The effect of trichlorfon and other organophosphates on prenatal brain development in the guinea pig

The organophosphates trichlorfon, dichlorvos, dimethoate, soman, triortho-cresyl phosphate (TOCP), and the diethoxy-analogue of trichlorfon (O,O-diethyl 2,2,2-trichloro-1-hydroxyethylphosphonate, ethyl-trichlorfon), were administrated to guinea pigs between day 42 and 46 of gestation. When the offsprings were examined at birth, there was a severe reduction in brain weight in the case of trichlorfon and dichlorvos, but not after treatment with the other organophosphates. The reduction in weight was most pronounced for cerebellum, medulla oblongata, thalamus/hypothalamus and quadrigemina. The effect was less marked for cerebral cortex and hippocampus. Since soman, a potent anticholinesterase, and TOCP, an inhibitor of neuropathy target esterase, did not show any effects, this excludes that the brain hypoplasia can be caused by inhibition of these two enzymes. Further, the lack of effect with ethyl-trichlorfon has shed some light on the part of the trichlorfon molecule which could be involved in the formation of the hypoplasia. It is suggested that alkylation of DNA may be involved in the development of the lesion. The possible consequences for a teratogenic effect of trichlorfon and dichlorvos on humans are discussed.Special issue dedicated to Dr. Bernard W. Agranoff.     

Activity of organophosphorus insecticides in bacterial tests for mutagenicity and DNA repair — Direct alkylation vs. metabolic activation and breakdown. I. Butonate,vinylbutonate, trichlorfon,dichlorvos, demethyl dichlorvos and demethyl vinylbutonate

The following organophosphates were tested for their ability to induce DNA damage in a rec-type repair test with Proteus mirabilis strains PG713 (rec^? hcr^?) and PG273 (wild type) and point mutations in his^? strain TA100 of Salmonella typhimurium — butonate: O,O-dimethyl-(1-n-butyryloxy-2,2,2-trichloroethyl)-phosphonate; vinylbutonate: O,O-dimethyl-(n-butyryloxy-2,2-dichlorovinyl)-phosphonate; trichlorfon: O,O-dimethyl-(1-hydroxy-2,2,2-trichloroethyl)-phosphonate; dichlorvos: O,O-dimethyl-O-(2,2-dichlorovinyl)-phosphate; the demethylated derivatives — demethyldichlorvos: O-methyl-O-(2,2-dichlorovinyl)-phosphoric acid; demethyl vinylbutonate: O-methyl-(1-n-butyryloxy-2,2-dichlorovinyl)phosphonic acid. Of the six compounds tested, dichlorvos and trichlorfon induced base pair substitutions and DNA damage. No mutagenicity and DNA damage were found in experiments with butonate, vinylbutonate, demethyl vinylbutonate and demethyl dichlorvos. Genotoxic activity for dichlorvos and the absence of both mutagenic and DNA damaging properties for its non-alkylating demethyl derivative favors the hypothesis that alkylation of DNA is the essential step for mutation induction by this organophosphate. Furthermore, the absence of genetic effects after treatment with vinylbutonate and demethyl dichlorvos does not support a crucial role of vinyl or allyl groups in side chains of organophosphates for genetic activity. Microsomal enzymes decreased genetic activity of dichlorvos and trichlorfon in vitro. No evidence for a role of metabolic activation in the mutagenic activity of any of these compounds was found.     

Fate of radioactivity in Atlantic Salmon (Salmo salar) following intragastric administration of (methyl-14C)-trichlorfon

The antiparasitic drug Neguvon® (Bayer), with the active component trichlorfon (0,0-dimethyl-(1-hydroxy-2,2,2-trichloroethyl)-phosphonate), is extensively used in fish farms in Norway. The fate of (methyl-¹⁴C)-trichlorfon was tested in Atlantic salmon (Salmo salar) by liquid scintillation counting at day 1, 4, 7, 14, and 30 post administration, and by autoradiography on selected organs 24 h after administration. The remaining radioactivity was found to be high compared to earlier measurements of the trichlor)fon content made by gas chromatography (Brandal 1977). The grains in autoradiographic preparations of muscle were unevenly distributed both in the muscle as a whole and within muscle fibers. In liver the grains were evenly distributed, but were absent from fat vacuoles. The study indicate that the radioactive residues in salmon muscle do not represent trichlorfon or its derivative dichlorvos (0,0-dimethyl-0-(2,2-dichlorovinyl)-phosphate), but rather hydrophilic metabo)lites of these compounds.     

