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H Glatt

Publications and source records attributed to H Glatt.

At least 37 records · Page 2Linked to original sources
PubMed1998

Chiral inversion of 1-hydroxyethylpyrene enantiomers mediated by enantioselective sulfotransferases.

The benzylic alcohol 1-hydroxyethylpyrene (1-HEP) is activated to a mutagen by sulfotransferases. The sulfuric acid ester formed is difficult to detect, as it is rapidly hydrolysed back to the alcohol. Incubation of the individual enantiomers of 1-HEP with human hydroxysteroid sulfotransferase (hHST) or estrogen sulfotransferase (hEST), expressed in bacteria, led to the formation of the other enantiomer. The rates of sulfation were determined from the initial rates of chiral inversion of the alcohol, knowing that hydrolysis follows an SN1 mechanism and therefore produces racemic alcohol. hEST showed high enantioselectivity for S-1-HEP, whereas hHST strongly preferred the R-enantiomer. The rates of sulfation of the preferred enantiomers were high, similar to those for the prototype substrates of hEST (beta-estradiol) and hHST (dehydroepiandrosterone). Moreover, after a 30-min incubation of S-1-HEP with hEST, 95% of the recovered alcohol showed the R-configuration, indicating that several cycles of sulfation and hydrolysis had led to the depletion of one enantiomer and to the enrichment of the other enantiomer.

Benzyl Alcohols↗
PubMed1998

Sulfotransferase-mediated activation of mutagens studied using heterologous expression systems.

Sulfation is a common final step in the biotransformation of xenobiotics and is traditionally associated with inactivation. However, the sulfate group is electron-withdrawing and may be cleaved off heterolytically in some molecules leading to electrophilic cations which may form adducts with DNA and other important cellular structures. Since endogenous sulfotransferases do not appear to be expressed in indicator cells of standard mutagenicity tests, rat and human sulfotransferases have been stably expressed in his- Salmonella typhimurium strain TA1538 and Chinese hamster V79 cells. Using these recombinant indicator cells, sulfotransferase-dependent genotoxic activities were detected with N-hydroxy-2-acetylaminofluorene, 2-acetylaminofluorene (in the presence of co-expressed rat cytochrome P450 1A2), hycanthone, 1'-hydroxysafrole, alpha-hydroxytamoxifen and various benzylic alcohols derived from polycyclic aromatic hydrocarbons. In several cases, it was critical that the reactive sulfuric acid conjugates were formed directly within the indicator cells, owing to the inefficient penetration of cell membranes. In other cases, spontaneous benzylic substitution reactions with medium components, such as halogenide ions or amino acids, led to secondary, membrane-penetrating reactive species. Different sulfotransferases, including related forms from rat and human, substantially differed in their substrate specificity towards the investigated promutagens. It is known that some sulfotransferases are expressed with high tissue and cell type specificities. This site-dependent expression together with the limitations in the distribution of reactive sulfuric acid conjugates may explain organotropic effects of compounds activated by this metabolic pathway.

Animals↗
PubMed1998

Expression of human estrogen sulfotransferase in Salmonella typhimurium: differences between hHST and hEST in the enantioselective activation of 1-hydroxyethylpyrene to a mutagen.

Various human sulfotransferases (hP-PST, hM-PST, hHST) and rat sulfotransferases (rPST-IV, rHSTa) have already been expressed in Ames' Salmonella strains (in particular in TA1538). Now a further strain, TA1538-hEST, which expresses the human estrogen sulfotransferase (hEST), has been constructed. This strain activated the primary benzylic alcohol 1-hydroxymethylpyrene (1-HMP) and the secondary benzylic alcohol 1-hydroxyethylpyrene (1-HEP) to mutagens. Human sulfotransferases hEST and hHST both activated 1-HEP, but they differed substantially in their enantioselectivity for this compound.

Animals↗
PubMed1998

Characterization of thyroid hormone sulfotransferases.

