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D P Ringer

Publications and source records attributed to D P Ringer.

At least 19 recordsLinked to original sources
PubMed1995

Homodimeric and heterodimeric aryl sulfotransferases catalyze the sulfuric acid esterification of N-hydroxy-2-acetylaminofluorene.

Three aryl sulfotransferases (ASTs) isolated from rat liver catalyze the sulfuric acid esterification of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-2AAF). These three ASTs were separated by high resolution anion exchange chromatography and were designated Q1, Q2, and Q3. Q1 and Q2 had high N-OH-2AAF sulfonation activity, whereas Q3 showed low activity. Reversed phase high performance liquid chromatography/mass spectrometry analysis showed Q1-Q3 to be comprised of 33,945- and 35,675-Da protein subunits. Q1 contained only the 35,675-Da protein subunit, Q2 contained equal quantities of 33,945- and 35,675-Da subunits, and Q3 contained only the 33,945-Da subunit. The subunit compositions of Q1-Q3 were confirmed by immunochemical analysis. Size exclusion high performance liquid chromatography confirmed that the active quaternary structure of the three isoenzymes was dimeric. Analysis of liver cytosols for the relative contributions of Q1-Q3 to total cytosolic N-OH-2AAF sulfotransferase activity indicated the Q1, Q2, and Q3 accounted for 44, 46, and 10% of the activity, respectively. These results demonstrate the existence of both homodimeric and heterodimeric aryl sulfotransferases and show that two ASTs, a homodimer of 35,675-Da subunits and a heterodimer of a 33,945- and a 35,675-Da subunit, are primarily responsible for hepatic N-OH-2AAF sulfotransferase activity.

Amino Acid Sequence↗
PubMed1995

Scanning microfluorometric analysis of proliferating cell nuclear antigen in formalin-fixed sections of hyperplastic and neoplastic rat liver.

Scanning laser cytometric analysis of fluorochrome-labeled cells was used to survey and quantitate the distribution of proliferating cells in formalin-fixed, paraffin-embedded tissue sections from hyperplastic and neoplastic rat livers. The technique used fluorescent immunochemical staining of proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells and propidium iodide as a fluorescent nuclear counterstain. Use of an antigen retrieval treatment improved detection of PCNA and treatment of tissue sections with crystal violet improved the sensitivity of the method by quenching background autofluorescence. PCNA evaluation of cell proliferation in regenerating rat liver 0-48 h post-partial hepatectomy showed that 3-43% of cells stained positively for PCNA, a pattern closely correlating with previously reported rates of maximum DNA synthesis. The PCNA staining patterns observed among cells in neoplastic nodules were more focal in distribution and indicated that from 5 to 25% of the cell nuclei per nodular region stained positively for PCNA. This use of image analysis for the rapid identification of proliferating cell areas in fixed, paraffin-embedded tissue active in neoplastic growth will expedite in situ cytochemical and molecular studies attempting to identify key differences between hyperplastic and neoplastic growth.

Animals↗
PubMed1995

Rat phenol-preferring sulfotransferase genes (Stp and Stp2): localization to mouse chromosomes 7 and 17.

The phenol-preferring sulfotransferases aryl sulfotransferase IV and N-hydroxyarylamine sulfotransferase catalyze sulfate conjugation of N-hydroxy-2-acetylaminofluorene, a metabolite capable of causing hepatocarcinogenesis in rats. We utilized published cDNA sequences of these sulfotransferases to type the progeny of two multilocus crosses and determined that the genes, aryl sulfotransferase (Stp) and N-hydroxyarylamine sulfotransferase (Stp2), map to positions on mouse chromosomes 7 and 17.

Animals↗
PubMed1995

Quantitative in situ image analysis of apoptosis in well and poorly differentiated tumors from rat liver.

