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Biomedical subjects

D H Phillips

Publications and source records attributed to D H Phillips.

At least 163 records · Page 9Linked to original sources
PubMed1989

Enzyme-mediated phosphorylation of polycyclic hydrocarbon metabolites: detection of non-adduct compounds in the 32P-postlabelling assay.

32P-Postlabelling analysis is a sensitive method of detecting covalent modification of DNA by chemical carcinogens. We demonstrate that tetrol derivatives of the polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene (BP) and chrysene become 32P-labelled in the assay in the absence of nucleic acids. The transfer of 32P from [gamma-32P]ATP to the PAH derivatives requires T4 polynucleotide kinase. Phosphorylated dihydrodiols, phenols, triols and parent hydrocarbons were not detected under standard TLC conditions. Labelling of the non-nucleotide substrates was at least 2000-fold less efficient than labelling of a synthetic BP - DNA adduct. Using 75 microCi[gamma-32P]ATP, the detection limit for BP tetrols was 100-200 pg. Labelling of non-adduct substrates is unlikely to interfere with the analysis of DNA isolated from mammalian tissues, but DNA modified by electrophiles in vitro may, if inadequately purified, give rise to spurious radioactive products.

Adenosine Triphosphate↗
PubMed1989

DNA adduct formation by 7-methoxy-2-nitro-naphtho[2,1-b]furan (R7000), an extremely potent mutagen.

The effects on DNA, in bacteria, of 7-methoxy-2-nitro-naphtho[2,1-b]furan (R7000), a very potent genotoxic product from the 2-nitronaphthofuran series, were investigated with two different approaches: (i) measurement of the binding of the radiolabelled mutagen to DNA and (ii) detection by the '32P-postlabelling' method of DNA adducts following treatment with unlabelled mutagens. The covalent binding of R7000 to DNA in Escherichia coli was demonstrated by both methods, and in the latter case it was found to involve the formation of nine different adducts. Formation of adducts by R7000 was shown to require metabolic activation of the compound.

Chromatography, Thin Layer↗
PubMed1988

Formation of DNA adducts in human skin maintained in short-term organ culture and treated with coal-tar, creosote or bitumen.

Adult and foetal human skin samples maintained in short-term organ culture were treated topically with solutions of coal-tar, creosote or bitumen, then DNA was isolated and analysed by 32P-post-labelling for the presence of aromatic DNA adducts. Autoradiographs of the 32P-labelled adducts resolved on polyethyleneimine-cellulose tlc sheets revealed a band of radioactivity indicative of the formation of adducts by a large number of components in these complex carcinogenic mixtures. Single doses of the materials, similar to those used to initiate tumours in experimental animals, resulted in the formation of approximately 0.3 fmol total adducts/micrograms DNA. The levels of adducts formed in human skin are thus similar to those formed in mouse skin after administration of doses known to be carcinogenic to the latter. The results provide direct evidence of DNA damage in human skin by materials strongly suspected of being carcinogenic to humans, and point to a method for evaluating other complex mixtures of aromatic chemicals for their potential carcinogenic hazard.

Coal Tar↗
PubMed1988

Monitoring occupational exposure to carcinogens: detection by 32P-postlabelling of aromatic DNA adducts in white blood cells from iron foundry workers.

