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W Pfau

Publications and source records attributed to W Pfau.

34 records · Page 2Linked to original sources
PubMed1994

Characterization of DNA adducts formed by aristolochic acids in the target organ (forestomach) of rats by 32P-postlabelling analysis using different chromatographic procedures.

We report the analysis of DNA adducts in the target organ (forestomach) of male Sprague-Dawley rats treated orally with two doses (10 mg/kg body wt) per week for 2 weeks of either aristolochic acid I (AAI), aristolochic acid II (AAII) or the plant extract aristolochic acid (AA). DNA adducts were detected and quantitated using the nuclease P1-enhanced version of the 32P-postlabelling assay. For identification of adducts, reference compounds were prepared by reaction of enzymatically activated AAI and AAII with 3'-purine phosphonucleosides and analysed by the n-butanol enrichment procedure. These reference compounds were assigned to the previously characterized DNA adducts of AAI [7-(deoxyguanosin-N2-yl)-aristolactam I = dG-AAI, 7-(deoxyadenosin-N6-yl)-aristolactam I = dA-AAI] and AAII [7-(deoxyadenosin-N6-yl)-aristolactam II = dA-AAII]. Cross referencing of the carcinogen-modified nucleoside bisphosphates obtained from forestomach DNA with the synthetic standard compounds by ion-exchange chromatography and reversed-phase HPLC demonstrated that the major DNA adducts formed by AAI and AA were identical to dG-AAI and dA-AAI. Likewise, forestomach DNA isolated from AAII-treated rats showed two purine-derived adduct spots, the major one being dA-AAII, the minor one being tentatively identified as 7-(deoxyguanosin-N2-yl)-aristolactam II. A minor adduct detected in forestomach DNA of rats treated with AAI was found to be chromatographically indistinguishable from the adduct identified as dA-AAII, indicating a possible demethoxylation reaction of AAI. Quantitation of DNA adducts revealed that in in vitro reactions with 3'-phosphonucleosides the adduct levels were approximately one order higher for both AAI- and AAII-derived adducts than in forestomach DNA modified with AAI or AAII in vivo. In vitro as well as in vivo adduction by AAI was more efficient than adduction by AAII. The pattern of adduct spots obtained from forestomach DNA of rats treated with the plant extract AA reflected the composition of the extract determined by HPLC analysis. Irrespective of the aristolochic acid used to induce DNA adducts, deoxyadenosine is the major target of modification, pointing to the general importance of deoxyadenosine adducts for chemical carcinogenesis of these naturally occurring products. This study shows that the combination of two independent chromatographic systems considerably enhances the fidelity of identification of DNA adducts with the 32P-postlabelling assay.

Animals↗
PubMed1993

Covalent binding of polycyclic aromatic hydrocarbon components of coal tar to DNA in mouse skin.

Treatment of mouse skin with coal tar is known to initiate tumour formation, with the carcinogenic activity associated mainly with polycyclic aromatic hydrocarbons (PAHs). A sample of pharmaceutical coal tar was analysed by gas chromatography and 19 major PAHs were identified. 32P-postlabelling analysis was used to characterize those PAHs that are responsible for the DNA binding of coal tar and, by inference, its biological activity. PAHs were grouped according to their reported carcinogenic activities and applied as mixtures to mouse skin. Group A contained all of the 19 PAHs, group B seven PAHs for which there is sufficient evidence for carcinogenicity and group C 12 PAHs with only limited or inadequate evidence of carcinogenicity in experimental animals. 32P-Labelled DNA adducts formed by coal tar were resolved on TLC into a pattern of three discrete spots (2, 4 and 6) and four areas of diffuse radioactivity (1, 3, 5 and 7). By comparison of the pattern of adducts formed by coal tar with those formed by the synthetic mixtures it appeared that PAHs in group B formed coal tar-DNA adduct spots 4 and 6, and that adduct spot 2 was formed by PAHs in group C. Attempts to identify those PAHs responsible for the formation of coal tar-DNA adducts 4 and 6 were made by comparing the chromatographic mobilities of 32P-labelled coal tar-derived DNA adducts formed in mouse skin, using TLC and HPLC, with those formed by PAHs in group B. As benzo[ghi]perylene (B[ghi]P), a component of group C, has been demonstrated to exhibit significant DNA binding ability previously, the chromatographic mobility of coal tar-DNA adduct spot 2 was compared to that of the major DNA adducts formed by B[ghi]P in vivo and in vitro. It appeared that coal tar adduct spot 2 was the major adduct formed by B[ghi]P in vitro and that benzo[a]pyrene, benzo[b]fluoranthene, benzo [j]fluoranthene and benzo[k]fluoranthene contributed to the formation of adduct spot 6. None of the PAHs examined appeared to be responsible for the formation of adduct spot 4.

