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M C MacLeod

Publications and source records attributed to M C MacLeod.

At least 55 records · Page 3Linked to original sources
PubMed1985

Separation of histones by reverse-phase high-performance liquid chromatography: analysis of the binding of carcinogens to histones.

Reverse-phase high-performance liquid chromatography (RP-HPLC) has been examined as an approach to the rapid analysis of carcinogen-modified histones. H1 and core histone fractions were prepared by differential acid extraction of 0.35 M NaCl-extracted rat liver nuclei previously exposed to [3H]-7r,8t-dihydroxy-9t, 10t-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene [( 3H]BPDE-I). Using a sodium perchlorate-phosphate (PCP)/acetonitrile solvent system, the H1 histone fraction was eluted from an Aquapore RP-300 column in five peaks (P1-P5). The core histone fraction was resolved into eight peaks (C1-C8) using a PCP/acetonitrile-methanol solvent system. The histones of each peak were identified by sodium dodecyl sulfate and Triton/acid/urea gel electrophoresis or amino acid analysis as follows: P1, H1 degrees; P2-P5, four different H1 variant fractions; C1, H4 + A24; C2, H2B; C3, H2A X 2 + to one H2A variant; C4, H2A.1; C5, H2A.1 + two H2A variants; C6, H3.2; C7, H3.3; C8, H3.1. The bulk of radioactivity was covalently bound to histone H2A, which had higher specific activities of BPDE-I than other histones. Significant amounts of radioactivity were observed in histones H3 and H1, but not in histones H2B and H4. These RP-HPLC systems have the advantages of an analysis time within 60 min, the identification of H1, H2A, and H3 variants, and the quantitative analysis of radioactive histones. These results indicate that these RP-HPLC systems are very useful to analyze the binding of carcinogens to histones.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1985

Catalysis of carcinogen-detoxification by DNA: comparison of enantiomeric diol epoxides.

The interactions of the (+)- and (-)-enantiomers of 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) with purified DNA have been studied in vitro. These compounds are formed by cellular metabolism of the potent environmental carcinogen benzo[a]pyrene, and the (+)-enantiomer is thought to be the ultimate carcinogenic metabolite. Non-covalent, intercalative binding was measured spectrophotometrically, hydrolysis was measured spectrofluorometrically and covalent binding was detected by liquid scintillation counting. No significant differences were found in the association constants for intercalative binding or in the ability of DNA to catalyse the hydrolysis of the two enantiomers. Covalent DNA binding was 4.5-fold higher for the (+)-enantiomer. When DNA was pretreated with a molar equivalent of the (-)-enantiomer, its subsequent ability to enhance the rate of BPDE-I hydrolysis and to bind covalently to (+)-BPDE-I was unimpaired. This suggests that the participation of the DNA in the hydrolysis reaction does not alter the DNA and therefore that the rate-enhancement is true catalysis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1985

Involvement of the exocyclic amino group of deoxyguanosine in DNA-catalysed carcinogen detoxification.

The interactions of 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a] pyrene (BPDE-I) with purified DNAs and synthetic polynucleotides of varying base composition have been studied. The ability of these polynucleotides to catalyse BPDE-I hydrolysis is strongly base-composition dependent, increasing with increasing (% G + C). Covalent binding of [3H](+)-BPDE-I to DNA shows a similar, linear dependence on base composition. Association constants for non-covalent binding exhibit no clear base-composition dependence. Poly(dI-dC) is relatively ineffective in catalysing hydrolysis, suggesting that the exocyclic amino group of deoxyguanosine is involved in catalysis. Several plausible models for this dependence are presented, ranging from conformational changes with changing base composition to general acid catalysis by the exocyclic amino group of deoxyguanosine.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1985

Interactions of benzo(a)pyrene diol-epoxides with linear and supercoiled DNA.

Previous spectroscopic studies of the major adduct formed by reaction of (+/-)-7r,8t-dihydroxy-9t, 10t-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-I) with linear DNA have been interpreted to suggest that the adduct is not intercalated in the double helix. However, studies of the electrophoretic mobility of supercoiled DNA treated with BPDE-I suggest that the adduct is intercalated. To resolve these interpretations, we have studied the reaction of BPDE-I with supercoiled and linear DNA. The kinetics of DNA-catalyzed hydrolysis and of covalent binding are similar for the two DNAs; supercoiled DNA exhibits a 20% increase in the rate of hydrolysis of BPDE-I at low DNA concentration compared to linear DNA. Fluorescence excitation spectra and fluorescence quenching experiments provide no support for a model in which BPDE-I adducts are intercalated in supercoiled DNA. When deoxyribonucleoside adducts were analyzed by high-performance liquid chromatography, identical distributions of BPDE-I adducts were found for supercoiled and linear DNA. These data are consistent with a previously proposed model (Hogan, M. E., Dattagupta, N., and Whitlock, J.P., Jr. J. Biol. Chem., 256: 4504-4513, 1981; Taylor, E.R., Miller, K. J., and Bleyer, A. J. J. Biomol. Struct. Dyn., 1: 883-904, 1983), in which the major BPDE-I adduct in both linear and supercoiled DNA exists in a conformation which allows stacking with the neighboring base pair and introduces a "kink" into the path of the helical axis. Although this model provides an explanation for all available experimental data, there are undoubtedly other DNA adduct conformational models which are also consistent with the data.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1985

