Detection of 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage, in white blood cells of workers occupationally exposed to styrene

Styrene-7,8-oxide (SO), the major in vivo metabolite of styrene, is a genotoxic compound and a potential carcinogenic hazard to occupationally exposed workers. The aim of the present work was to investigate the ability of styrene exposure to induce formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in white blood cells (WBC) of boatbuilders occupationally exposed to styrene. The study of these adducts was conducted to see if styrene exposure can cause oxidative damage of DNA. The 8-OHdG/10(5) dG ratio from 17 styrene-exposed workers showed significant increases (mean +/- SD, 2.23 +/- 0.54, median 2.35, P < 0.001) in comparison to the controls (1.52 +/- 0.45, median 1.50). However, 11 out of 17 workers who were between the ages of 32 and 60 years and had been occupationally exposed to styrene for > 10 years showed higher 8-OHdG/10(5) dG ratios (2.31 +/- 0.62, median 2.37) in comparison to 6 workers with < 6 years of occupational styrene-exposure (2.11 +/- 0.36, median 2.05; P > 0.05, no significant difference between the two groups of workers). The studies presented here provide an indication that styrene exposure can result in oxidative DNA damage.     

Induction and repair of double-strand breaks in the replicating DNA of HeLa cells

The photochemical (lambda < 400 nm) decomposition of some monocyclic and polycyclic nitramines produces .NO2, which can be detected in the respective nitramine crystals at 77 K by EPR (electron paramagnetic resonance). In solutions of perdeutero-dimethylsulfoxide (DMSO-d6) the .NO2 produced by photolytic decomposition of dissolved nitramines can be spintrapped by the solvent to give a radical having the structure CD3-(SO2)-(NO.)-CD3. In this article, we examine this reaction for two nitramines: cyclotrimethylenetrinitramine (RDX) and hexanitrohexaazaisowurzitane (HNIW), which are energetic materials. The decay of the spin-adduct radical (I) follows first-order kinetics for both nitramines studied, having a rate constant (k) of congruent to 7.1 x 10(-4) s-1. The net growth in spin concentration of (1) measured from EPR spectra is fitted by a first-order rate equation taking into account the simultaneous competitive decay rate of spin adduct (I). Using the rate data and EPR spin concentration data, the ratio of free .NO2 produced per parent nitramine molecule is estimated as 1:1 for RDX and 4:1 for HNIW. Biological implications of trapping of .NO2 by dimethyl sulfoxide are discussed.     

Glutathione modulates the formation of 8-hydroxydeoxyguanosine in isolated DNA and mutagenicity in Salmonella typhimurium TA100 induced by mineral fibres

Treatment of isolated DNA with crocidolite and man-made vitreous fibre-21 (MMVF-21) significantly increased the concentration of 8-hydroxydeoxyguanosine (8-OHdG) in isolated DNA above background levels and co-treatment with glutathione (GSH) eliminated this effect. Crocidolite, MMVF-21 and chrysotile fibres increased the number of revertants in Salmonella typhimurium TA100 and GSH-deficient strains, TA100/NG-54 and TA100/NG-57, over background levels. This increase was small in TA100 but was greater in the GSH-deficient strains. When these bacterial strains were further depleted of GSH by co-culture with buthionine sulfoximine, all fibres tested caused a significant increase in the number of revertants over the parent strains. Pre-treatment with the GSH precursor N-acetyl-L-cysteine reduced the number of revertants to below that of the parent strain. Previous studies have shown a mechanistic role for iron-catalyzed production of oxygen radicals in the mutagenicity of fibres and this study suggests a protective role for GSH against such oxidative damage possibly by acting as a radical scavenger.     