Tetraenoic and pentaenoic mycolic acids from Mycobacterium thamnopheos. Structure, taxonomic and biosynthetic implications

On the basis of the analysis of mycolates, the type strain of Mycobacterium thamnopheos has been considered as a member of the genus Nocardia. In a comparative study conducted on mycobacterial species we found that M. thamnopheos synthesized two types of mycolate having the same mobilities on thin-layer chromatography as those of mycobacteria, but different from nocardomycolates. Mass spectrometry analyzes showed that the major series of both types consisted of polyunsaturated mycolic acids, ranging from C72 to C78 with four or five double bonds. On pyrolytic mass spectrometry or gas chromatography, the least polar mycolates released mainly monounsaturated C22 esters whereas the other type yielded saturated C20 and C22 esters. These results suggested that M. thamnopheos might be more related to the Aurantiaca taxon than to mycobacteria and Nocardia. The permanganate-periodate oxidation products of esters obtained by pyrolysis of the least polar mycolates showed that they contained docosen-4-oic and docosen-6-oic acids. Both types of mycolate esters yielded the same set of long-chain meroaldehydes on pyrolysis. These meroaldehydes were significantly distinct from those of mycobacterial mycolates in the location of the double bonds. After hydrogenation of the double bond located in the alkyl-branched chain, the two types of mycolates had the same mobility on thin-layer chromatography, indicating that the difference of migration was due to the additional double bond found in the least polar mycolates. Based on stereochemical data, the relative configuration of both mycolates was found to be threo, like that established for all mycolates studied so far.     

A rapid synthesis of a DNA fragment using an unprotected nucleoside and a phosphine derivative

A rapid synthesis of DNA fragment from unprotected nucleoside and phosphine derivative, morpholinophosphordichloridite, has been studied, demonstrating a d(T-T) and its amino-phosphonate derivative syntheses. A high selectivity of this reagent eliminates the protection of nucleoside hydroxyl groups. The P-N bond in the resulting dinucleoside phosphite can readily be converted to a phosphite triester with alcohol and to the corresponding aminophosphonate by a non-aqueous oxidation with m-chlorobenzoic acid. The P-N bond in the phosphate link is very stable and so provides a protection for the phosphoryl group which has many potential uses. Deprotection can be achieved by a simple treatment with NH2OH.     

Effects of nucleotide bromination on the stabilities of Z-RNA and Z-DNA: a molecular mechanics/thermodynamic perturbation study

The structures of ZI- and ZII-form RNA and DNA oligonucleotides were energy minimized in vacuum using the AMBER molecular mechanics force field. Alternating C-G sequences were studied containing either unmodified nucleotides, 8-bromoguanosine in place of all guanosine residues, 5-bromocytidine in place of all cytidine residues, or all modified residues. Some molecules were also energy minimized in the presence of H2O and cations. Free energy perturbation calculations were done in which G8 and C5 hydrogen atoms in one or two residues of Z-form RNAs and DNAs were replaced in a stepwise manner by bromines. Bromination had little effect on the structures of the energy-minimized molecules. Both the minimized molecular energies and the results of the perturbation calculations indicate that bromination of guanosine at C8 will stabilize the Z forms of RNA and DNA relative to the nonbrominated Z form, while bromination of cytidine at C5 stabilizes Z-DNA and destabilizes Z-RNA. These results are in agreement with experimental data. The destabilizing effect of br5C in Z-RNAs is apparently due to an unfavorable interaction between the negatively charged C5 bromine atom and the guanosine hydroxyl group. The vacuum-minimized energies of the ZII-form oligonucleotides are lower than those of the corresponding ZI-form molecules for both RNA and DNA. Previous x-ray diffraction, nmr, and molecular mechanics studies indicate that hydration effects may favor the ZI conformation over the ZII form in DNA. Molecular mechanics calculations show that the ZII-ZI energy differences for the RNAs are greater than three times those obtained for the DNAs. This is due to structurally reinforcing hydrogen-bonding interactions involving the hydroxyl groups in the ZII form, especially between the guanosine hydroxyl hydrogen atom and the 3'-adjacent phosphate oxygen. In addition, the cytidine hydroxyl oxygen forms a hydrogen bond with the 5'-adjacent guanosine amino group in the ZII-form molecule. Both of these interactions are less likely in the ZI-form molecule: the former due to the orientation of the GpC phosphate away from the guanosine ribose in the ZI form, and the latter apparently due to competitive hydrogen bonding of the cytidine 2'-hydroxyl hydrogen with the cytosine carbonyl oxygen in the ZI form. The hydrogen-bonding interaction between the cytidine hydroxyl oxygen and the 5'-adjacent guanosine amino group in Z-RNA twists the amino group out of the plane of the base. This may be responsible for differences in the CD and Raman spectra of Z-RNA and Z-DNA.     

