In vitro metabolism of 12-methylbenz(a)anthracene: Effect of the methyl group on the stereochemistry of a 5,6-dihydrodiol metabolite

While metabolism of benz[a]anthracene by rat liver microsomes produced a (+)5R,6R-dihydrodiol as the major enantiomer, metabolism of 12-methylbenz[a]anthracene under similar conditions gave a (?)5S,6S-dihydrodiol as the major enantiomer. This is the first example indicating that the methyl substituent of a polycyclic aromatic hydrocarbon can drastically alter the stereoselective preference of the microsomal drug-metabolizing enzyme systems toward a substrate molecule in the formation of a dihydrodiol metabolite at an unsubstituted aromatic double bond.     

Some properties of vicinal diol-epoxides derived from benzo(a)anthracene and benzo(a)pyrene

The alkylating properties of pairs of syn- and anti-isomers of 2 diol-epoxides derived from benzo(a)pyrene (BP) and of 1 derived from benz(a)anthracene (BA) have been investigated. Of the anti-diol-epoxides, anti-BP 7,8-diol-9,10-oxide was the most reactive compound towards DNA, towards sodium p-nitrothiophenolate in a non-aqueous solvent system, and towards 4-(p-nitrobenzyl)pyridine in aqueous solution; anti-BP 9,10,-diol-7,8-oxide was of intermediate reactivity and anti-BA 8,9-diol-10,11-oxide was least reactive. The syn-diol-epoxides gave unsatisfactory results with DNA and 4-(p-nitrobenzyl)pyridine because of their rapid solvolysis in aqueous solution, but with sodium p-nitrothiophenolate showed the order of reactivity syn-BP 7,8-diol-9,10-oxide greater than syn-BA 8,9-diol-10,11-oxide greater than syn-BP 9,10-diol-7,8-oxide. The products of the reaction between diol-epoxides and nucleic acids were examined by Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC) and the diol-epoxides were shown to react principally with the guanosine and adenosine moieties of RNA.     

Neuromedin B: a novel bombesin-like peptide identified in porcine spinal cord

Metabolism of 1-nitrobenzo(a)pyrene (1-nitro-BaP) by rat liver microsomes yielded 1-nitro-BaP trans-7,8-dihydrodiol, 1-nitro-BaP trans-9,10-dihydrodiol and 1-nitro-BaP 7,8,9,10-tetrahydrotetrol. Formation of these metabolites suggests that a vicinal 7,8,9,10-dihydrodiol-epoxide is a metabolite of 1-nitro-BaP.     

Effect of a peri fluoro substituent on the conformation of dihydrodiol derivatives of polycyclic aromatic hydrocarbons

$\text{Trans}$-3,4-, 5,6-, 8,9-, and 10,11-dihydrodiols formed from the metabolism of 7-fluorobenz[a]anthracene by rat liver microsomes were isolated by reversed-phase high performance liquid chromatography. Ultraviolet absorption, mass, and NMR spectral analyses indicated that the 5,6- and 8,9-dihydrodiols were preferentially in quasi-diaxial conformations, whereas the 3,4- and 10,11-dihydrodiols were preferentially in quasi-diequatorial conformations. CPK space-filling models suggest that the quasi-diaxial conformation is primarily the result of electronic repulsion between the fluorine and the $\text{peri}$ hydroxyl oxygen. These findings provide a structural basis in the interpretation of the carcinogenic potencies of some fluorinated polycyclic aromatic hydrocarbons.     

The cyanobacterium Gloeobacter violaceus PCC 7421 uses bacterial-type phytoene desaturase in carotenoid biosynthesis

Carotenoid composition and its biosynthetic pathway in the cyanobacterium Gloeobacter violaceus PCC 7421 were investigated. beta-Carotene and (2S,2'S)-oscillol 2,2'-di(alpha-L-fucoside), and echinenone were major and minor carotenoids, respectively. We identified two unique genes for carotenoid biosynthesis using in vivo functional complementation experiments. In Gloeobacter, a bacterial-type phytoene desaturase (CrtI), rather than plant-type desaturases (CrtP and CrtQ), produced lycopene. This is the first demonstration of an oxygenic photosynthetic organism utilizing bacterial-type phytoene desaturase. We also revealed that echinenone synthesis is catalyzed by CrtW rather than CrtO. These findings indicated that Gloeobacter retains ancestral properties of carotenoid biosynthesis.     

