Binding of azobenzene and p-diaminoazobenzene to the human voltage-gated sodium channel Nav1.4.

The activity of voltage-gated ion channels can be controlled by the binding of photoswitches inside their internal cavity and subsequent light irradiation. We investigated the binding of azobenzene and p-diaminoazobenzene to the human Nav1.4 channel in the inactivated state by means of Gaussian accelerated molecular dynamics simulations and free-energy computations. Three stable binding pockets were identified for each of the two photoswitches. In all the cases, the binding is controlled by the balance between the favorable hydrophobic interactions of the ligands with the nonpolar residues of the protein and the unfavorable polar solvation energy. In addition, electrostatic interactions between the ligand and the polar aminoacids are also relevant for p-diaminoazobenzene due to the presence of the amino groups on the benzene moieties. These groups participate in hydrogen bonding in the most favorable binding pocket and in long-range electrostatic interactions in the other pockets. The thermodinamically preferred binding sites found for both photoswitches are close to the selectivity filter of the channel. Therefore, it is very likely that the binding of these ligands will induce alterations in the ion conduction through the channel.

Interactions of 4,4'-diaminoazobenzene derivatives with telomeric G-quadruplex DNA.

The development of small molecules to stabilize the G-quadruplex structure has garnered significant attention for anticancer drug discovery. Herein, we report the synthesis of several 4,4'-diaminoazobenzene derivatives containing different substituent groups and their ability to bind and stabilize telomeric G-quadruplex DNA. Circular dichroism (CD) spectroscopy was performed to characterize the quadruplex topologies, measure stabilization effects, and evaluate their capabilities for conformational photoregulation. 4,4'-Diaminoazobenzene derivatives were found to moderately stabilize quadruplex structures but not affect conformational photoregulation. This work further develops the design and general understanding of the stabilization effects of small molecules with telomeric G-quadruplex DNA.

The N-hydroxy metabolites of N-methyl-4-aminoazobenzene and related dyes as proximate carcinogens in the rat and mouse.

The carcinogenicities for rats and mice of N-methyl-4-aminoazobenzene (MAB) and its hepatic microsomal metabolite N-hydroxy-N-methyl-4-aminoazobenzene (N-hydroxy-MAB) were compared under several conditions. N-Ethyl-4-aminoazobenzene, 4-aminoazobenzene, and their N-hydroxy derivatives were also included in some of the assays. About 25% of the rats given MAB or N-hydroxy-MAB (3 to 5 mmol/kg body weight) by stomach tube over a 5-week period developed hepatic tumors by 18 to 22 months. Similarly treated rats subsequently given phenobarbital in the drinking water until the termination of the experiment developed about twice as many hepatic tumors. N-Hydroxy-MAB, administered p.o., but not MAB, also induced multiple papillomas and extensive carcinomas of the forestomach in approximately 50% of the rats. Only low incidence of hepatocellular carcinomas occurred in partially hepatectomized rats given a single i.p. injection of 180 mumol/kg body weight of MAB or N-hydroxy-MAB with or without subsequent administration of phenobarbital. Although repeated s.c. doses of N-benzoyloxy-N-methyl-4-aminoazobenzene induced sarcomas at the injection site in 90% of the rats, only 3 of 20 rats developed sarcomas at the site of s.c. injections of N-hydroxy-MAB. N-Ethyl-4-aminoazobenzene, 4-aminoazobenzene, and their N-hydroxy derivatives did not induce significant numbers of tumors in any of the above assay systems. Administration to preweaning male mice of MAB, N-hydroxy-MAB, N-hydroxy-N-ethyl-4-aminoazobenzene, and N-hydroxy-4-aminoazobenzene resulted in high incidences and high multiplicities of hepatic tumors (averages of 5 to 7 tumors/mouse) within 1 year. N-Ethyl-4-aminoazobenzene and 4-aminoazobenzene also induced hepatic tumors under the same conditions, but they were less active. These data support the conclusion that the N-hydroxy metabolites of these aminoazo dyes are proximate carcinogens.

