Activation of carcinogenic N-nitrosopropylamines to mutagens by lung and pancreas S9 fractions from various animal species and man

The mutagenic potential of 7 carcinogenic N-nitrosopropylamines was examined by the Ames liquid incubation assay, using lung and pancreas 9000 × g supernatant (S9) fractions from rats, hamsters, mice, rabbits, monkeys and humans for metabolic activation. N-Nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP), N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitrosomethyl(2-oxopropyl)amine (MOP) showed positive mutagenicity in strain TA100 in the presence of lung S9 from each of the uninduced animals and humans. Besides the 3 N-nitrosopropylamines, N-nitrosomethyl(2-hydroxypropyl)amine (MHP) was also positive in the presence of lung S9 from polychlorinated biphenyl (PCB)-induced rats, hamsters and mice. On the other hand, in the presence of pancreas S9 from uninduced or PCB-induced animals, only HPOP and BOP showed positive mutagenicity. In contrast, N-nitrosobis(2-hydroxypropyl)amine (BHP), N-nitrosobis (2-acetoxypropyl)amine (BAP) and N-nitroso-2,6-dimethylmorpholine (NDMM) showed negative mutagenicity in the presence of lung and pancreas S9 from either uninduced or PCB-induced animals and humans. HPOP was a direct-acting mutagen, and lung and pancreas S9 from 5 animal species and man did not affect the activity. BOP was mutagenic even in the presence of bovine serum albumin. The mutagenic activation of MHP by lung S9 from PCB-induced rats, hamsters and mice was completely inhibited by preincubation in an atmosphere of carbon monoxide or by addition of cytochrome c or metyrapone to the S9 mixture, whereas 7,8-benzoflavone totally lacked this effect. However, that of MOP was insensitive to these inhibitors. These results of mutagenicity assay indicate that only the methyl derivatives of N-nitrosopropylamines, MHP and MOP are activated by the lung from 5 animal species and man, whereas the pancreas from all the tested animals did not activate the 7 N-nitrosopropylamines to mutagens, and that the phenobarbital-inducible major cytochrome P-450 in the lung of rodents is involved in the mutagenic activation of MHP.     

Oxidation of bovine serum albumin initiated by the Fenton reaction--effect of EDTA, tert-butylhydroperoxide and tetrahydrofuran

Oxidation of bovine serum albumin (BSA) was investigated using different oxidants: The water-soluble azo-initiator 2,2'azo-bis-(2-amidinopropane) hydrochloride (AAPH), a combination of FeCl₃ and ascorbate or the Fenton oxidant consisting of FeCl₂, H₂O₂ and EDTA. In addition, the effects of exogenous compounds such as tert-butyl hydroperoxide (tBuOOH) or solvents such as tetrahydrofuran (THF), often used in model systems, was evaluated. The extent of protein damage was studied by measuring protein carbonyl groups and protein hydroperoxides. The interaction between Fenton oxidant and EDTA, THF or tBuOOH was further characterized using spin trapping electron spin resonance (ESR) spectroscopy. The results showed that the extent of protein oxidation depended on the oxidant used. The Fenton oxidant was the most reactive of the initiators tested. However, in the absence of EDTA, the Fenton system produced protein carbonyl groups on BSA equivalent to that obtained with the other oxidants, however, significantly more protein hydroperoxide was produced. Surprisingly, it was also found that addition of tBuOOH or THF to BSA reduced protein damage when the oxidation was initiated with the Fenton oxidant. ESR investigation showed that EDTA played a key role in the generation of free radicals. It was also revealed that in an EDTA containing system both tBuOOH and THF were able to react with radicals without inducing protein damage in effect protecting BSA from oxidative damage.     