The identification of 3-methylcholanthrene-9,10-dihydrodiol as an intermediate in the binding of 3-methylcholanthrene to DNA in cells in culture

Two metabolites of 3-methylcholanthrene (3MC), previously shown to be precursors of the DNA-bound form of 3MC observed in embryo cells in culture, were prepared from 3MC by microsomal metabolism and isolated by high pressure liquid chromatography (HPLC). From HPLC analysis of the metabolites of 3MC, from mass spectrometric analysis and from comparison with the fluorescence spectra of all 5 possible dihydrodiols of the alkylated benzanthracenes, it was deduced that one of the precursor metabolites was a 9,10-dihydrodiol of 3MC while the other was a 1 or 2-hydroxy derivative thereof.     

Blood group A glycolipid (Ax) with globo-series structure which is specific for blood group A1 erythrocytes: one of the chemical bases for A1 and A2 distinction

A new blood group A-active glycolipid fraction, termed Ax, showing a chromatographic mobility between Aa and Ab was found in blood group A1 erythrocytes but not in A2 erythrocytes. Ax was identified by its conversion to "globo H" by alpha-N-acetylgalactosaminidase and by 1H-NMR spectroscopy as GalNAc alpha l----3[Fuc alpha l----2]Gal beta l----3GalNAc beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer. Globo-H (Fuc alpha l----2Gal beta l----3GalNac beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer) was found in blood group A, and O but not in A1 erythrocytes. Thus, one of the A1-specific determinants must be an A determinant carried by globo-series structure.     

Binding of benzo[a]pyrene at the 1,3,6 positions to nucleic acids in vivo on mouse skin and in vitro with rat liver microsomes and nuclei

Loss of tritium from specific positions in [³H,¹⁴C] aromatic hydrocarbons can elucidate their binding site(s) to DNA and RNA and indicate the mechanism of activation. Studies of tritium loss from [6-³H,¹⁴C]benzo[a]pyrene(B[a]P), [1,3-³H,¹⁴C]B[a]P, [1,3,6-³H,¹⁴C]B[a]P, [6,7-³H,¹⁴C]B[a]P, and [7-³H,¹⁴C]B[a]P were conducted in vitro using liver nuclei and microsomes from 3-methylcholanthrene-induced Sprague-Dawley rats and in vivo on the skin of Charles River CD-1 mice. The relative loss of tritium from $\text{[}{}^3\text{H}\text{,}{}^{14}\text{C}\text{]}\text{B}\text{[a]}\text{P}$ was measured after binding to skin DNA and RNA, to nuclear DNA, and to native and denatured calf thymus and rat liver DNA's and poly(G) by microsomal activation. In skin, nuclei, and microsomes plus native DNA, virtually all B[a]P binding occurred at positions 1,3 and 6; while with microsomes plus denatured DNA or poly(G), B[a]P showed no binding at the 6 position and a small amount at the 1 and 3 positions. In vivo and with nuclei, binding at the 6 position predominated. Little loss of tritium from the 7 position was seen; this was expected because binding at this position is not thought to occur. This confirms the interpretation of loss of tritium as an indication of binding at a given position. These results demonstrate that the use of microsomes to activate B[a]P is not a valid model system for delineating the in vivo mechanism of B[a]P activation, and support previous evidence for one-electron oxidation as the mechanism of activation of hydrocarbons in binding to nucleic acids.     

The binding of benz(a)anthracene derivatives to glyceraldehyde-3-phosphate dehydrogenase and mammary tissue following exposure to laboratory light.

7,12-Dimethylbenz(a)anthracene (DMBA) and 7-methoxymethyl-12-methylbenz(a)anthracene (MeO-DMBA) are converted to a number of products during short exposures in aqueous suspension to laboratory illumination. The mixture of products binds to glyceraldehyde-3-phosphate dehydrogenase (GPDH) while inhibiting its activity but there is no apparent relationship between the binding and inhibition of enzyme activity. There is little, or no, binding or enzyme inhibition when the compounds are protected from light. 7-Bromomethyl-12-methylbenz(a)anthracene (Br-DMBA) binds to GPDH whether photoactivated or not but enzyme inhibition depends upon light exposure. The binding of light-exposed DMBA by surviving rat mammary tissue was five-times greater than with the unchanged hydrocarbon. Binding of MeO-DMBA products also occurred after light exposure but not in the dark.     

