Effects of species, MFO inducers and conjugation agents on the in vitro covalent binding of 14C-3-methylindole metabolite in liver and lung tissues

The degree of tissue covalent binding of 14C-3-methylindole metabolite in goat and rat pretreated with phenobarbital or 3-methylcholanthrene was compared. The effect of conjugating agents, i.e. glutathione (GSH), cysteine and sulfate, in reducing the degree of tissue covalent binding was measured. The degree of tissue covalent binding was significantly higher in the lung than the liver of goats. In rats, covalent binding was higher in the liver than the lung. Glutathione and cysteine were effective in decreasing the degree of in vitro covalent binding in both liver and lung tissues of goat and rat.     

Covalent binding of perfluorinated fatty acids to proteins in the plasma, liver and testes of rats.

Perfluorinated fatty acids alter hepatic lipid metabolism and are potent peroxisome proliferators in rodents. Two such perfluorinated acids, perfluorodecanoic acid (PFDA) and perfluorooctanoic acid (PFOA), were examined to determine if they covalently bind cellular proteins. PFDA and PFOA were found to covalently bind proteins when administered to rats in vivo. The liver, plasma and testes of male rats treated with [1-14C]PFDA or PFOA (9.4 mumol/kg) contained detectable levels of covalently bound 14C (0.1-0.5% of the tissue 14C content). Characterization of PFDA covalent binding to albumin in vitro showed that cysteine significantly decreased binding with no effect of methionine, suggesting protein sulfhydryl groups are involved. In cytosolic and microsomal incubation there was no effect of the addition of CoA, ATP or NADPH on the magnitude of the covalent binding of PFDA. Therefore PFDA need not be metabolically activated to form covalent adducts. Despite demonstration of covalent binding of PFDA and PFOA to proteins both in vivo and in vitro, the role of this macromolecular binding in perfluorinated fatty acid toxicity is not known.     

Binding modes of 6,7 di-substituted 4-anilinoquinoline-3-carbonitriles to EGFR

4-Anilino-3-cyanoquinolines were reported to have irreversible binding to epidermal growth factor receptor kinase (EGFRK) by forming a covalent linkage to C773. Our initial docking studies gave results inconsistent with the in vitro data and showed two different binding modes. To perceive the exact mode of binding of these ligands, two models of the ligand-EGFR complexes were considered: (1) reversible binding mode in which the ligand had hydrogen bond interactions at the binding site and (2) irreversible binding mode wherein the ligand's Michael acceptor side chain has proximity to the sulfhydryl group of C773 of EGFR, thereby enabling a covalent interaction. The irreversible binding mode correlated better than reversible binding mode with respect to in vitro data. However, our results indicate that both modes are being adopted by the ligands and could be utilized to design more potent EGFRK inhibitors.     

Predictability of the covalent binding of acidic drugs in man

Although metabolism via glucuronide conjugation has generally been considered a detoxification route for carboxylic acids, the newly discovered chemical reactivity of these conjugates, leading to covalent binding with proteins, is consistent with the toxicity observed for drugs containing the carboxylic acid moiety. Here we report that degradation rates (intramolecular rearrangement and hydrolysis) for 9 drug glucuronide metabolites show an excellent correlation (r²=0.995) with the extents of drug covalent binding to albumin in vitro. Furthermore, this binding capacity is predictable based on chemical structure of the acid and depends on the degree of substitution at the carbon alpha to the carboxylic acid. The in vivo covalent binding in humans for these drugs is also predictable (r²=0.873) when the extent of adduct formation is corrected for the measured plasma glucuronide concentrations. These results suggest that the structure of a carboxylic acid drug may predict the degree to which the corresponding acyl glucuronides will form covalent adducts that probably/possibly lead to toxicity. This information could be a useful adjunct in drug design.     

