Biomedical subjects

W J Bodell

Publications and source records attributed to W J Bodell.

At least 37 records · Page 2Linked to original sources

PubMed1994

DNA adduct formation by tamoxifen with rat and human liver microsomal activation systems.

Using microsomal preparations from rat and human liver, we investigated the activation of the anti-estrogen compound tamoxifen (TMX) to form DNA adducts. Pretreatment of rats with phenobarbital increased DNA adduct formation by microsomal activation of TMX 3- to 6-fold, depending on the cofactors used. When reduced nicotinamide-adenine dinucleotide phosphate (NADPH) was used as a cofactor in human and rat microsomal activation systems, the relative DNA adduct levels were 2.9 and 5.2 x 10(-8) respectively and 1-3 TMX-DNA adducts were detected by 32P-postlabeling; DNA adduct 1 was the same in both microsomal systems. When cumene hydroperoxide (CuOOH) was used as a cofactor, activation of TMX produced four major DNA adducts and several minor DNA adducts in both rat and human liver microsomes; the relative adduct levels were 11.1 and 23.1 x 10(-8) respectively. TMX-DNA adducts 1, 4, 5 and 6 were similar in both human and rat microsomal systems with CuOOH as the cofactor. The TMX-DNA adducts formed with NADPH as the cofactor were clearly different from those formed with CuOOH as the cofactor, which implies that the metabolites leading to the individual DNA adducts were different. Addition of a P450 inhibitor, either n-octylamine or alpha-naphthylisothiocyanate, to the activation system reduced adduct formation by 70-93%. We propose that the TMX-DNA adducts formed with NADPH as the cofactor result from P450 acting as a mono-oxygenase, whereas the adducts formed with CuOOH as the cofactor result from P450 acting as a peroxidase. Our findings suggest that further studies may be required to establish the safety of TMX treatment of women for purposes other than chemotherapy.

Animals↗

PubMed1994

32P-post-labeling detection of DNA adducts in mononuclear cells of workers occupationally exposed to styrene.

32P-Post-labeling was used to analyze for the presence of DNA adducts in 47 workers exposed to styrene in a boat manufacturing facility. Individual airborne exposures measured several times over the course of 1 year ranged from 1 to 235 mg/m3 with a mean value of 65.6 mg/m3. Two adducts were detected in the DNA of mononuclear cells of these workers. The following levels of adducts were detected: adduct 1, range 0.6-102 x 10(-8) (mean 15.8 x 10(-8); adduct 2, range 0.1-70.9 x 10(-8) (mean 14.2 x 10(-8). Significant linear relationships were found between styrene exposure and both DNA adducts (adduct 2, r = 0.330, P = 0.012; adduct 1, r = 0.244, P = 0.049). Co-chromatography experiments identified DNA adduct 1 in the exposed samples as N2-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine-3',5'-bisphosphate. DNA adduct 2 remains unidentified. No significant linear relationships were observed between the level of DNA adducts and sister chromatid exchanges, possibly because of the poor precision of the 32P-post-labeling assay (the estimated coefficients of variation for adducts 1 and 2 were 2.54 and 1.96, respectively). These results demonstrate that occupational exposure to styrene results in the formation of DNA adducts in human mononuclear cells.

DNA↗

PubMed1994

Synthesis of a 25 base oligonucleotide containing a styrene oxide modification at the O6 position of 2'-deoxyguanosine at a defined site and incorporation studies of the similarly modified 2'-deoxyguanosine-5'-triphosphate.

A diastereomeric mixture of the regioisomers O6-(2-hydroxy-2-phenylethyl)-2'-deoxyguanosine (st6G, beta-isomer) and O6-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine (alpha-isomer) was site-specifically placed in a 25 base oligonucleotide template 5'-CCGCTAst6GCGGGTACCGAGCTCGAAT-3' using CED phosphoramidite chemistry. Using 32P-post-labeling we found the oligonucleotide to contain 95% of the beta-isomer and 5% of the alpha-isomer of st6G. st6G as the 3'-phosphate was found to be considerably more acid labile than O6-methyl-2'-deoxyguanosine-3'-phosphate, leading to dealkylation during oligonucleotide synthesis. The diastereomeric mixture of O6-(2-hydroxy-2-phenylethyl)-2'-deoxy-guanosine-5'-triphosphate (st6dGTP) was chemically synthesized and used as a substrate for the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. This study demonstrated that st6dGTP could be incorporated opposite deoxycytidine and did not completely block replication.

