Showing papers by "Franz Oesch published in 1993"

Genotoxicity characteristics of reverse diol-epoxides of chrysene

Abstract: Trans-3,4-dihydroxy-3,4-dihydrochrysene (chrysene-3,4-diol), a major metabolite of chrysene, is further metabolized by rat liver enzymes to products which effectively revert the his- Salmonella typhimurium strain TA98 to histidine prototrophy, but are only weakly mutagenic in strain TA100 and in Chinese hamster V79 cells (acquisition of resistance to 6-thioguanine). The liver enzyme mediated mutagenicity of chrysene-3,4-diol is substantially enhanced in the presence of 1,1,1-trichloropropene 2,3-oxide, an inhibitor of microsomal epoxide hydrolase. The predominant metabolites of chrysene-3,4-diol, namely the anti- and syn-isomers of its 1,2-oxide (termed reverse diol-epoxides), proved to be extraordinarily effective mutagens in S.typhimurium strain TA98, but were only moderately active in strains TA100 and TA104, and in the SOS induction in Escherichia coli PQ37. These genotoxicity spectra in bacteria are completely different from those observed with the bay-region diol-epoxides of chrysene and 3-hydroxychrysene. In V79 cells, the reverse diol-epoxides formed low levels of DNA adducts and were very weak inducers of gene mutations. In M2 mouse prostate cells, however, high numbers of transformed foci were induced by chrysene-3,4-diol and its diastereomeric 1,2-oxides. Chrysene-3,4-diol was somewhat more potent than chrysene-1,2-diol. The potency of both reverse diol-epoxides was similar to that of the syn-diastereomers of the bay-region diol-epoxides of chrysene and 3-hydroxychrysene, but lower than that of their anti-diastereomers. The reverse diol-epoxides of chrysene, unlike the bay-region diol-epoxides, were inactivated by purified microsomal epoxide hydrolase. Noteworthy findings were also made with regard to the chemical stability of the diol-epoxides in buffer, determined from the decline in mutagenicity after preincubation in the absence of the target cells. Despite its lower delta Edeloc/beta value for the formation of the benzylic carbocation, anti-chrysene-3,4-diol 1,2-oxide was shorter-lived (t1/2 = 46 min) than anti-chrysene-1,2-diol 3,4-oxide (t1/2 = 74 min). Unlike other investigated diastereomeric pairs of diol-epoxides, it was also shorter-lived than its syn-diastereomer (t1/2 = 340 min).

Evidence for the involvement of a bis-diol-epoxide in the metabolic activation of dibenz[a,h]anthracene to DNA-binding species in mouse skin

Abstract: Dibenz[a,h]anthracene (DB[a,h]A) and its microsomal metabolites, trans-3,4-dihydro-3,4-dihydroxydibenz[a,h]anthracene (DBA-3,4-diol), trans,trans-3,4:8,9-tetrahydro-3,4:8,9-tetrahydroxydibenz[a,h]anth racene, trans,trans-3,4:10,11-tetrahydro-3,4:10,11-tetrahydroxydibenz[a,h] - anthracene (DBA-3,4,10,11-bis-diol) and trans,trans-3,4:12,13-tetrahydro-3,4:12,13- tetrahydroxydibenz[a,h]anthracene were each applied topically to mouse skin and the epidermal DNA isolated 24 h later. 32P-postlabeling analysis of each of the DNA samples was performed. DNA from mice treated with DB[a,h]A produced an adduct map on TLC consisting of one major and three minor adduct spots. A similar pattern of spots was produced by DBA-3,4-diol. No detectable DNA adducts were produced by trans,trans-3,4:12,13-tetrahydro-3,4:12,13-tetrahydroxy- dibenz[a,h]anthracene, although a single, minor adduct spot was produced by trans,trans-3,4:8,9-tetrahydro-3,4:8,9-tetrahydroxydibenz[a,h]- anthracene. However, DBA-3,4,10,11-bis-diol was found to produce a major single adduct that comigrated on thin layer chromatography with the major adduct produced by both DB[a,h]A and DBA-3,4-diol. In addition, this adduct was present at a level 10 times higher than the corresponding adduct produced by treatment with the parent hydrocarbon. Coelution of the major adducts formed from DB[a,h]A and DBA-3,4-diol with that formed from DBA-3,4,10,11-bis-diol was also demonstrated on reverse-phase high performance liquid chromatography. Thus, we propose that, in mouse skin, the major pathway of DB[a,h]A activation to DNA binding products is via a 3,4-diol to the 3,4,10,11-bis-diol and ultimately to a bis-diol-epoxide (potentially the 3,4,10,11-bis-dihydrodiol-1,2-oxide).