Isolation and characterization of a tetrasialoganglioside from mouse brain, containing 9-O-acetyl,N-acetylneuraminic acid

A new ganglioside, containing an alkali-labile linkage, was extracted from mouse brain and purified. It represents 3.6% of total lipid-bound sialic acid in the tissue and was obtained in pure form with a yield of about 35%. It contains sphingosine, glucose, galactose, N-acetylgalactosamine and sialic acid in the molar ratio 1:1:2:1:4 and, upon exhaustive sialidase treatment gives the monosialoganglioside GM1. Partial acid hydrolysis, methylation analysis, gas-liquid chromatography-mass spectrometry and chromium trioxide oxidation studies showed its basic neutral glycosphingolipid core to be ganglio-N-tetraose-ceramide. Three of the four sialic acid residues are N-acetylneuraminic acid and one, as shown by gas-liquid chromatography-mass spectrometry, is 9-O-acetyl,N-acetylneuraminic acid, which contains the alkali labile linkage. 9-O-acetyl,N-acetylneuraminic acid is -ketosidically linked to position 8 of the N-acetylneuraminic acid residue bound to position 3 of the internal galactose. The other two N-acetylneuraminic acid residues form a disialosyl residue linked to position 3 of external galactose. The complete structure of the studied ganglioside is as follows: NeuAc2–8NeuAc2–3Galβ1–3GalNAcβ1–4(9-O-Ac-NeuAca2–8NeuAc2-1′-N-acylsphingosine, and it can be considered as a derivative of the tetrasialoganglioside GQ1b.     

Dyestuff wastewater treatment using chemical oxidation,physical adsorption and fixed bed biofilm process

Fenton’s oxidation and activated carbon adsorption were examined as pretreatment processes for dyestuff wastewater having high salinity, colour, and non-biodegradable organic concentrations. In this work, each wastewater stream produced by individual production processes was classified as streams R1, R2, and R3. The stream having a value of BOD₅/COD lower than 0.4 was pretreated by Fenton’s oxidation or activated carbon adsorption to increase the ratio of BOD₅/COD which indicates biodegradability. For Fenton’s oxidation with one stream having a value of BOD₅/COD lower than 0.4, the optimal reaction pH was 3.0 and the minimum dosing concentration (mg l⁻¹) of H₂O₂:FeSO₄·7H₂O was 700:3500. Stream R3, which consisted mainly of methanol was efficiently treated by activated carbon adsorption. The ratio of BOD₅/COD was also increased to 0.432 and 0.31 from 0.06 in Fenton’s oxidation and activated carbon adsorption, respectively. A biological treatment system using a fixed bed reactor was also investigated to enhance biological treatment efficiency at various hydraulic retention times, pretreatment conditions by Fenton’s reagent and salt concentrations by dyestuff wastewater. In addition, the efficiency of Fenton’s oxidation as a post-treatment system was also investigated to present a total treatment process of dyestuff wastewater. As the influent COD and salinity were increased, the effluent SS and COD were consequently increased. However, as the microorganisms became adapted to the changed influent condition, the treatment efficiency of the fixed bed reactor quickly recovered under the high COD and salinity since the microorganisms were well adapted to toxic influent conditions. A wastewater treatment process consisting of chemical oxidation, activated carbon adsorption, fixed bed biofilm process and Fenton’s oxidation as a post-treatment system can be useful to treat dyestuff wastewater having high salinity, colour, and non-biodegradable organic concentration.     

Treatment of Diesel-Contaminated Soil by Fenton and Persulfate Oxidation with Zero-Valent Iron

The objective of this research is to investigate Fenton and persulfate oxidation with zero-valent iron [Fe(0)] as a batch type ex-situ remediation technology for the treatment of diesel-contaminated soil. Results from batch experiments indicate that Fe(0) is a better catalyst for H₂O₂ and persulfate than Fe²⁺ for the enhancement of Fenton and persulfate oxidation in a batch system. Maximum removal was obtained after 12 h when 1 and 2 g of Fe(0) were added to hydrogen peroxide (250 mg/L) and persulfate (250 mg/L), respectively, in a soil-water system. As the amounts of Fe(0) and persulfate were increased three times at the optimal ratio, the removal of total petroleum hydrocarbon (TPH) was enhanced accordingly. More than 90% of the TPH was removed in 3 h, and the treated soil met the Korean regulation level (500 mg/kg) for TPH. Increased amounts of Fe(0) and hydrogen peroxide (up to 10 g and 1250 mg/L, respectively) also significantly enhanced degradation under the optimal conditions. The results of our study suggest that Fe(0)-assisted Fenton and persulfate oxidation in a batch reactor may be an alternative option to treat diesel-contaminated soil.     