Androgen-dependent renal microsomal cytochrome P-450 responsible for N-hydroxylation and mutagenic activation of 3-methoxy-4-aminoazobenzene in the BALB/c mouse.

A murine renal microsomal enzyme responsible for the mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) was characterized by its catalytic activity for the mutagenic and metabolic conversion of 3-MeO-AAB. Incubation of 3-MeO-AAB with a renal or hepatic microsome fraction from male BALB/c mice in the presence of NADPH and NADH yielded N-hydroxy and 4'-hydroxy metabolites of 3-MeO-AAB as determined by two-dimensional thin layer chromatography, and the enzyme responsible for the N-hydroxylation was named 3-MeO-AAB N-hydroxylase. A mutagenicity test using Salmonella typhimurium TA98 bacteria as a tester strain has revealed that N-hydroxy-3-MeO-AAB is a potent direct mutagen but that 4'-hydroxy-3-MeO-AAB is not mutagenic. Although 3-MeO-AAB N-hydroxylase activity in liver microsomes showed no sex difference, the enzyme activity in the kidney was detected from male mice but not from females. However, administration of testosterone to female mice induced the enzyme in the kidney. Castration of male mice depressed the activity of 3-MeO-AAB N-hydroxylase in renal microsomes but it little affected the hepatic activity, and on administration of testosterone to the castrated mice the depressed renal microsomal activity recovered to a normal level. The activity of 3-MeO-AAB hydroxylase and the amount of cytochrome P-450 in renal microsomes showed a close correlation. Both renal and hepatic microsomes required NADPH as a main cofactor to mutagenize 3-MeO-AAB and to yield N-hydroxy-3-MeO-AAB from 3-MeO-AAB, and the enzyme activity was strongly inhibited by 7,8-benzoflavone. When the activities of renal and hepatic 3-MeO-AAB N-hydroxylase were compared on the basis of the amount of cytochrome P-450, the renal type enzyme showed about 8 times greater activity than hepatic type enzyme. These results indicate that the kidney contains an androgen-dependent microsomal 3-MeO-AAB hydroxylase which is different from an isozyme present in the liver and which is a new type of cytochrome P-450 isozyme.

4-aminoazobenzene and N,N-dimethyl-4-aminoazobenzene as equipotent hepatic carcinogens in male C57BL/6 X C3H/He F1 mice and characterization of N-(Deoxyguanosin-8-yl)-4-aminoazobenzene as the major persistent hepatic DNA-bound dye in these mice.

In contrast to the well-established requirement for an N-methyl group for efficient hepatic tumor induction by dietary administration of derivatives of 4-aminoazobenzene (AB) to adult rats, we have now observed that AB and its N-methyl and N,N-dimethyl derivatives have high and approximately equal hepatocarcinogenicity when given as a single i.p. dose to male 12-day-old C57BL/6 X C3H/ HeF1 (B6C3F1) mice. The hepatoma multiplicity induced by these dyes was approximately linearly related to the dose from 0.017 to 0.15 mumol/g body weight; at the high dose, an average of 11 hepatomas/mouse was observed at 10 months. Female B6C3F1 mice were resistant to tumor induction under these conditions. AB and its N-methyl derivative also induced the same incidences of hepatomas on administration of a single dose of 0.45 mumol/g body weight to 12-day-old male C3H/He mice (about 15 hepatomas/mouse) or C57BL/6 mice (about 1 hepatoma/mouse). Infant male Fischer rats were much less susceptible; less than 25% of the rats given 4 i.p. injections (0.3 to 0.4 mumol/g of body weight/injection) of N-methyl-4-amino-azobenzene and less than or equal to 5% of those given these doses of N,N-dimethyl-4-aminoazobenzene or AB before 22 days of age developed hepatic carcinomas by 24 months. Reverse-phase high-performance liquid chromatography of enzymatically hydrolyzed hepatic DNA from 12-day-old male B6C3F1 mice or Fischer rats given an i.p. dose (0.08 or 0.3 mumol/g of body weight) of [prime-ring-3H]AB showed a single major adduct which was chromatographically identical to N-( deoxyguanosin -8-yl)-4-aminoazobenzene synthesized by reaction at pH 7 of N-acetoxy-4-aminoazobenzene (formed in situ from N-hydroxy-4-aminoazobenzene and acetic anhydride) with deoxyguanosine. Mouse and rat liver DNA contained 20 and 0.5 pmol, respectively, of this adduct per mg 24 hr after administration of 0.3 mumol of [prime-ring-3H]AB/g of body weight. At 24 hr after administration of N,N-[prime-ring-3H]dimethyl-4-aminoazobenzene to male B6C3F1 mice, N-( deoxyguanosin -8-yl)-4-aminoazobenzene, N-( deoxyguanosin -8-yl)-N-methyl-4-aminoazobenzene, and 3-( deoxyguanosin -N2-yl)-N-methyl-4-aminoazobenzene were present in a ratio of approximately 4:2:1, respectively. Unlike the N-( deoxyguanosin -8-yl)-N-methyl-4-aminoazobenzene adducts, the N-( deoxyguanosin -8-yl)-4-aminoazobenzene adducts were relatively stable in the DNA; the level of the latter adducts decreased about 60% between 24 hr and 21 days.(ABSTRACT TRUNCATED AT 400 WORDS)