Formation of DNA adducts from 1-hydroxymethylpyrene in liver cells in vivo and in vitro

Abstract: The binding of 1-hydroxymethylpyrene (HMP) and its active metabolites, 1-hydroxymethylpyrene sulfate (SMP) and 1-chloromethylpyrene (CMP), to DNA was studied. In the liver of rats, maximum adduct levels were observed 1.5 h after i.p. injection of HMP, followed by a relatively rapid decrease. Separate exposure of different liver cell types in vitro to HMP led to high adduct levels in parenchymal cells, intermediate levels (1/10) in endothelial cells and low levels (1/200) in Kupffer cells. The adduct patterns were similar in the different cell types. The same pattern was also obtained when isolated DNA was incubated with SMP or CMP. One of the four major spots co-chromatographed on TLC with a dG adduct, one with a dA adduct and one with both dG and dA adducts. The fourth spot did not co-chromatograph with any of the adducts observed in reactions with any nucleic acid homopolymer.

Characterization of highly polar bis-dihydrodiol epoxide--DNA adducts formed after metabolic activation of dibenz[a,h]anthracene.

Abstract: Dibenz[a,h]anthracene as well as a biologically important metabolite of dibenz[a,h]anthracene, namely the M-region dihydrodiol trans-3,4-dihydroxy-3,4-dihydrodibenz[a,h]anthracene were in addition to further metabolism to a bay region diol epoxide, extensively transformed to a distal bisdihydrodiol, 3,4,10,11-tetrahydroxy-3,4,10,11-tetrahydro-dibenz[a,h]anthracene, which exhibited after renewed metabolic activation high DNA binding efficiency, leading to a new class of very polar DNA adducts. After incubation of dibenz[a,h]anthracene with DNA in the presence of liver microsomes from Aroclor 1254 treated male Sprague-Dawley rats highly polar DNA adducts probably originating from 3R,4R,10R,11R-tetrahydroxy-3,4,10,11-tetrahydrodibenz[a,h]an thracene and 3R,4R,10S,11S-tetrahydroxy-3,4,10,11-tetrahydrodibenz[a,h]anthr ace ne were identified by reversed phase HPLC and by the 32P-postlabeling method. The adducts obtained were further characterized by comparing their fluorescence spectra with those obtained from 3,4,10,11-tetrahydroxy- 3,4,10,11-tetrahydrodibenz[a,h]anthracene and from 3,4-dihydroxy-3,4-dihydrodibenz[a]anthracene, the putative chromophore of the polar adduct. DNA adducts formed via 3S,4S,10S,11S-tetrahydroxy-3,4,10,11-tetrahydro- dibenz[a,h]anthracene were not found. After incubation of 14C-labelled dibenz[a,h]anthracene highly polar DNA adducts derived from the bisdihydrodiol contributed 38% to the adducts found in the HPLC profile. Bay region diol epoxide adducts represented a fraction of 25%. Using the 32P-postlabelling technique a higher DNA binding yield for the racemic bisdihydrodiol (38 +/- 12 pmol/mg DNA) was calculated than for the most active 3,4-dihydrodiol enantiomer, 3R,4R-dihydroxy-3,4- dihydrodibenz[a,h]anthracene (23 +/- 6 pmol/mg DNA).

Some substrates and inhibitors of cytosolic epoxide hydrolase induce sister-chromatid exchanges in mammalian cells, but do not induce gene mutations in Salmonella typhimurium and V79 cells

Abstract: Trans -stilbene oxide, trans -β-methylstyrene, 7,8-oxide, trans -β-ethylstyrene, 7,8-oxide, trans -β-propylstyrene 7,8-oxide and 4-fluorochalcone oxide were investigated for genotoxic activity in bacterial and mammalian cells, in the absence of external xenobiotic-metabolising systems. All compounds strongly enhanced the frequency of sister-chromatid exchanges (SCE) in cultured human lymphocytes. None of them was mutagenic in Salmonella typhimurium (reversion of the his − strains TA98, TA100 and TA104). The limit of detection was 1 20,000 to 1 10 6 of the activity of the positive control, benzo[ a ]pyrene 4,5-oxide, depending on the compound and the bacterial strain. Trans -β-methylstyrene 7,8-oxide and 4-fluorochalcone oxide were additionally tested for induction of SCE and gene mutations in the same target cells, namely Chinese hamster V79 cells. Their influence on the level of SCE was similar to that observed in human lymphocytes, whilst gene mutations (at the hprt locus) were not induced. The four investigated styrene oxide derivatives are known to be excellent substrates for a mammalian enzyme, cytosolic epoxide hydrolase (cEH). 4-Fluorochalcone oxide is a potent selective inhibitor of this enzyme and is structurally similar to the investigated styrene oxide derivatives. These properties of the test compounds however cannot explain the observed discrepancies in the results, since the genetic end point (SCE versus gene mutations) was decisive, and SCE were induced in cEH-proficient human lymphocytes as well as in cEH-deficient V79 cells.

Characterization of DNA adducts at the bay region of dibenz[a,h]anthracene formed in vitro.