Oxidative breakdown and conversion of urocanic acid isomers by hydroxyl radical generating systems.

cis-Urocanic acid (cis-UCA), formed from trans-urocanic acid (trans-UCA) by photoisomerization, has been shown to mimic suppressive effects of UV on the immune system. It is our hypothesis that UCA oxidation products in the skin play a role in the process of immunosuppression. Recently, both UCA isomers were found to be good hydroxyl radical scavengers and in this context we investigated the formation of products resulting from the interaction of hydroxyl radicals with UCA. Hydroxyl radicals were generated by (1) UV/H(2)O(2) (photooxidation), (2) ferrous ions/H(2)O(2) (Fenton oxidation) and (3) cupric ions/ascorbic acid. Oxidation products were identified by spectrometric methods and assessed by reversed-phase HPLC analysis. The photooxidation of UCA was induced by UV-B and UV-C, but not by UV-A radiation. Photooxidation and Fenton oxidation of trans-UCA, as well as of cis-UCA yielded comparable chromatographic patterns of UCA oxidation products. Several of the formed products were identified. The formation of three identified imidazoles was shown in UV-B exposed corneal layer samples, derived from human skin.     

Bulky adducts detected by P-postlabeling in DNA modified by oxidative damage in vitro. Comparison with rat lung I-compounds

Oxygen free radicals, such as the hydroxyl radical generated by interaction of Fe²⁺ and H₂O₂ (Fenton reaction), are produced in mammalian cells as a result of aerobic metabolism and under various pathological conditions and are known to elicit mutations and potentially other adverse effects by reacting with DNA bases. Several products thus formed have recently been characterized as hydroxylated derivatives of cytosine, thymine, adenine, and guanine and imidazole-ring-opened derivatives of adenine and guanine in DNA. As shown herein by ³²P-postlabeling, incubation of DNA under Fenton reaction conditions led to additional products which, by virtue of resistance to nuclease P1 catalyzed 3′-dephosphorylation and chromatographic behavior, appeared to be bulky adducts rather than small polar, hydroxylated or ring-opened nucleotide derivatives. Two major and five minor DNA derivatives were measured after ³²P-postlabeling and TLC mapping of DNA oxidized in vitro under conditions known to lead to formation of reactive oxygen species. Amounts of products formed depended on Fe²⁺ and H₂O₂ concentrations and increased in the presence of -ascorbic acid. One of the two major products was also detected in lung DNA of rats where its amount increased with animal age. Thus, at least one I-compound appeared to have its origin in the interaction of DNA with reactive oxygen species.     

Oxidative breakdown and conversion of urocanic acid isomers by hydroxyl radical generating systems

cis-Urocanic acid (cis-UCA), formed from trans-urocanic acid (trans-UCA) by photoisomerization, has been shown to mimic suppressive effects of UV on the immune system. It is our hypothesis that UCA oxidation products in the skin play a role in the process of immunosuppression. Recently, both UCA isomers were found to be good hydroxyl radical scavengers and in this context we investigated the formation of products resulting from the interaction of hydroxyl radicals with UCA. Hydroxyl radicals were generated by (1) UV/H₂O₂ (photooxidation), (2) ferrous ions/H₂O₂ (Fenton oxidation) and (3) cupric ions/ascorbic acid. Oxidation products were identified by spectrometric methods and assessed by reversed-phase HPLC analysis. The photooxidation of UCA was induced by UV-B and UV-C, but not by UV-A radiation. Photooxidation and Fenton oxidation of trans-UCA, as well as of cis-UCA yielded comparable chromatographic patterns of UCA oxidation products. Several of the formed products were identified. The formation of three identified imidazoles was shown in UV-B exposed corneal layer samples, derived from human skin.     