Blood samples were volunteered by workers in a Finnish iron foundry who were occupationally exposed to polycyclic aromatic hydrocarbons and from control subjects not known to be occupationally exposed to this class of chemical carcinogens. DNA was isolated from peripheral white blood cells and digested with micrococcal nuclease, spleen phosphodiesterase and nuclease P1. The DNA digest was then incubated with [gamma-32P]ATP and polynucleotide kinase. Aromatic adducts present in the digest that were resistant to nuclease P1 were thus 32P-labelled while unmodified nucleotides were not. The 32P-labelled adducts were resolved by t.l.c. and detected by autoradiography. Foundry workers were classified as belonging to high, medium or low exposure groups according to their exposure to airborne benzo[a]pyrene (high greater than 0.2, medium 0.05-0.2, low less than 0.05 microgram BP/m3 air). Aromatic adducts were found to be present in DNA from 3/4 samples from the high exposure group, 8/10 samples from the medium exposure group. 4/18 samples from the low exposure group and 1/9 samples from the unexposed controls. The levels of adducts found in the high and medium group samples ranged up to 1 adduct in 10(7) nucleotides but the levels formed in the low exposure group samples were not significantly different from those in unexposed controls. No differences related to the smoking habits of the subjects were observed. Most of the DNA adducts detected had chromatographic mobilities distinct from those formed when the 7,8-diol 9,10-oxide of BP reacted with DNA. The results indicate that highly-exposed individuals are more likely to contain aromatic DNA adducts in their white blood cells, but large interindividual variations were evident. In addition, multiple samples from the same subjects indicate that qualitative and quantitative changes in adduct patterns occur with time. This pilot study suggests that 32P-postlabelling may be useful in monitoring human exposure to known and to previously unidentified environmental genotoxic agents.

Carcinogens↗
PubMed1988

Metabolic activation of 7-ethyl- and 7-methylbenz[a]anthracene in mouse skin.

The carcinogen 7-methylbenz[a]anthracene (7-MBA) is considered to be metabolically activated via its bay-region dihydrodiol-epoxide, trans-3,4-dihydro-3,4-dihydroxy-7-methyl-benz[a]anthracene 1,2-oxide (7-MBA-3,4-diol 1,2-oxide). When tested on mouse skin, a target tissue for polycylic aromatic hydrocarbon carcinogenesis, 7-ethylbenz[a]anthracene (7-EBA) was much less active than 7-MBA, and this difference may be due to differences in the pathways by which the two compounds are metabolized and activated. In the present work, the metabolism by mouse-skin microsomes of both hydrocarbons to dihydrodiols has been examined. Both were metabolized to a similar extent with the 8,9-dihydrodiols being detected as the predominant metabolites. The 3,4-, 5,6-and 10,11-dihydrodiols of 7-MBA and the 3,4- and 10, 11-dihydrodiols of 7-EBA, were also detected. 7-MBA was found to bind covalently to microsomal protein at 10 times the level of 7-EBA. The covalent binding of benz[a]anthracene (BA), 7-EBA and 7-MBA to DNA in mouse skin following topical application was determined using the 32P-postlabelling assay. The results correlated with the relative carcinogenic activities of the compounds with 7-MBA binding at five and nine times the level of 7-EBA and BA respectively. For all three hydrocarbons, the major hydrocarbon: 32P-labelled nucleoside bisphosphate, eluted in the same area of the TLC maps, suggesting the involvement of a common type of bay-region dihydrodiol-epoxide intermediate.

Animals↗
PubMed1988

Further metabolism of diol-epoxides of chrysene and dibenz[a,c]anthracene to DNA binding species as evidenced by 32P-postlabelling analysis.

Incubation of r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-chrysene-1,2-diol 3,4-oxide), the bay-region diol-epoxide of chrysene, with rat liver microsomes in the presence of NADP+ and DNA, followed by 32P-postlabelling analysis of the DNA, revealed the presence of at least two adducts not detected when anti-chrysene-1,2-diol 3,4-oxide was incubated with DNA alone. The formation of these adducts was not blocked by the epoxide hydrolase inhibitor 1,1,1-trichloropropane-2,3-oxide. One of the adducts cochromatographed with the adduct spot obtained when authentic 9-hydroxy-r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-9-OH-chrysene-1,2-diol 3,4-oxide) was reacted with DNA. Evidence suggested that a second adduct could also be formed by further metabolism of anti-9-OH-chrysene-1,2-diol 3,4-oxide. In addition, evidence was obtained for the further metabolism of the syn-isomer of chrysene 1,2-diol 3,4-oxide and the anti-isomer of a non-bay-region diol-epoxide of dibenz[a,c]anthracene to DNA binding species, but not for that of either the anti- or syn-isomers of the bay-region diol-epoxide of benzo[a]pyrene, the anti-isomers of the bay-region or a non-bay-region diol-epoxide of benz[a]anthracene, or the anti-isomer of the bay-region diol-epoxide of benzo[b]fluoranthene.

