Characterization and quantitation of 3-alkylthymidines from reactions of mutagenic propylene oxides with thymidine

Thymidine was reacted in methanol with four epoxides of varying mutagenicities: propylene oxide, glycidol, epichlorohydrin and trichloropropylene oxide. A single product was detected with each epoxide, and these products had the same retention times on silica high pressure liquid chromatography (HPLC). UV spectra of the products identified them as 3-alkylthymidines, and this was confirmed by infrared (IR) and nuclear magnetic resonance (NMR) spectra. Mass spectra (MS) analysis showed the products to be consistent with attachment at the least substituted carbon of the epoxide. Formation of 3-alkylthymidines correlated to Taft [sigma]* electron withdrawing values for the substituents on the epoxides and mutagenicities in strain TA100 of the Ames Assay.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24612/1/0000022.pd

Isomerization of trans-diethylstilbestrol to pseudo-diethylstilbestrol

This study reports the formation and isolation of a diethyl-stilbestrol-dimethylsulfoxide (DES-DMSO) adjunct and Z-3, 4-di(p-hydroxyphenyl)-2-hexene ([psi]-DES) from -DES. The presence of [psi]-DES was indicated by NMR and mass spectrometry and confirmed by direct comparison to a reference sample. High resolution NMR(360 MHz) along with the comparison of the chemical shift values of methine and methyl protons attached to carbon-carbon double bonds in Z and E isomers of 3-substituted-2-pentenes and dienestrol derivatives were used in postulating the Z-stereochemistry for [psi]-DES. A NMR additive increment method was useful for the comparison of the chemical shift values of methine protons in [psi]-DES and other literature compounds. Nuclear Overhauser Enhancement (NOE) confirmed the Z-stereochemistry of [psi]-DES.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24220/1/0000479.pd

High-performance thin-layer chromatography assay for epoxide hydrolase activity and the determination of phenoxypropane-1,2-diols

A simple, rapid and sensitive high-performance thin-layer chromatographic assay for the determination of epoxide hydrolase activity in rat liver homogenates is described. It is extended to the determination of a series of phenoxypropne-1,2-diols. The hydrolase assay has the advantages of using a readily available substrate, 2,3-epoxypropyl 4-methoxyphenyl ether, of multiple sample application, and of the simultaneous determination of reaction product (diol) as well as unchanged substrate (epoxide). The use of an internal standard, 4-nitroanisole, results in high sensitivity and good reproducibility of the proposed method. The limit of dial detection is 20 pmol.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25891/1/0000454.pd

Mechanisms for the biodehalogenation of iodocompounds

The deiodination of [125I]-4-iodobiphenyl, [125I]-4-iodonitrobenzene and [125I]-4-iodoaniline was investigated. No deiodination was detected in rat thyroid homogenates. However, at least three biodeiodination mechanisms were indicated for substrates in rat liver subcellar fractions. Microsomal dehalogenation occurred to a minor extent with increased dehalogenation taking place in the cytosol fraction. The cytosol deiodination was extensive for 4-iodonitrobenzene and was mediated by glutathione. A second cytosol deiodination mechanism, not mediated by glutathione, was evident when 4-iodobiphenyl was the substrate. This soluble enzyme system could be enhanced by Arochlor 1254 or 4-iodobiphenyl pretreatment.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22572/1/0000117.pd

Mutagenicity in Salmonella assays of cyclohexane epoxide derivatives

15 Cyclohexane epoxide derivatives were synthesized and compared for direct mutagenicity an bacterial toxicity using Salmonella typhimurium strain TA100 in the liquid suspension and spot-test version of the Ames procedure. While no general correlations could be established for position and stereochemistry of the hydroxylated derivatives, an increase in mutagenicity was noted for the presence of electron-withdrawing groups and unsaturation in conjugation with the oxirane groups.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25651/1/0000203.pd

Substituent effects on the mutagenicity of phenyl glycidyl ethers in Salmonella typhimurium

Phenyl glycidyl ether and 6 para-substituted derivatives, the methoxy, tert-butyl, methyl, chloro, bromo and nitro compounds, were tested in the Ames' test for mutagenicity. With the exception of the tert-butyl derivative in TA1535, all 7 compounds were mutagenic in both strains TA100 and TA1535. Electron-donating groups in the para position decreased mutagenicity while electron-withdrawing groups increased this mutagenicity. The mutagenicity of the series of compounds in both strains could be correlated to the Hammett substituent constants for the para-substituent groups. The glycidyl ether results might best be considered in terms of a normal reaction with bionucleophiles as compared to the literature report for the correlation of mutagenicity with abnormal product formation for a styrene oxide series.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24019/1/0000268.pd

The in vivo and in vitro genotoxicity of aromatic amines in relationship to the genotoxicity of benzidine