Structure of dihydroxyacetone phosphate dimethyl acetal, a stable dihydroxyacetone phosphate precursor, in the crystalline state

Crystal and molecular structures of four different salts of a dihydroxyacetone phosphate (DHAP) precursor, its dimethyl acetal [2,2-dimethoxy-1,3-propanediol phosphate, C(5)H(13)O(7)P, (MeO)(2)DHAP]: (cha)(2)[(MeO)(2)DHAP].H(2)O (6a), (cha)[(MeO)(2)DHAP] (6b), Na(2)[(MeO)(2)DHAP].5.75H(2)O (6c) and K(2)[(MeO)(2)DHAP].H(2)O (6d), along with the cyclohexylammonium (cha) salt of its phenyl ester (cha)[(MeO)(2)DHAP(Ph)] (6e) are described. In the (MeO)(2)DHAP mono- and dianions, slightly different orientation of the phosphate group in relation to the acetal carbon atom is observed, with a delicate tendency of phosphate group to be located antiperiplanar in the monoanions and anticlinal in the dianions. The 2,2-dimethoxy-1,3-propandiol moiety, (MeO)(2)DHA, seems to be very rigid and its conformation is independent of phosphorylation, the ionization state of the inserted phosphate group and its additional substitution. The overall structures of the cyclohexylammonium (6a,b) and potassium salts (6d) have a double-layered architecture, while the sodium cation network in 6c forms the system of channels, which are filled up with the [(MeO)(2)DHAP](2-) ions. The different architectures of 6c and 6d crystals result from the different ways in which the relevant dianions coordinate to sodium and potassium ions and affect also the hydrogen bonding system observed in 6c and 6d crystals.     

The modified nucleosides of tRNAs. II. Synthesis of 2''-O-methylcytidylyl (3''-5'') cytidine.

The synthesis of 2'-O-methylcytidylyl (3'-5')cytidine by the triester method using as protecting groups, 2,2,2-trichloroethyl for phosphate hydroxyl group, p-chlorophenyoxyacetyl for 5-hydroxyl group, methoxymethylidene for 2',3'-cis-diol system, and benzoyl for the exo-amino group of cytidine is presented. The obtained product was characterised by UV, electrophoresis, chromatography and an enzymatic digestion.     

A theoretical investigation of the interactions between hydroxyl-functionalized ionic liquid and water/methanol/dimethyl sulfoxide

Density functional calculations have been used to investigate the interactions of 1-(2-hydroxyethyl)-3-methylimidazolium ([C₂OHmim]⁺)-based ionic liquids (hydroxyl ILs) with water (H₂O), methanol (CH₃OH), and dimethyl sulfoxide (DMSO). It was found that the cosolvent molecules interact with the anion and cation of each ionic liquid through different atoms, i.e., H and O atoms, respectively. The interactions between the cosolvent molecules and 1-ethyl-3-methylimizolium ([C₂mim]⁺)-based ionic liquids (nonhydroxyl ILs) were also studied for comparison. In the cosolvent–[nonhydroxyl ILs] systems, a furcated H-bond was formed between the O atom of the cosolvent molecule and the C2-H and C6-H, while there were always H-bonds involving the OH group of the cation in the cosolvent–[hydroxyl ILs] systems. Introducing an OH group on the ethyl side of the imidazolium ring may change the order of solubility of the molecular liquids.     

Direct evidence for inhibition of free radical formation from Cu(I) and hydrogen peroxide by glutathione and other potential ligands using the EPR spin-trapping technique.

