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E Bresnick

Publications and source records attributed to E Bresnick.

At least 91 records · Page 5Linked to original sources

Modulation of the interaction of benzo[a]pyrene with a hamster tracheal epithelial cell line.

A hamster tracheal cell line rapidly absorbed and subsequently metabolized benzo[a]pyrene (B[a]P). At 10(-7) M B[a]P, less than 5% of the applied hydrocarbon was retained by the cell after 8 h incubation. However, at higher concentrations up to 20% was retained. During a subsequent 24 h incubation in the absence of polycyclic hydrocarbon, the residual B[a]P was metabolized and, for the most part, excreted. Analysis of B[a]P bound to DNA showed that alkylation increased as a consequence of metabolism of the B[a]P retained after replacement of the culture medium. Analysis of DNA repair is therefore markedly affected by the contribution from this continued alkylation. At 10(-7) M B[a]P, DNA alkylation was rapidly completed, and within 24 h a marked reduction in total adducts was observed. During this phase, a specific removal was observed of two adducts that have been tentatively identified as derived from deoxyadenosine. A slower second phase of repair was followed for up to 5 days at which time 25% of the adducts still remained in DNA. During this slow phase, the repair appeared to show preference for two of the four remaining adducts. In cells that were incubated for 8 h with 10(-6) M B[a]P, no rapid early phase of repair was seen during the following 24 h because of the continued alkylation. Thereafter, only a slow repair was observed. The deoxyadenosine adducts were still detectable 5 days after treatment suggesting that their repair was inhibited or saturated at this high concentration of B[a]P.

Alkylation↗

A system in mouse liver for the repair of O6-methylguanine lesions in methylated DNA.

An activity from mouse liver with catalyzes the disappearance of O6-methylguanine from DNA methylated with methylnitrosourea has been partially purified by ammonium sulfate fractionation and DNA-cellulose chromatography. The activity does not require divalent metal ions and is not affected by EDTA. It is specific for the repair of O6-methylguanine lesions and does not affect the removal of 7-methylguanine, 7-methyladenine or 3-methyladenine. The disappearance of O6-methylguanine is linear with respect to the concentration of protein and is dependent on incubation temperature. The kinetics and substrate dependence experiments suggest that the protein factor is product-inactivated. Amino acid analysis of hydrolysates of protein obtained after incubation of methylated DNA with the protein factor indicates the presence of radiolabeled S-methyl-L-cysteine, suggesting that during the repair of O6-methylguanine from methylated DNA, the methyl group is transferred to a sulfhydryl of a cysteine residue of a protein. This represents the first such demonstration in a mammalian system.

Amino Acids↗

Effect of 3-methylcholanthrene administration on hepatic ribonucleic acid polymerase activities.

The effect of the in vivo administration of 3-methylcholanthrene upon rat hepatic RNA polymerase activities was investigated. Aggregate RNA polymerase activity assayed in liver nuclei was stimulated by 33% over control. Characterization of the individual RNA polymerase activities by virtue of their differential sensitivity to alpha-amanitin revealed that RNA polymerase I activity was maximally increased by 70% at approx. 16 h post-administration of the polycyclic hydrocarbon; RNA polymerase II activity was stimulated by 33%. The kinetics of RNA polymerases I and II stimulation differed in that the nucleolar enzyme's activity increased earlier and peaked later. RNA polymerase III activity was not significantly different from control. Phenobarbital, another inducer of the mixed function oxidases, had essentially no effect on the activity of hepatic RNA polymerases. Solubilization of the RNA polymerases followed by separation on diethylaminoethyl (DEAE)-Sephadex allowed for a comparison of the treated and control enzymatic activities using a common exogenous template. While no qualitative difference was evident, RNA polymerases I and II isolated from 3-methylcholanthrene-treated rats again were more active than control, indicating an effect of the polycyclic hydrocarbon at the level of the enzyme.

Animals↗

Further characterization of the effects of 3-methylcholanthrene administration upon hepatic ribonucleic acid polymerase activities.

The administration of 3-methylcholanthrene (MC) to rats results in a marked increase in the specific activities of hepatic RNA polymerases I and II. In the present study, we were able to show that this increase was not caused by a shift in the ratio of 'free' to 'template-engaged' RNA polymerase. By means of binding studies with [3H]amatoxin, we were unable to demonstrate any increase in the number of RNA polymerase II molecules in liver after MC administration to the rats. RNA polymerase I was purified in excess of 3000-fold from hepatic nuclei isolated both from control and MC-treated rats. The stimulation in activity was demonstrated at each step in the purification scheme until glycerol sedimentation analysis. Results from cation-exchange chromatography on phosphocellulose indicated that the polycyclic hydrocarbon increased the enzyme activity of RNA polymerase Ib somewhat specifically. Subsequent to glycerol gradient centrifugation, this stimulatory advantage was no longer evident. Reconstitution experiments revealed the presence of a stimulatory component, which was demonstrated in low molecular weight fractions from both control and experimental preparations.

