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A Kootstra

Publications and source records attributed to A Kootstra.

At least 19 recordsLinked to original sources
PubMed1994

Protection from UV-B-induced DNA damage by flavonoids.

In this study an in vitro run-off transcription assay was used to determine if flavonoids can prevent the accumulation of UV-B-induced DNA damage. Template plasmid DNA was irradiated with UV-B light, which resulted in a decreased capacity to support transcription. Purified flavonoids naringenin and rutin as well as flavonoid extracts from apple skin prevented the accumulation of DNA damage. The results support the hypothesis that flavonoids protect DNA from UV-induced DNA damage.

Adenine Phosphoribosyltransferase↗
PubMed1992

Analysis of point mutations in murine c-Ha-ras of skin tumors initiated with dibenz[a,j]anthracene and derivatives.

This study was designed to evaluate the point mutations in the murine c-Ha-ras gene of skin papillomas induced by initiation with dibenz[a,j]anthracene (DB[a,j]A), its bay-region anti-diol epoxide ((+/-)anti-DB[a,j]A-DE), and a 7,14-dimethyl analogue (7,14-diMeDB[a,j]A). Recent studies (Nair RV, et al., Chem Res Toxicol 4:115-122, 1991) in our laboratory have revealed both deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts formed from the anti- and syn-diol epoxides of DB[a,j]A in cultured mouse epidermal cells after exposure to this hydrocarbon. Using PCR amplification and direct sequencing, we found specific A182----T transversion mutations (eight of 10 tumors) in codon 61 of c-Ha-ras in papillomas induced by initiation with DB[a,j]A. Analysis of papillomas generated by initiation with the more biologically potent analogue 7,14-diMeDB[a,j]A revealed that five of five tumors exhibited A182----T transversions in codon 61. The nature of the changes in the two DB[a,j]A tumors not showing codon 61 mutations in Ha-ras is currently not known since these tumor DNAs also did not possess c-Ha-ras mutations at codons 12, 13, or 59. Interestingly, papillomas produced by initiation with (+/-)anti-DB[a,j]A-DE also possessed A182----T transversion mutations in codon 61 of c-Ha-ras (five of five tumors). These data suggest that dAdo adducts derived from both parent hydrocarbons may play an important role in their tumor-initiating activity and possibly implicate a specific diol epoxide-dAdo adduct in this process.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

Preferential modification of GC boxes by benzo[a]pyrene-7,8-diol-9,10-epoxide.

The distribution of binding sites for the ultimate carcinogen anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE-l) in the 5' region of the Chinese hamster ovary aprt gene has been determined. A plasmid (pGAL) containing the entire hamster aprt gene including the 3' and 5' flanking regions was inserted into the BamHI site of the multiple cloning site of pGEM so that the T7 promoter was 5' to the aprt gene. In vitro transcription of BPDE-I-modified pGAL, using the T7 RNA polymerase, revealed two prominent transcriptional stop sites. One of these sites was located in the first exon of the aprt gene, whereas the second transcriptional stop was located approximately 150 bp upstream from the translational start site. This latter region contains two perfect GC-box consensus sequences that are potential Sp1 binding sites. Using a specific laser cutting technique to map BPDE-I DNA binding sites in the 5' flanking region of the aprt gene, we found that the DNA region containing the GC-box consensus sequences was indeed a hot spot for BPDE-I modification.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1986

The dynamics of chromatin carcinogen interactions in the human cell.

Human lung epithelioid cells were treated with Benzo (a) pyrene diol epoxide (anti) in order to establish the binding and removal of covalent adducts in chromosomal components. Isolating two different classes of mononucleosomes, it was found that their DNA contained different concentrations of B(a)PDE-DNA adducts, while in both these mononucleosomal preparations only histones H2A and H3 contained detectable amounts of the carcinogen. Further analysis showed that in the intact human cell the carcinogen-DNA adduct distribution is constantly changing as a function of differential accessibility and repair. These results emphasize the dynamics of chromatin-carcinogen modifications.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1982

Isolation of high mobility group-containing mononucleosomes from avian erythrocyte nuclei and their sensitivity to DNase I.