Animals↗
PubMed1988

Covalent binding of components of coal-tar, creosote and bitumen to the DNA of the skin and lungs of mice following topical application.

In order to assess the DNA damaging ability of complex carcinogenic mixtures, male Parkes mice were treated topically with solutions of (i) pharmaceutical coal-tar, (ii) creosote, a blend of coal-tar fractions or (iii) bitumen, a product of oil-refining. DNA was isolated from the treated skin and analysed by 32P-post-labelling. A band of radioactivity was obtained on polyethyleneimine--cellulose TLC indicating the formation of DNA adducts by a large number of different chemical compounds present in these fossil fuel products. The chromatographic properties of the adducts were broadly characteristic of those formed by polycyclic aromatic hydrocarbons. The levels of DNA binding were approximately 0.4 fmol total adducts/micrograms DNA 24 h after treatment for coal-tar and creosote and 0.09 fmol/micrograms DNA for bitumen treatment. The persistence of adducts in mouse skin following a single dose of either coal-tar or creosote was found to exhibit a phase of rapid removal, in which one half to two thirds of the initial levels of adducts, detected at 24 h after treatment, were removed by 7 days followed by a second phase in which one half to two thirds of the remainder was removed in the succeeding 25 days. When mice were treated topically with multiple carcinogenic doses of coal-tar, creosote or bitumen for up to 5 weeks, a steady accumulation of adducts was seen in skin DNA during the course of the treatment, approaching a steady-state level towards the end of the treatment period in some instances. A similar accumulation of adducts was also evident in lung DNA, the levels being approximately half those attained in skin. The results demonstrate the application of 32P-post-labelling to the detection of DNA adducts formed in vivo by complex carcinogenic mixtures of the type to which humans are exposed.

Administration, Topical↗
PubMed1988

Activating mutations in human c-Ha-ras-1 gene induced by reactive derivatives of safrole and the glutamic pyrolysis product, Glu-P-3.

Foci of transformed NIH3T3 cells were observed after transfection of plasmids containing the c-Ha-ras-1 protooncogene modified in vitro either with the 3-N,N-acetoxyacetyl derivative (N-AcO-AGlu-P-3) of the mutagenic L-glutamic acid pyrolysis product 3-amino-4,6-dimethyldipyrido-[1,2-a:3',2'-d]imidazole (Glu-P-3) or with 1'-acetoxysafrole (AcO-S), a reactive derivative of the carcinogen safrole. DNA isolated from these foci were used in a second round of transfection, and the DNA obtained from the secondary transformants was analysed to determine the nature of mutations responsible for activating the protooncogene. The polymerase chain reaction method was used to amplify sequences of the gene likely to contain activating mutations, and these regions were then subjected to selective hybridization with specific oligonucleotides to locate and identify the point mutations. Five out of six transformants induced by N-AcO-AGlu-P-3 contained mutations at codon 61. Three of the codon 61 mutations were at the first base and the other two were at the third base, all were GC----TA transversions. Two AcO-S-induced transformants contained a GC----TA transversion, in one case at the first base of codon 61, in the other at the first base of codon 12. Another AcO-S-induced transformant, and the sixth transformant induced by N-AcO-AGlu-P-3 were apparently not mutated in codon 12, 61 or 117. Both N-AcO-AGlu-P-3 and AcO-S react predominantly with guanine residues in DNA, and all the mutations identified here were at GC base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Aromatic DNA adducts in white blood cells of foundry workers.