Sulfation is an intriguing pathway of thyroid hormone metabolism since it facilitates the degradation of the hormone by the type I deiodinase (D1). This study reports the preliminary characterization of iodothyronine sulfotransferase activities of rat and human liver cytosol and recombinant rSULT1C1 and hSULT1A1 isoenzymes. All these enzyme preparations catalyzed the sulfation of--in decreasing order of efficiency--3,3'-diiodothyronine (3,3'-T2) > 3,3',5-triiodothyronine (T3) approximately 3,3',5'-triiodothyronine (rT3) > thyroxine (T4). 3,3'-T2 sulfotransferase activity was found to be higher in male than in female rat liver, which has also been shown by others for the expression of rSULT1A1 and rSULT1C1. No sulfation of iodothyronines was observed with rSULT1A1. Different phenol derivatives were found to be potent inhibitors of the sulfation of 3,3'-T2 by native and recombinant sulfotransferases, with pentachlorophenol and 2,4,6-tribromophenol being the most potent. The inhibitions exerted by the different phenols on 3,3'-T2 sulfation by rSULT1C1 correlated better with the effects observed in male than with those in female liver. A strong correlation was also observed between the inhibition profiles of human liver cytosol and hSUL1T1A1. These results suggest that: (1) rSULT1C1 is an important isoenzyme for the sulfation of thyroid hormone in male rat liver; (2) another isoenzyme with similar properties, perhaps rSULT1B1, is responsible for thyroid hormone sulfation in female rat liver and may also contribute to this process in male rat liver; and (3) hSULT1A1 is an important isoenzyme for thyroid hormone sulfation in human liver.

Animals↗
PubMed1998

Clonal heterogeneity at allelic methylation sites diagnostic for Prader-Willi and Angelman syndromes.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are developmental disorders resulting from the absence of the paternal or maternal contribution to the 15q11-13 region, respectively. Allele-specific methylation at D15S63 (PW71) has routinely been used as a diagnostic indicator of PWS and AS in DNA samples derived from peripheral blood. Extensive variation in allele-specific methylation patterns, however, has been observed at this site in different tissues, but the frequency or mechanism of this variation has remained uncharacterized. Herein, we have investigated the cellular basis of variation in methylation patterns at four sites of allelic methylation near SNRPN by using DNA samples derived from a panel of primary T lymphocyte clones. Interclonal variability was observed at three of these sites, including the diagnostic PW71 site. Changes in allele-specific methylation patterns occurred at a frequency of about one change in 50% of the cells every 22-25 doublings. In contrast, stable allele-specific methylation was observed in these clonal populations at exon 1 of SNRPN and the androgen receptor locus on the inactive X chromosome, suggesting that methylation at some CpG sites is more faithfully maintained than others. Clonal heterogeneity at PW71 was not an artifact of cell culture because the absence of allelic methylation was also observed in about 20% of the alleles in unstimulated peripheral blood. These results demonstrate that variation in allele-specific methylation at PW71 and other sites in the PWS/AS region appear to depend on the clonal complexity of the particular tissue and on the lack of strict maintenance of methylation within clones.

Alleles↗
PubMed1998

Rat, but not human, sulfotransferase activates a tamoxifen metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture.