Induction of oxidative DNA damage by ferric iron in mammalian cells

Ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-citrate) were compared with respect to their potential to induce oxidative DNA damage in V79 Chinese hamster cells. DNA base modifications, including 8-hydroxyguanine (7,8-dihydro-8-oxoguanine), were quantified by the frequency of lesions recognized by the bacterial Fpg protein (formamidopyrimidine-DNA glycosylase) in combination with the alkaline unwinding assay. Fe-NTA induced oxidative DNA damage in a time- and dose-dependent manner, yielding significant increases in Fpg-sensitive sites above background after incubation for 24 or 48 h with 500 and 250 microM respectively. At both time points the frequency of DNA base modifications exceeded the number of DNA strand breaks. In contrast, neither DNA strand breaks nor Fpg-sensitive sites were detected after treatment with Fe-citrate at concentrations up to 2 microM for 24 or 48 h; this inactivity of Fe-citrate was independent of the molar ratio of iron to ligand (1:1, 1:2, 1:10 or 1:20). The results indicate that the cellular damage induced by ferric iron depends strongly on the actual complex applied, possibly due to differences in the intracellular distribution, which in turn may affect the availability of iron for redox reactions at or in close proximity to the DNA.     

Oxidation of NG-hydroxy-L-arginine by nitric oxide synthase: evidence for the involvement of the heme in catalysis

The involvement of the protoporphyrin IX heme iron of macrophage nitric oxide synthase (NOS) in the oxidation of NG-hydroxy-L-arginine (L-NHA) to nitric oxide (NO) and citrulline was investigated by carbon monoxide (CO) inhibition studies and binding difference spectroscopy. A CO:oxygen mixture (80:20) was found to inhibit the reaction by 33% with L-NHA as a substrate compared to 57% with L-arginine. Spectral perturbations were observed upon the addition of L-NHA to oxidized NOS, producing a type I binding difference spectrum with a maximum at 384 nm and minimum at 420 nm. In addition, L-NHA was incapable of reducing anaerobic oxidized NOS in the absence of NADPH. These studies support the involvement of the heme in the oxidation of L-NHA to NO and citrulline, indicating that the heme functions in both of the currently characterized oxidative steps of the NOS reaction.     

Human intestinal epithelial cells respond to Cryptosporidium parvum infection with increased prostaglandin H synthase 2 expression and prostaglandin E2 and F2alpha production

Cryptosporidium parvum is an important cause of diarrhea in humans and several animal species. Prostaglandins play a central role in regulating intestinal fluid secretion in animal models of cryptosporidiosis, but their cellular sources and mechanisms of induction are unclear. Here, we show that C. parvum infection directly activates prostaglandin H synthase 2 expression and prostaglandin E2 and F2alpha production in human intestinal epithelial cells.     

Viral DNA synthesis in vitro with the inclusions isolated from adenovirus 12-infected cells

A fraction defined as the inclusions was isolated by banding in CsC1 gradients from nuclei of adenovirus 12-infected KB cells. When examined by electron microscopy, the isolated inclusions were relatively homogeneous, finely granular materials of moderate electron density, possibly representing the disintegrated type II or IV inclusions. The conditions of endogenous DNA synthesis in vitro with the inclusions were determined. The product of DNA synthesis in vitro with the inclusions was mainly viral and scarcely cellular, as revealed by DNA-DNA hybridization and methylated albumin kieselgur column chromatography. However, viral DNA synthesized in vitro was smaller (18S, 22S) than viral DNA in virions (31 S, 34 S) in neutral and alkaline sucrose gradients. Effects of various treatment of the inclusions on the DNA-synthesizing activity showed that phospholipase C inhibited the activity efficiently. The in vitro DNA synthesis was stimulated by addition of the cytoplasmic extract from adenovirus 12-infected cells and not that from unifected cells. The analysis of the composition of the inclusions showed that the inclusions contained DNA, protein, phospholipid and a small amount of RNA and carbohydrate.     

Phenylhydrazones as new good substrates for the dioxygenase and peroxidase reactions of prostaglandin synthase: formation of iron(III)-sigma-phenyl complexes