Blood samples were obtained from volunteers working in a Finnish iron foundry who were occupationally exposed to polycyclic aromatic hydrocarbons (PAH) and from control subjects not known to be occupationally exposed to this class of chemical carcinogens. Foundry workers were classified as belonging to high, medium or low exposure groups according to their exposure to airborne benzo[a]pyrene: high, greater than 0.2: medium, 0.05-0.2: low, less than 0.05 micrograms benzo[a]pyrene/m3 air). Aromatic adducts were found to be present in white blood cell DNA from most of the exposed workers using the enzyme-linked immunosorbent assay (ELISA) to detect aromatic DNA adducts and the 32P-postlabelling technique. There was a dose-response relationship between the estimated exposure and adduct levels by both methods, and a reasonable correlation between the results of the immunoassay and postlabelling carried out in two laboratories. The levels of adducts found in the samples from the high and medium exposure groups by ELISA ranged up to five adducts in 10(7) nucleotides: the aromatic adducts detected by the postlabeling assay were at a level of two adducts/10(8) nucleotides in the high and medium exposure categories. No effect due to age, sex or the smoking habits of the subjects was observed. The results indicate that DNA extracted from white blood cells of highly exposed workers is more likely to contain aromatic DNA adducts than that from workers without occupational exposure to PAH, but large interindividual variations were evident. This study suggests that the antibody and 32P-postlabelling assays may be useful in monitoring human exposure to known and previously unidentified environmental genotoxic agents.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1988

A comparison of 32P-postlabelling and immunological methods to examine human lung DNA for benzo[a]pyrene adducts.

Human lung DNA isolated from surgical specimens has been examined for the presence of polycyclic aromatic hydrocarbon-DNA adducts using both 32P-postlabelling and immunological methods. Of 12 samples examined to date, five had detectable amounts of benzo[a]pyrene diol epoxide-DNA adducts (BPDE-DNA) as determined by the enzyme-linked immunosorbent assay (ELISA), after immunoaffinity concentration. Values ranged from 3.5 to 11.5 fmol/mg DNA. When the same group of samples was analysed using the 32P-postlabelling technique, adducts could be detected in all the samples examined. There was generally not a good correspondence between the two methods. The number of adducts measured by 32P-postlabelling ranged from 1-100 per 10(8) nucleotides, which is some two orders of magnitude higher than with the immunological method, indicating that the BPDE-DNA adduct is probably not the major adduct present in these samples.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1988

An aromatic DNA adduct in colonic mucosa from patients with colorectal cancer.

The incidence of colorectal cancer in the western hemisphere is though to be the result, in part, of environmental agents, and many studies strongly implicate diet as a determining factor. It is conceivable that the ingestion of genotoxic chemicals present in food or the endogenous formation of such substances in the gut may initiate colorectal cancer in humans. In the present study, 32P-postlabelling has been used to examine DNA from normal-appearing colonic mucosa obtained from (i) patients undergoing surgery for colorectal cancer and (ii) adult and fetal controls for the presence of aromatic DNA adducts.

Colon↗
PubMed1987

Formation of DNA adducts in mouse skin treated with metabolites of chrysene.

Analysis by 32P-postlabelling of DNA isolated from mouse skin that had been treated in vivo with the polycyclic hydrocarbon chrysene revealed the presence of 7 adducts. All 7 adducts were also present in DNA from mice treated with trans-1,2-dihydro-1,2-dihydroxychrysene (chrysene-1,2-diol), and one of them, adduct 2, was formed from the triol derivative 9-hydroxy-trans-1,2- dihydro-1,2-dihydroxychrysene (9-hydroxychrysene-1,2-diol) and from 3-hydroxychrysene. Adducts were not detected in DNA from mice treated with trans-3,4-dihydro-3,4-dihydroxychrysene (chrysine-3,4-diol) or with 1-, 2-, 4-, 5- or 6-hydroxychrysene. In vitro modification of DNA by the anti-isomer of the bay-region diol-epoxide yielded adducts 3-7, while the corresponding triol-epoxide yielded adducts 2. It is concluded that chrysene activation in mouse skin proceeds principally via the bay-region diol-epoxide and to a lesser extent via the related bay-region triol-epoxide.

Animals↗
PubMed1986

Mutations activating human c-Ha-ras1 protooncogene (HRAS1) induced by chemical carcinogens and depurination.