Microsomal N-oxidation of the hepatocarcinogen N-methyl-4-aminoazobenzene and the reactivity of N-hydroxy-N-methyl-4-aminoazobenzene.

The N-oxidation of the hepatocarcinogen N-methyl-4-aminoazobenzene (MAB) was catalyzed by hepatic microsomes in a reduced pyridine nucleotide- and oxygen-dependent reaction. The initial N-oxidation product, N-hydroxy-N-methyl-4-aminoazobenzene (N-HO-MAB), was readily oxidized to a second product that yielded N-hydroxy-4-aminoazobenzene upon subsequent acid treatment. The secondary N-oxidation product may be formed nonenzymatically and is presumed to be N-HO-MAB N-oxide or its dehydrated derivative, N-(p-phenylazophenyl)nitrone. Under the same conditions, MAB was also oxidatively N-dealkylated to 4-aminoazobenzene, which was N-oxidized to N-hydroxy-4-aminoazobenzene. Unlike the latter reactions, the microsomal N-oxidation of MAB was independent of cytochrome P-450, as shown by its lack of sensitivity to inhibition by 2-[(2,4-dichloro-6-phenyl)phenoxy]ethylamine and its inability to utilize cumene hydroperoxide in place of reduced pyridine nucleotides and oxygen. The N-oxidation of MAB was also catalyzed by the purified microsomal flavoprotein mixed-function amine oxidase of Ziegler et al. The noncarcinogenic dye N-ethyl-4-aminoazobenzene was metabolized similarly to MAB. For male animals the hepatic levels of MAB N-oxidase activity were in the order: rat greater than hamster, guinea pig greater than mouse, rabbit. Little or no MAB N-oxidase activity was present in several extrahepatic rat tissues. N-HO-MAB, N-hydroxy-N-ethyl-4-aminoazobenzene, and N-hydroxy-4-aminoazobenzene catalyzed the aerobic oxidation of cysteine and glutathione. These hydroxylamines also bound covalently to proteins. The binding of N-HO-MAB with nucleic acids was only 3 to 6% that observed with serum albumin. Under anhydrous conditions the nitrone generated aerobically from N-HO-MAB reacted with carbon-carbon or carbon-nitrogen double bonds, or both, in fatty acids, retinol, purines, and pyrimidines to yield isoxazolidine and/or oxadiazolidine addition products. The nitrone from N-hydroxy-N-ethyl-4-aminoazobenzene was much less reactive under these conditions. Syntheses of N-HO-MAB and N-hydroxy-N-ethyl-4-aminoazobenzene are reported.