Copper-induced oxidative damage is generally attributed to the formation of the highly reactive hydroxyl radical by a mechanism analogous to the Haber-Weiss cycle for Fe(II) and H2O2. In the present work, the reaction between the Cu(I) ion and H2O2 is studied using the EPR spin-trapping technique. The hydroxyl radical adduct was observed when Cu(I), dissolved in acetonitrile under N2, was added to pH 7.4 phosphate buffer containing 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Formation of the hydroxyl radical was dependent on the presence of O2 and subsequent formation of H2O2. The kscav/kDMPO ratios obtained were below those expected for a mechanism involving free hydroxyl radical and reflect the interference of nucleophilic addition of H2O to DMPO to form the DMPO/.OH adduct in the presence of nonchelated copper ion. Addition of ethanol or dimethyl sulfoxide to the reaction suggests that a high-valent metal intermediate, possibly Cu(III), was also formed. Spin trapping of hydroxyl radical was almost completely inhibited upon addition of Cu(I) to a solution of either nitrilotriacetate or histidine, even though the copper was fully oxidized to Cu(II) and H2O2 was formed. Bathocuproinedisulfonate, thiourea, and reduced glutathione all stabilized the Cu(I) ion toward oxidation by O2. Upon addition of H2O2, the Cu(I) in all three complexes was oxidized to varying degrees; however, only the thiourea complex was fully oxidized within 2 min of reaction and produced detectable hydroxyl radicals. No radicals were detected from the bathocuproinedisulfonate or glutathione complexes. Overall, these results suggest that the deleterious effects of copper ions in vivo are diminished by biochemical chelators, especially glutathione, which probably has a major role in moderating the toxicological effects of copper.     

Molecular dynamics simulation of the antimicrobial salivary peptide histatin-5 in water and in trifluoroethanol: a microscopic description of the water destructuring effect.

The results of 520 ps molecular dynamics simulation of histatin-5, a small peptide present in human saliva and possessing antimicrobial activity, dissolved in water and in 2,2,2-trifluoroethanol, are reported. The simulations indicate that histatin-5 is destabilized in water and begins to unfold after 250 ps, while in organic solvent it maintains a regular secondary structure throughout the trajectory. Analysis of the peptide-solvent hydrogen bonds indicates that 2,2,2-trifluoroethanol is a poorer proton acceptor than water. The fluorine atom of the alcohol is almost never engaged in a hydrogen bond and the organic solvent interacts mainly with the peptide through its hydroxyl group. For some residues analysis of the solvent residence time indicated longer values for 2,2,2-trifluoroethanol than for water. The most striking difference is related to the number of times the solvent enters and leaves the first coordination shell of the peptide. This value was more than one order of magnitude higher for water than for the alcohol, suggesting that this may be the main cause of alpha-helix destabilization perpetrated by water.     

Mechanistic study of the structure–activity relationship for the free radical scavenging activity of baicalein

Density functional theory calculations were performed to evaluate the antioxidant activity of baicalein. The conformational behaviors of both the isolated and the aqueous-solvated species (simulated with the conductor-like polarizable continuum solvation model) were analyzed at the M052X/6-311 + G(d,p) level. The most stable tautomers of various forms of baicalein displayed three IHBs between O4 and OH5, O5 and OH6, and O6 and OH7. The most stable tautomer of the baicalein radical was obtained by dehydrogenating the hydroxyl at C6, while the most stable anion tautomer was obtained by deprotonating the C7 hydroxyl in gaseous and aqueous phases. The expected antioxidant activity of baicalein was explained by its ionization potentials (IPs) and homolytic O–H bond dissociation enthalpies (BDEs), which were obtained via the UM052X optimization level of the corresponding radical species. Heterolytic O–H bond cleavages (proton dissociation enthalpies, PDEs) were also computed. The calculated IP, BDE, and PDE values suggested that one-step H-atom transfer, rather than sequential proton loss–electron transfer or electron transfer–proton transfer, would be the most favorable mechanism for explaining the antioxidant activity of baicalein in the gas phase and in nonpolar solvents. In aqueous solution, the SPLET mechanism was more important.     

Melanostatin conformations in solution.

The conformations of melanostatin have been studied experimentally using CD spectroscopy and via calculations. In aqueous solution and 2,2,2-trifluoroethanol (TFE) there is no evidence that monomers of the tripeptide exist in an ordered (β-bend) structure. In water and TFE solutions (3–6 × 10^?4M) the neutral molecules aggregate very slowly, taking about 3 days to attain equilibrium at room temperature. At equivalent concentrations in TFE, although not in water, the cationic molecules also slowly aggregate, although to a lesser extent. Calculations using rotational isomeric state theory give the most probable unperturbed end-to-end distance of the molecule at 9.3 ± 0.1 Å and indicate that a vast majority of the molecules exist in some extended conformation, end-to-end distance ≥6 Å. Only 0.4% of the molecules are calculated to have O…?H separations compatible with a β-bend structure. An intramolecular hydrogen bond must have an energy at least 2 kcal/mol lower than that of an intermolecular hydrogen bond to solvent if a β-bend is to be experimentally observable.     