Animals↗
PubMed1993

Separation of 32P-labelled nucleoside 3',5'-bisphosphate adducts by HPLC.

Relatively few reported attempts have been made to substitute HPLC for the thin-layer ion-exchange chromatography (TLC) conventionally used in the 32P-postlabelling assay. Using a reverse-phase phenyl-modified silica gel column and a gradient of methanol in 0.5 M sodium phosphate buffer (pH 2.0), we were able to improve the resolution of very similar adducts. Combined with on-line detection of Cerenkov radiation, this method allows separation of sub-femtomole quantities of 32P-labelled nucleoside 3',5'-bisphosphates modified by bulky carcinogens. Using this method, we were able to separate nine of the ten major adducts formed by reaction of the diol-epoxides of ten polycyclic aromatic hydrocarbons with DNA, and resolve different adducts formed by a single carcinogen. The major adducts formed by benzo[b]fluoranthene (BbF) or dibenz[a,h]anthracene in mouse skin in vivo have been shown to be distinct from the adducts formed directly by the bay-region diol-epoxides. The heterocyclic amines IQ and MeIQ have each been shown to form one major DNA adduct in several in vitro and in vivo systems; using HPLC we were able to resolve the two adducts formed by these food mutagens. HPLC is especially useful for the identification of adducts by means of chromatographic comparisons and in the analysis of the multiple adducts formed by complex mixtures of environmental carcinogens. The major adducts formed by benzo[a]pyrene (BaP) and BbF in mouse skin in vivo that were not resolved on TLC were well separated by HPLC and thus a major DNA adduct formed in the skin of mice treated topically with coal tar was found to be derived from BaP rather than BbF.

Animals↗
PubMed1992

HPLC separation of 32P-postlabelled benzo[b]fluoranthene-DNA adducts.

Analysis using 32P-postlabelling and a recently developed HPLC method resolved the adduct formed by reaction of the benzo[b]fluoranthene (BbF) anti-bay-region diol-epoxide with DNA from the more polar major adduct produced by the hydrocarbon in three different biological systems. In each case, the adduct formed from the anti-bay-region diol-epoxide constituted only a minor proportion of the total DNA modification. Comparisons of the DNA adducts formed from the hydrocarbon with those formed in microsomal incubations from the putative metabolites BbF-9,10-diol, anti-BbF-9,10-diol-11,12-oxide and the 5,9,10- and 6,9,10-BbF-triols indicate that the predominant pathway for BbF activation in skin probably involves a bay-region triol-epoxide possessing a phenolic OH-group on the peninsula ring.

Animals↗
PubMed1992

HPLC separation of 32P-postlabelled DNA adducts formed from dibenz[a,h]anthracene in skin.

Mouse skin and human skin have been treated in vivo or in short-term organ culture with dibenz[a,h]anthracene (DB[a,h]A), the related 3,4- or 5,6-diols or the anti- or syn-3,4-diol 1,2-oxides. DNA hydrolysates have been 32P-postlabelled and the adducts present examined by HPLC using a phenyl-modified reverse phase column and, for comparison, by PEI-cellulose TLC and autoradiography. The adducts formed when the diol-epoxides were reacted with salmon sperm DNA were also examined. The results show that in mouse skin treated in vivo, the major adducts formed from DB[a,h]A and the 3,4-diol were the same and that two of them were more polar than those formed in skin or in DNA that had been treated with the related anti- or syn-diol epoxides. Human skin treated with DB[a,h]A in culture yielded an adduct profile that was qualitatively similar to the profiles obtained with mouse skin.

Animals↗
PubMed1992

Metabolic activation of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) to DNA binding species in human mammary epithelial cells.

When incubated in suspension with the heterocyclic aromatic amine food mutagens 2-amino-3-methylimidazo [4,5-f]-quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), human mammary epithelial cell aggregates were found, by 32P-postlabelling analysis, to yield DNA that contained adducts. Analysis by HPLC of the 32P-labelled digests of mammary cell DNA indicated that in each case a major adduct peak corresponded to that produced in DNA in vitro by activated derivatives of the two compounds. The patterns of adducts obtained when DNA digests were separated by TLC on polyethyleneimine-cellulose plates were found to resemble those previously shown to be present in DNA of tissues of mice fed IQ or MeIQ. These results demonstrate the ability of human mammary epithelial cells to activate carcinogenic heterocyclic compounds known to be present in the human diet to DNA binding derivatives.