Binding mode of chemically activated semiquinone free radicals from quinone anticancer agents to DNA

Chemical reduction of the highly active quinone-containing antitumor drugs, adriamycin and daunorubicin formed the same partially reduced free radical previously reported [9] by microsomal activation. In vitro incubation of the chemically activated free radical intermediates with DNA resulted in covalent binding of these drugs to DNA. The adriamycin semiquinone radical has a greater affinity for DNA and covalent complexes up to one adriamycin per 12 nucleotides were obtained. The daunorubicin semiquinone radical, on the other hand, showed a lesser binding affinity and gave rise to complexes in which one drug molecule was covalently bound per 135 nucleotides. The stronger covalent binding of adriamycin to DNA may account for more severe DNA damage induced by this drug.     

Interaction of heme with variants of the heme chaperone CcmE carrying active site mutations and a cleavable N-terminal His tag

Cytochrome c maturation in the periplasms of many bacteria requires the heme chaperone CcmE, which binds heme covalently both in vivo and in vitro via a histidine residue before transferring the heme to apocytochromes c. To investigate the mechanism and specificity of heme attachment to CcmE, we have mutated the conserved histidine 130 of a soluble C-terminally His-tagged version of CcmE (CcmEsol-C-His6) from Escherichia coli to alanine or cysteine. Remarkably, covalent bond formation with heme occurs with the protein carrying the cysteine mutation, and the process occurs both in vivo and in vitro. The yield of holo-H130C CcmEsol-C-His6 produced in vivo is low compared with the wild type. In vitro heme attachment occurs only under reducing conditions. We demonstrate the involvement of one of the heme vinyl groups and a side chain at residue 130 in the bond formation by showing that in vitro attachment does not occur either with the heme analogue mesoheme or when alanine is present at residue 130. These results have implications for the mechanism of heme attachment to the histidine of CcmE. In vitro, CcmEsol lacking a His tag binds 8-anilino-1-naphthalenesulphonate and heme, the latter both noncovalently and via a covalent bond from the histidine side chain, similarly to the tagged proteins, thus countering a recent proposal that the His tag causes the heme binding. However, the His tag does appear to enhance the rate of in vitro covalent heme binding and to affect the heme ligation in the ferric b-type cytochrome form.     

In vivo and in vitro binding of 2,4-dichlorophenoxyacetic acid to a rat liver mitochondrial protein

2,4-dichlorophenoxyacetic acid (2,4-D) is a hormonal herbicide widely used in the world because of its efficacy in the control of broadleaf and woody plants. In this study we have demonstrated in vivo covalent binding of the phenoxyherbicide 2,4-D to a single protein of 52 kD (from rat liver mitochondrial preparation) detected through immunoblotting studies with the specific antiserum for 2,4-D. The direct involvement of 2,4-D in the formation of the adduct has also been demonstrated in vitro, using liver mitochondrial preparations exposed to 14C-UL-2,4-D. Radiolabeled protein separated by SDS-PAGE and afterwards electroeluted showed a single labeled protein of 52 kD. When mitochondria exposed to radiolabeled xenobiotic were devoid of their outer membrane, the specific activity observed suggest that protein involved in covalent interaction belongs to the inner mitochondrial membrane. We propose that covalent binding of the phenoxyherbicide 2,4-D to a very specific single protein of 52 kD observed in vitro and in vivo may be related to known alterations of the mitochondrial function.     

Indole-3-carbinol protects against covalent binding of benzo[a]pyrene and N-nitrosodimethylamine metabolites to mouse liver macromolecules

Benzo[a]pyrene (BaP) and N-nitrosodimethylamine (NDMA) are carcinogens and indirect acting mutagens. A naturally occurring dietary indole, indole-3-carbinol (I-3-C), has been shown to decrease the incidence of aryl hydrocarbon induced neoplasia in experimental animals. We examined the relationship between the ability of I-3-C to alter the rate of carcinogen oxidation and its ability to decrease the rate of covalent binding of carcinogen metabolites to DNA and protein. We found that I-3-C inhibited the covalent binding of NDMA oxidation products to DNA in vitro in proportion to its ability to inhibit carcinogen metabolism. Pretreatment of mice by gavage with I-3-C resulted in no change in the rate of aryl hydrocarbon hydroxylase or NDMA demethylase in hepatic post-mitochondrial supernatant. However, this pretreatment resulted in a 60-90% decrease in the ability of carcinogen oxidative metabolites to bind covalently to DNA or protein in vitro. Similarly, in in vivo experiments, gavage with I-3-C, followed by gavage with BaP or NDMA, resulted in a 63-85% decrease in covalent binding to macromolecules, with no concomitant change in carcinogen metabolism. The results suggest that the in vivo administration of I-3-C may confer protection for hepatic macromolecules against covalent binding of the metabolites of these two indirect acting mutagens.     