X-ray structure of the dipotassium salt of D-mannose 1-phosphate 3.25 hydrate

The first crystal structure of mannose 1-phosphate is described. The dipotassium hydrate salt crystallizes in the P2(1)2(1)2 space group. There are two independent dianions (I and II) in the asymmetric unit, which are alpha anomers adopting the 4C(1) chair conformation. The main difference between the two mannose 1-phosphate dianions is the orientation of the phosphate group with relation to the pyranosyl ring. In I, one of the phosphate oxygen atoms is antiperiplanar positions with respect to carbon atom C-1, whereas the two others are situated synclinally. The corresponding orientations of the terminal phosphate oxygen atoms in II are synperiplanar and anticlinal. The potassium cations are six- and seven-coordinate, mainly with O atoms of hydroxyl groups and water molecules. There are potassium channels extending along the c-axis. In the packing arrangement, water molecules and mannose phosphate groups also define two different types of layers parallel to a-axis. Within water channels there are extensive hydrogen-bonding networks.     

Role of superoxide in radiation-killing of Escherichia coli and in thymine release from thymidine

The role of superoxide and hydroxyl radicals in gamma-radiation-killing of Escherichia coli K12 was studied in aerated suspensions supplemented with formate, phosphate, superoxide dismutase, catalase and saturated with nitrous oxide. Nitrous oxide, which converts e-aq to .OH, caused decreased radiosensitivity. On the other hand, formate, which results in conversion of .OH to .O2-, resulted in an increased radiosensitivity. The results implicated .O2- as a major cause of radiation-mediated cell-killing. The addition of the enzymes, superoxide dismutase or catalase to the E. coli suspensions prior to and during irradiation had no effect on cell survival, indicating that the biologically significant site of generation and action of .O2- is an intracellular one. Further studies were undertaken to examine the role of superoxide in DNA damage. The release of thymine from the DNA base, thymidine was studied as a result of gamma-irradiation and of chemically generated superoxide (using KO2 in dimethyl sulfoxide). Thymine was identified by HPLC and mass spectrometry. C-13 NMR analysis of the reaction mixture of thymidine with KO2 in dimethyl sulfoxide provided evidence for attack of .O2 at the ribosyl Cl' atom.     

Destruction of cytochrome P-450 by vinyl fluoride, fluroxene, and acetylene. Evidence for a radical intermediate in olefin oxidation

Vinyl fluoride, vinyl bromide, fluroxene (2,2,2-trifluoroethyl vinyl ether), and acetylene alkylate the prosthetic heme group of cytochrome P-450 enzymes which catalyze their metabolism. The alkylated heme moiety has been identified in all four cases, after carboxyl group methylation and demetalation, as the dimethyl easier of N-(2-oxoethyl)protoporphyrin IX. The dimethyl acetal derivative of the aldehyde group in this structure is also isolated. The formation of the same prosthetic heme adduct with the four substrates requires introduction of an oxygen at the trifluoroethoxy or halide-substituted terminus of the pi bond and reaction of the unsubstituted terminus with a heme nitrogen atom. This reaction orientation is consistent with a radical intermediate, possibly formed by way of an initial pi-bond radical cation, but is difficult to reconcile with a cationic intermediate. The occurrence of a radical intermediate in the oxidation of olefins by cytochrome P-450 is thus suggested.     

Crystal structures of dihydroxyacetone and its derivatives

The crystal and molecular structures of three crystalline forms of the dihydroxyacetone dimer, C6H12O6, DHA-dimer: alpha (1a), beta (1b), and gamma (1c), the hydrated calcium chloride complex of dihydroxyacetone monomer, CaCl2(C3H6O3)(2) x H2O, CaCl2(DHA)2 x H2O (2a), the tetrahydrated calcium chloride complex of dihydroxyacetone monomer, CaCl2(C3H6O3) x 4H2O, CaCl2(DHA) x 4H2O (2b), the dihydroxyacetone monomer, C3H6O3, DHA (2c), and dihydroxyacetone dimethyl acetal, C5H12O4, (MeO)2DHA (3) are described. Compounds 1a and 2b crystallize in the triclinic system, and 1b,c, 2a,c, and 3 are monoclinic. Molecules of all forms of dihydroxyacetone dimer 1a,b, and 1c are the trans isomers, with the 1,4-dioxane ring in the chair conformation and the hydroxyl and hydroxymethyl groups in axial and equatorial dispositions, respectively. The Ca2+ ions in 2a and 2b are bridged by the carbonyl O atoms from two symmetry-related DHA molecules to form centrosymmetric dimers with Ca...Ca distance of 4.307(2)A in 2a and 4.330(2) and 4.305(2)A in two crystallographically independent dimers in 2b. DHA molecules coordinate to the Ca2+ ions by hydroxyl and carbonyl oxygen atoms. The eight-coordinate polyhedra of Ca2+ are completed by water molecule and Cl- ion in 2a and by four water molecules in 2b. The dihydroxyacetone molecules in 2a,b, and 2c are in an extended conformation, with both hydroxyl groups being synperiplanar (sp) to the carbonyl O atom. All hydroxyl groups in 2c (along with water molecules in 2a and 2b) are involved as donors in medium strong and weak intermolecular O-H...O hydrogen bonding. Some of them, as well as carbonyl O atoms or Cl- ions in 2a and 2b, act as acceptors in C-H...O (and C-H...Cl) hydrogen interactions.     