N,N-diethylaminobenzaldehyde (DEAB) as a substrate and mechanism-based inhibitor for human ALDH isoenzymes.

N,N-diethylaminobenzaldehyde (DEAB) is a commonly used "selective" inhibitor of aldehyde dehydrogenase isoenzymes in cancer stem cell biology due to its inclusion as a negative control compound in the widely utilized Aldefluor assay. Recent evidence has accumulated that DEAB is not a selective inhibitory agent when assayed in vitro versus ALDH1, ALDH2 and ALDH3 family members. We sought to determine the selectivity of DEAB toward ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH2, ALDH3A1, ALDH4A1 and ALDH5A1 isoenzymes and determine the mechanism by which DEAB exerts its inhibitory action. We found that DEAB is an excellent substrate for ALDH3A1, exhibiting a Vmax/KM that exceeds that of its commonly used substrate, benzaldehyde. DEAB is also a substrate for ALDH1A1, albeit an exceptionally slow one (turnover rate ∼0.03 min(-1)). In contrast, little if any turnover of DEAB was observed when incubated with ALDH1A2, ALDH1A3, ALDH1B1, ALDH2 or ALDH5A1. DEAB was neither a substrate nor an inhibitor for ALDH1L1 or ALDH4A1. Analysis by enzyme kinetics and QTOF mass spectrometry demonstrates that DEAB is an irreversible inhibitor of ALDH1A2 and ALDH2 with apparent bimolecular rate constants of 2900 and 86,000 M(-1) s(-1), respectively. The mechanism of inactivation is consistent with the formation of quinoid-like resonance state following hydride transfer that is stabilized by local structural features that exist in several of the ALDH isoenzymes.

Arylhydroxamic acid bioactivation via acyl group transfer. Structural requirements for transacylating and electrophile-generating activity of N-(2-fluorenyl)hydroxamic acids and related compounds.

The synthesis of a series of 12 N-(2-fluorenyl)hydroxamic acids, N-(2-fluorenyl)-N-hydroxyureas, and N-(2-fluorenyl)-N-hydroxycarbamates is reported. The compounds were evaluated for their ability to serve as substrates for a partially purified hamster hepatic arylhydroxamic acid N,O-acyltransferase preparation. Transacylating activity was measured spectrophotometrically with 4-aminoazobenzene as the acyl group acceptor, and electrophile-generating activity was quantified by the N-acetylmethionine trapping assay. Only the N-acetyl, N-propionyl, and N-methoxyacetyl derivatives exhibited relatively high levels of activity as measured by either of the assay methods. These results are generally consistent with previously reported conclusions regarding the steric and electronic characteristics of acyl groups that are required for activation by this enzyme system. N,O-Acyltransferase inactivation by N-hydroxy-2-acetamidofluorene depressed the bioactivation of the N-acetyl compound to a greater extent than either the N-propionyl or N-methyloxyacetyl derivative.

DNA adduct formation of hepatocarcinogenic aromatic amines in rat liver: effect of cytochrome P450 inducers.

F344 rats were treated with an i.p. injection of 2-amino-6- methyldipyrido[1,2-a:3',2'-d]imidazole (Glu P-1) or 3-methoxy-4-aminoazobenzene (3-MeO-AAB) and examined for the formation of the DNA adduct in the liver. To examine the effect of pretreatment with a cytochrome P450 (CYP) inducer on the formation of DNA adduct, these rats were pretreated with 3-methylcholanthrene (MC; CYP1A1/1A2 inducer) or phenobarbital (PB; CYP2B inducer). Administration of Glu P-1 and 3-MeO-AAB gave 2 and 5 adducts, respectively, as determined by 32P-postlabeling assay. By Glu P-1 administration, pretreatment of rats with MC, but not with PB, increased the total amount of DNA adducts including 3 new adducts as minor products. In contrast, pretreatment of rats with PB increased the total amount of DNA adducts derived by 3-MeO-AAB. The increase of aromatic amine DNA adducts by pretreatment with a CYP inducer was proportional to the activity of induced CYP isozyme(s) responsible for the mutagenic activation of each aromatic amine.

