Effect of 7,8-benzolfavone on the formation of benzo(alpha)pyrene-DNA-bound products in hamster embryo cells.

The hydrocarbon-deoxyribonucleoside products present in enzyme digests of DNA from hamster embryo cultures that had been treated with[3H]-benzo[alpha]pyrene (BP) were isolated by chromatography on Sephadex LH20 columns. The products isolated from cells treated with 7,8-benzoflavone (7,8-BF) for 18 h prior to the addition of [3H] BP were indistinguishable from the products isolated from untreated cultures, but the amounts of these products decreased with increasing concentrations of 7,8-BF. The amount of BP metabolized was also decreased in 7,8-BF-treated cultures. The decrease in the amounts of hydrocarbon-deoxyribonucleoside products per mg DNA was logarithmic with respect to the decrease in BP metabolism. The findings are consistent with the hypothesis that 7,8-BF inhibits both an initial and a later metabolic step involved in the conversion of BP to a reactive species that binds to cellular DNA.     

Differences in the repair of DNA strand breaks induced by 9-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene in cultured human fibroblasts

Two benzo(a)pyrene metabolites were found to induce DNA strand breaks in cultured human fibroblasts. DNA strand breaks induced by the non- or weakly carcinogenic 9-hydroxy-benzo(a)pyrene were repaired within two hours, while those induced by the strongly carcinogenic trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene were repaired at a much slower rate.     

Formation of 7,12-dimethylbenz[alpha] anthracene-DNA adducts in 7,8-benzoflavone-treated hamster embryo cells.

Pretreatment of secondary cultures of Syrian hamster embryo cells with 7,8-benzoflavone (7,8-BF) inhibited both the metabolism of 7,12-dimethylbenz[alpha] anthracene (DMBA) and the formation of DMBA-DNA adducts. The DMBA-deoxyribonucleoside adducts from 7,8-BF-treated cultures had the same elution profiles on Sephadex LH-20 columns as those from cultures exposed to DMBA alone, but 7,8-BF-treated cultures contained smaller amounts of DMBA-DNA adducts per mg DNA. As the concentration of 7,8-BF was increased, the decrease in the amount of DMBA-DNA adducts per mg DNA was logarithmic with respect to the decrease in the amount of DMBA metabolized. The results suggest that more than one metabolic step is required for the binding of DMBA to DNA in hamster embryo cells.     

8-Oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine levels in human urine do not depend on diet

In the present study, we used the method involving HPLC pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection for the determination of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in human urine. The mean levels of 8-oxoGua and 8-oxodGuo in the urine samples of the subjects on unrestricted diet were respectively 1.87 nmol/kg 24 h (±0.90) and 0.83 nmol/kg 24h (±0.49), and in the case of the groups studied, they did not depend on the applied diet. The sum of the amounts of both compounds in urine can give information about the formation rate of 8-oxoGua in cellular DNA. It is also likely that the levels of modified nucleo-base/side in urine sample are reflective of the involvement of different repair pathways responsible for the removal of 8-oxodGuo from DNA, namely base excision repair (BER) and nucleotide excision repair (NER).     

The effect of manganese and ferric ions on the in vitro formation of dihydrodihydroxy metabolites of benzo(a)pyrene

The effect of ferric and manganese ions on the in vitro metabolism of benzo(a)pyrene (BP) to dihydrodihydroxy (diol) metabolites by rat liver microsomal preparations was studied. Of the 3 diols separated by high-pressure liquid chromatography (HPLC) and called diols 1, 2 and 3 in order of elution, diol 1 was identified by its U.V. spectrum as the 9,10-diol; diols 2 and 3 have not yet been identified positively but are probably the 4,5- and 7,8-diols respectively. Higher concentrations of both metals altered the diol profile; 10 and 50 mumol Fe3+ per incubation caused the disappearance of diols 1 and 2 and an increase in diol 3; 10 mumol Mn2+ caused a significant decrease in diol 2 while 50 mumol reduced diol 2 to a negligible amount and inhibited the formation of diol 1; both concentrations caused a relative increase in diol 3. If the tentative identification of diol 3 as the 7,8-diol is correct, manganese and ferric ions could be significant in the metabolism of BP to the active metabolite, the 7,8-diol-9,10-epoxide.     