Conformation of the glucotriose unit in the lipid-linked oligosaccharide precursor for protein glycosylation.

The conformation of the glucotriose unit of the protein glycosylation precursor Glc3Man9GlcNAc2 was assessed by deuterium exchange studies on the model tetrasaccharide alpha Glc----2 alpha Glc----3 alpha Glc----3 alpha Man----OCH2CH2CH3 dissolved in deuterated dimethyl sulfoxide. The hydroxyl proton on C-2 of the nonreducing end glucose and on C-4 of the glucose attached to mannose both show dramatic isotope shifts indicative of a strong hydrogen bond between these two hydroxyl groups. Such a hydrogen bond requires a fixed conformation of the glucotriose unit that brings these hydroxyl groups within 3 A of each other, a conformation that is supported by molecular modeling based on hard-sphere exo-anomeric (HSEA) calculations. The temperature dependence of the hydroxyl proton chemical shifts supports the postulated hydrogen bond, and the torsional angles between the three glucose units derived from the HSEA calculations are consistent with results from related studies on other saccharides. The results support a model for biochemical function in which the glucotriose unit could modulate the activity of the oligosaccharyltransferase by binding in a fixed conformation to a specific effector site in the enzyme.     

A Cellular Automata Model of Proton Hopping Down a Channel

Proton hopping is the process where a H‐atom on a hydronium ion forms a H‐bond with the O‐atom of a neighboring H₂O molecule. There is then an exchange of bonding forces when that covalent bond of the H‐atom in the hydronium ion changes to a H‐bond, and the previous H‐bond changes to a covalent bond with the neighboring O‐atom. The neighboring molecule now becomes a hydronium (H₃O⁺) ion. This process repeats itself very rapidly among neighboring hydronium and H₂O molecules. There is a flow of protonic character through bulk H₂O, referred to as proton hopping. This process carries information through living systems where H₂O is present. A cellular automata model of proton hopping down a channel has been created and studied. Variations in the rate of proton entry into the channel and the effects of the polar character of the channel walls was studied using the model. The behavior of the models corresponds to experimental results.     

Effect of thiourea on microsomal oxidation of alcohols and associated microsomal functions.

Thiourea and diethylthiourea, two compounds which react with hydroxyl radicals, inhibited NADPH-dependent microsomal oxidation of ethanol and 1-butanol. Inhibition by both compounds was more effective in the presence of the catalase inhibitor, azide. Inhibition by thiourea was noncompetitive with respect to ethanol in the absence of azide but was competitive in the presence of azide. Urea, a compound which does not react with hydroxyl radicals or H2O2, was without effect. Thiourea had no effect on NADH- and NADH-cytochrome c reductase, NADPH oxidase, and NADH- and NADPH-dependent oxygen uptake. Thiourea inhibited the activities of aniline hydroxylase and aminopyrine demethylase. Thiourea, but no other hydroxyl radical scavengers, e.g., dimethyl sulfoxide, mannitol, and benzoate, reacted directly with H202 and decreased H2O2 accumulation in the presence of azide. Therefore the actions of thiourea are complex because it can react with both hydroxyl radicals and H2O2. Differences between the actions of thiourea and those previously reported for dimethyl sulfoxide, mannitol, and benzoate, e.g., effects on drug metabolism, effectiveness of inhibition in the absence of azide, or kinetics of the inhibition, probably reflect the fact that thiourea reacts directly with H2O2 whereas the other agents do not. The current results remain consistent with the concept that microsomal oxidation of alcohols involves interactions of the alcohols with hydroxyl radicals generated from microsomal electron transfer.