The evaluation of apoptosis is an important aspect in the study of chemical carcinogenesis. Methods were developed employing the ApopTag kit (Oncor, Gaithersburg, MD) and automated image analysis to quantitate the distribution of apoptosis in formalin-fixed, paraffin-embedded liver tumor sections from rats induced by 2-acetylaminofluorene. Specific treatments of tissue sections were developed that permitted quenching of background tissue autofluorescence with crystal violet and permitted permeating the fixed tissue sections by trypsin digestion. Tissue sections were stained by using the ApopTag kit for detection of in situ apoptosis and with propidium iodide as a counterstain for tissue nuclei. Automated statistical evaluation of the percentage of tissue nuclei also staining positively for apoptosis was determined by using dual fluorescence detection and imaging laser microscopy. The quantitative results indicated that the staining index for apoptosis in normal liver was 0.14 +/- 0.04% whereas well and poorly differentiated tumors showed increases of 3.48 +/- 0.59% and 7.41 +/- 0.81%, respectively. The staining indexes for apoptosis showed a tight correlation between fluorescent and peroxidase-diaminobenzidine detection in sequential tissue sections. The use of in situ apoptosis staining and automated image analysis for rapid identification and quantitation of cells undergoing death in fixed tissue will expedite additional studies to evaluate the in situ tissue pathobiology of tumors and aid in the study of molecular mechanisms associated with cancer development and treatment.

2-Acetylaminofluorene↗
PubMed1994

Evidence of two separate mechanisms for the decrease in aryl sulfotransferase activity in rat liver during early stages of 2-acetylaminofluorene-induced hepatocarcinogenesis.

Enzymatic and immunohistochemical experiments were conducted to evaluate the mechanistic basis for the downregulation of the important detoxication/bioactivation enzyme aryl sulfotransferase IV (AST IV) during 2-acetylaminofluorene (2AAF)-induced hepatocarcinogenesis. To distinguish between possible genotoxic and cytotoxic actions of 2AAF, three different dietary protocols were used in these experiments: group 1 received 2AAF for 12 wk, group 2 received 2AAF for 3 or 6 wk and then a control diet lacking xenobiotics for 3 or 6 wk, and group 3 received 2AAF for 3 or 6 wk and then phenobarbital for 3 or 6 wk. When hepatic AST IV activity was assessed, N-hydroxy-2AAF sulfotransferase activity was found to decrease 80-90% in response to 2AAF feeding, but activity recovered to essentially normal levels in the livers of rats subsequently placed on either control diets or diets with phenobarbital, suggesting a reversible cytotoxic mechanism for loss of AST IV activity. However, when liver sections from the rats were evaluated immunohistochemically, two distinct patterns were detected for the downregulation of AST IV activity. In the livers of rats administered only 2AAF (group 1), a general pattern of overall downregulation of AST IV expression was observed throughout the liver and among most but not all newly developed nodules. In tissue sections from rats initially fed 2AAF and then placed on a control diet (group 2) or a diet with phenobarbital (group 3), the nodules continued to show low levels of AST IV expression, while expression in the areas surrounding nodules returned to the normal, high levels. In addition, among those rats fed 2AAF for just 3 wk and then control diet or diet containing phenobarbital for 6 wk, only rats fed phenobarbital developed altered foci that stained weakly for AST IV expression. These results show that there were two kinds of 2AAF-mediated decrease in hepatic AST IV activity: a general overall loss of AST IV expression dependent on administration of 2AAF and reversible upon removal of 2AAF from the diet and a loss of AST IV expression among newly developed liver foci and nodules that persisted in the absence of 2AAF administration and appeared to be a property of 2AAF-induced subpopulations of cells. These patterns may correspond, respectively, to cytotoxic and genotoxic mechanisms of 2AAF action.

2-Acetylaminofluorene↗
PubMed1994

Sulfation of carcinogenic aromatic hydroxylamines and hydroxamic acids by rat and human sulfotransferases: substrate specificity, developmental aspects and sex differences.

Sulfation of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and structurally related hydroxamic acids by rat and human sulfotransferases was studied. There was a clear sex and age difference in the sulfation of N-OH-AAF and the other hydroxamic acids by rat liver cytosols; adult male rats had the highest sulfation activity. Experiments with purified aryl sulfotransferase IV (AST IV) indicated that the high expression of this enzyme in male rat liver may be responsible for these differences. No such sex or age difference was found for the sulfation of aromatic hydroxylamines. In cytosols of adult human livers, sulfation activity towards aromatic hydroxamic acids and hydroxylamines was clearly present, but activities were much lower than in rat liver cytosols. Sulfation activity towards these compounds was also found in fetal and neonatal liver and adrenals. These compounds probably are sulfated by several different sulfotransferases in humans.