The covalent binding of acetaminophen to protein. Evidence for cysteine residues as major sites of arylation in vitro

Covalent binding of the reactive metabolite of acetaminophen has been investigated in hepatic microsomal preparations from phenobarbital-pretreated mice. Low molecular weight thiols (cysteine and glutathione) were found to inhibit this binding, whereas several other amino acids which were tested did not. Bovine serum albumin (BSA), which contains a single free sulfhydryl group per molecule and which thus represents a macromolecular thiol compound, inhibited covalent binding of the reactive acetaminophen metabolite to microsomal protein in a concentration-dependent manner. The acetaminophen metabolite also became irreversibly bound to BSA in these experiments, although this binding was reduced by approx. 47% when the thiol function of BSA was selectively blocked prior to incubation. Covalent binding of the acetaminophen metabolite to bovine alpha s1-casein, a soluble protein which does not contain any cysteine residues, was found to occur to an extent of 37% of that which became bound to native BSA. These results were taken to indicate that protein thiol groups are major sites of covalent binding of the reactive metabolite of acetaminophen in vitro. The covalent binding characteristics of synthetic N-acetyl-p-benzoquinoneimine (NAPQI), the putative electrophilic intermediate produced during oxidative metabolism of acetaminophen, paralleled closely those of the reactive species generated metabolically. These findings support the contention that NAPQI is indeed the reactive arylating metabolite of acetaminophen which binds irreversibly to protein.     

Covalent and non-covalent interactions of betaig-h3 with collagen VI. Beta ig-h3 is covalently attached to the amino-terminal region of collagen VI in tissue microfibrils

Transforming growth factor-beta induced gene-h3 (betaig-h3) was found to co-purify with collagen VI microfibrils, extracted from developing fetal ligament, after equilibrium density gradient centrifugation under both nondenaturing and denaturing conditions. Analysis of the collagen VI fraction from the non-denaturing gradient by gel electrophoresis under non-reducing conditions revealed the present of a single high molecular weight band that immunostained for both collagen VI and betaig-h3. When the fraction was analyzed under reducing conditions, collagen VI alpha chains and betaig-h3 were the only species evident. The results indicated that betaig-h3 is associated with collagen VI in tissues by reducible covalent bonding, presumably disulfide bridges. Rotary shadowing and immunogold staining of the collagen VI microfibrils and isolated tetramers indicated that betaig-h3 was specifically and periodically associated with the double-beaded region of many of the microfibrils and that this covalent binding site was located in or near the amino-terminal globular domain of the collagen VI molecule. Using solid phase and co-immunoprecipitation assays, recombinant betaig-h3 was found to bind both native and pepsin-treated collagen VI but not individual pepsin-collagen VI alpha chains. Blocking experiments indicated that the major in vitro betaig-h3 binding site was located in the pepsin-resistant region of collagen VI. In contrast to the tissue situation, the in vitro interaction had the characteristics of a reversible non-covalent interaction, and the Kd was measured as 1.63 x 10(-8) m. Rotary shadowing of immunogold-labeled complexes of recombinant betaig-h3 and pepsin-collagen VI indicated that the in vitro betaig-h3 binding site was located close to the amino-terminal end of the collagen VI triple helix. The evidence indicates that collagen VI may contain distinct covalent and non-covalent binding sites for betaig-h3, although the possibility that both interactions use the same binding region is discussed. Overall the study supports the concept that betaig-h3 is extensively associated with collagen VI in some tissues and that it plays an important modulating role in collagen VI microfibril function.     