Tamoxifen increases the risk of human endometrial cancer and is a potent carcinogen in rat liver, in which it produces DNA adducts and cytogenetic damage. Nevertheless its prophylactic use against breast cancer in healthy women is under investigation in several large trials. To investigate whether rat hepatocarcinogenicity predicts human hepatocarcinogenicity we used genetically engineered bacterial and mammalian target cells to investigate how alpha-hydroxy-tamoxifen, a major phase I metabolite of tamoxifen, is further metabolised by rat and human phase II enzymes, sulfotransferases, to mutagenic and DNA-adduct-forming species. We expressed rat hydroxysteroid sulfotransferase a, a liver-specific enzyme, and corresponding human sulfotransferase in bacteria (Salmonella typhimurium) and in a mammalian cell line (Chinese hamster V79 cells) and tested alpha-hydroxytamoxifen for DNA adduct formation and mutagenicity in these systems, using unmodified cells as controls. In cells that expressed rat hydroxysteroid sulfotransferase, alpha-hydroxytamoxifen was mutagenic and formed the same pattern of DNA adducts as that found in the liver of tamoxifen-treated rats. Alpha-hydroxytamoxifen was not activated, or was at least 20 times less active in cells expressing human hydroxysteroid sulfotransferase. All the other six known human xenobiotic-metabolising sulfotransferases were also expressed in S. typhimurium. None activated alpha-hydroxytamoxifen to a mutagen. These results suggest that the risk of DNA adduct formation, and cancer, in the human liver is low and explain why tamoxifen is a powerful carcinogen to the rat liver, and why standard short-term tests fail to detect its mutagenicity.

Animals↗
PubMed1998

Detoxification of optically active bay- and fjord-region polycyclic aromatic hydrocarbon dihydrodiol epoxides by human glutathione transferase P1-1 expressed in Chinese hamster V79 cells.

Dihydrodiol epoxides (DEs) are important carcinogenic metabolites of polycyclic aromatic hydrocarbons (PAHs). The metabolic formation of four stereoisomeric DEs (a pair of optically active diastereomers termed as syn- and anti-form) is possible. Glutathione tranferases (GSTs) have been demonstrated to catalyze the detoxification of DEs. Purified GSTs display remarkable differences in catalytic efficiencies towards bay- and fjord-region DEs along with a high degree of regio- and stereoselectivity. Here we determined to which extent heterologously expressed human GSTP1-1, a major GST isoform in lung, affects the mutagenicity of stereoisomeric bay-region DEs of benzo[a]pyrene in Chinese hamster V79 cells. To evaluate the influence of sterical crowding in the substrate on the activity of GSTP-1, the study was extended to the strongly mutagenic fjord-region (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene. GSTP1-1,reduced preferentially the mutagenicity (studied at the hprt locus) of (+)-anti and (+)-syn-DEs of benzo[a]pyrene (by 66 and 67%) as compared with the corresponding (-)-anti- and (-)-syn-enantiomers (by 15 and 13%). These results are in line with previous studies on the enantioselectivity of purified GSTP1-1 towards the DE isomers of benzo[a]pyrene and benzo[c]phenanthrene showing that enantiomers with (R)-configuration at the benzylic oxiranyl carbon are better substrates than those with (S)-configuration. Interestingly, the (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene were efficiently detoxified by GSTP-1-1 in the constructed cell line (reduction of mutagenicity by 66 and 64%). This study demonstrates that differences in the caalytic activity seen for purified GST towards individual mutagens do not necessarily reflect the detoxification of DEs by the same enzyme in a living cell and provides further evidence that specific human GSTs play a role in the detoxification of DEs of PAHs.

Animals↗
PubMed1997

Human gamma-aminobutyric acid-type A receptor alpha5 subunit gene (GABRA5): characterization and structural organization of the 5' flanking region.

The gamma-aminobutyric acid-type A receptor alpha5 subunit gene (GABRA5) is widely expressed in brain and localized to the imprinted human chromosome 15q11-q13. A combination of cDNA library screening and 5' RACE analysis led to identification of three distinct mRNA isoforms of GABRA5 in human adult and fetal brain tissues, each of which differs only in the noncoding 5' UTR sequence. Alignment of the genomic and cDNA sequences of GABRA5 revealed that the mRNA isoforms resulted from three alternative first exons 1A, 1B, and 1C. Northern blot analysis showed that the expression of GABRA5 was not only tissue specific but region specific in brain. CAT reporter assays revealed promoter elements in the 5' proximity of each first exon. The GABRA5 promoter regions lacked TATA and CCAAT boxes but contained several other consensus transcriptional factor recognition sequences. These findings suggest that the differential exon 1 usage of GABRA5 arises as a consequence of alternative promoter activation.