Urinary nucleic acid oxidation product levels show differential associations with pharmacological treatment in patients with type 2 diabetes

The relationship between RNA and DNA oxidation and pharmacological treatment has not been systematically investigated in patients with type 2 diabetes (T2D). We aimed to investigate the association between pharmacological treatments and levels of urinary markers of nucleic acid oxidation in T2D patients. Vejle Diabetes Biobank cohort data was nested into nationwide registry data. Multiple logistic regression was used to associate drug usage with risk of high (above median) RNA and DNA oxidation. Data from 2664 T2D patients (64% male, age range: 25–75) were included. Questionnaire-validated lipid lowering drug use was associated with low RNA oxidation (Odds ratio, OR 0.71, 95% CI: [0.59–0.87]). Insulin and non-specific antidiabetic drugs were associated with low DNA oxidation (insulin: OR 0.60, 95% CI [0.49–0.73]). Oral antidiabetics were associated with high DNA oxidation and RNA oxidation (OR 1.30, 95% CI [1.10–1.53] and OR 1.26, 95% CI [1.07–1.29]). Our findings indicate that diabetes-related drugs are associated with RNA and DNA oxidation and further studies are required to determine causality in T2D patients.     

Crystal structure of the bifunctional dihydroneopterin aldolase/6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Streptococcus pneumoniae

The enzymes dihydroneopterin aldolase (DHNA) and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyse two consecutive steps in the biosynthesis of folic acid. Neither of these enzymes has a counterpart in mammals, and they have therefore been suggested as ideal targets for antimicrobial drugs. Some of the enzymes within the folate pathway can occur as bi- or trifunctional complexes in bacteria and parasites, but the way in which bifunctional DHNA-HPPK enzymes are assembled is unclear. Here, we report the determination of the structure at 2.9 A resolution of the DHNA-HPPK (SulD) bifunctional enzyme complex from the respiratory pathogen Streptococcus pneumoniae. In the crystal, DHNA is assembled as a core octamer, with 422 point group symmetry, although the enzyme is active as a tetramer in solution. Individual HPPK monomers are arranged at the ends of the DHNA octamer, making relatively few contacts with the DHNA domain, but more extensive interactions with adjacent HPPK domains. As a result, the structure forms an elongated cylinder, with the HPPK domains forming two tetramers at each end. The active sites of both enzymes face outward, and there is no clear channel between them that could be used for channelling substrates. The HPPK-HPPK interface accounts for about one-third of the total area between adjacent monomers in SulD, and has levels of surface complementarity comparable to that of the DHNA-DHNA interfaces. There is no "linker" polypeptide between DHNA and HPPK, reducing the conformational flexibility of the HPPK domain relative to the DHNA domain. The implications for the organisation of bi- and trifunctional enzyme complexes within the folate biosynthesis pathway are discussed.     

Cytotoxic metabolites from the leaves of the mangrove Rhizophora apiculata

One new 8,4’-oxyneolignan (1) and one new 7,8-dihydrobenzofuranone (2), together with twenty-one known compounds (3‒23) were isolated from the methanol extract of Rhizophora apiculata leaves. The structures of new compounds, as well as their absolute configuration, were determined by a combination of spectroscopic methods and quantum chemical calculations of NMR and ECD data. All isolated compounds were evaluated for their cytotoxicity, and only 2,6-dimethoxy-1,4-benzoquinone (8) exhibited inhibitory effects against SK-LU-1, HepG2, and MCF7 cells with IC₅₀ values in the range of 8.33–14.82 μM.     