Aging↗
PubMed1994

Molecular mechanisms for the regulation of aryl sulfotransferase IV expression during 2-acetylaminofluorene-induced hepatocarcinogenesis in rat.

The dietary administration of 2-acetylaminofluorene to male rats to induce hepatocarcinogenesis causes a reversible as well as persistent down-modulation of N-hydroxy-2AAF sulfotransferase activity. Studies are presented which indicate that several molecular mechanisms may be involved in the down-regulation of sulfotransferase activity and expression. These include carcinogen-mediated inactivation of sulfotransferase mRNA or protein, interference with hormonal regulation of sulfotransferase expression, and, mutation of the sulfotransferase gene.

2-Acetylaminofluorene↗
PubMed1994

Hypomethylation of the rat aryl sulfotransferase IV gene and amplification of a DNA sequence during multistage 2-acetylaminofluorene hepatocarcinogenesis.

Rat hepatic aryl sulfotransferase IV (AST IV), which catalyses sulfuric acid esterification of N-hydroxy-2-acetylaminofluorene to its ultimate carcinogenic form, is differentially expressed during multistep 2-acetylaminofluorene (AAF) hepatocarcinogenesis. Two molecular mechanisms associated with this effect involve modulation of mRNA translational capacity at the early stages, and gene transcription at the late stages of the carcinogenic process. To characterize further the molecular mechanisms that may be involved in the transient regulation of the enzyme expression, an AST IV cDNA was used to assess the change in methylation profile and restriction fragment length polymorphism (RFLP) in the gene domain of genomic DNA derived from rats at different stages of carcinogenesis. The onset of hypomethylation of the AST IV gene domain and amplification of a 5.3-kb DNA sequence was found to correlate with the stage in AAF hepatocarcinogenesis, where rats begin to exhibit irreversible loss in hepatic enzyme expression and the liver becomes committed to hepatoma formation. This represents the first observation of both altered methylation status of AST IV gene domain and amplification of a DNA sequence whose expression may play a role in the genesis and/or progression of neoplastic transformation of initiated cells during AAF hepatocarcinogenesis.

2-Acetylaminofluorene↗
PubMed1994

Molecular approach to rapid assessment of p53 tumor suppressor mutations in esophageal tumors from stained histological slides.

The analysis of the tumor suppressor gene, p53, is of fundamental importance in prognosis and staging in many cancers; however, the molecular techniques required to analyze this gene have been expensive, time consuming, and unrelatable to the histological appearance of the samples. This research explored one model of clinically testing for specific mutations in the p53 gene by scraping selected areas of stained histological slides and analyzing for "hot-spot" p53 mutations. Selectively removing samples from the stained histological slide will be of special value in examining suspicious regions in adenomas, potential metastatic regions, and the margins of resected area. A polymerase chain reaction (PCR)-mediated restriction fragment length polymorphism (RFLP) analysis approach in which naturally occurring or primer-mediated mutagenesis-induced restriction enzyme sites were utilized to test seven hot-spot mutations. These assays were able to detect one mutated sequence in 100, and therefore, were sufficiently sensitive to be used with very heterogeneous tumors. Several of the assays could be multiplexed to reduce the number of PCRs necessary to screen for the seven mutational hot spots. Furthermore, an exact determination of the base change could be obtained by direct sequencing of the PCR products. Although this form of analysis may be applicable only to certain types of cancers (e.g., bladder, brain, colon, esophageal, gastric, thyroid, and ovarian tumors), this approach can obtain detailed mutational information from specific regions of a histological slide in a cost-effective and timely manner.

Adenoma↗
PubMed1993

Genomic structure of rat liver aryl sulfotransferase IV-encoding gene.

This report contains the first description of the genomic structure for a sulfotransferase (ST). The gene (ASTIV) encodes rat hepatic aryl ST IV, also known as tyrosine-ester ST (EC 2.8.2.9). A phage genomic clone containing 70% of the 3' AST gene coding sequence was isolated after screening a rat genomic library with an ASTIV cDNA. The remaining 5' sequence was determined from a PCR product obtained from rat genomic DNA and ASTIV cDNA-specific primers. ASTIV spans 3.5 kb and contains eight exons and seven introns. The fourth intron of this gene contains sequences homologous to rodent B1 repetitive elements and an Alu repeat found in rat. An alignment of the primary structures of ten different ST revealed several conserved regions, as well as a putative binding site for the cofactor for enzymatic sulfation reactions, 3'-phosphoadenosine-5'-phosphosulfate.