Antioxidant effects of isoflavonoids and lignans, and protection against DNA oxidation

The antioxidant capability of a series of isoflavonoid and lignan compounds in both cellular and cell-free systems has been investigated, and related to structure. Nordihydroguaiaretic acid exhibited a potent antioxidant activity in both HepG2 and MDA-MB-468 cells (IC₅₀ 5.3 and 1.1 μM respectively), as determined by inhibition of 2',7'-dichlorofluorescin oxidation via t-BOOH, although no inhibition was observed with other compounds tested in this system. All compounds inhibited the formation of 8-oxodeoxyguanosine in DNA exposed to hydroxyl radicals via gamma irradiation or the Fenton reaction. Whilst almost complete inhibition of gamma irradiation-induced damage was achieved (IC₅₀ ranged from 0.2 to 0.8 μM), inhibition was less pronounced with the Fenton system. The ability of all compounds to interact with DNA (as well as with reactive oxygen and iron) was also demonstrated by scanning UV spectroscopy, suggesting that the compounds may inhibit DNA oxidation at least in part by binding to DNA. Hydroxyl radical-scavening, iron-chelating and DNA-binding activity of these compounds supports their proposed role as natural cancer-protective agents.     

Biocatalytic oxidation of S-alkylcysteine derivatives by chloroperoxidase and Beauveria species

Treatment of N-methoxycarbonyl C-carboxylate ester derivatives of S-methyl- -cysteine by chloroperoxidase (CPO)/hydrogen peroxide resulted in oxidation at sulfur to produce the (R_S) sulfoxide in moderate to high diastereomeric excess (DE). The (S_S) natural product sulfoxide chondrine was obtained via biotransformation of the N-t.boc derivative of -4-S-morpholine-2-carboxylic acid using Beauveria bassiana or Beauveria caledonica.     

Production of hydroxyl radical by the synergistic action of fungal laccase and aryl alcohol oxidase

A mechanism for the production of hydroxyl radical (*OH) during the oxidation of hydroquinones by laccase, the ligninolytic enzyme most widely distributed among white-rot fungi, has been demonstrated. Production of Fenton reagent (H2O2 and ferrous ion), leading to *OH formation, was found in reaction mixtures containing Pleurotus eryngii laccase, lignin-derived hydroquinones, and chelated ferric ion. The semiquinones produced by laccase reduced both ferric to ferrous ion and oxygen to superoxide anion radical (O2*-). Dismutation of the latter provided the H2O2 for *OH generation. Although O2*- could also contribute to ferric ion reduction, semiquinone radicals were the main agents accomplishing the reaction. Due to the low extent of semiquinone autoxidation, H2O2 was the limiting reagent in Fenton reaction. The addition of aryl alcohol oxidase and 4-methoxybenzyl alcohol (the natural H2O2-producing system of P. eryngii) to the laccase reaction greatly increased *OH generation, demonstrating the synergistic action of both enzymes in the process.     

A method to determine RNA and DNA oxidation simultaneously by HPLC-ECD: greater RNA than DNA oxidation in rat liver after doxorubicin administration

We developed a novel method for the simultaneous extraction and analysis of total tissue RNA and DNA to quantify the RNA and DNA oxidation products 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine using HPLC coupled to electrochemical detection (HPLC-ECD). The protein denaturing agents guanidine thiocyanate and phenol/chloroform at neutral pH were found to be very efficient for the isolation of RNA and DNA from rat brain, liver and muscle. The method is very fast, allows extraction at 0 degrees C, gives high yields of pure RNA and DNA with low background oxidation levels, and also determines the RNA/DNA ratio. Experiments with isolated RNA and DNA exposed to the Fenton reagents H2O2/ascorbate/Fe3+ (or Cu2+) resulted in significantly greater RNA oxidation. The RNase inhibitor 2-mercaptoethanol, commonly used for RNA extraction, acted as a pro-oxidant during nucleic acid extraction, an effect attenuated by the inclusion of the metal chelator deferoxamine mesylate. In vivo, administration of doxorubicin (an oxidant generator) to Fisher-344 rats resulted in a significant increase in liver RNA oxidation, but no significantly increased DNA oxidation. This new method could be useful to assess oxidatively damaged RNA and DNA simultaneously, and our data show that RNA is more susceptible to oxidative stress than DNA in vivo and in vitro.     