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.     

Dynamic ligation properties of the Escherichia coli heme chaperone CcmE to non-covalently bound heme

The cytochrome c maturation protein CcmE is an essential membrane-anchored heme chaperone involved in the post-translational covalent attachment of heme to c-type cytochromes in Gram-negative bacteria such as Escherichia coli. Previous in vitro studies have shown that CcmE can bind heme both covalently (via a histidine residue) and non-covalently. In this work we present results on the latter form of heme binding to a soluble form of CcmE. Examination of a number of site-directed mutants of E. coli CcmE by resonance Raman spectroscopy has identified ligands of the heme iron and provided insight into the initial steps of heme binding by CcmE before it binds the heme covalently. The heme binding histidine (His-130) appears to ligate the heme iron in the ferric oxidation state, but two other residues ligate the iron in the ferrous form, thereby freeing His-130 to undergo covalent attachment to a heme vinyl group. It appears that the heme ligation in the non-covalent form is different from that in the holo-form, suggesting that a change in ligation could act as a trigger for the formation of the covalent bond and showing the dynamic and oxidation state-sensitive ligation properties of CcmE.     

Irreversible binding with biological macromolecules and effects in bacterial mutagenicity tests of the radical cation of promethazine and photoactivated promethazine. Comparison with chlorpromazine

The irreversible binding of the radical cation of promethazine (PMZ+.) to DNA and protein in vitro and bacterial macromolecules in situ has been studied. Binding experiments were performed with synthesized [35S] promethazine. The results are compared to those with the chlorpromazine radical cation (CPZ+.). Secondary reaction products which result from fission of the alkylamino side chain are involved in the macromolecular binding of PMZ+. Compared to CPZ+. the covalent DNA binding of PMZ+. is significantly less. A larger amount of PMZ+. binds to single-stranded DNA than to double-stranded DNA. The extent of binding to proteins and RNA is of the same order as that of CPZ+. Bacterial mutagenicity tests show that the low genotoxicity of PMZ+. is related to the low DNA binding. The bacterial cytotoxicity is possibly related to the covalent protein binding. Similar results have been obtained with photoactivated promethazine (PMZ) and chlorpromazine (CPZ). The role of radical cations in the photosensitization and metabolic activation of phenothiazine drugs is discussed.     

Binding of enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene to polynucleotides

DNA covalent binding studies with enantiomers of trans-7,8-dihydroxy- anti-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (anti-BPDE) have been carried out by means of spectroscopic techniques (UV, CD, and fluorescence). Synthetic polynucleotides are employed to investigate binding differences between the G.C and A.T base pairs and to elucidate the bases for the stereoselective covalent binding of DNA toward anti-BPDE. The results indicate that of all the polynucleotides studied, only poly(dA-dT).poly(dA-dT) exhibits predominant intercalative covalent binding towards (+)-anti-BPDE and suffers the least covalent modification. Only minor intercalative covalent contributions are found in alternating polymer poly(dA-dC).poly(dG-dT). These observations parallel the DNA physical binding results of anti-BPDE and its hydrolysis products. They support the hypothesis that intercalative covalent adducts derive from intercalative physical binding while the external covalent adducts derive from external bimolecular associations. In contrast to the A.T polymers, the guanine containing polymers exhibit pronounced reduction in covalent modification by (-)-anti-BPDE. The intercalative covalent binding mode becomes relatively more important in the adducts formed by the (-) enantiomer as a consequence of decreased external guanine binding. These findings are consistent with the guanine specificity, stereoselective covalent binding at dG, the absence of stereoselectivity at dA for anti-BPDE, and the enhanced binding heterogeneity for the (-) enantiomer as found in the native DNA studies. The possible sequence and/or conformational dependence of such stereoselective covalent binding is indicated by the opposite pyrenyl CD sign exhibited by (+)-anti-BPDE bound to polynucleotides with pyrimidine on one strand and purine on another vs. that bound to polymers containing alternating purine-pyrimidine sequences.     