Conditions have been established which have led to the isolation of mononucleosomes which contain the high mobility group (HMG) proteins, in particular HMG 14, from mature chicken erythrocyte nuclei after extended micrococcal nuclease digestion. This selective enhancement of HMG-containing mononucleosomes appears to be due to their preferential solubilization at a time when other mononucleosomes, i.e. those containing H5 and H1 which represent the bulk of the mononucleosomes, were no longer soluble. Isolation of "early" mononucleosomes and subsequent analysis of these mononucleosomes after DNase I digestion showed that the DNA of these "early" mononucleosomes was more accessible to DNase I and that those mononucleosomes which contained HMG 14 were more soluble when the DNA became extensively degraded by DNase I. The resulting pattern of single-stranded DNA fragments suggests that the NH2 termini of the core histones no longer bind strongly to the nucleosomal DNA of the "early" mononucleosomes, and thus enhance the rate of DNase I digestion, while the presence of HMG 14 increased the solubility of these mononucleosomes. These two properties are probably the basis for the increased DNase I sensitivity of the transcriptionally active chromatin.

Animals↗
PubMed1982

Effect of histone acetylation on the formation and removal of B(a)P chromatin adducts.

The modification of core histone proteins in mouse 10T1/2 cells and human lung epitheloid (A549) cells by B(a)PDE in vivo and in vitro was found to be similar. Only histones H2A and H3 were extensively modified. Also other proteins, possibly A24 protein and the minor histone H1 species seem to be binding relatively high levels of this ultimate carcinogen. Butyrate treatment which causes hyperacetylation of the core histones, did not change the specificity of B(a)PDE binding to core histones, nor did it affect the initial level of DNA modification. The acetylated species of histone H3 were all accessible to B(a)PDE, suggesting that these epsilon-amino-groups of the lysine residues are not the targets of the B(a)PDE. The rate of removal of B(a)P-DNA adducts was not affected by butyrate treatment in either normal human or XP fibroblasts. Furthermore the B(a)P-core histones were not preferentially removed from normal human fibroblast chromatin during a 24 h post-treatment incubation.

Acetylation↗
PubMed1982

Selective modification of nuclear proteins by polycyclic aromatic hydrocarbons and by benzo[a]pyrene diol epoxides.

Metabolites of benzo[a]pyrene (B[a]P) have been shown to modify chromosomal proteins with great specificity. Using the (+) and (-) enantiomers of anti-B[a]P diol epoxide to label isolated nuclei we found a remarkable difference in the capacity of these two compounds to modify histones H2A and H3. The (+) enantiomer modified histones H2A and H3, while the (-) enantiomer, which was shown to modify mainly histone H2A, had a much lower affinity for histone H3. We have also examined the selective, modification of chromosomal proteins by different polycyclic aromatic hydrocarbons and it was observed that 7,12-dimethylbenz[a]-anthracene (DMBA), 3-methylcholanthrene (3-MC) and B[a]P showed qualitative similarities in terms of their protein binding. This suggests that stereospecific interactions leading to binding of reactive metabolites of DMBA, B[a]P and 3-MC to chromosomal proteins share common features.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1982

Formation and removal of B[a]P diol epoxide--DNA adducts in human fibroblasts.

Using xeroderma pigmentosum fibroblasts, deficient in excision repair, as controls to measure the initial rate of (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE)--DNA adducts removal in normal human fibroblasts, it was found that the maximum amount of carcinogen DNA adducts occurred 1 h after the addition of B[a]PDE, and that during the first hour approximately 12% of the DNA--carcinogen adducts had already been removed. Thus the formation and removal of DNA--carcinogen adducts occurred simultaneously within the first hour after B[a]PDE addition to confluent fibroblasts. Examination of excision repair over an extended period showed that during a further 6 h, DNA adducts were removed at a rate four times slower than that observed during the first hour. Since the maximum level of B[a]PDE--DNA adducts was observed 1 h after the addition of B[a]PDE to the cells in culture, this suggested that the rate of breakdown of B[a]PDE was much slower than that observed in vitro. Further experiments indeed indicated that the rate of hydrolysis of B[a]PDE within the cell was significantly decreased. Thus, the stability of B[a]PDE inside the cell is governed by very different parameters than those observed in vitro.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1981

Binding of benzo(a)pyrene derivatives to specific proteins in nuclei of intact hamster embryo cells.