Animals↗

Administration of 3-methylcholanthrene to rats increases the specific hybridizable mRNA coding for cytochrome P-450c.

Poly(A)+-RNA obtained from the livers of 3-methylcholanthrene (3MC)-treated rats was translated into cytochrome P-450c in a cell-free reticulocyte system. In this translational system, no precursor cytochrome P-450c was observed. The mRNA responsible for the synthesis of this cytochrome was isolated by immunoprecipitation of liver polyribosomes obtained at 15 hr after 3MC treatment, and a cDNA was constructed by the reverse transcriptase reaction. The cDNA was further purified by hybridizing at a high R0t (product of RNA concentration and incubation time) to poly(A)+-RNA isolated from control rat liver, and the nonhybridized, single-stranded cDNA was isolated by hydroxylapatite chromatography. This cDNAp-450c was employed in hybridization reactions with poly(A)+-RNA isolated from the livers of rats treated with 3MC for various times. These studies indicated a maximal induction of mRNAp-450c at about 15 hr after 3MC injection, although levels of this mRNA were significantly increased by 7 hr. The mRNAp-450c concentration had diminished by 24 hr but remained higher than control levels for at least 48 hr. These studies establish an effect of 3MC upon the accumulation of mRNAp-450c in rat liver.

Animals↗

Phospholipase activity in skin after application of phorbol esters and 3-methylcholanthrene.

Topical administration of the promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), is accompanied by an increased incorporation of [3H]arachidonic acid into mouse skin and a very significant activation of epidermal cell membrane phospholipase A2 without affecting intracellular acid phospholipase. Similar enhancement in epidermal cell phospholipase A2 was observed after application of phorbol-12,13-didecanoate or 3-methylcholanthrene to mouse skin. A small but significant increase in phospholipase A2 activity is also seen after application of the nonpromoter irritant, acetic acid. The elevated levels of the prostaglandins observed in mouse skin after topical application of promoters are probably triggered by the activation of this membrane enzyme.

Animals↗

Formation and removal of benzo(a)pyrene adducts of DNA in hamster tracheal epithelial cells.

A cloned cell line derived from normal hamster tracheal epithelium has been characterized with respect to its response to the environmental pollutant and carcinogen benzo(a)pyrene [B(a)P]. These cells metabolize B(a)P to ultimate reactive forms as assayed by alkylation of DNA. Alkylation with radiotracer amounts of B(a)P was maximum at 8 hr, at which time 70% of the applied hydrocarbon had been converted to water-soluble forms. At longer incubation times, the rate of removal of adducts exceeded the rate of formation. When B(a)P-containing medium was replaced with fresh medium at two or four hr, a subsequent biphasic removal of adducts occurred, a rapid removal for the first four hr postincubation and then a slower repair. About 50% of the DNA-bound hydrocarbon remained in DNA after 48 hr. Cells were able to divide in the presence of these lesions, undergoing five doublings (five days), while only 60% of the adducts were removed from the DNA. Integrity of DNA during this period was monitored by the alkaline elution technique. A toxic dose of B(a)P was required to cause any increase in the rate of elution. Minimal single-strand breakage was observed from two to eight hr of B(a)P treatment, but at 15 hr DNA appeared normal. Comparison was made with a nontoxic dose of methyl methanesulfonate which caused very rapid elution of DNA after only one hr treatment. At least 15 deoxyribonucleoside-bound B(a)P adducts were separated by high-pressure liquid chromatography. Four adducts, probably deoxyadenosine-B(a)P, were removed almost completely in 24 hr, while the others appeared to be poorly removed. The possible significance to neoplasia of persistent and repairable lesions is discussed.

Animals↗

Nuclear uptake and subsequent nuclear metabolism of benzo(a)pyrene complexed to cytosolic proteins.

The binding of [3H]benzo(a)pyrene to proteins of rat liver cytosol, the nuclear uptake of cytosolic protein-bound [3H]-benzo(a)pyrene, and the subsequent nuclear metabolism of the polycyclic hydrocarbon were investigated. The binding of [3H]benzo(a)pyrene to cytosol had a saturable high affinity component with a Kd of 2.54 nM and a capacity of 530 fmol/mg protein. Specific binding of [3H]benzo(a)pyrene to cytosol was also assayed using sucrose density gradient analysis. Nuclear uptake of protein-bound [3H]benzo(a)pyrene was demonstrated both directly and by sucrose density gradient analysis. The nuclear benzo(a)pyrene was readily converted to metabolites which were qualitatively and quantitatively no different from nuclear metabolites of exogenously (not protein associated) added [3H]benzo(a)pyrene.

Animals↗

Correlation of induction of aryl hydrocarbon hydroxylase in cultured rat hepatocytes with saturable high-affinity binding of 3-methylcholanthrene to a 4S cytoplasmic protein.