Renal and hepatic microsomal enzymes responsible for bioactivation of 3-methoxy-4-aminoazobenzene in the rodent.

Activities of the renal and hepatic microsomal enzymes responsible for the N-hydroxylation and mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) were examined in male mice, rats, hamsters and guinea pigs. In all these rodent species, hepatic microsomes showed definite N-hydroxylation of 3-MeO-AAB, whereas the renal activity was detected only in mice. The hepatic enzyme responsible for N-hydroxylation of 3-MeO-AAB (3-MeO-AAB N-hydroxylase) was induced in all species except mice by phenobarbital and selectively in mice and hamsters by 3-methylcholanthrene, whereas these cytochrome P450 inducers did not affect the renal enzyme in mice, rats or hamsters. In individual microsome samples, activities for N-hydroxylation and mutagenic activation of 3-MeO-AAB correlated well. These results indicate that the renal and hepatic enzymes responsible for the metabolic activation of 3-MeO-AAB differed among different species of rodent animals in terms of their activity and inducibility with cytochrome P450 inducers.

Characterization of DNA adducts of the carcinogen N-methyl-4-aminoazobenzene in vitro and in vivo.

Since the susceptibility of specific tissues to tumor formation has been correlated with the persistence of DNA-carcinogen adducts, the identity and persistence of DNA adducts formed from the hepatocarcinogen N-methyl-4-aminoazobenzene (MAB) has been determined. The synthetic ultimate carcinogen N-benzoyloxy-N-methyl-4-aminoazobenzene (N-BxO-MAB) was reacted in vitro with either calf thymus or rat liver DNA to yield approx. 1 bound residue per 1000 nucleotides. After enzymatic hydrolysis of the DNA and high pressure liquid chromatographic analysis, at least six MAB adducts were detected. Two of the products cochromatographed with MAB-DNA adducts formed in rat liver in vivo following oral administration of the precarcinogen MAB. These two adducts were identified by mass, UV and nuclear magnetic resonance (NMR) spectroscopy as N-(deoxyguanosin-8-yl)- and 3-(deoxyguanosin-N2-yl)-MAB. The former adduct was initially the predominant product in vivo, but it could not be detected 7 days following treatment. The latter adduct remained at a constant level for 14 days and therefore appears to be a persistent lesion.

Formation of N-(glutathion-S-methylene)-4-aminoazobenzene following metabolic oxidation of the N-methyl group of the carcinogen, N-methyl-4-aminoazobenzene.

A major biliary metabolite of the hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB), in the rat was identified as N-(glutathion-S-methylene)-4-aminoazobenzene (GS-CH2-AB). This conjugate was prepared synthetically by a Mannich condensation of 4-aminoazobenzene (AB), formaldehyde (CH2O) and glutathione (GSH) and has been characterized by chemical analysis and by ultraviolet, visible and 13C-NMR spectroscopy. The same conjugate was also formed in vitro by incubating N-methyl-4-aminoazobenzene (MAB), NADPH, NADH and GSH with rat hepatic microsomes. Evidence is presented that GSH reacted with an intermediate resulting from a cytochrome P-450-dependent oxidation of the N-methyl substituent. This reactive intermediate is presumed to be either an N-methylol or a methimine derivative of AB. The significance of this detoxification mechanism is discussed. The presence of an additional major aminoazo-dye GSH conjugate is also noted.

Formation of 3-(glutathion-S-YL)-N-methyl-4-aminoazobenzene and inhibition of aminoazo dye-nucleic acid binding in vitro by reaction of glutathione with metabolically-generated N-methyl-4-aminoazobenzene-N-sulfate.