Benzidine and 12 related aromatic amines have been studied for the effects of substituent groups and [pi] orbital conjugation on their genotoxicity as measured by their mutagenicity in vitro with Salmonella and by chromosomal aberrations (CA) in vivo in the bone-marrow cells of mice. The in vitro studies indicated increases in mutagenicity with increases in the electron withdrawing ability of para' substituents. Mutagenicity also increases with increased conjugation as shown by the degree of planarity of the biphenyl compounds and by comparing the mutagenecities of biphenyl amines to stilbenes as well as to ethylene bridged diphenyl compounds. The relative in vitro mutagenicity results were not predictive of relative in vivo CA results. The 3 most genotoxic compounds in vivo were the conjugated amines without substituents in the para' position. The CA values for 4-aminostilbene were exceptionally high. These in vivo results indicate increased genotoxicity for benzidine analogs without substitution in the para' position.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29922/1/0000279.pd

Comparison of the isolation of adducts fo 2'-deoxycytidine and 2'-deoxyguanosine with phenylglycidyl ether by high-performance liquid chromatography on a reversed-phase column and a polystyrene-divinylbenzene column

2'-Deoxycitidine (dCyd) and 2'-deoxyguanosine (dGuo) were subjected to reaction with phenylglycidyl ether (PGE) in methanol in order to study the formation of the corresponding 2'-deoxynucleoside adducts. Separation methods were developed on analytical and semi-preparative scales using high-performance liquid chromatography with photodiode-array detection on a reversed-phase column and on a polystyrene-divinylbenzene column. The use of the latter column was prompted by decomposition of the preparatively isolated dGuo-PGE adducts on the reversed-phase column. The use of a polystyrene-divinylbenzene column solved this problem and also revealed the presence of one more peak in both the dCyd-and dGuo-PGE reaction mixtures.The adducts of dCyd and dGuo were isolated on preparative reversed-phase and polystyrene-divinylbenzene columns and characterized by UV, fast atom bombardment mass and 360 MHz 1H NMR spectrometry. The adducts of dCyd were the diastereomers of N-3-(2-hydroxy-3-phenoxypropyl)-2'-deoxycytidine and N4-(2-hydroxy-3-phenoxypropyl)-2'-deoxycytidine whereas those of dGuo were the two diastereomers of N-7-(2-hydroxy-3-phenoxypropyl)-2'-deoxyguanosine and a third peak which appeared to be mainly (N2-(2-hydroxy-3-phenoxypropyl)-2'-deoxyguanosine.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29601/1/0000690.pd

The genotoxicity of enantiomeric aliphatic epoxides

The (R)- and (S)-optical isomers of 9 epoxides, benzyloxymethyloxirane, epichlorohydrin, glycidol, glycidyl 3-nitrobenzenesulfonate, glycidyl 4-nitrobenzoate, glycidyl tosylate, styrene oxide, glycidyl 1-naphthyl ether and glycidyl 4-nitrophenyl ether, have been compared for their in vivo and in vitro genotoxicity. The in vitro short-term test employed was the Ames mutagenicity assay with Salmonella strain TA100. The in vivo tests were chromosomal aberrations (CA) as well as sister-chromatid exchange (SCE) in bone-marrow cells of mice following intraperitoneal administration of these epoxides. Differences in mutagenicity between isomers were established with TA100 for all the compounds. While 13 of the isomers were genotoxic compared to a negative control by CA measurements, only in the case of glycidyl 4-nitrobenzoate could a significant difference be found between isomers by this test. However, with SCE evaluations, differences were detected between the (R)- and (S)-isomers for all the pairs of compounds with the exception of those for benzyloxymethyloxirane and glycidyl 4-nitrophenyl ether. At least in part, differences in the patterns of genotoxicity among compounds can be related to their differences in reaction pathways.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31061/1/0000738.pd

Adduct formation identification between phenyl glycidyl ether and 2'-deoxyadenosine and thymidine by chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy

Thymidine 2'-deoxyadenosine were reacted with phenyl glycidyl ether in order to study the formation of the corresponding 2'-deoxynucleoside adducts. Separation methods were elaborated using either reversed-phase high-performance liquid chromatography with photodiode-array detection, or centrifugal circular thin-layer chromatography. The adducts were isolated on a preparative scale and were fully characterized by UV spectroscopy, desorption chemical ionization and fast atom bombardment mass spectrometry and 270- and 360-MHz 1H NMR spectrometry. For thymidine the main adduct was characterized as N-3-(2-hydroxy-3-phenoxypropyl)thymidine. With 2'-deoxyadenosine, predominantly N-l-(2-hydroxy-3-phenoxypropyl)-2'-deoxyadenosine was formed. With longer reaction times, the formation of a minor amount of dialkylated 2'-deoxyadenosine was observed. These nucleoside adducts will be used as marker compounds for studies of DNA adduct formation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28807/1/0000641.pd