The binding of 3-methylcholanthrene (3-MC), a potent inducer of aryl hydrocarbon hydroxylase activity, to cytoplasmic proteins of a cloned rat hepatocyte culture, RL-PR-C, was studied by sucrose gradient centrifugation. Time course and dose-binding experiments performed on late-passage aryl hydrocarbon hydroxylase-inducible cultures indicate the presence of a saturable pool of high-affinity (average Kd, 3.6 nm) binding sites in the cytosol of these cells. The number of binding sites varied from 20,000 to 80,000 per late-passage hepatocyte with a total capacity of approximately 2.2 pmol of 3-MC bound per mg of cytosolic protein. The complex sedimented at 4.0 +/- 0.2S regardless of the ionic strength of the homogenization buffer or gradient solutions. It was sensitive to denaturation by sodium dodecyl sulfate and trypsin but not by DNase I, RNase A, or the nonionic detergent Nonidet P-40. The binding of 3-MC to the protein was inhibited by 1,2-benzanthracene, benzo(a)pyrene, 5,6-benzoflavone, and 7,8-benzoflavone but not by a series of steroids, aflatoxin B1, phenobarbital, or Aroclor 1254. Elevating the temperature of cultures cells to 37 degrees after the standard ligand-binding incubation at 4 degrees resulted in a rapid decrease in cytoplasmic saturable binding and a concomitant increase in nuclear- and chromatin-associated ligand. A portion of this nuclear-associated ligand was extractable with 400 mM KCl. Adsorption of the [3H]-3-MC binding complex by nuclei in vitro suggested that the 4S binding protein facilitated the entry of 3-MC into the nucleus. The presence of the 4S binding species correlated with the level of inducibility of aryl hydrocarbon hydroxylase throughout its development in RL-PR-C and therefore may be involved in the process of induction of this enzyme.

Animals↗

Colon carcinogenesis: an overview.

The mechanisms for activation of procarcinogens and some of the factors that affect these reactions are considered. It is proposed that the nucleus of the colonic cell may actually be responsible for the formation of ultimate carcinogenic forms of exogenous procarcinogens. The contributions of the intestinal microbial flora to activation and detoxification of the carcinogens are discussed, and the suggestion that inactive glucuronides of procarcinogens are hydrolyzed to proximate carcinogens by the microbial flora is proposed. Finally, the two-stage mechanism for carcinogenesis is discussed; large bowel cancer is the focal point.

Animals↗

Interaction of DNA with cytosolic 3-methylcholanthrene binding proteins from either rat or mouse liver.

The ability of cytosolic 3-methylcholanthrene binding proteins from rat liver to interact with DNA was studied using DNA-cellulose chromatography. Two DNA binding fractions, eluting in 0.15 M KCl (peak 1) and 0.33 M KCl (peak 2), were observed on salt elution from a denatured DNA-cellulose column which had been incubated with rat liver cytosol containing radiolabelled 3-methylcholanthrene. No detectable DNA binding fractions were found when columns containing cellulose alone or native DNA-cellulose were used. Temperature activation of the cytosolic proteins containing 3-methylcholanthrene did not result in a significant difference in DNA binding characteristics when compared with a non-treated sample. The pretreatment of rats with Aroclor 1254 induced peak 1 3.7 fold over control values. An analysis of the proteins present in peaks 1 and 2 from control and induced rats was carried out using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Comparative DNA-cellulose chromatography of cytosolic liver proteins from a cytochrome P-448 inducible mouse strain (DBA/6J) and non-inducible mouse strain (DBA/2J) showed much higher levels of DNA binding by protein bound 3-methylcholanthrene from C57BL/6J hepatic cytosol.

Animals↗

Malignant transformation of cultured rat hepatocytes by (+)- and (-)-trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene.

Rat hepatocyte cultures (RL-PR-C) were tested with the (+)- or (-)-trans-7,8-dihydrodiol isomers of benzo(a)pyrene [B(a)P] and were assessed for growth inhibition, chromosomal damage, growth in soft agar, and tumor formation. Because early-passage cells have a noninducible low specific activity, aryl hydrocarbon hydroxylase parallel inhibition studies were performed on aryl hydrocarbon hydroxylase-inducible late-passage cultures. Early-passage cells exhibited little inhibition in the presence of B(a)P or either isomer. Comparable later-passage cells demonstrated inhibitory effects with B(a)P and the (+)- and (-)-trans-7,8-dihydrodiol metabolites. No treated cultures grew in soft agar, and the modal number of chromosomes was unaffected by carcinogen treatment. However, both (+) and (-) isomer-treated early-passage cells formed tumors in isogeneic animals, the (+) isomer being more efficient in this regard. These results indicate that noninhibitory doses of either the (+)- or (-)-trans-7,8-dihydrodiol isomer of B(a)P are nonetheless capable of malignantly transforming hepatocyte cells in vitro.

Animals↗