Adult↗
PubMed1997

Structure and organization of GABRB3 and GABRA5.

The genes encoding the gamma-aminobutyric acid (GABA) type-A receptor subunits beta 3 (GABRB3), alpha 5 (GABRA5), and gamma 3 (GABRG3) map to chromosome 15q11-q13. The three genes are contained within roughly 800 kb of the distal part of the imprinted Prader-Willi and Angelman syndrome region. A 570-kb contig encompassing GABRB3 and GABRA5 has been constructed in P1, lambda phage, and PAC clones. GABRB3 spans 250 kb of DNA and is organized into 9 exons that range from 68 to 504 bp, while GABRA5 is encoded by 11 exons (65 to 924 bp in length) within 86 kb. The exon/intron borders for both genes have been characterized and, primers have been designed to amplify each of the individual exons. Two reference STR markers have been positioned in the contig. The reference STR for GABRB3 is in fact located at least 60 kb beyond the 3' terminus of GABRB3, while D15S97 is contained within intron 4 of GABRB3. The detailed physical map of this GABAA receptor subunit gene cluster should not only be useful in genetic studies of the 15q11-q13 region, but will also be important for investigating the evolution and expression of the GABAA receptor gene superfamily.

Base Sequence↗
PubMed1997

Metabolic activation of the (+)-S,S- and (-)-R,R-enantiomers of trans-11,12-dihydroxy-11,12-dihydrodibenzo[a,l]pyrene: stereoselectivity, DNA adduct formation, and mutagenicity in Chinese hamster V79 cells.

Polycyclic aromatic hydrocarbons require metabolic activation in order to exert their biological activity initiated by DNA binding. The metabolic pathway leading to bay or fjord region dihydrodiol epoxides as ultimate mutagenic and/or carcinogenic metabolites is thought to play a dominant role. For dibenzo[a,l]pyrene, considered as the most potent carcinogenic polycyclic aromatic hydrocarbon, the formation of the fjord region syn- and/or anti-11,12-dihydrodiol 13,-14-epoxide (DB[a,l]PDE) diastereomers has been found to be the principal metabolic activation pathway in cell cultures leading to DNA adducts. In order to further elucidate the stereoselectivity involved in this activation pathway via the formation of the trans-11,12-dihydrodiol, we have synthesized the enantiomerically pure 11,12-dihydrodiols of dibenzo[a,l]-pyrene and investigated their biotransformation in rodents. Incubations with liver microsomes of Sprague-Dawley rats and CD-1 mice pretreated with Aroclor 1254 revealed that the enzymatic conversion to the fjord region DB[a,l]PDE strongly depends on the absolute configuration of the 11,12-dihydrodiol enantiomers. While oxidation at the 13,14-position of the (+)-(11S,12S)-dihydrodiol is limited to a small extent, the (-)-11R,12R-enantiomer is metabolized to its fjord region dihydrodiol epoxides in considerably higher amounts. Moreover, this substrate is transformed with high stereoselectivity to the corresponding (-)-anti-dihydrodiol epoxide by liver microsomes of Aroclor 1254-treated rodents. The metabolism results were in good accordance with the extent of stable adduct formation in calf thymus DNA as investigated by the 32P-postlabeling technique and with the mutagenicity in Chinese hamster V79 cells of the two enantiomeric 11,12-dihydrodiols mediated by hepatic postmitochondrial preparations of Aroclor 1254-treated rats. The results indicate that both genotoxic events occurred predominantly by the stereoselective activation of the (-)-(11R,12R)-dihydrodiol to the (-)-anti-DB[a,l]PDE with R,S,S,R-configuration.

Animals↗
PubMed1997

Sulfation and sulfotransferases 4: bioactivation of mutagens via sulfation.