Amino Acid Sequence↗
PubMed1993

DNA precursors are channelled to nuclear matrix DNA replication sites.

Studies of replicative DNA synthesis using DNA precursors have shown that the DNA that was replicated most recently is that associated with the nuclear matrix. Consequently, precursors arising via the salvage and the de novo metabolic pathways are first incorporated into a small percentage of the total nuclear DNA that is termed nuclear matrix-associated DNA. These results have been substantiated in cell culture, as well as in intact mammalian systems. Furthermore, when DNA precursors were injected intravenously into regenerating rat liver, a significant lag in the incorporation of orotic acid-derived nucleotides (de novo pathway precursors) into nuclear DNA was observed, when compared with deoxythymidine-derived nucleotides (salvage pathway precursors). This lag in incorporation kinetics was also evident at the nuclear matrix level, although, once incorporated into nuclear matrix-associated DNA, the distribution patterns of both precursors into extra-matrix nuclear DNA fractions were identical. To determine the basis for this kinetic lag, we compared the incorporation kinetics of orotic acid and of deoxythymidine into dTTP and into nuclear matrix-associated DNA, respectively. Orotic acid-derived nucleotides entered the cytosolic dTTP pool before being incorporated into nuclear matrix-associated DNA, that is, traversing the classical metabolic route of DNA precursors. Conversely, deoxythymidine-derived nucleotides by-passed the soluble dTTP cellular pool and engaged directly in DNA synthesis at the nuclear matrix. Not only is this the first evidence for nucleotide channelling in an intact mammalian system, but it also forms direct evidence that salvage pathway DNA precursors are channelled to nuclear matrix-associated sites of DNA replication.

Animals↗
PubMed1993

High resolution fractionation and characterization of ADP-ribose polymers.

Methods are described for the high resolution fractionation and characterization of ADP-ribose polymers. Polymers prepared in vitro using purified poly(ADP-ribose) polymerase were isolated free from interfering nucleic acids and salts using dihydroxyboronyl-Bio-Rex 70 chromatography and fractionated using anion exchange high-pressure liquid chromatography. The homogeneity of isolated polymer fractions was characterized by gel electrophoresis and polymer size was determined by analysis following enzymatic digestion to nucleosides. The method allows isolation of oligomers up to 50 mer as single species and larger polymers can be isolated free from oligomers according to size and branching frequency. The ability to isolate individual species of ADP-ribose polymers should prove useful for the study of the polymers and their noncovalent interactions with other components of chromatin. Microheterogeneity of individual oligomers was studied and shown to be due to differences at the protein proximal ends resulting from the chemical method of release of polymers from protein. The method also was applied to fractionate polymers generated in intact cultured mouse cells in response to treatment with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine.

Animals↗
PubMed1993

Changes in levels of ADP-ribose polymers in rat liver during 2-acetylaminofluorene-induced hepatocarcinogenesis.

The exposure of rats to the carcinogen 2-acetylaminofluorene (2-AAF) results in the accumulation of DNA-damaging adducts. The inability of cells to repair such damage adequately is a putative causal event in chemical carcinogenesis. It has been shown that one cellular response to DNA damage that leads to DNA repair is poly(ADP-ribosyl)ation of nuclear proteins. To examine the possible existence of an altered poly(ADP-ribosyl)ation response to 2-AAF-mediated damage of rat liver DNA, tissue ADP-ribose polymer levels were determined during various stages of 2-AAF-mediated carcinogenesis. 2-AAF was administered to rats in a discontinuous feeding regimen comprised of five consecutive cycles, each cycle consisting of 3 weeks on 2-AAF diet followed by 1 week of recovery on a control diet without 2-AAF. During cycle one of 2-AAF administration, rat liver ADP-ribose polymer levels increased 3-fold over that found in livers of rats fed only the control diet. In contrast, when rats were administered the non-genotoxic liver mitogen 4-AAF for one cycle, no significant elevation occurred in ADP-ribose polymer levels. Elevated ADP-ribose polymer production was also observed during cycles two and three of 2-AAF administration. However, during cycles four and five of 2-AAF administration, a period when rats administered 2-AAF acquire a high risk for hepatocarcinogenesis, an altered pattern of ADP-ribose polymer production occurred in rat livers. ADP-ribose polymer levels in these rat livers remained low, similar to levels found in control rat livers, despite the administration of 2-AAF. When the livers from rats fed either one or five cycles of 2-AAF were analyzed for possible decreases in the levels of tissue NAD+, the substrate for poly(ADP-ribose) polymerase, no changes in relative abundance were found. In addition, analysis of poly(ADP-ribose) polymerase activity showed no decrease at five cycles of 2-AAF administration. These results indicated that at late stages of 2-AAF-induced hepatocarcinogenesis, 2-AAF does not induce an expected increase in ADP-ribose polymer levels, and suggested that significant changes in DNA repair may occur at a time just preceding an increased risk for developing liver cancer.