Binding of Enantiomers of Trans-7, 8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydro-benzo [a]pyrene to Polynucleotides

Abstract

DNA covalent binding studies with enantiomers of trans-7,8-dihydroxy- anti-9,10-epoxy- 7,8,9,10-tetrahydro-benzo [a] pyrene (anti-BPDE) have been carried out by means of spectroscopic techniques (UV, CD, and fluorescence). Synthetic polynucleotides are employed to investigate binding differences between the G · C and A · T base pairs and to elucidate the bases for the stereoselective covalent binding of DNA toward anti-BPDE. The results indicate that of all the polynucleotides studied, only poly(dA-dT) · poly(dA-dT) exhibits predominant intercalative covalent binding towards (+)-anti-BPDE and suffers the least covalent modification. Only minor intercalative covalent contributions are found in alternating polymer poly(dA-dC) · poly(dG-dT). These observations parallel the DNA physical binding results of anti-BPDE and its hydrolysis products. They support the hypothesis that intercalative covalent adducts derive from intercalative physical binding while the external covalent adducts derive from external bimolecular associations. In contrast to the A · T polymers, the guanine containing polymers exhibit pronounced reduction in covalent modification by (-)-anti-BPDE. The intercalative covalent binding mode becomes relatively more important in the adducts formed by the (-) enantiomer as a consequence of decreased external guanine binding. These findings are consistent with the guanine specificity, stereoselective covalent binding at dG, the absence of stereoselectivity at dA for anti-BPDE, and the enhanced binding heterogeneity for the (-) enantiomer as found in the native DNA studies. The possible sequence and/or conformational dependence of such stereoselective covalent binding is indicated by the opposite pyrenyl CD sign exhibited by (+)-anti-BPDE bound to polynucleotides with pyrimidine on one strand and purine on another vs. that bound to polymers containing alternating purine-pyrimidine sequences.     

Effect of a disulfide-interchange enzyme on the assembly of human secretory immunoglobulin A from immunoglobulin A and free secretory component.

A disulfide-interchange enzyme from rat liver microsomes was found to promote binding in vitro of human free secretory component (SC) to dimeric serum-type IgA containing J chain, as assessed by immune precipitation and gel filtration. This effect was greater withe native than with partially reduced SC. Most of the bound SC was covalently linked, as determined by electrophoresis in polyacrylamide gels in detergent. The enzyme did not promote binding of native or partially reduce SC to IgG, IgA monomer, IgA dimer without J chain, or IgM. In the case of IgM, the enzyme did, however, promote covalent bonding of previously non-covalently linked SC. The results overall suggest that a disulfide-interchange enzyme could play a role in vivo in the cell-associated assembly of secretory IgA by promoting the covalent attachment of SC to a dimer of serum-type IgA and that the J chain in the IgA dimer contributes to the enzyme effect.     

Studies of mechanisms of niridazole-elicited embryotoxicity: evidence against a major role for covalent binding

Studies reported here were designed to examine the hypothesis that covalent binding of reactive intermediates to macromolecules of the conceptus represents a major mechanism for the embryotoxicity of niridazole (NDZ). The roles of embryonic thiol content and oxygenation on: 1) malformation incidence; 2) reductive metabolism; and 3) covalent binding to embryonic macromolecules of metabolites resulting from reductive biotransformation of NDZ were studied. Results were compared with those from studies with the nondysmorphogenic analog of NDZ, 4'-methylniridazole (MNDZ). Day 10 rat embryos were pretreated for 5 hours in vitro with either L-buthionine-S, R-sulfoximine (BSO) or N-acetylcysteine (NAC) to modulate their glutathione (GSH) content. BSO reduced GSH levels, but NAC was ineffective. Following pretreatment, embryos were cultured for an additional 15 hours in the presence of [14C]NDZ or [14C]MNDZ with an initial oxygen concentration of 5%. At the end of the culture period (day 11, AM), those embryos with active heartbeat and vitelline circulation were examined for asymmetric malformations. Drug metabolites were subjected to multiple extractions from the culture medium and subjected to quantitative high-performance liquid chromatography (HPLC) analysis. Homogenates of the embryos were extracted with trichloroacetic acid (TCA) to estimate the covalent binding of radiolabeled parent compound/metabolites. Autoradiographic analyses were performed on other embryos. BSO pretreatment, which reduces embryonic GSH tissue levels, dramatically increased both the conversion of NDZ to 1-thiocarbamoyl-2-imidazolidinone (TCI) (generated via reductive metabolism of NDZ) and covalently bound label but failed to increase embryotoxicity. NAC, by contrast, did not significantly affect embryonic GSH levels, TCI generation, or covalent binding. Because both rates of metabolism of NDZ to TCI and covalent binding could vary independently of malformation incidence, we concluded that they do not represent critical mechanistic factors for the embryotoxicity of NDZ and related nitroheterocycles.     