Phenylhydrazones of various aromatic and aliphatic aldehydes or ketones act as good substrates of the dioxygenase reaction of prostaglandin synthase (PGHS). Corresponding alpha-azo hydroperoxides are formed as intermediates with maximum initial rates of O2 consumption between 8 and 230 mol (mol of PGHS)-1 s-1 for benzophenone and hexanal phenylhydrazone, respectively. The Km values for these reactions vary from 100 to 300 microM. These alpha-azo hydroperoxides are then converted to the corresponding alpha-azo alcohols by the peroxidase reaction of PGHS. During such oxidations of phenylhydrazones by PGHS, a new complex of this hemeprotein characterized by peaks at 438 and 556 nm is formed. This complex was obtained both by direct reaction of PGHS Fe(III) with phenyldiazene and by reaction of PGHS Fe(III) with phenylhydrazine in the presence of O2. By analogy to results previously reported for hemoglobin, myoglobin, catalase, and cytochrome P450, this species should be a sigma-phenyl PGHS FeIII-Ph complex. The PGHS FeIII-Ph complex should derive from an oxidation of the intermediate alpha-azo alcohol by PGHS Fe(III), cleavage of the resulting alkoxy radical with formation of a ketone (or aldehyde) and Ph*, and combination of PGHS Fe(II) with Ph*. Such an oxidation of alpha-azo alcohols by lipoxygenase-FeIII with formation of Ph* was reported previously. The formation of Ph* and of PGHS FeIII-Ph is likely the cause of the inhibitory effects previously reported for arylhydrazones toward PGHS.     

Competitive reactions of peroxynitrite with 2'-deoxyguanosine and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG): relevance to the formation of 8-oxodG in DNA exposed to peroxynitrite

We have examined the formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) in reactions of peroxynitrite with 2'-deoxyguanosine (dG) and calf-thymus DNA. Peroxynitrite reacts with dG at neutral pH, but this reaction does not result in the buildup of 8-oxodG. We also do not find any evidence for the formation of 8-oxodG in calf-thymus DNA upon exposure to peroxynitrite. When 8-oxodG is mixed with 1000-fold excess dG and then allowed to react with peroxynitrite, about 50% of the 8-oxodG is destroyed. The preferential reaction of 8-oxodG is also evident when dG in calf-thymus DNA is partially oxidized in an Udenfriend system and then allowed to react with peroxynitrite. We suggest that 8-oxodG is not produced in peroxynitrite-mediated oxidations of dG and DNA or that it is produced but then is rapidly consumed in further reactions with peroxynitrite. Oxidized DNA bases frequently can be more oxidation sensitive than their corresponding progenitors and, therefore, may be present at] low steady-state concentrations and not represent stable markers of oxidative stress status. The importance of the 8-oxodG/peroxynitrite reaction is discussed in relation to the formation of more stable, secondary oxidation products that might be more useful markers of DNA damage.     

In vivo evidence for the involvement of anionic phospholipids in initiation of DNA replication in Escherichia coli

In vitro, anionic phospholipids can reactivate inactivated DnaA protein, which is essential for initiation of DNA replication at the oriC site of Escherichia coli [Sekimizu, K. & Kornberg, A. (1988) J. Biol. Chem. 263, 7131-7135]. Mutations in the pgsA gene (encoding phosphatidylglycerophosphate synthase) limit the synthesis of the major anionic phospholipids and lead to arrest of cell growth. We report herein that a mutation in the rnhA gene (encoding RNase H) that bypasses the need for the DnaA protein through induction of constitutive stable DNA replication [Kogoma, T. & von Meyenburg, K. (1983) EMBO J. 2, 463-468] also suppressed the growth arrest phenotype of a pgsA mutant. The maintenance of plasmids dependent on an oriC site for replication, and therefore DnaA protein, was also compromised under conditions of limiting anionic phospholipid synthesis. These results provide support for the involvement of anionic phospholipids in normal initiation of DNA replication at oriC in vivo by the DnaA protein.     

Further evidence for central histamine H2-receptor involvement in the hypotensive effect of clonidine in the rat

Arteriosclerotic aneurysms of the abdominal aorta constitute a common clinical entity. Rarely are they associated with retroperitoneal fibrosis and ureteral obstruction requiring ureterolysis. Fifteen such cases have been reported, with resection successful in 5 of 7. A sixteenth case is presented complicated by the presence of a persistent left cardinal vein. It is the third aneurysm resected with such an anomaly, and to our knowledge the first to be associated with retroperitoneal fibrosis and ureteral obstruction. Ureterolysis with resection of the aneurysm was performed. The difficulties presented by these pathologic entities, as well as the anomalous venous pattern, are reviewed. Complete preoperative evaluation, including intravenous pyelogram, retrograde pyelography, aortography, and venacavography, for the definition of anatomic relationships and planning of the surgical approach is stressed.     