In hamster embryo cells incubated for 24 hr with 4 microM [3H]benzo(a)pyrene, a major portion of the nonextractable radioactivity in nuclear preparations copurifies with the protein fraction. When these proteins are analyzed by sodium dodecyl sulfate:polyacrylamide gel electrophoresis, significant variations in the labeling intensities of the various proteins are seen. Control experiments demonstrate that the labeling is due to covalent binding to protein. Histones H3 an H2A are heavily labeled while the other histones of the nucleosome core, H2B and H4, are devoid of radioactivity. Large amounts of label are associated with proteins with mobilities similar to the very lysine-rich histones H1. However, the results of differential extraction experiments suggest that the labeled proteins do not belong to either the H1 or the high-mobility-group class of chromosomal proteins. During 6 hr of inhibition of protein synthesis by cycloheximide, the metabolism of [3H]benzo(a)pyrene, as monitored by high-pressure liquid chromatography, remained normal. Patterns of labelling of nuclear proteins after 3 or 6 hr were identical in the presence and absence of cycloheximide. This finding strongly suggests that binding of benzo(a)pyrene derivatives to nuclear proteins occurs in situ.

Animals↗
PubMed1980

Specificity in interaction of benzo[a]pyrene with nuclear macromolecules: implication of derivatives of two dihydrodiols in protein binding.

Benzo[a]pyrene (B[a]P), 7,8-dihydroxy-7,8-dihydro-B[a]P, and 9,10-dihydro-B[a]P are metabolized by hamster embryo cells to derivatives that bind to nuclear macromolecules. The selectivity for different classes of macromolecules varies depending on the compound analyzed. The ratio of DNA specific activity to protein specific activity (pmol bound/mg of macromolecules) is high (1.51) for 7,8-dihydroxy-7,8-dihydro-B[a]P, extremely low (0.03) for 9,10-dihydroxy-9,10-dihydro-B[a]P, and intermediate (0.26) for B[a]P. Histones H3 and H2A are the major targets of 7,8-dihydroxy-7,8-dihydro-B[a]P; a protein(s) with a mobility similar to that of histone H1 is heavily labeled by 9,10-dihydroxy-9,10-dihydro-B[a]P, with minor labeling of other (nonhistone) bands. The labeling pattern seen with B[a]P is a combination of the patterns seen with the two dihydrodiol metabolites studied. Analysis of the ethyl acetate-soluble metabolites suggests that hamster embryo cells produce 9,10-dihydroxy-7,8-oxy-7,8,9,10-tetrahydro-B[a]P from 9,10-dihydroxy-9,10-dihydro-B[a]P and raise the possibility that this vicinal diol epoxide is an intermediate in the binding of 9,10-dihydroxy-9,10-dihydro-B[a]P to nuclear proteins. The differences seen suggest that factors other than the intrinsic chemical reactivity of the epoxide group are extremely important in the interaction of potential ultimate carcinogens with biological systems.

Animals↗
PubMed1979

Interaction of benzo[alpha]pyrene diol-epoxide with nuclei and isolated chromatin.

Chicken erythrocyte chromatin and nuclei were labeled with benzo[alpha]-pyrene (B[alpha]P) diol-epoxide (anti) and digested with micrococcal nuclease to mono- and dinucleosomes. Analysis of the distribution of the carcinogen showed that the internucleosomal region bound 3-4 times more carcinogen per unit DNA than did nucleosomes. The enhanced binding of the 'ultimate' carcinogen to the internucleosomal region was similar when isolated chromatin or nuclei were used for in vitro labeling. Furthermore, isolation of the histone core proteins, H2A, H2B, H3 and H4, revealed that only 15% of the carcinogen was associated with the histones and that the majority of the carcinogen was bound to chromosomal DNA. Fluorography of purified nucleosomal histones showed that the covalent association of the carcinogen was mainly with histones H3 and H2B.

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