EFFECTS OF A 8-OXOADENOSINE INCORPORATION ON QUADRUPLEX STRUCTURES: THERMAL STABILITIES AND STRUCTURAL STUDIES

The effects of incorporation of 8-oxoadenosine in two different truncations of human telomeric sequence forming quadruplex structures are reported. In order to characterise their structures, a combination of NMR and UV spectroscopy and computational techniques were used. Both oligonucleotides have been found to form fourfold symmetric quadruplex structures. As a tautomeric equilibrium between keto and enol forms of 8-oxoadenosine may establish in solution and intrinsic stabilities effects, such as internal H-bonds, for example, may determine the predominance of some particular tautomer, molecular modelling studies were performed on quadruplex structures containing both the tautomeric forms. Both molecules resulted to be thermally less stable than the natural.     

7,8-secolignans from Schisandra wilsoniana and their anti-HIV-1 activities

Two new 7,8‐secolignans, marphenols A and B ( 1 and 2 , resp.), together with a known related derivative, 7,8‐secoholostylone B ( 3 ), were isolated from the stems of Schisandra wilsoniana. The structures of 1 and 2 were elucidated by spectroscopic methods, including extensive 1D‐ and 2D‐NMR techniques. The anti‐HIV‐1 activities of 1 – 3 were evaluated. Compound 1 inhibited HIV‐1_IIIB‐induced syncytia formation with an EC₅₀ value of 0.55 μg ml^?1. It reduced p24 antigen expression in acutely HIV‐1_IIIB‐infected C8166 cells and primary isolate HIV‐1_TC‐2‐infected peripheral blood mononuclear cells (PBMCs), with EC₅₀ values of 3.34 and 0.52 μg ml^?1, respectively. It showed no effects on the HIV‐1_IIIB replication in chronically infected H9 cells as well as fusion inhibition.     

Two new iridoid glucosides from Ixora chinensis

From leaves and twigs of Ixora chinensis, two new iridoid glucosides, ixoroside (1) and ixoside (7,8-dehydroforsythide) (2) along with known geniposidic acid (3) have been isolated and their structures have been established.     

A class of strong inhibitors of microsomal monooxygenases: the ellipticines.

Ellipticine (E) and its 9-hydroxy derivative inhibit strongly various liver monooxygenase activities mediated by microsomes from control and phenobarbital (PB), benzo[alpha]pyrene (BP) or Aroclor 1254 (Aroclor)-pretreated rats. The inhibition constants, Ki, are remarkably low, and often smaller than 1 micron, particularly in the case of microsomes containing cytochrome P-448. The inhibitory potency (I50) of 9-hydroxyellipticine (9-OHE) is larger (about ten-fold) than the one of classical inhibitors (metyrapone or 7,8-benzoflavone (7,8-BF)), whatever the activities studied and the induction of microsomes. Differences exist between the mechanisms of inhibition according to the form of cytochrome P-450 present in microsomes of differently pretreated rats; whichever the activities studied, one observes: (a) a competitive inhibition towards the activity of non-induced or PB-induced microsomes and (b) a non-competitive inhibition towards the activity of Aroclor or BP-induced microsomes, at variance with 7,8-BF. These results are in good agreement with the interaction properties of the ellipticines with microsomal cytochromes P-450.     

Ring test for the determination of N-terminal valine adducts of styrene 7,8-oxide with haemoglobin by the modified Edman degradation technique

A ring-test was organised between three laboratories using different versions of the modified Edman degradation technique for the gas chromatographic-mass spectrometric determination of N-terminal valine adducts of styrene 7,8-oxide. The analyses were performed on a sample of human haemoglobin reacted in vitro with styrene 7,8-oxide and on a set of five haemoglobin samples from mice dosed by i.p. injection of styrene. Strong correlations between the haemoglobin adduct determinations of the different laboratories were observed. However, covariance analysis revealed different slopes for the dose-response curves, indicating differences for the calibration of the reference globin or reference peptide.     