The reaction of glutathione (GSH) with metabolically-formed N-methyl-4-aminoazobenzene-N-sulfate (MAB-N-sulfate), a presumed ultimate carcinogenic metabolite of N,N-dimethyl-4-aminoazobenzene (DAB), was investigated using a hepatic sulfotransferase incubation mixture containing GSH and the proximate carcinogen, N-hydroxy-N-methyl-4-aminoazobenzene (N-HO-MAB). Under these conditions, 6--16% of the MAB-N-sulfate formed could be trapped as an aminoazo dye-GSH adduct. Upon subsequent purification, the adduct was shown to be chromatographically and spectrally identical to 3-(glutathion-S-yl)-N-methyl-4-aminoazobenzene (3-GS-MAB), a known biliary metabolite of DAB and a product of the reaction of the synthetic ultimate carcinogen, N-benzoyloxy-N-methyl-4-aminoazobenzene(N-BzO-MAB), with GSH. Neither 2'- nor 4'-GS-MAB, both products of the latter reaction, were detected in the sulfotransferase incubation mixture. GSH-S-transferases did not appear to be involved in the reaction of MAB-N-sulfate of N-BzO-MAB with GSH. The addition of triethyltin, a potent GSH-S-transferase inhibitor, had no effect on the yield of 3-GS-MAB in (N-HO-MAB sulfotransferase)-GSH incubations; and the addition of cytosol or purified GSH transferases A and B to a (N-BzO-MAB)-GSH reaction mixture did not increase the amount of 3-GS-MAB formed.

Peroxidase-mediated irreversible binding of arylamine carcinogens to DNA in intact polymorphonuclear leukocytes activated by a tumor promoter.

Addition of the tumor promoter phorbol myristate acetate to polymorphonuclear leukocytes results in the oxidation of the arylamine carcinogens; [14C]benzidine, N-[14C]methylaminoazobenzene and [14C]aminofluorene to reactive intermediate(s) that bind irreversibly to the leukocyte DNA. The binding was dependent on oxygen and was decreased by sulfhydryl inhibitors and phenolic antioxidants that inhibit the respiratory burst triggered by the phorbol myristate. Both the binding and the respiratory burst were increased by azide, presumably as a result of intracellular catalase inhibition. However higher concentrations of azide and cyanide prevented binding without affecting the respiratory burst indicating that myeloperoxidase is a catalyst for the binding. Granules isolated from the activated leukocytes and H2O2 catalyzed a cyanide sensitive benzidine binding to calf thymus DNA. Myeloperoxidase and H2O2 also catalysed extensive binding of these arylamines to calf thymus DNA. The leukocytes appear to be a useful model cell for studying one electron oxidation-catalyzed carcinogen activation.

Lack of release from hepatocytes in vitro or excretion in vivo of mutagenic chrysoidine metabolites.

Rat liver postmitochondrial supernatant (S9) converted the azo dyes chrysoidine Y and R to products that were mutagenic towards Salmonella typhimurium strain TA100. No such release of mutagens was demonstrated using intact rat hepatocytes as an activation system despite the fact that chrysoidine dyes cause unscheduled DNA synthesis in these cells. It appears that genotoxic products produced within hepatocytes either react within the cell or are detoxified prior to release. Following intraperitoneal administration of chrysoidine Y to rats (100 mg/kg i.p.) there was also no evidence of mutagenic or por-mutagenic products excreted in bile or urine. The S9-derived mutagens appear to be largely independent of bacterial acetylation since they were active in the acetylation-deficient strain TA98/1,8-DNP6 in addition to strain TA98. The ultimate mutagenic form(s) are therefore unlikely to be acetoxyarylamines.

Bacterial mutagenesis and hepatocyte unscheduled DNA synthesis induced by chrysoidine azo-dye components.