Oxidation of the ketoxime acetoxime to nitric oxide by oxygen radical-generating systems.

Ketoximes undergo a cytochrome P450-catalyzed oxidation to nitric oxide and ketones in liver microsomes. In addition, nitric oxide synthase (NOS) can catalyze the oxidative denitration of the >C=N-OH group of amidoximes. The objective of this work was to characterize the oxidation of a ketoxime (acetoxime) and to assess the ability of NOS to catalyze the generation of nitric oxide/nitrogen monoxide (*NO) from acetoxime. Acetoxime was oxidized to NO2- (and NO3-) by microsomes enriched with several P450 isoforms, including CYP2E1, CYP1A1, and CYP2B1. Nitric oxide was identified as an intermediate in the overall reaction. Superoxide dismutase and catalase significantly inhibited the reaction. Exogenous iron increased the microsomal generation of NO2- from acetoxime, while metal chelators (desferrioxamine, EDTA, DTPA) inhibited it. A Fenton-like system (Fe2+ plus H2O2, pH 7.4) consumed acetoxime with production of NO2- and NO3-, whereas oxidation by superoxide or by H2O2 was inefficient. The results presented suggest a role for hydroxyl radical-like oxidants in the oxidation of acetoxime to nitric oxide. O-Acetylacetoxime and O-tert-butylacetoxime were not oxidized by a Fenton system or by liver microsomes to any significant extent. Formation of the 5,5'-dimethyl-1-pyrroline-N-oxide/. OH adduct by a Fenton system was significantly inhibited by acetoxime, while O-acetylacetoxime and O-tert-butylacetoxime were inactive. These results suggest that the. OH-dependent oxidation of acetoxime initially proceeds via abstraction of a hydrogen atom from its hydroxyl group, as opposed to the oxidation of its >C=N- function. HepG2 cells with low levels of expression of P450 did not significantly produce NO2- from acetoxime, while HepG2 cells expressing CYP2E1 did, and this generation was blocked by a CYP2E1 inhibitor. Acetoxime was inactive either as a substrate or as an inhibitor of iNOS activity. These results indicate that reactive oxygen species play a key role in the oxidation of acetoxime to. NO by liver microsomes by a mechanism involving H abstraction from the OH moiety by hydroxyl radical-like oxidants and suggest the possibility that acetoxime may be an effective producer of. NO primarily in the liver by a pathway independent of NOS.     

An electrochemical biosensor for the detection of tyrosine oxidation induced by Fenton reaction

A simple and sensitive electrochemical biosensor was used to detect tyrosine oxidation induced by hydroxyl radicals generated by Fenton reaction (Fe(2+)/H(2)O(2)). Poly(glu, tyr) (4:1) peptides were immobilized on indium tin oxide (ITO) electrode surface via layer-by-layer assembly technique, and Os(bpy)(3)(2+)-mediated tyrosine oxidation current was employed as the signal reporter of the biosensor. It was found that the electrochemical signal of the peptide decreased markedly after incubation with Fenton reagents. Interestingly, L-dopa, the oxidation product of tyrosine, was likely to form complexes with Fe(III), which could suppress the electro-oxidation of L-dopa and resulted in decrease of current response. Our results indicate that the peptide damage involved two steps and was a second-order reaction. X-ray photoelectron spectroscopy was used to quantitatively determine nitrogen elemental percentage on peptide-coated electrode surface, which eliminated the possibility that signal decrease was caused by peptide backbone cleavage. Moreover, the lowest concentration of Fenton reagents that could be detected was 10 μM Fe(2+) or H(2)O(2), similar to the level in vivo. We suggest that the biosensor can be used to detect protein damage induced by Fenton reaction.     