Base Sequence↗

PubMed1994

The effects of incorporation of bromodeoxyuridine into mammalian DNA on the migration patterns of DNA fragments subjected to pulsed-field gel electrophoresis after X irradiation or cutting with a restriction enzyme.

Incorporation of bromodeoxyuridine (BrdU) into DNA in both Chinese hamster ovary (CHO) cells and human melanoma (U1) cells reduced the rate of DNA migration in transverse alternating-field electrophoresis (TAFE) agarose gel optimized for separating molecules larger than 2 Mb. This "BrdU migration effect" was independent of the method of damaging the DNA; i.e., the effect was observed after irradiation of the cells or treatment of the plugs containing DNA with the restriction enzyme, Mlu-1, and similar results were found for CHO and U1 cells. However, when the amount of cutting of DNA was estimated from the amount of DNA migrating from the plugs, a difference between U1 cells and CHO cells was observed in that incorporation of BrdU enhanced the cutting of DNA by either irradiation or Mlu-1 digestion for U1 cells but not for CHO cells. Therefore, the "BrdU migration effect" could not be attributed to an increase in large molecules because of reduced cutting of the BrdU-labeled DNA. The decrease in migration rate during pulsed-field gel electrophoresis when BrdU replaces thymidine in the DNA is hypothesized to result from the increase in negative charge on BrdU-labeled DNA. An increase in electron charge density is expected to increase the reorientation time of BrdU-labeled DNA fragments during each voltage pulse due to an increase in the elongation length of the DNA caused by an increase in electronegativity.

Animals↗

PubMed1993

Peroxidase activation of hydroquinone results in the formation of DNA adducts in HL-60 cells, mouse bone marrow macrophages and human bone marrow.

Metabolism of benzene results in the formation of multiple metabolites, including hydroquinone (HQ). HQ is a reducing co-substrate for peroxidase enzymes, and the resultant semiquinone and para-benzoquinone (p-BQ) may bind to DNA. The role of peroxidase activation in the formation of DNA adducts by benzene metabolites has not been established. In this study we investigated the role of peroxidase activation in the formation of DNA adducts by HQ and p-BQ in HL-60 cells, human bone marrow (HBM) cells, mouse bone marrow macrophages (MBMM) and the U-937 and Raji leukemia cell lines. Adduct formation was measured by P1-enhanced 32P-postlabeling; peroxidase activity was measured with a spectrophotometric assay. Treatment with p-BQ resulted in the formation of two DNA adducts in all of the cell lines. The DNA adducts were identical in all of the cells, however, the adduct level varied by 80-fold. Treatment with HQ produced one DNA adduct in HL-60 cells, HBM and MBMM; no adducts were detected in U-937 or Raji cells. The HQ-DNA adducts in the three cell lines were identical. The adduct level was highest in the HL-60 cells, followed by HBM and MBMM. There was a statistically significant correlation between peroxidase activity and the formation of HQ-DNA adducts. These results suggest that peroxidase-mediated metabolism is involved in the activation of HQ to form DNA adducts in mouse bone marrow and HBM.

Animals↗

PubMed1993

DNA crosslinking, sister chromatid exchange and cytotoxicity of N-2-chloroethylnitrosoureas tethered to minor groove binding peptides.