Intracellular aflatoxin B1-binding proteins in rat liver

Intracellular aflatoxin B1 binding in rat liver was studied under both in vitro and in vivo conditions. Binding in vivo appeared similar to that observed in vitro except that some covalent adduct formation was detected. Participation of previously described carcinogen-binding proteins such as the Ah receptor, h2-5S protein, 4-5S receptor for 3-methylcholanthrene and the Z-protein fraction was discounted on the grounds of competition binding studies and gel-permeation chromatography. The molecular weight of 45,000 was estimated for the major aflatoxin B1-binding component. Aflatoxin B1 co-eluted with the glutathione S-transferases during gel-permeation and separation of the various isozymes by cation-exchange chromatography indicated interactions with the YaYa and YaYc-forms. These proteins, however, account for less than 20% of the total intracellular aflatoxin binding. A protein of apparent monomeric structure appears to form the major in vitro/in vivo complex with aflatoxin B1.     

In vitro macromolecular binding of 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane (o,p'-DDD) in the mouse lung and liver

The activation and covalent binding of 14C-labelled 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane (o,p'-DDD) in mouse lung and liver S-9 preparations were examined in vitro. These results showed an oxidative cytochrome P-450 mediated transformation of o,p'-DDD to metabolite(s) that bind covalently to proteins, phospholipids and to added naked DNA in both lung and liver. The apparent Km-values for the covalent binding of o,p'-DDD to protein were 0.25 microM and 3.30 microM in lung and liver, respectively. Addition of glutathione to the incubation medium decreased the binding of o,p'-DDD more efficiently in the liver than in the lung. Thus, the selective lung binding of o,p'-DDD previously observed in vivo seems to result from an in situ activation. The tissue selectivity in vivo is suggested to be due to the low apparent Km in the lung favouring bioactivation at low, ecotoxicologically relevant doses, as well as to a less pronounced protection by glutathione in the lung.     

Enhancement by butylated hydroxytoluene of the in vitro activation of 3,3'-dichlorobenzidne

Mutagenicity to Salmonella TA98 and covalent binding to DNA of 3,3'-dichlorobenzidine (DCB) were used to assess the influence of di-tert.-butylated hydroxytoluene (BHT) on the in vitro activation of the arylamine by rat hepatic S9 metabolic systems. BHT at a concentration of 4 or 20 μM enhanced the mutagenicity of DCB by 32 or 21%, respectively, and the covalent binding of DCB to added DNA by 76 or 328%, respectively. The antioxidant altered the HPLC profile of isolable DCB metabolites, causing a decrease in the formation of three metabolites, an increase in the formation of one metabolite, and the formation of an entirely new metabolite. BHT inhibited the mutagenicity of the promutagen 2-acetylaminofluorene (2-AAF) but had no effect on that of the direct-acting mutagen 2,4-dinitrophenylhydrazine (DNPH). The results show that BHT enhances the mutagenicity of and DNA binding by DCB, in contrast with the predominantly inhibitory effect of the antioxidant on the mutagenicity of other chemicals that require bioactivation.