Exogenous N-linoleoyl tyrosine marker as a tool for the characterization of cellular oxidative stress in macrophages

Oxidative stress and its resultant products continue to attract investigators. Numerous endogenous substances have been suggested as potential markers for the identification of oxidative stress in tissues and organisms. In this study, we present a novel concept whereby an exogenous marker is designed and synthesized for the characterization of oxidative stress. The designed marker is constructed from tyrosine (Tyr) and linoleic acid (LA), which are attached covalently to form N-linoleoyl tyrosine (N-LT). Each of the two components (Tyr and LA) is known to be easily oxidized upon exposure to different types of reactive species. Combining the two allows their distinction from the endogenous Tyr and LA in the tested biological samples. The ability of the N-LT marker to characterize oxidative stress in macrophage cell lines was first studied using different types of ROS/RNS. N-LT was found to interact with macrophages, binding to the cell membrane. Upon treatment of J-774 A.1 macrophages with N-LT (40 μM) and with various oxidants; HOCl (0.2, 0.4 mM), copper ions (20 μM), SIN-1 (0.1, 1.0 mM), specific oxidized N-LT (Ox-N-LT) products were formed, depending on the type of oxidant used. Exposing cells to HOCl (0.2 mM) resulted in exclusive attack of the LA residue of N-LT, preferentially forming an adduct of HOCl to the LA double bond (N-L(HOCl)T, 4.3%). In contrast, when SIN-1 (0.1 mM) was applied as the oxidant, the Tyr moiety of N-LT was most reactive, yielding a nitration product of the Tyr aromatic ring (N-LT(NO₂), 1.8%). Similar N-LT oxidation in cell-free systems yielded a significantly higher content of Ox-N-LT (10.8% N-L(HOCl)T, 7% N-LT(NO₂)). The designed marker was then tested with peritoneal macrophages taken from atherosclerotic apolipoprotein-deficient (E⁰) mice showing specific and selective oxidation of N-LT to yield N-LT-hydroperoxide (1.9% N-L(OOH)T), at significantly higher levels than resulted from similar experiments using peritoneal macrophages harvested from control BalbC mice (0.0% N-L(OOH)T). In contrast, the differences in N-L(epoxy)T level between BalbC and E⁰ mice were not significant using both types of peritoneal macrophages (E⁰ and BalbC), suggesting that N-L(OOH)T is characteristic of the atherosclerotic state. Thus, we show that the designed marker is sufficiently sensitive to detect oxidative stress imposed on cells and cell-free systems and to react selectively with the various ROS/RNS induced. Such a marker may be useful for characterizing oxidative stress in general, and possibly also in oxidative-stress-associated diseases.     

Membrane potential generation by two reconstituted mitochondrial systems: Liposomes inlayed with cytochrome oxidase or with ATPase

Formation of a membrane potential in two types of liposomes, one inlayed with cytochrome c + cytochrome oxidase, and another, with oligomycin-sensitive ATPase, has been demonstrated. To detect a membrane potential, phenyl dicarbaundecaborane (PCB⁻), a penetrating anion probe, was used.

The first type of liposome was reconstituted from a solution of purified cytochrome oxidase, mitochondrial phospholipids and cytochrome c, the latter being enclosed inside liposomes. Cytochrome c bound to the outer surface of the liposome membrane was removed by washing with NaCl. Such liposomes catalyzed oxidation of ascorbate by oxygen in the presence of phenazine methosulfate or N,N,N′,N′-tetramethyl-p-phenylenediamine. The oxidation was found to support the PCB⁻ uptake by liposomes. The PCB⁻ response was prevented and reversed by cyanide, protonophorous uncouplers and external cytochrome c.

Liposomes of the second type were prepared from a solution of mitochondrial phospholipids, coupling factors F₁and F_c, and the hydrophobic proteins of the oligomycin-sensitive ATPase. These liposomes catalyzed ATP hydrolysis coupled with the PCB⁻ uptake. The latter effect was prevented and reversed by oligomycin and uncouplers.

The conclusion is made that membrane potential can be independently formed by enzymic reactions of two different kinds: (1) redox (e.g. cytochrome c oxidase) and (2) hydrolytic (ATPase).     