Biotransformation↗
PubMed1991

Improved reversed-phase high-performance liquid chromatographic separation of 32P-labelled nucleoside 3',5'-bisphosphate adducts of polycyclic aromatic hydrocarbons.

32P-Postlabelling is a sensitive technique for the detection and analysis of carcinogen-DNA adducts. In this paper we describe the development of an improved high-performance liquid chromatography (HPLC) method for the separation of 32P-labelled 3',5'-bisphosphates of nucleosides modified by reactive derivatives of carcinogenic polycyclic aromatic hydrocarbons (PAH). Optimal resolution of the major 32P-postlabelled DNA adducts formed by the anti-diol-epoxides of ten PAH was achieved using a phenyl-modified silica gel column with a gradient of methanol in phosphate buffer at low pH and high ionic strength. Use of a radioactivity flow detector coupled to the HPLC apparatus allowed detection of subfemtomole quantities of labelled adducts.

Carcinogens↗
PubMed1990

The structural basis for the mutagenicity of aristolochic acid.

Molecular orbital calculations with aristolochic acid I (AAI) and the model compounds 8-nitro-1-naphthoic acid (1,8NNA) and 3-nitro-2-naphthoic acid (2,3NNA) confirm a similar conformation of the nitro and carboxyl groups in these molecules. The ortho isomer 2,3NNA is not mutagenic in the Salmonella strains TA 100 or TA 1537, but the peri-substituted 1,8NNA shows mutagenic activity similar to AAI in TA 100, although it is only weakly active in TA 1537. We propose a mechanism of activation via a cyclic nitrenium ion with an aristolactam structure which is possible only in peri-substituted nitro carboxylic acids.

Aristolochic Acids↗
PubMed1990

Aristolochic acid activates ras genes in rat tumors at deoxyadenosine residues.

Aristolochic acid I (AAI), a nitrophenanthrene derivative, is the major component of the carcinogenic plant extract aristolochic acid, which has been used as a medicine since antiquity. Long term oral administration of AAI to male Wistar rats induces multiple tumors, mainly in the forestomach, ear duct, and small intestine. The presence of activated transforming genes was investigated in various tumors of 18 AAI treated rats, namely in 14 squamous cell carcinomas of the forestomach, 7 squamous cell carcinomas of the ear duct, 8 tumors of the small intestine, 3 tumors of the pancreas, 1 adenocarcinoma of the kidney, 1 lymphoma, and 2 metastases in the lung and the pancreas. By utilizing the tumorigenicity assay and Southern blot analysis, we have detected an activated c-Ha-ras gene in the DNAs of 5 of 5 squamous cell carcinomas of the forestomach. Direct sequencing of amplified material revealed an AT----TA transversion mutation at the second position of codon 61 of the c-Ha-ras gene (CAA to CTA) in all transfectants as well as in the 5 original rat tumors. Enzymatic amplification of ras sequences followed by selective oligonucleotide hybridization detected identical mutations in 93% (13 of 14) of forestomach tumors, in 100% (7 of 7) of ear duct tumors, and in the lung metastasis. Among those tumors tested, we had 4 cases in which the forestomach tumors and the ear duct tumors originated from the same rat, showing the same mutation in both tissues. Moreover, similar mutations were demonstrated at c-Ki-ras codon 61 in 1 of 7 ear duct tumors (CAA to CAT) and in 1 of 8 tumors of the small intestine (CAA to CTA) as well as at c-N-ras 61 (CAA to CTA) in a pancreatic metastasis. Additional transfection experiments of some tumors scoring negative for ras gene mutations in dot blot analyses revealed a CAA to CTA transversion at codon 61 of the c-Ha-ras gene in 1 forestomach tumor as well as at codon 61 of the c-N-ras in 1 hyperplasia of the pancreas and in 1 lymphoma. The apparent selectivity for mutations at adenine residues in AAI induced tumors is consistent with the identification of an N6-deoxyadenosine-AAI adduct formed by reaction of AAI with DNA in vitro, suggesting that carcinogen-deoxyadenosine adducts are the critical lesions in the tumor initiation by aristolochic acid.

Animals↗
PubMed1990

Aristolochic acid binds covalently to the exocyclic amino group of purine nucleotides in DNA.