Chloroethylnitrosoureas (CENU) are clinically important chemotherapeutic agents whose mechanism of action involves the formation of interstrand DNA crosslinks via an ethane bridge between N1-G and N3-C. CENU generally alkylate G at the N7- and O6-positions, with the latter lesion being the precursor to the interstrand crosslink. In previous studies, we reported the synthesis of CENU appended by a C2H4 linker to the N-terminus of DNA minor groove binding dipeptides (lex, information reading peptides) based on N-methylpyrrole-carboxamide subunits. Because of the dipeptide structure, these CENU-lex's react with DNA at adenines associated with lex equilibrium binding sites. No other CENU has been reported to yield A adducts. The biological evaluation of these CENU-lex's show that they are somewhat less cytotoxic than their simpler counterparts. In addition, in vitro studies show that the minor groove binding CENU-lex's afford a lower level of sister chromatid exchange (SCE) in 9L cells that are sensitive to CENU. There is no difference between CENU-lex in SCE induction in 9L-2 cells that are resistant to CENU. Formation of DNA interstrand crosslinks from the CENU-lex's is lower than for their nonaffinity binding analogs in low ionic strength buffer, but similar in the same buffer containing 200 mM NaCl. Salt inhibits crosslinking for all CENU, but distamycin, a competitive inhibitor of lex minor groove binding, uniquely enhances crosslinks for the CENU-lex's. These results are consistent with the novel minor groove adduction being a 'detoxification' pathway for the CENU-lex's since this lesion is formed at the expense of the cytotoxic major groove interstrand crosslink.

Alkylation↗

PubMed1993

Detection of dopamine--DNA adducts: potential role in Parkinson's disease.

Oxidation of catecholamines may lead to the formation of o-semiquinones and o-quinones in catecholaminergic brain tissues, and these reactive molecules may form DNA or protein adducts. In this study, cultured cells were treated with dopamine (DA) for 24 h and 32P-postlabelling was used to detect DA-DNA adducts. In HL-60 cells, 250 microM DA induced 8.5 DNA adducts/10(8) nucleotides; adduct formation was dose-dependent up to 500 microM DA. Addition of H2O2 increased the relative adduct levels 7- to 13-fold, but no adducts were detected when DA and ascorbic acid were added simultaneously. In human glioblastoma cell lines U87, U251, SF-763 and SF-767, 1000 microM DA produced 0.98-2.31 adducts/10(8) nucleotides. These results suggest that the formation of DNA adducts by DA may contribute to the development of certain neurodegenerative diseases such as Parkinson's disease.

Ascorbic Acid↗

PubMed1993

Restriction endonuclease recognition and southern hybridization of bromodeoxyuridine-substituted genomic DNA.

Human glioma cell lines exposed to various concentrations of bromodeoxyuridine (BrdUrd) were studied to determine the effect of BrdUrd substitution on restriction endonuclease recognition and Southern hybridization of genomic DNA. BrdUrd substitution had no effect on the recognition of restriction endonucleases. When the exposure to BrdUrd was 2h or less and the BrdUrd substitution rate was less than 40%, there was no difference in the density of hybridized bands after Southern hybridization using human non-recombinant complementary DNA as a probe. Hybridization was suppressed significantly by exposures longer than 24 h or BrdUrd substitution rates greater than 40%. These results suggest that the BrdUrd substitution rate and the exposure time to BrdUrd influence the hybridization reaction by a DNA probe. Brief exposure (up to 2 h) to BrdUrd does not influence restriction endonuclease recognition or Southern hybridization of genomic DNA.

Base Sequence↗

PubMed1993

Investigation of benzene-DNA adducts and their detection in human bone marrow.

We have examined DNA adduct formation in HL-60 cells and human bone marrow treated with either hydroquinone or p-benzoquinone and have found that these treatments produce the same DNA adduct in both cell types. The DNA adduct level from these treatments varied from 0.05 to 7.5 adducts per 10(7) nucleotides as a function of treatment time and concentration for both compounds. Reaction of calf thymus DNA with p-benzoquinone produced three adducts as detected by 32P-postlabeling. These adducts have been identified as (3'-hydroxy)-3,N4-benzetheno-2'-deoxycytidine-3'-phosphate; (3'-hydroxy)-1,N6-benzetheno-2'-deoxyadenosine-3'-phosphate; and (3'-hydroxy)-1,N2-benzetheno-2'-deoxyguanosine-3'-phosphate. The DNA adduct formed in HL-60 cells did not correspond to any of the principal adducts formed in DNA reacted with p-benzoquinone, suggesting that cellular environment modifies DNA adduct production by p-benzoquinone. These studies demonstrate that DNA adduct formation occurs in human bone marrow treated with benzene metabolites and suggest that P1-enhanced 32P-postlabeling may be used to detect DNA adducts resulting from benzene exposure.