The TrkB agonist, 7,8-dihydroxyflavone,impairs fracture healing in mice

Objectives:To study the effects of the selective TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF), on fracture healing in mice and on an osteoprogenitor cell line, Kusa4b10, in vitro.Methods:Mice received unilateral closed mid-shaft tibial fractures and treated for two weeks with vehicle or 5 mg/kg/day DHF and euthanised at 28 days post-fracture. Calluses were analysed by micro-computed tomography (µCT) and three-point bending biomechanical test. Kusa4b10 cells were cultured with 50nM of 7,8-DHF or vehicle for 3-, 7-, 14-days for RT-PCR, and 21 days for mineralization.Results:µCT found 7,8-DHF calluses had decreased tissue volume (p=0.042), mean polar moment of inertia (p = 0.004), and mean cross-sectional area (p=0.042) compared to controls. At 28 days biomechanical analyses showed 7,8-DHF treatment decreased peak force (p=0.011) and stiffness per unit area (p=0.012). 7,8-DHF treatment did not change Kusa4b10 gene expression of Runx2 and alkaline phosphatase at all time points, nor mineralization.Conclusions:7,8-DHF treatment had a negative impact on fracture healing at 28 days post-fracture via an unknown mechanism. 7,8-DHF may have had a central role in impairing fracture healing.     

Transformation of 2-chloroquinoline to 2-chloro-cis-7,8-dihydro-7,8-dihydroxyquinoline by quinoline-grown resting cells of Pseudomonas putida 86

Abstract Resting cells of Pseudononas putida strain 86 were grown on quinoline transformed 2-chloroquinoline to 2-chloro- cis -7,8-dihydro-7,8-dihydroxyquinoline which was not converted further. 7,8-Dioxygenating activity was present when the enzymes of quinoline catabolism were induced. Quinoline-grown cells of strain 86 treated simultaneously with 2-chloroquinoline and D-(-)- threo -chloramphenicol to prevent protein biosynthesis also formed the cis -7,8-dihydrodiol of 2-chloroquinoline. Succinate-grown resting cells did not oxidize 2-chloroquinoline. Acid-catalyzed decomposition of 2-chloro- cis -7,8-dihydro-7,8-dihydroxyquinoline predominantly yielded 2-chloro-8-hydroxyquinoline. By analogy, accumulation of the putative dead-end metabolite 1 H -8-hydroxy-2-oxoquinoline during growth of P. putida 86 on quinoline is suggested to likewise result from dehydration of the 7,8-dihydrodiol of 1 H -2-oxoquinoline.     

An alternative 7-ethoxyresorufin o-deethylase activity assay: A continuous visible spectrophotometric method for measurement of cytochrome P-450 monooxygenase activity

A procedure to directly measure the cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase activity with a visible spectrophotometer is described and compared to the standard fluorometric method. The two assays yielded identical results with both β-naphthoflavone-treated mammalian (rat) and fish (scup, Stenotomus chrysops) liver microsomes. The assay takes advantage of a clean distinction in visible absorption spectra obtained for highly purified 7-ethoxyresorufin (substrate) and resorufin (enzymatic product). The purification and characterization of resorufin, the enzymatic product, are detailed, and its extinction coefficient (ε₅₇₂ = 73 mm^?1 cm^?1) provides for an accurate quantitation of enzyme activity. The large visible extinction coefficient of the product chromophore provides a high sensitivity for low-activity samples. The application of this enzyme assay in a visible spectrophotometer, along with the considerable evidence that a single aromatic hydrocarbon-inducible cytochrome P-450 isozyme is responsible for the catalysis, enhances the utility of this substrate in microsomal monooxygenase assays. The utility of the visible assay is further demonstrated by the simple determinations of the coupling ratio for 7-ethoxyresorufin oxidation in scup liver microsomes and the K_I for 7,8-benzoflavone and phenylimidazole inhibition of the enzymatic reaction.