The plant extract aristolochic acid (AA) has been used as a herbal drug in many cultures since antiquity. In 1982 AA was shown to be mutagenic and a strong carcinogen in Wistar rats. The crude mixture consists of five nitrophenanthrene carboxylic acid derivatives with aristolochic acid I [AA I; 8-methoxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxyli c acid] being the major component. The isolated compound has been found to be mutagenic in the Ames assay. The major metabolite of AA I formed under anaerobic conditions in vitro and excreted in vivo in several species including man, is the reduction product aristolactam I. Using the 32P-postlabeling assay, we could show that AA I forms covalent DNA adducts upon metabolic activation in vitro and in vivo in different organs in the rat. Xanthine oxidase, a mammalian nitroreductase, has served as a sufficient model system mimicking the reductive route of in vivo activation of carcinogenic nitroarenes. This paper reports on two major fluorescent adducts of AA I formed by in vitro reaction of AA I with xanthine oxidase and deoxyguanosine or deoxyadenosine. After isolation and purification by preparative HPLC the adducts were characterized by 1H-NMR, FAB mass, UV/Vis and fluorescence spectroscopy. Their structures were elucidated as 7-(deoxyguanosin-N2-yl)-aristolactam I and 7-(deoxyadenosin-N6-yl)-aristolactam I. These findings are in marked contrast to the results reported for other nitroaromatic carcinogens, where C8-modified deoxyguanosine adducts predominate and N2-substituted deoxyguanosine derivatives are found as minor reaction products. Our results suggest a cyclic N-acylnitrenium ion with delocalized positive charge as the ultimate carcinogenic species, binding preferentially to the exocyclic amino group of purine nucleotides in DNA.

Aristolochic Acids↗
PubMed1990

32P-postlabelling analysis of the DNA adducts formed by aristolochic acid I and II.

We report the quantitation of DNA adducts in target and nontarget organs of male Wistar rats treated orally with five daily doses (10 mg/kg body wt) aristolochic acid I (AAI) or aristolochic acid II (AAII), the major components of the herbal drug aristolochic acid, a forestomach carcinogen in the rat. DNA adducts were detected and analysed using the nuclease P1-enhanced variation of the Randerath 32P-postlabelling assay. The highest level of DNA adducts formed was by AAI in the target organ, forestomach (330 +/- 30 adducts/10(8) nucleotides), but high levels were also observed in a non-target tissue, the glandular stomach (180 +/- 15). Lower amounts of adducts were detected in liver, kidney and urinary bladder epithelium. With AAII the binding levels were generally lower than the AAI, the highest level of adducts being detected in kidney (80 +/- 20 adducts/10(8) nucleotides) and lower levels in liver, stomach and urinary bladder epithelia. Adduct patterns similar to those in vivo were observed in two new in vitro assays. Rat faecal bacteria were shown to be able to activate AAI and AAII to reactive species, which were trapped with exogenous calf thymus DNA and analysed by postlabelling. Incubation of AAI and AAII in explanted rat stomach held in short-term organ culture resulted in DNA adduct formation in the epithelia of both forestomach and glandular stomach. To assign the recently characterized in vitro nucleoside adducts of AAI to the bisphosphate derivatives, a new ion-pair HPLC procedure on a reversed-phase column was developed. By monitoring Cerenkov radiation on-line, a good separation of AAI adducts was observed, demonstrating that adducts formed in vivo were chromatographically indistinguishable with those formed in vitro, and previously characterized as an aristolactam I moiety bound covalently to the exocyclic amino groups of deoxyadenosine and deoxyguanosine.

Animals↗
PubMedDate not supplied

N6-adenyl arylation of DNA by aristolochic acid II and a synthetic model for the putative proximate carcinogen.

Aristolochic acid II (AAII), one of the major components of the carcinogenic plant extract aristolochic acid, is known to be mutagenic and to form DNA adducts in vitro and in vivo. The major fluorescent DNA adduct formed upon xanthine oxidase mediated reduction in the presence of calf thymus (CT-) DNA or deoxyadenosine was isolated by means of preparative HPLC and identified by fluorescence, UV/vis absorbance, and 1H NMR spectroscopy as 7-(deoxy-adenosin-N6-yl)aristolactam II. As a model proximate carcinogen, N-chloroaristolactam II was prepared chemically from aristolactam II, the reduction product of AAII. This model compound was spectroscopically characterized and found to react directly with CT-DNA without any activation, forming the same deoxyadenosine adduct. HPLC analysis with fluorescence monitoring detected this adduct in vivo in the liver DNA of Wistar rats treated orally with AAII. These results confirm the anticipated metabolic activation mechanism of AAII as occurring via a cyclic nitrenium ion.

Adenine↗