In vitro modification of plasmids containing the human c-Ha-ras1 protooncogene (HRAS1) with the ultimate carcinogens N-acetoxy-2-acetylaminofluorene and r-7, t-8-dihydroxy-t-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (anti-BPDE) generated a transforming oncogene when the modified DNA was transfected into NIH 3T3 cells. The protooncogene was also activated by heating the plasmid at 70 degrees C, pH 4, to generate apurinic/apyrimidinic sites in the DNA. DNA isolated from transformed foci was analyzed by hybridization with 20-mer oligonucleotides designed to detect single point mutations within two regions of the gene commonly found to be mutated in tumor DNA. Of 23 transformants studied, 7 contained a mutation in the region of the 12th codon, whereas the remaining 16 were mutated in the 61st codon. Of the codon-61 mutants, 6 were mutated at the first base position (C X G), 5 at the second (A X T), and 5 at the third (G X C). The point mutations induced by anti-BPDE were predominantly G X C----T X A and A X T----T X A base substitutions, whereas four N-acetoxy-2-acetylaminofluorene-induced mutations were all G X C----T X A, and a single depurination-induced activation that was analyzed contained an A X T----T X A transversion. Together, these methods provide a useful means of determining point mutations produced by DNA-damaging agents in mammalian cells.

Acetoxyacetylaminofluorene↗
PubMed1986

Mutagenic potential of DNA adducts formed by diol-epoxides, triol-epoxides and the K-region epoxide of chrysene in mammalian cells.

The syn- and anti-isomers of chrysene-1,2-diol-3,4-oxide (syn-diol-epoxide and anti-diol-epoxide) and of 9-hydroxychrysene-1,2-diol-3,4-oxide (syn-triol-epoxide and anti-triol-epoxide), and chrysene-5,6-oxide, the K-region epoxide, were tested for their ability to induce 6-thioguanine-resistant mutants in V79 Chinese hamster cells. The levels of DNA adducts formed by each compound in the V79 cells were determined by 32P-post-labelling analysis. The most potent mutagen, in terms of the mutation frequency/nmol compound administered, was the anti-triol-epoxide, which was 1.7 times as active as the anti-diol-epoxide. The anti-diol-epoxide was approximately 10 times more active than both the syn-triol-epoxide and the syn-diol-epoxide, which in turn were several times more active than the K-region epoxide. However, when the results were expressed as mutations/pmol total adducts formed, the anti-triol-epoxide and anti-diol-epoxide were shown to be of similar potency and approximately twice as active as the other three compounds. Thus differences in the conformation of adducts formed with DNA by syn- and anti-isomers may be responsible for their different mutagenic potentials; the presence of a phenolic OH-group at the 9-position of a chrysene-1,2-diol-3,4-oxide appears to increase its chemical reactivity.

Cell Line↗
PubMed1986

Aromatic DNA adducts in human bone marrow and peripheral blood leukocytes.

DNA from normal human bone marrow mononuclear and non-mononuclear cells was analysed by 32P-post-labelling for the presence of aromatic adducts. Ten out of 10 individuals showed the presence of adducts at levels of 1-9 adducts per 10(8) nucleotides that were not detected in four samples of human foetal bone marrow. Inter-individual variations in the patterns of these presumed aromatic adducts were observed. Similar adducts were also present in the DNA of peripheral white blood cells of both smokers and non-smokers, although at lower levels than in bone marrow. The data suggest that the adducts result from environmental exposure to an as-yet-unidentified genotoxic agent or agents.

Adenosine Triphosphate↗
PubMed1985

Metabolic activation of 7,12-dimethylbenz[a]anthracene in rat mammary tissue: fluorescence spectral characteristics of hydrocarbon-DNA adducts.

The hydrocarbon-deoxyribonucleoside adducts present in DNA isolated from the mammary glands of rats that had been treated with 7,12-dimethylbenz[a]anthracene (DMBA) were separated by Sephadex LH20 column chromatography, purified by high performance liquid chromatography (HPLC), and examined by photon-counting spectrophotofluorimetry. The adducts were found to have anthracene-like fluorescence spectra which is consistent with the reaction of diol-epoxides formed in the 1,2,3,4-ring of DMBA with mammary gland DNA.

9,10-Dimethyl-1,2-benzanthracene↗