Biohydroxylation of N,N-dialkylarylamines by the isolated topsoil bacterium Bacillus megaterium

Topsoil microorganisms were screened for their acceptability of the standard substrate N,N-dimethylaniline in bacterial ‘whole-cell’ incubations. One bacterium converted N,N-dimethylaniline and was identified as Bacillus megaterium by 16S rDNA analysis and DNA/DNA-hybridization. In contrast to the well-known C-hydroxylation by liver microsomes, leading to p-hydroxylation, B. megaterium formed o- and p-monohydroxylated products, i.e. N,N-dimethyl-2-aminophenol and N,N-dimethyl-4-aminophenol, both identified by gas chromatography–mass spectrometry (GC–MS) using synthesized reference compounds. The observed hydroxylation showed slight regioselectivity in favour of the o-hydroxylated product. Two further substrates, N,N-diethylaniline and N-ethyl-N-methylaniline, were also successfully biohydroxylated by B. megaterium with corresponding regioselectivity. Interestingly, aniline, known to be transformed easily by cytochrome P-450meg into p-aminophenol, was not accepted as substrate.     

Progress in arylpiperazine synthesis by the catalytic amination reaction

Careful base and solvent optimization for catalytic amination is described. A Pd-catalyzed amination between some arylbromide and unprotected piperazine (1 equiv) was efficiently carried out with Pd/BINAP catalyst in a toluene–DBU solvent system, which is useful for the one-pot preparation of unsymmetrical piperazine through amination and in-situ N-protection. Reaction with N-BOC-piperazine was also successful in toluene–DBU or more polar NMP with Cs₂CO₃ as a key base. No reports have previously reported such solvent and base optimization in arylpiperazine synthesis.     

Difference in 2-thenoyltrifluoroacetone sensitivity of electron transport with and against the redox potential gradient

The effect of 2-thenoyltrifluoroacetone on electron transport with and against the redox potential gradient, with succinate or ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) as electron donor, was studied in rat liver mitochondria. It was found that 2-thenoyltrifluoroacetone inhibited succinate-linked intramitochondrial pyridine nucleotide reduction at low concentrations, which neither affected succinate oxidation in the controlled state nor interfered with intramitochondrial pyridine nucleotide reduction in the ascorbate plus TMPD case. The effect of 2-thenoyltrifluoroacetone on succinate-linked intramitochondrial pyridine nucleotide reduction is not attributable either to blocking of the overall rate of electron flow in the succinate dehydrogenase branch of the respiratory chain or to interference with energy transformation. Transition from the controlled to the active state enhanced the inhibitory effect of 2-thenoyltrifluoroacetone on succinate-linked respiration, and it became as sensitive to 2-thenoyltrifluoroacetone as the succinate-linked intramitochondrial pyridine nucleotide reduction. In the light of the above findings, the possibility is discussed that electrons from succinate enter the main branch of the respiratory chain by different routes, according to whether the flow is with or against the potential gradient.     

Formation of ribonucleotides in DNA modified by oxidative damage in vitro and in vivo. Characterization by 32P-postlabeling.

Oxygen free radicals generated by the interaction of Fe2+ and H2O2 (Fenton reaction) are capable of reacting with DNA bases, which may induce premutagenic and precarcinogenic lesions. Products formed in DNA by such reactions have been characterized as hydroxylated derivatives of cytosine, thymine, adenine, and guanine and imidazole ring-opened derivatives of adenine and guanine. As shown here by 32P-postlabeling, incubation of DNA under Fenton reaction conditions gave rise to additional oxidation products in DNA that were characterized as putative ribonucleosides by enzymatic hydrolysis of the oxidized DNA, 32P-postlabeling, and co-chromatography in multiple systems with authentic markers. Formation of these products in DNA was enhanced by the presence of L-ascorbic acid in the reaction mixtures and their total amounts were similar to those of the major DNA oxidation product, 8-hydroxy-2'-deoxyguanosine. The ribonucleoside guanosine was also formed in kidney DNA of male rats treated with ferric nitrilotriacetate, a renal carcinogen. It is postulated that ribonucleotides alter conformation and function of DNA and thus their presence in DNA may lead to adverse health effects.