rat stomach fundus the water-soluble metabolites accounted for over 50% of the total metabolites. Conditions which would tend to increase or decrease the levels of free prostaglandin endoperoxides during the incubation of arachidonic acid with the microsomes gave increased or decreased levels of covalent binding. Our data suggest that the prostaglandin endoperoxides are responsible for the covalent binding observed during prostaglandin biosynthesis. This covalent binding to tissue macromolecules may be of physiological and pathological significance.     

Effects of ascorbic acid on the nonenzymatic binding to DNA and the mutagenicity of N-hydroxylated metabolite of a tryptophan-pyrolysis product

Ascorbic acid enhanced the nonenzymatic binding of the mutagen 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2) to DNA with a concomitant increase in the mutagenicity of N-OH-Trp-P-2. The covalent binding of N-OH-Trp-P-2 to DNA was higher at pH 7.4 than pH 6.2 or 5.0. Ascorbic acid increased the binding of N-OH-Trp-P-2 at all pH levels examined. These results indicate that ascorbic acid enhances the DNA damage caused by N-OH-Trp-P-2.     

Comparative metabolism, covalent binding and toxicity of BHT congeners in rat liver slices.

The metabolism, covalent binding and hepatotoxicity of butylated hydroxytoluene (BHT, 4-methyl-2,6-di-t-butylphenol) and two congeners (E-BHT, 4-ethyl-2,6-di-t-butylphenol; I-BHT, 4-isopropyl-2,6-di-t-butylphenol) were compared using precision-cut liver slices prepared from phenobarbital (PB)-treated male Sprague-Dawley rats. At equimolar concentrations (1 mM) BHT was the most toxic of the three compounds, causing an 80% decrease in cell viability over a 6 h incubation period. E-BHT was intermediate in toxicity while the isopropyl derivative was relatively nontoxic. Intracellular glutathione levels decreased prior to the onset of cytotoxicity. The cytochrome P450 inhibitor metyrapone completely inhibited the toxicity of all three compounds. The rates of metabolism of the three compounds to glutathione conjugates were compared in both PB-treated microsomes and PB-induced liver slices. In both models, the rate of formation was greatest for BHT, followed by E-BHT and I-BHT. Synthetic quinone methides (QMs) were prepared from each parent phenol and the rates of reactivity with three nucleophiles (water, methanol and glutathione) were compared. With each nucleophile, BHTQM was the most reactive, while I-BHTQM was the least reactive. Finally, covalent binding to protein was assessed in two ways. First, alkylation of an isolated model protein (bovine insulin) was measured in a microsomal enzyme activation system by mass spectrometry. Incubations with BHT produced the greatest extent of protein alkylation, followed by E-BHT, while no alkylation was observed with I-BHT. In the second system, covalent binding to cellular protein was assessed in rat liver PB microsomes and tissue slices by Western blotting using an antibody specific for the tert-butylphenol portion of the compounds. Binding was greatest for BHT, intermediate for E-BHT and could not be detected for I-BHT. The alkylation pattern for E-BHT was strikingly similar to that of BHT, suggesting that both compounds bound similar proteins. In summary, our results suggest that for hindered phenols such as BHT, increasing the length of the 4-alkyl substituent retards the rate of formation of reactive intermediates, significantly reduces the electrophilicity of the reactive intermediate, and greatly reduces the amount but not the selectivity of covalent binding to cellular protein, thereby reducing the toxicity of the parent compound.     

Metabolites of procainamide and practolol inhibit complement components C3 and C4.

Drug-induced systemic lupus erythematosus arises from toxic side-effects of administration of hydralazine, isoniazid, procainamide and practolol. Hydralazine and isoniazid are nucleophilic drugs and inhibit the covalent binding reaction of complement components, C3 and C4, an effect likely to lead to deposition of immune complexes (a feature of systemic lupus erythematosus). Procainamide and practolol do not themselves inhibit C3 and C4. A range of metabolites and putative metabolites of procainamide and practolol were synthesized, and tested for their ability to inhibit the covalent binding reactions of C3 and C4. The highly nucleophilic hydroxylamine metabolite of procainamide was strongly inhibitory in both tests, as was a putative hydroxylamine metabolite of practolol. These studies indicate a potential role for the hydroxylamine metabolites in mediating the toxic side-effects of procainamide and practolol, and emphasize the need for adequate measurements of hydroxylamine metabolites in human tissue.