Thromboxane synthase inhibitor, UK 38485, prevents renal injury in the rabbit isolated perfused kidney exposed to cold ischemia

Recent studies have indicated that, the administration of thromboxane A2 (TxA2) inhibitors improved renal functions in experimental renal allograft transplantation. Thus TxA2, a vasoconstrictor metabolite of arachidonic acid, may play a role in renal function and blood flow during hypothermic storage. The aim of the present study was to evaluate the cytoprotective effect of TxA2 synthase inhibitor, UK 38485, on altered renal function due to cold ischemia for 24 and 72 h. Experiments were performed in isolated perfused kidney from adult rabbits. Kidneys were perfused with Euro-Collins (EC) containing UK 38485 and incubated with the same solution in a beaker exposed to cold ischemia for 24 and 72 h. The same procedure was applied to the control kidneys in EC solution alone. Vascular responses and urinary output to noradrenaline, angiotensin II, endothelin-1, acetylcholine, and sympathetic stimulation were assessed as the functional activity of kidney. The addition of UK 38485 to EC solution increased the preservation time of kidney and protects the vascular endothelial regulatory functions and urine excretion when compared to EC alone. The results of the present study can be taken as an evidence of the cytoprotective effect of the UK 38485 and might be useful for kidney preservation.     

Endogenous oncornaviral DNA sequences: evidence for two classes of viral DNA sequences in guinea pig cells

The nature of the endogenous viral DNA sequences in guinea pig cells was studied by hybridization. A segment of the viral RNA (r-VRNA) hybridizing to abundant (or reiterated) DNA sequences (R-VDNA) was isolated by recycling to a Cot of 300. The hybridization of the recycled VRNA, as well as the total VRNA, was followed by determining their kinetics and by Wetmur-Davidson analysis. The kinetics of hybridization of total VRNA were complex, did not follow a second-order kinetics, and revealed two slopes by Wetmur-Davidson analysis. The recycled RNA, on the other hand, had a second-order reaction rate expected of the hybridization between a single species of RNA and DNA sequences and yielded a single straight line in a Wetmur-Davidson plot. The Cot1/2 and slope of the recycled r-VRNA was almost identical to that of the abundant VDNA sequences obtained from the hybridization data of the total VRNA. Guinea pig 28S rRNA with or without recycling was used in monitoring hybridization rate. The kinetics of hybridization of 28S RNA followed a second-order reaction and produced a single straight line by Wetmur-Davidson plot, with a second-order reassociation rate constant of 9.6 x 10(-3) liters/mol-s, a Cot1/2 of 104 mol-s/liter, and reiteration frequency of 146. There was no difference in the kinetics of hybridization of 28S RNA before and after recycling. These experiments showed that guinea pig cells contain two classes of VDNA sequences. (i) R-VDNA sequences with a second-order reassociation rate constant of 8.2 x 10(-4) liters/mol-s, a Cot1/2 of 1,219 mol-s/liter, and a reiteration frequency of 12 represent 37.5% of the viral genome. (ii) Unique VDNA sequences with a second-order reassociation rate constant of 1.2 x 10(-4) liters/mol-s, a Cot1/2 of 7,692 mol-s/liter, and a reiteration frequency of 2 represent 62.5% of the viral genome.     

Vaccinia locomotion in host cells: evidence for the universal involvement of actin-based motility sequences ABM-1 and ABM-2