The metabolism of 7,12-dimethylbenz[a]anthracene and 7-hydroxymethyl-12-methylbenz[a]anthracene by rat liver and adrenal homogenates and by rat adrenocortical cells

The metabolism of 3H-labelled 7,12-dimethylbenz[a]anthracene (DMBA) and of 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA) into solvent- and water-soluble and protein-bound derivatives has been examined in rat liver and adrenal homogenates and in rat adrenocortical cells in culture. Although the overall extents of metabolism of the substrates by the two types of homogenate were similar, there was twice as much binding to protein in incubations with the 7-hydroxymethyl derivative. Rat adrenal cells in culture metabolized DMBA more extensively than 7-OHM-12-MBA and converted much more of the parent hydrocarbon into water-soluble derivatives. Both hydrocarbons were metabolized to yield dihydrodiols that were separated and identified by high performance liquid chromatography (HPLC). The 8,9-dihydrodiol was the major dihydrodiol formed from DMBA but, with 7-OHM-12-MBA as substrate, metabolism was diverted to the 10,11- and 3,4-positions in adrenal and hepatic preparations respectively. The viability of rat adrenocortical cells in culture, as measured by trypan blue exclusion, did not appear to be affected by treatment with DMBA, 7-OHM-12-MBA, the sulphate ester of 7-OHM-12-MBA or by 3,4-dihydro-3,4-dihydroxy-7-hydroxymethyl-12-methylbenz[a]anthracene.     

Chemoenzymatic synthesis and properties of Schiff bases containing (R)-1-(9-anthryl)ethylamine

Racemic 1-(9-anthryl)ethylamine (10), obtained in 70% overall yield from commercial 9-cyanoanthracene, was kinetically resolved by the Candida antarctica A lipase-catalyzed acetylation with isopropyl acetate as acyl donor, affording (R)-(+)-10 with 95.8% enantiomeric excess (e.e.) (E-value 43.5), which afforded Schiff bases (R)-4 and(R)-8. (1)H-NMR, CD, and MM2 calculations offer a consistent picture of the conformational properties of these potential ligands and an explanation for the limited enhancement of enantioselectivity in cyclopropanation of styrene by their Cu(I) complexes, as compared with previously studied ligands in this series.     

Paeonol exhibits anti-tumor effects by apoptotic and anti-inflammatory activities in 7,12-dimethylbenz(a)anthracene induced oral carcinogenesis

We investigated the preventive potential of paeonol on 7,12-dimethylbenz(a)anthracene (DMBA) induced oral carcinogenesis. Oral tumors were developed in the buccal pouches of Syrian golden hamsters using topical application of 0.5% DMBA three times/week for 10 weeks. DMBA treated hamsters developed hyperplasia, dysplasia and well-differentiated squamous cell carcinoma. The animals also exhibited increased lipid oxidation, decreased antioxidant status and altered levels of detoxification agents. Paeonol treatment of DMBA treated hamsters for 14 weeks decreased tumor incidence, volume and burden Paeonol treatment also increased antioxidant activity and decreased lipid oxidation to near normal levels. Histomorphology and the expression patterns of mutant p53, cyclo-oxygenase (COX-2) and caspase-9 were investigated in the oral buccal mucosa. Paeonol exhibited protective effects against DMBA induced oral carcinogenesis owing to its antitumor, antioxidant, anti-inflammatory and apoptosis inducing properties.     

Chronic unpredictable stress (CUS) enhances the carcinogenic potential of 7,12-dimethylbenz(a)anthracene (DMBA) and accelerates the onset of tumor development in Swiss albino mice