Abstract: Bay region diolepoxide-DNA adducts of dibenz[a,h]anthracene (DBA) formed in vitro were identified and their absolute stereochemistry was assigned. After activation of [5,12-14C]DBA with liver microsomes obtained from Aroclor 1254 treated male Sprague-Dawley rats in the presence of calf thymus DNA for 1 h, the amount of DNA adducts was found to be 9.9 +/- 2.4 pmol/mg DNA, calculated on the basis of the portion of radioactivity eluted from the HPLC reversed-phase column with a water/acetonitrile gradient. Bay region diolepoxide-DNA adducts represented 27.5% of radioactivity associated with DNA adducts. The absolute configuration of the various adducts was determined from the reaction of the (+)- and (-)-3,4-dihydrodiol after metabolic activation and the reaction of the anti- and syn-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracen e with DNA or with the individual deoxyribonucleotides. The main bay region adduct was identified as a deoxyguanosine adduct of (anti)-3S,4R-dihydroxy-1R,2S-epoxy-1,2,3,4-tetrahydrodibenz [a,h]anthracene, a metabolite of (-)-3,4-dihydroxy-3,4-dihydrodi- benz[a,h]anthracene. Anti bay region diolepoxide-deoxyguanosine adducts of DBA contributed to 17.7% and syn diolepoxide-derived deoxyguanosine adducts to 5.8% of adduct-associated radioactivity. The amount of bay region deoxyadenosine adducts was calculated to be 4%. For six of probably eight different deoxyadenosine adducts absolute stereochemistry could be assigned. 32P-Postlabelling experiments revealed a binding of 23 +/- 6 pmol/mg DNA for (-)-3,4-dihydrodiol and of 1.5 +/- 0.4 pmol/mg DNA for (+)-3,4-dihydrodiol of DBA.

Effects of sodium butyrate on DNA content, glutathione S-transferase activities, cell morphology and growth characteristics of rat liver nonparenchymal epithelial cells in vitro

Abstract: The effects of sodium butyrate, which has been shown to act as a differentiation promoting agent in several different tumor cell lines, were studied in a rat liver nonparenchymal epithelial cell line. Exposure of these cells to 3.75 mM butyrate resulted in an inhibition of cell proliferation and, at the same time, an increase in cell diameter (2- to 6-fold) and size of the nuclei (approximately 2-fold) after 3 days in culture. Binucleated cells arose, comprising approximately 12% of the cells investigated, and the number of cells with an abnormal set of chromosomes was increased. Intercellular communication, measured by dye transfer of Lucifer Yellow, was unchanged. From the various xenobiotic metabolizing enzyme activities measured, only those of glutathione S-transferases were significantly altered (increases of 4- to 9-fold) by butyrate treatment. These increases were mainly due to the predominant rise in the pi class isoenzyme which is a well-known tumour marker in rat hepatocarcinogenesis. Thus, our results cannot be interpreted as being either due to promotion of differentiation or due to transformation. The state and type of cell under study has to be considered and investigations of further differentiation parameters are needed to obtain a deeper insight into the biological activity and the underlying mechanisms of cell state modifying agents like butyrate.

Characterization of Highly Polar Dna Adducts Derived from Dibenz[A,H]Anthracene (DBA), 3,4-Dihydroxy-3,4-Dihydro-DBA, and 3,4,10,11-Tetrahydroxy-3,4,10,11-Tetrahydro-DBA:

Abstract: Two highly polar DNA adducts were found after metabolic activation of 3,4,10,11-tetrahydroxy-3,4,10,11-tetrahydrodibenz[a,h]anthracene (DBA-3,4,10,11-bisdiol) by liver microsomes isolated from male Sprague-Dawley rats pretreated with Aroclor 1254 in presence of calf thymus DNA. These DNA adducts could be assigned to the metabolites of dibenz[a,h]anthracene (DBA), of 3R,4R,10R,11R-tetrahydroxy-3,4,10,11-tetrahydro-DBA and of 3R,4R,10S,11S-tetrahydroxy-3,4,10,11-tetrahydro-DBA. DNA adducts derived from metabolites of 3S,4S,10S,11S-tetrahydroxy-3,4,10,11-tetrahydro-DBA were not found. These highly polar adducts also could be detected by reversed phase HPLC after incubation of dibenz[a,h]anthracene, 3R,4R-dihydroxy-3,4-dihydro-DBA ((-)-DBA-3,4-diol) and 3S,4S-dihydroxy-3,4-dihydro-DBA ((+)-DBA-3,4-diol) with DNA in presence of the activating system. After incubation of 14C labelled DBA DNA adducts derived from DBA-3,4,10,11-bisdiol were found in a fraction of 38% and bay region 3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-DBA-DNA adducts at a level of 25%. DBA-3,4,10,11-bisdiol exhibited a higher DNA binding yield (38 +/- 12 pmol/mg DNA) than (-)-DBA-3,4-diol (23 +/- 6 pmol/mg DNA), the most mutagenic 3,4-diol enantiomer. For (+)-DBA-3,4-diol the highly polar DNA adducts derived from DBA-3,4,10,11-bisdiol were by far the most predominant adducts in vitro.

Synthesis of Oligodeoxynucleotides Containing Diastereomeric Dihydrodiol Epoxide-N6-Deoxyadenosine Adducts of Polycyclic Aromatic Hydrocarbons.

Abstract: A generally applicable route is reported for the synthesis of oligodeoxynucleotides which contain structurally defined N 6 -deoxyadenosine adducts, derived from sterically highly hindered dihydrodiol epoxides of polycyclic aromatic hydrocarbons (PAH).