Benzene↗

PubMed1992

Potentiation of DNA adduct formation in HL-60 cells by combinations of benzene metabolites.

Using P1 nuclease enhanced 32P postlabeling, we investigated DNA adduct formation in HL-60 promyelocytic leukemia cells treated with the benzene metabolites hydroquinone, catechol, and 1,2,4-benzenetriol. Comparison of the slopes of the dose-response curves showed that hydroquinone was 7-9 times more effective than 1,2,4,-benzenetriol and catechol at inducing DNA adducts. Comparison of hydroquinone with catechol showed a good correlation between adduct formation and cytotoxicity. Similar comparisons of hydroquinone and 1,2,4,-benzenetriol suggest that cellular processes in addition to DNA adduct formation contributed to cytotoxicity. In cells treated with the combination of hydroquinone and either catechol or 1,2,4,-benzenetriol, DNA adduct formation was 3-6 times greater than the sum of adduct formation produced by single-agent treatments. Treatment with hydroquinone and 1,2,4,-benzenetriol produced DNA adducts not detected after treatment with either metabolite alone. The synergistic interaction of benzene metabolites in the production of DNA adducts may play an important role in the genotoxic effects of benzene in vivo.

Autoradiography↗

PubMed1992

Iododeoxyuridine incorporation and radiosensitization in three human tumor cell lines.

Iododeoxyuridine is a halogenated pyrimidine and non-hypoxic cell radiosensitizer currently being used in clinical trials. The amount of radiosensitization by IdUrd is related to the amount of incorporation of the drug into a cell's DNA. These experiments were carried out in three human tumor cell lines (lung, glioma, and melanoma) in monolayer culture exposed to concentrations of IdUrd from 0.1-10 microM for one and three cell cycles before irradiation to determine incorporation and sensitization as a function of drug exposure. Except for the lung cell line, which required greater than 1 microM IdUrd, these cells demonstrate radiosensitization when exposed to 0.1 microM or greater of IdUrd. Maximum sensitization occurred at 10 microM IdUrd for all the cell lines at three cell cycles. The percent thymidine replacement by IdUrd increased with increasing concentrations, but was cell line dependent. Maximum percent replacement occurred at 10 microM at three cell cycles for all the cell lines: lung = 22.4%, glioma = 32.0%, and melanoma = 39.1%. The relationships between percent thymidine replacement and sensitization are not identical across these human tumor cell lines. If IdUrd is going to be a successful radiosensitizer in clinical trials, sustained plasma levels of 10 microM or greater for at least three cell cycles should be achieved during irradiation. This may be best accomplished with repeated short exposures to IdUrd (three cell cycles or approximately 4 days in these cell lines) every 1-2 weeks during radiation. Measurements of thymidine replacement in a tumor biopsy should be attempted prior to radiation to develop a predictive assay for radiosensitization.

Cell Survival↗

PubMed1992

Peroxidative activation of o-phenylhydroquinone leads to the formation of DNA adducts in HL-60 cells.

Using 32P-postlabeling we studied DNA adduct formation in HL-60 cells treated with the o-phenylphenol metabolites o-phenylhydroquinone (o-PHQ) and o-phenylbenzoquinone (o-PBQ). Treatment with 25-500 microM o-PHQ for 8 h produced one principal and three minor adducts with a relative distribution of 80, 10, 6 and 4%. The relative adduct levels from these treatments were 0.26-2.31 adducts/10(7) nucleotides. Treatment with 25-250 microM o-PBQ for 2 h resulted in a similar level of DNA modification and adduct distribution. Reaction of purified calf thymus DNA with o-PBQ produced one DNA adduct, which did not correspond to the major adduct produced in HL-60 cells. These results show that o-PHQ and o-PBQ can form DNA adducts. Peroxidase activation of o-phenylphenol may therefore play a role in the carcinogenic effect of this compound.