2-Acetylaminofluorene↗
PubMed1992

Characterization of a complementary DNA for rat liver aryl sulfotransferase IV and use in evaluating the hepatic gene transcript levels of rats at various stages of 2-acetylaminofluorene-induced hepatocarcinogenesis.

A complementary DNA (cDNA) for rat hepatic aryl sulfotransferase IV (AST IV) was isolated, characterized, and used as a hybridization probe to evaluate the molecular basis for the differential expression of AST IV during 2-acetylaminofluorine (2AAF)-induced hepatocarcinogensis. The AST IV cDNA clone was obtained by immunochemical screening of a male Sprague-Dawley rat liver cDNA library. The AST IV cDNA was found to be 1.3 kilobases long and to encode a fusion protein which was reactive with an antibody to AST IV and enzymatically able to generate the sulfuric acid ester of N-hydroxy-2AAF. Sequence analysis of the AST IV cDNA showed it to be 1127 residues in length and to have essentially complete homology with PST-I cDNA, a previously reported (S. Ozawa, et al., Nucleic Acids Res., 18: 4001, 1990), 1028-base cDNA for an uncharacterized rat liver aryl sulfotransferase. Comparison of the PST-I/AST IV cDNA-deduced amino acid sequence with data from a partial (51%) amino acid sequence analysis of purified AST IV showed complete amino acid homology, confirming the identity of the cDNA and establishing that AST IV was an N-blocked, 291-amino acid protein with a molecular mass of 33,909 daltons. The AST IV cDNA sequence differed from the PST-I cDNA in two principal ways: the 5' end lacked 18 coding bases, and the 3' end contained a 190-base extention in the untranslated region, including a consensus sequence for signalling polyadenylation. Studies of AST IV gene transcript levels showed that the livers of rats fed 2AAF for 3 wk (early stage hepatocarcinogenesis) and hyperplastic nodules from the livers of rats fed 2AAF for 19 wk (intermediate stage hepatocarcinogenesis) displayed transcript levels similar to those of livers from normal rats. This contrasted with the 60 to 70% lower than normal capacity of the mRNA fractions to express AST IV observed during in vitro translation. These results indicated that modulation of AST IV expression at early and intermediate stages of hepatocarcinogenesis involved regulatory mechanisms at the translational level. In contrast, mRNA fractions isolated from some 2AAF-induced liver tumors or from known chemical carcinogen-derived rat hepatoma cell lines showed losses of both AST IV transcript level and in vitro translation capacity, suggesting that regulation at the transcriptional level may become important at late stages of 2AAF-induced hepatocarcinogenesis. These results indicated that the molecular mechanisms for the 2AAF-mediated down regulation of AST IV expression during 2AAF-induced hepatocarcinogenesis involved alterations in regulation at both translational and transcriptional levels.

2-Acetylaminofluorene↗
PubMed1992

Reaction product inactivation of aryl sulfotransferase IV following electrophilic substitution by the sulfuric acid ester of N-hydroxy-2-acetylaminofluorene.