Social stressors evolving from individual and population interactions produce stress reactions in many organisms (including humans), influencing homeostasis, altering the activity of the immunological system, and thus leading to various pathological states including cancer and their progression. The present study sought to validate the effectiveness of chronic unpredictable stress (CUS) in cancer promotion and to assess oxidative stress outcomes in terms of various in vivo biochemical parameters, oxidative stress markers, DNA damage, and the development of skin tumors in Swiss albino mice. Animals were randomized into different groups based on their exposure to CUS alone, 7,12-dimethylbenz(a)anthracene (DMBA) alone (topical), and DMBA-12-O-tetradecanoylphorbol-13-acetate (TPA) (topical) and exposure to CUS prior to DMBA or DMBA-TPA treatments and sacrificed after 16 weeks of treatment. Prior exposure to CUS significantly increased the pro-oxidant effect of carcinogen, depicted by compromised levels of antioxidants in the circulation and skin, accompanied by enhanced lipid peroxidation, plasma corticosterone, and marker enzymes as compared to DMBA-alone or DMBA-TPA treatments. DNA damage results corroborated the above biochemical outcomes. Also, the development of skin tumors (in terms of their incidence, tumor yield, and tumor burden) in mice in the presence and absence of stress further strongly supported our above biochemical measurements. CUS may work as a promoter of carcinogenesis by enhancing the pro-oxidant potential of carcinogens. Further studies may be aimed at the development of interventions for disease prevention by identifying the relations between psychological factors and DNA damage.     

Effect of various chemicals on the metabolism of benzo(a)pyrene by cultured rat colon

The effect of various co- and anti-carcinogens of colon carcinogenesis on the metabolism of benzo(a)pyrene (BP) in cultured rat colon is reported. Rat colon enzymatically converted BP into metabolites which bind to cellular macromolecules i.e., DNA and protein. Activity of aryl hydrocarbon hydroxylase (AHH) activity and binding levels of BP to macromolecules were higher in the descending colon when compared to other segments. The major metabolites of BP, extractable with ethylacetate, were quinones, tetrols, 7,8-diol and a peak containing 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene and 7,8,9-trihydroxy-7,8-dihydrobenzo(a)pyrene. The binding levels of BP to DNA and protein in the explant was lowered by co-incubation with 7,8-benzoflavone (7,8-BF) (3.6 and 18.0 μM), a known inhibitor of AHH, and with disulfiram (100 μM), an anti-oxidant. The absence of vitamin A in the media also resulted in a lower level of BP binding to DNA and protein and in lower activity of AHH. Pretreatment with known inducers of AHH such as phenobarbital (PB) or benz(a)anthracene (BA), did not have any significant effect on the binding levels of BP to DNA or on the AHH activity. of the bile acids investigated only taurodeoxycholic acid significantly increased the binding level of BP to DNA.     

Evidence for the involvement of a diol-epoxide in the binding of 7,12-dimethylbenz(a)anthracene to DNA in cells in culture.

1,1,1-Trichloropropene 2,3-oxide (TCPO), a known inhibitor of the enzyme epoxide hydrase, inhibits binding of the carcinogen, 7,12-dimethylbenz(a)anthracene (DMBA), to the DNA of secondary mouse embryo cell cultures under conditions which do not appreciably decrease the overall metabolism of this carcinogen. This suggests that the formation of a transdihydrodiol is a necessary step in the metabolic pathway leading to DNA binding and that binding probably occurs through the generation of a reactive diol-epoxide. In concert with this, the major DMBA-DNA product isolated by chromatography on Sephadex LH-20 eluted with a methanol-water gradient is resolved into two separate components in a methanol-sodium borate solution gradient suggesting that, as is known for benzo(a)pyrene, two stereoisomeric diol-epoxides are involved in the binding of DMBA to DNA.     

The metabolism of 6-(2,3,4-trihydroxy-3-methylbutylamino) purine by soybean callus

6-(2,3,4-trihydroxy-3-methylbutylamino) purine (trihydroxyzeatin) applied to soybean callus is metabolised slowly. After 48 h only one peak of biological activity which co-eluted with the applied cytokinin was detected. When the callus was incubated on a medium which contained 10^–5 M trihydroxyzeatin, spiked with 8 {¹⁴C} trihydroxyzeatin, for 28 days, three peaks of biological activity and three peaks of radioactivity were detected. One of the biologically active and radioactive peaks co-eluted with zeatin. Another of the radioactive peaks co-eluted with N-(purin-6-yl) glycine. From the data obtained it apears that trihydroxyzeatin can be both oxidized and reduced by soybean callus. The potential to be converted to zeatin may explain why trihydroxyzeatin and its parent compound, which is usually rapidly metabolised by living material, are equally active in the soybean callus bioassay. From the radioactive data obtained it appears that trihydroxyzeatin is susceptible to oxidation to form N-(purin-6-yl) glycine.     