Benzoquinones↗

PubMed1992

Identification of N2-substituted 2'-deoxyguanosine-3'-phosphate adducts detected by 32P-postlabeling of styrene-oxide-treated DNA.

Styrene-7,8-oxide, a metabolite of the industrial chemical styrene, was reacted with calf thymus DNA. Six adducts were detected by 32P-postlabeling. The two diastereomers of N2-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine-3'-phosphate and the corresponding N-1 substituted compounds were isolated from the aqueous reaction mixture of 2'-deoxyguanosine-3'-phosphate and styrene-7,8-oxide (pH 10.5) and characterized by liquid secondary-ion and four-sector tandem mass spectrometry, ultraviolet, circular dichroism, and fluorescence spectrophotometry, and 32P-postlabeling. Co-chromatography of the DNA-styrene-7,8-oxide reaction products with the synthetic standards showed that adduct no. 6 arose as a result of aralkylation at the N2-exocyclic site of the guanine base. The recovery of the N2-adduct was dependent on the concentration of the solvent used during octadecylsilyl chromatography. These studies revealed that the N2-guanosine derivatives are the major products of the reaction of DNA and styrene-7,8-oxide in vitro detected by 32P-postlabeling.

Autoradiography↗

PubMed1991

3-Methyladenine DNA glycosylase activity in a glial cell line sensitive to the haloethylnitrosoureas in comparison with a resistant cell line.

Extracts of a glial cell line (SF-126) which is sensitive to the cytotoxic effect of the haloethylnitrosoureas and of a cell line (SF-188) which is resistant to these agents have been tested for their ability to release methylated bases from a DNA substrate which has been modified with [3H]dimethyl sulfate. In comparison with the sensitive cell line, extracts from the resistant cell line have 2-3-fold higher enzymatic activity. High performance liquid chromatography profiles of the bases which are released by these extracts show that the activity is specific for 3-methyladenine, suggesting that the resistant cells contain elevated levels of 3-methyladenine DNA glycosylase. Previous studies have shown that these cells also contain elevated levels of O6-alkylguanine-DNA alkyl-transferase, suggesting that both enzyme activities may be involved in the resistance of this cell line to the haloethylnitrosoureas.

DNA↗

PubMed1991

Positive therapeutic interaction between thiopurines and alkylating drugs in human glioma xenografts.

We used human anaplastic glioma xenografts to evaluate the therapeutic efficacy of combinations of alkylating drugs, either 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-(2,5-dioxo-3-piperidyl)-1-nitrosourea (PCNU), or procarbazine, and thiopurines, either 6-mercaptopurine (6MP) or 6-thioguanine (6TG). Using growth delay as the endpoint in subcutaneous (s.c.) tumors and increased life span as the endpoint in intracranial (i.c.) tumors, we found that combinations of chloroethylnitrosoureas (CENUs) and thiopurines were significantly more active than either type of agent alone. In contrast, combinations of procarbazine and thiopurines were not significantly more active than procarbazine alone. The therapeutic potentiation of the CENU was greater when the latter was given on the 4th day of the thiopurine treatment cycle than when it was given on the 1st day. Characterization of the interaction between CENUs and thiopurines also revealed a supraadditive therapeutic response at higher BCNU doses in combination with 6TG. Interaction between the nitrosoureas and the thiopurines probably occurs in the guanine base of tumor DNA and has important therapeutic implications.

Alkylating Agents↗

PubMed1991

Detection of DNA adducts in HL-60 cells treated with hydroquinone and p-benzoquinone by 32P-postlabeling.