Rat liver N-hydroxy-2-acetylaminofluorene (N-OH-2AAF) sulfotransferase activity is mediated by aryl sulfotransferase IV (AST IV) and causes the bioactivation of N-OH-2AAF to a highly reactive sulfuric acid ester form putatively capable of inducing liver cancer. Dietary administration of 2-acetylaminofluorene (2AAF) to induce hepatocarcinogenesis in rats has been shown to cause a rapid loss in N-OH-2AAF sulfotransferase activity. A possible mechanism for the in vivo loss in sulfotransferase activity may be the PAPS-dependent, sulfotransferase-catalyzed, reaction product inactivation of the enzyme by covalent reaction with the N-OH-2AAF sulfuric acid ester. In vitro studies to evaluate this possibility utilized a highly purified form of AST IV and measured the extent of PAPS-dependent interaction between the enzyme and N-OH-2[9-14C]AAF. The results showed the presence of a adenosine-3'-phospho-5'-phosphosulfate (PAPS)-dependent 14C-labeling of AST IV. The labeling could be blocked if the sulfotransferase inhibitor pentachlorophenol was present. Analysis of 14C-labeled AST IV following alkaline digestion and chromatography of digestion products indicated that AST IV cysteine and methionine residues were primary sites of 2[9-14C]AAF adduction. Studies involving the pretreatment of AST IV with PAPS and N-OH-2AAF prior to the measurement of N-OH-2AAF sulfotransferase activity showed a close parallel between formation of the AST IV cysteine-2AAF adduct and loss of activity. Similar studies showed that enzyme inactivation and cysteine-2AAF adduct formation could be blocked when excessive amounts of a competing nucleophile, methionine, were present during the pretreatment step, suggesting that inactivation does not proceed by a mechanism-based process. Finally, experiments involving prior reaction of AST IV with the thiol-blocking agent, N-ethylmaleimide, before measurement of enzyme activity showed essentially full loss of sulfotransferase activity and suggested that formation of AST IV cysteine-2AAF adducts could be a mechanism for enzyme inactivation. These results indicate that the in vitro inactivation of AST IV by the reactive N-OH-2AAF sulfuric acid ester is accompanied by covalent binding to AST IV, possibly through the formation of cysteine-2AAF adducts, and suggests that this mechanism merits further consideration as a basis for the loss of N-OH-2AAF sulfotransferase activity in vivo.

Animals↗
PubMed1992

Sulfation of hydroxylamines and hydroxamic acids in liver cytosol from male and female rats and purified aryl sulfotransferase IV.

Sulfation activity towards hydroxamic acids and hydroxylamines was determined in liver cytosols for juvenile and adult males and female rats, as well as in purified rat liver aryl sulfotransferase IV preparations. Sulfation activity towards the hydroxamic acids N-hydroxy-2-acetylaminofluorene, N-hydroxy-2-acetylaminophenanthrene, N-hydroxy-4-acetylaminobiphenyl, N-hydroxy-4'-fluoro-4-acetylaminobiphenyl, N-hydroxy-2-acetylamino-5-phenylpyridine, was higher in cytosols derived from adult males (two or three times) than in those from adult females and juveniles (both sexes). N-Hydroxy-2-acetylamino-3-methyl-5-phenylpyridine (N-OH-2AAMPP), however, was poorly sulfated by any of the cytosols. Sulfation activity towards the hydroxylamines N-hydroxy-2-aminofluorene, N-hydroxy-2-aminophenanthrene, N-hydroxy-4-aminobiphenyl, N-hydroxy-4'-fluoro-4-aminobiphenyl was much lower. N-Hydroxy-2-amino-5-phenylpyridine (N-OH-2APP), however, was sulfated much better than the other hydroxylamines. No higher sulfation activity in adult male cytosols for hydroxylamines was found, except for N-OH-2APP and N-hydroxy-2-amino-3-methyl-5-phenylpyridine (N-OH-2AMPP). Purified aryl sulfotransferase IV (AST IV) converted all hydroxamic acids; N-OH-2AAMPP was a poor substrate. Of the hydroxylamines only N-OH-2APP and N-OH-2AMPP were conjugated. These results suggest that hydroxylamines and hydroxamic acids are converted by different sulfotransferases in the rat in vivo. They also indicate that AST IV may be the major enzyme responsible for sulfation of a variety of aromatic hydroxamic acids in the male rat liver. The results presented here are discussed in relation to the carcinogenic effects of some of these compounds.

Aging↗