Vaccinia uses actin-based motility for virion movement in host cells, but the specific protein components have yet to be defined. A cardinal feature of Listeria and Shigella actin-based motility is the involvement of vasodilator-stimulated phosphoprotein (VASP). This essential adapter recognizes and binds to actin-based motility 1 (ABM-1) consensus sequences [(D/E)FPPPPX(D/E), X = P or T] contained in Listeria ActA and in the p90 host-cell vinculin fragment generated by Shigella infection. VASP, in turn, provides the ABM-2 sequences [XPPPPP, X = G, P, L, S, A] for binding profilin, an actin-regulatory protein that stimulates actin filament assembly. Immunolocalization using rabbit anti-VASP antibody revealed that VASP concentrates behind motile virions in HeLa cells. Profilin was also present in these actin-rich rocket tails, and microinjection of 10 microM (intracellular) ABM-2 peptide (GPPPPP)3 blocked vaccinia actin-based motility. Vinculin did not colocalize with VASP on motile virions and remained in focal adhesion contacts; however, another ABM-1-containing host protein, zyxin, was concentrated at the rear of motile virions. We also examined time-dependent changes in the location of these cytoskeletal proteins during vaccinia infection. VASP and zyxin were redistributed dramatically several hours before the formation of actin rocket tails, concentrating in the viral factories of the perinuclear cytoplasm. Our findings underscore the universal involvement of ABM-1 and ABM-2 docking sites in actin-based motility of Listeria, Shigella, and now vaccinia.     

Characterization and evaluation by 32P-postlabelling of psoralen-type DNA adducts in HeLa cells

Samples of DNA irradiated at 405 and/or 365 nm in the presence of 8-methoxypsoralen (8-MOP) were analysed via a modified postlabelling assay using three hydrolysis enzymes other than those employed previously. These enzymes (deoxyribonucleaseI, venom phosphodiesterase and alkaline phosphatase) liberated 3'-adducted dinucleotide monophosphate instead of the 5'-modified dinucleotide monophosphate normally obtained. The first separation chromatography (D1) of samples irradiated in the presence of 8-MOP showed a single spot above the origin, and the next separation (D2) resolved this spot into two components (spots I and II). Double irradiation experiments in which samples of DNA were first irradiated at 405 nm before being irradiated at 365 nm showed that spot II could be transformed into spot I. The use of 6,4,4'-trimethylangelicin, which induced only photomonoadducts under UVA irradiation, gave only spot II. These two results indicated that spots I and II were respectively due to interstrand cross-links and monoadducts. Dose-effect experiments showed that spots I and II were dose dependent, and low-dose irradiations permitted us to measure one interstrand cross-link and two monoadducts per 10(8) base pairs.     

Aspirin inhibition and acetylation of the plant cytochrome P450, allene oxide synthase, resembles that of animal prostaglandin endoperoxide H synthase

The enzymatic reactions leading to octadecanoid lipid signaling intermediates in plants are similar to those of animals and are inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs) such as salicylic acid and aspirin. In animals, NSAIDs inhibit the cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthase, which ultimately blocks the formation of prostaglandins. In plants, NSAIDs block the formation of 12-oxo-phytodienoic acid and jasmonates, which are the equivalent signaling compounds. In this study we show that NSAIDs act as competitive inhibitors of allene oxide synthase (AOS), the cytochrome P450 that initiates plant oxylipin synthesis. We also show that aspirin causes the time-dependent inhibition and acetylation of AOS, which leads the irreversible inactivation of this enzyme. This inhibition and acetylation superficially resembles that observed for the inactivation of COX in animals. In AOS, aspirin acetylates three serine residues near the C-terminal region that appear to be highly conserved among AOS sequences from other plants but are not conserved among "classical" type P450s. The role of these serine residues is unclear. Unlike animal COX, where acetylation of a single serine residue within the substrate channel leads to inactivation of prostaglandin endoperoxide H synthase, the three serine residues in AOS are not thought to line the putative substrate channel. Thus, inhibition by aspirin may be by a different mechanism. It is possible that aspirin and related NSAIDs could inhibit other P450s that have motifs similar to AOS and consequently serve as potential biochemical targets for this class of drugs.     

Brain self-stimulation: direct evidence for the involvement of dopamine in the prefrontal cortex

Rats were trained to self-stimulate the medial prefrontal cortex, a region rich in dopaminergic terminals. After the region adjacent to the electrode site was labeled with [14C]dopamine, it was perfused repeatedly by means of push-pull cannulas. Electrical stimulation of this cortical area in six animals enhanced the release of dopamine and its associated metabolites in nine of 16 experiments. Thus in vivo evidence is provided that dopamine is involved in the brain self-stimulation mechanism within the frontal cortex.