We have examined DNA adduct formation and cytotoxicity in HL-60 cells treated with either hydroquinone (HQ) or p-benzoquinone (p-BQ). Treatment of HL-60 cells with either HQ or p-BQ produced the same DNA adduct. The DNA adduct level varied from 0.05 to 10 adducts per 10(7) nucleotides as a function of treatment time and concentration for both compounds. To achieve the same DNA adduct level required higher concentrations and longer treatment times with HQ compared to p-BQ. p-BQ was also more cytotoxic to HL-60 cells than HQ. Reaction of calf thymus DNA with a p-BQ/HQ mixture produced five adducts as detected by 32P-postlabeling. Two isomers of (hydroxy)-1,N2-benzetheno-2'- deoxyguanosine-3'-phosphate were isolated from the reaction of 2'-deoxyguanosine-3'-phosphate with a p-BQ/HQ mixture and one of the isomers was identified as adduct no. 1 of the DNA reaction. The DNA adduct formed in HL-60 cells treated with HQ or p-BQ did not correspond to any of the principal adducts formed in DNA reacted with p-BQ/HQ. This result suggests that cellular mechanisms modify DNA adduct formation by HQ and p-BQ.

Benzoquinones↗

PubMed1991

Molecular dosimetry of sister chromatid exchange induction in 9L cells treated with 6-thioguanine.

The induction of sister chromatid exchanges (SCE) in 9L cells treated with 6-thioguanine (6-TG) has been investigated. A 24 h treatment with 0.2 microM 6-TG induced approximately 28 SCE/metaphase. The dose-response curve was linear at doses below 0.2 microM and had a slope of 139 SCE/metaphase/microM 6-TG. At concentrations of 0.023 to 1 microM, incorporation of 6-TG in DNA was linear with dose. The slope of the dose-response curve was 4135 mumol 6-TG/mol DNA/microM 6-TG. Comparison of these results with those obtained in our previous studies of the monofunctional alkylating agent ethylnitrosourea and the bifunctional alkylating agent 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea suggest that to accurately estimate the effectiveness of particular DNA modifications at inducing SCE, the extent of formation of these DNA modifications must be known.

Alkylating Agents↗

PubMed1990

Binding of benzo[a]pyrene to DNA by cytochrome P-450 catalyzed one-electron oxidation in rat liver microsomes and nuclei.

To investigate whether cytochrome P-450 catalyzes the covalent binding of substrates to DNA by one-electron oxidation, the ability of both uninduced and 3-methylcholanthrene (MC) induced rat liver microsomes and nuclei to catalyze covalent binding of benzo[a]pyrene (BP) to DNA and formation of the labile adduct 7-(benzo[a]pyren-6-yl)guanine (BP-N7Gua) was investigated. This adduct arises from the reaction of the BP radical cation at C-6 with the nucleophilic N-7 of the guanine moiety. In the various systems studied, 1-9 times more BP-N7Gua adduct was isolated than the total amount of stable BP adducts in the DNA. The specific cytochrome P-450 inhibitor 2-[(4,6-dichloro-o-biphenyl)oxy]ethylamine hydrobromide (DPEA) reduced or eliminated BP metabolism, binding of BP to DNA, and formation of BP-N7Gua by cytochrome P-450 in both microsomes and nuclei. The effects of the antioxidants cysteine, glutathione, and p-methoxythiophenol were also investigated. Although cysteine had no effect on the microsome-catalyzed processes, glutathione and p-methoxythiophenol inhibited BP metabolism, binding of BP to DNA, and formation of BP-N7Gua by cytochrome P-450 in both microsomes and nuclei. The decreased levels of binding of BP to DNA in the presence of glutathione or p-methoxythiophenol are matched by decreased amounts of BP-N7Gua adduct and of stable BP-DNA adducts detected by the 32P-postlabeling technique. This study represents the first demonstration of cytochrome P-450 mediating covalent binding of substrates to DNA via one-electron oxidation and suggests that this enzyme can catalyze peroxidase-type electron-transfer reactions.

Animals↗