5 azo dye components of Gurr chrysoidine 'Y' have been separated, synthesised and identified. Dyes with a methyl substitution (particularly between the two amino groups) were more mutagenic in Salmonella typhimurium strain TA100 with control rat liver S9 than the non-methylated counterpart (range 66-1992 revertants at 50 micrograms/plate). Mutagenicity was also catalysed by human-liver S9 and pre-treatment of rats with either phenobarbitone or beta-naphthoflavone enhanced the activation ability of S9 by greater than 4-fold. Using the most potent promutagenic component (2,4-diamino-3-methylazobenzene), the use of inhibitors of cytochrome P450 (metyrapone: 1.0 mM; alpha-naphthoflavone: 0.075 mM; DPEA: 0.125 mM) and of the flavin monooxygenase (methimazole: 0.75 mM) suggested a major role for cytochrome P448 in the activation of chrysoidine to mutagens. The ability of chrysoidine components to induce unscheduled DNA synthesis in rat hepatocytes in vitro was demonstrated and ranged between 11.92 and 23.5 net nuclear grains at a dose level of 2.5 micrograms/incubation. Since each dye was equi-potent, methyl substitution had little influence on genetic toxicity in hepatocytes.

3-Methoxy-4-aminoazobenzene, a unique carcinogenic aromatic amine as a substrate for cytochrome-P-450-mediated mutagenesis.

Rat liver microsomal enzyme(s) that catalyze mutagenic activation of a carcinogenic aminoazo dye, 3-methoxy-4-aminoazobenzene (3-MeO-AAB), was studied by virtue of the Salmonella typhimurium TA98 assay using o-aminoazotoluene (OAT) as the control. Male Wistar rats were pretreated with phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyl (PCB), and the liver microsomal activities for mutagenic activation of 3-MeO-AAB and OAT were examined. In agreement with the reported results on several carcinogenic aromatic amines, MC pretreatment resulted in greater activation of microsomal activity in the OAT mutagenesis (about a 4-fold increase as compared to the untreated control) than did PB (1.5-fold increase). By contrast, the mutagenic activation of 3-MeO-AAB is found to be more efficiently catalyzed by those enzyme(s) that are induced by PB pretreatment (4-fold increase) than by those that are induced by MC (1.8-fold increase). The induced enzymes that principally mediate the mutagenic activation of these azo dyes are indicated to be cytochrome P-450s, because the mutagenic activation was strongly inhibited by addition of cytochrome P-450 inhibitors such as 2-diethylaminoethyl-2,2-diphenylvalerate (SKF 525A) and 7,8-benzoflavone. These data suggest that 3-MeO-AAB is a unique carcinogenic aromatic amine as a substrate for mutagenic activation via catalysis of those cytochrome P-450s that are induced by PB pretreatment.

Characterization of the binding of chrysoidine, an illegal food additive to bovine serum albumin.

Chrysoidine is an industrial azo dye and the presence of chrysoidine in water and food has become an environmental concern due to its negative effects on human beings. Binding of dyes to serum albumins significantly influence their absorption, distribution, metabolism, and excretion properties. In this work, the interactions of chrysoidine with bovine serum albumin (BSA) were explored. Isothermal titration calorimetry results reveal the binding stoichiometry of chrysoidine to BSA is 1:15.5, and van der Waals and hydrogen bonding interactions are the major driving force in the binding of chrysoidine to BSA. Molecular docking simulations show that chrysoidine binds to BSA at a cavity close to Sudlow site I in domain IIA. However, no detectable conformational change of BSA occurs in the presence of chrysoidine as revealed by UV-vis absorption, circular dichroism and fluorescence spectroscopy studies.

Binding of chrysoidine to catalase: spectroscopy, isothermal titration calorimetry and molecular docking studies.

Chrysoidine is an industrial azo dye and the presence of chrysoidine in water and food has become an environmental concern due to its negative effects on human beings. In this work, the interactions between chrysoidine and bovine liver catalase (BLC) were explored. Obvious loss in catalytic activity was observed after incubation of BLC with chrysoidine, and the inhibition effect of BLC was found to be of the non-competitive type. No profound conformational change of BLC occurs in the presence of chrysoidine as revealed by UV-vis absorption, circular dichroism and fluorescence spectroscopy studies. Isothermal titration calorimetry results indicate that catalase has two sets of binding sites for chrysoidine. Further, molecular docking simulations show that chrysoidine is located within the bottleneck in the main channel of the substrate to the active site of BLC, which explain the activity inhibition of BLC by chrysoidine.