Differences in the binding of stereoisomeric benzo(a)pyrene-7,8-diol-9,10-epoxides to histones in rat liver nuclei.

We have compared the covalent binding of two stereoisomeric benzo(a)pyrene-7,8-diol-9,10-epoxides to histones. From rat liver nuclei exposed to carcinogenic [3H]-(+/-)-7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo(a)p yrene [( 3H]BPDE-I) or noncarcinogenic [3H]-(+/-)-7r,8t-dihydroxy-9c,10c-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene [( 3H]BPDE-II), H1 and core histone fractions were prepared by differential acid extraction. The specific activity (dpm/mg protein) of the core histone fraction for [3H]BPDE-I was much higher than that of [3H]BPDE-II. Alternatively in the H1 histone fraction, the binding level of [3H]BPDE-I was lower than that of [3H]BPDE-II. By reverse-phase high performance liquid chromatography, the analyses of BPDE isomers binding to histones showed that histones H2A and H1 were heavily labeled by [3H]BPDE-I and -II, respectively. In particular, the ratio of specific activities for BPDE-I to II in peak C3, which mainly contains H2A X 2 variant, was higher than those of other histone H2A variants and other core histones. These results indicate that the BPDE isomers have differential binding affinities to histones. The covalent binding of BPDE-I to histone H2A (especially H2A X 2 variant) may be important in the potential carcinogenic effects in nuclei.

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

Time course of metabolism of benzo[e]pyrene by hamster embryo cells and the effect of chemical modifiers.

In cultures of hamster embryo cells, benzo[a]pyrene (B[a]P) is metabolized primarily in the bay region. In contrast, little or no bay region metabolism of the noncarcinogenic isomer benzo[e]pyrene (B[e]P) could be detected during 12--96-h incubations of hamster embryo cells with 4 microM [3H]B[e]P. The upper limit to 9,10-dihydro-9,10-dihydroxy-B[e]P formation is about 0.2% of the ethyl acetate-soluble metabolites (less than 0.1% of the total metabolites). The major identified metabolites of B[e]P were 4,5-dihydro-4,5-dihydroxy B[e]P and the glucuronide conjugates of 3-OH-B[e]P and 4,5-dihydro-4,5-dihydroxy B[e]P. Simultaneous treatment of cells with either B[a]P or 7,8-benzoflavone (BF) did not induce bay region metabolism of [3H]B[e]P.

Animals↗
PubMed1982

Benzo(a)pyrene-binding proteins of hamster embryo cell nuclei: comparison of nuclear isolation procedures.

Hamster embryo cells metabolize benzo(a)pyrene to derivatives that covalently modify the nuclear macromolecules including proteins. Not all proteins are modified to the same extent nor by the same metabolites. In particular, a protein of apparent molecular weight 32,000 is highly modified by derivatives of trans-9,10-dihydro-9,10-dihydroxy B(a)P. This protein is shown here to be preferentially lost from nuclei during purification by centrifugation through high molarity sucrose solutions followed by osmotic shock. It does not appear to be a cytoplasmic contaminant, but shares many properties of an abundant protein from Xenopus laevis oocytes, nucleoplasmin.

Animals↗
PubMed1982

Physical interactions of isomeric benzo [a] pyrene diol-epoxides with DNA.

The physical interactions of two diol-epoxides derived from benzo [a] pyrene (B[a]P), 7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydro B[a]P (BPDE) and 9,10-dihydroxy-7,8-oxy-7,8,9,10-tetrahydro B[a]P, (reverse BPDE) with DNA have been studied in a simple, in vitro system. The effects of DNA on the rates of hydrolysis of BPDE and reverse BPDE were studied by fluorescence spectroscopy. For both compounds, interaction with DNA was indicated by an increase in the rate of hydrolysis in the presence of DNA. This increased hydrolysis was more marked for BPDE than for reverse BPDE. Direct confirmation of physical binding was obtained by u.v. spectroscopy, where a 10 nm redshift in absorbance maxima characteristic of polycyclic aromatic hydrocarbon.DNA intercalation complexes was observed. Using absorbance changes to monitor binding, association constants of 6580 L/mol and 5080 L/mol were determined for BPDE and reverse BPDE, respectively. Consistent with the intercalation model, binding was inhibited by low concentrations of Mg2+. The enhancement of hydrolytic rate by DNA for BPDE and reverse BPDE was also inhibited by low concentrations of Mg2+, suggesting involvement of intercalation complexes in the mechanism of enhanced hydrolysis.