The in vitro structure and functions of the disordered late embryogenesis abundant three proteins

Late embryogenesis abundant (LEA) proteins are produced during seed embryogenesis and in vegetative tissue in response to various abiotic stressors. A correlation has been established between LEA expression and stress tolerance, yet their precise biochemical mechanism remains elusive. LEA proteins are very rich in hydrophilic amino acids, and they have been found to be intrinsically disordered proteins (IDPs) in vitro. Here, we perform biochemical and structural analyses of the four LEA3 proteins from Arabidopsis thaliana (AtLEA3). We show that the LEA3 proteins are disordered in solution but have regions with propensity for order. All LEA3 proteins were effective cryoprotectants of LDH in the freeze/thaw assays, while only one member, AtLEA3‐4, was shown to bind Cu²⁺ and Fe³⁺ ions with micromolar affinity. As well, only AtLEA3‐4 showed binding and a gain in α‐helicity in the presence of the membrane mimic dodecylphosphocholine (DPC). We explored this interaction in greater detail using ¹⁵N‐heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance, and demonstrate that two sets of conserved motifs present in AtLEA3‐4 are involved in the interaction with the DPC micelles, which themselves gain α‐helical structure.     

The use of high pressure liquid chromatography to study chemically induced alterations in the pattern of benzo[a]pyrene metabolism.

The metabolism of radiolabeled benzo[a]pyrene (BP) by control, 3-methyl-cholanthrene (3-MC) induced, and 1,1,1-trichloropropene-2,3-oxide (TCPO)-inhibited rat liver microsomes was measured using fluorescence, radiometric, and high-pressure liquid chromatographic (HPLC) assays. Significant differences in the total measurable metabolism of BP by the three microsomal enzyme incubations resulted from the use of the three assay procedures. Appreciable differences in the concentration of the metabolite fractions after 3-MC induction and TCPO inhibition are clearly demonstrated. NMR analysis revealed that while the 3-hydroxy-BP fraction is greater than 90% pure, the 9-hydroxy fraction contains a number of metabolites having essentially identical retention times.     

Radioactive assay for aryl hydrocarbon hydroxylase. Improved method and biological importance

By addition of two volumes of a 1M aqueous KOH/dimethylsulfoxide ($\text{15}\text{85}$; v/v) mixture to the enzymatic incubation medium, it is possible to selectively extract the unmetabolized benzo(a)pyrene in hexane. Therefore, the radio-activity remaining in the water phase corresponds to all the in vitro synthesized metabolites. This isotopic method is very sensitive (2 × 10^?11 moles) and is almost insensitive to the room lighting. The aryl hydrocarbon hydroxylase activities found with this method are 2,3 and 10 times higher in the liver, lung and kidney respectively compared to those obtained with the fluorimetric method.     

太行菊不同器官中绿原酸和4种黄酮类物质含量研究

为深入研究我国特有植物太行菊,采用高效液相色谱法(HPLC),以ZORBAX Eclipse XDB-C18(4.6×150 mm,5μm)为分析柱,甲醇-0.01%磷酸水溶液梯度洗脱,流速1.0 mL/min,检测波长254 nm,柱温25℃;对太行菊和传统药用野菊不同器官的绿原酸、芦丁、槲皮素、木犀草素、芹菜素含量进行测定。此外,以芦丁为对照品,采用硝酸铝络合紫外分光光度法,以没食子酸为对照品,采用福林酚法,分别对太行菊和野菊不同器官中总黄酮和总多酚的含量进行研究。结果表明,基于上述HPLC检测方法,五种化合物的质量浓度在5.94～178.2、4.36～130.8、8.96～268.8、4.08～122.4、2.98～25.33 mg/L范围内与峰面积线性关系良好,相关系数r均大于0.9993,方法的精密度、重复性、稳定性及平均加样回收率试验结果均满足分析要求。太行菊叶中五种化合物的总含量高于其花、茎和野菊对应器官,与太行菊和野菊不同器官中总黄酮和总多酚含量分析结果一致。因此,太行菊整个植株,尤其是叶和花的开发利用潜力显著。