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

Benzo[a]pyrene metabolites: formation in rat liver cell-culture lines, binding to macromolecules, and mutagenesis in V79 hamster cells.

Benzo(a]pyrene was metabolized in liver cell lines derived from BC-IV and BD-IV rats which included several chemically-transformed lines (IAR-6-1; IAR-19; IAR-28), one spontaneous transformant (IAR-27) as well as one non-malignant line (IAR-20). Cultures were treated with tritiated benzo[a]pyrene over a 5-day period. The cells and medium were extracted with ethyl acetate and the distribution between organic-soluble and water-soluble metabolites determined. Organic-soluble metabolites consisting of dihydrodiols, phenols and quinones were determined by high-pressure liquid chromatography, and macromolecular binding of BP to each cell line was measured over a 24-h period. Comparisons between binding and overall metabolism were not directly proportional in these liver cell lines. However, there was a positive correlation for benzo[a]pyrene mutagenesis in the V-79 hamster cell assay with 8-azaguanine as a marker when the cell lines with the highest (IAR-20) and lowest (IAR-27) metabolic competence were used as activating cell layers.

Animals↗
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↗
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↗
PubMed1980

Metabolism of carcinogenic 2-hydroxybenz.

The rates of metabolism of the carcinogenic 2-hydroxybenzo[a]pyrene (2-OH-B[a]P) and the non-carcinogenic 3- and 9-hydroxybenzo[a]pyrenes in cultured cell systems have been studied and compared. While 70-80% of the non-carcinogens are converted to water-soluble derivatives by hamster embryo fibroblasts in 24 h, carcinogenic 2-OH-B[a]P is metabolized at a slower rate (45% in 24 h), comparable to that for the parent hydrocarbon, benzo[a]pyrene (B[a]P). Analysis of extracellular organic solvent-soluble metabolites of 2-OH-B[a]P in cultured hamster embryo fibroblasts, using h.p.l.c., indicates the presence of a single major metabolite, which has been identified by mass spectroscopy as a dihydroxy derivative of B[a]P. At least one additional major organic solvent-soluble metabolite is formed in cultures of either mouse epidermal epithelial cells or human foreskin fibroblasts, indicating a different balance of metabolic pathways in these cell systems. The greater persistence of carcinogenic 2-OH-B[a]P in cells and its higher concentration in the cell cytoplasm compared with the non-carcinogenic phenols may be related to its relatively high biological activity. Differences in metabolism of 2-OH-B[a]P in several cultured cell systems indicate the importance of an appropriate choice of activating system in understanding the relationship between metabolism and carcinogenesis.

Animals↗
PubMed1980

Metabolism and macromolecular binding of carcinogenic and noncarcinogenic metabolites of benzo(a)pyrene by hamster embryo cells.

The metabolism and macromolecular binding of four metabolites of benzo(a)pyrene in hamster embryo fibroblasts has been studied. Two noncarcinogenic phenolic derivatives, 3-hydroxybenz(a)pyrene and 9-hydroxybenzo(a)pyrene, are rapidly metabolized, primarily to their respective glucuronic acid conjugates and other H2O-soluble conjugates (78.4 to 80.8% of total radioactivity). Water-soluble conjugates were also formed from the carcinogenic phenol, 2-hydroxybenzo(a)pyrene, and from 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, but in lower amounts (36.8 to 43.8% of total radioactivity. With each of the compounds, from 10 to 20% of the radioactivity was converted to ethyl acetate-soluble metabolites. The amount of unmetabolized 2-hydroxybenzo(a)pyrene recovered intracellularly was 20-fold higher than that recovered in incubations with the other phenols. Covalent binding to nuclear macromolecules was monitored after isopyknic separation. Binding of the three phenols tested was similar and was lower than the binding of benzo(a)pyrene to nuclear DNA, RNA, and protein. In contrast to the results with the monohydroxybenzo(a)pyrenes, high levels of covalent binding were observed with 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene; binding to DNA was 8-fold higher (315 pmol bound per mg DNA) than binding of benzo(a)pyrene to DNA.

Animals↗