Sulfation is a common final step in the biotransformation of xenobiotics and is traditionally associated with inactivation. However, the sulfate group is electron-withdrawing and may be cleaved off heterolytically in some molecules, leading to an electrophilic cation. The stable heterologous expression of sulfotransferases in indicator cells of standard mutagenicity tests has substantially improved the accessibility of this activation pathway. Sulfotransferase-mediated genotoxic effects have been demonstrated for numerous benzylic alcohols derived from polycyclic aromatic hydrocarbons and various aromatic hydroxylamines. Also, hycanthone (a benzylic alcohol), alpha-hydroxytamoxifen (an allylic alcohol), 1'-hydroxysafrole (an allylic/benzylic alcohol), and 2-nitropropane are activated to genotoxicants by sulfotransferases. Various reactive sulfate conjugates show strong mutagenic effects only when they are generated directly within the indicator cell, due to their inefficient penetration of cell membranes. In other cases, secondary membrane-penetrating reactive species are formed from sulfuric acid esters by displacement reactions with medium components, such as chloride or amino acids. Reaction with water regenerates the alcohol, which becomes available for a new cycle of activation. Different sulfotransferases from the same species as well as related forms from rat and human differ in their substrate specificities and tissue distributions. These characteristics and reactivities of the sulfate conjugates formed may explain organotropic effects of the compounds activated via sulfotransferases.

Animals↗
PubMed1997

Angelman syndrome associated with an inversion of chromosome 15q11.2q24.3.

Angelman syndrome (AS) most frequently results from large (> or = 5 Mb) de novo deletions of chromosome 15q11-q13. The deletions are exclusively of maternal origin, and a few cases of paternal uniparental disomy of chromosome 15 have been reported. The latter finding indicates that AS is caused by the absence of a maternal contribution to the imprinted 15q11-q13 region. Failure to inherit a paternal 15q11-q13 contribution results in the clinically distinct disorder of Prader-Willi syndrome. Cases of AS resulting from translocations or pericentric inversions have been observed to be associated with deletions, and there have been no confirmed reports of balanced rearrangements in AS. We report the first such case involving a paracentric inversion with a breakpoint located approximately 25 kb proximal to the reference marker D15S10. This inversion has been inherited from a phenotypically normal mother. No deletion is evident by molecular analysis in this case, by use of cloned fragments mapped to within approximately 1 kb of the inversion breakpoint. Several hypotheses are discussed to explain the relationship between the inversion and the AS phenotype.

Angelman Syndrome↗
PubMed1996

Design, production and characterization of antibodies discriminating between the phenol- and monoamine-sulphating forms of human phenol sulphotransferase.

1. Phenol sulphotransferases (PSTs) are important enzymes in xenobiotic and endobiotic detoxication, and a key component of the body's chemical defence mechanism. 2. Human phenol-(P-PST) and monoamine-(M-PST) sulphating forms of PST share 93% amino acid sequence identity, and to date the various antibodies produced against PSTs all recognize both enzymes. 3. We have identified two peptides based on the cDNA-derived amino acid sequences of human P-PST and M-PST, which elicited for the first time antibodies capable of discriminating between these highly homologous enzymes. 4. These antibodies represent valuable tools for studying the expression, distribution and function of human phenol sulphotransferases.

Amino Acid Sequence↗
PubMed1995

Human dehydroepiandrosterone sulfotransferase. Purification, molecular cloning, and characterization.

Human tissues possess at least four distinct forms of cytosolic ST, three of which are involved in the sulfation of steroids. DHEA-ST is responsible for the majority of hydroxysteroid and bile acid sulfation in human tissues and abundant levels of the enzyme are present in human liver and adrenal tissues. In the adult human adrenal, DHEA-ST has been localized immunologically to the zona reticularis of the adrenal cortex. No age- or gender-related differences in the expression of DHEA-ST activity in adult human liver cytosols have been reported. The cDNA encoding DHEA-ST has been isolated from a human liver cDNA library and expressed in both mammalian COS cells and E. coli. Purification and molecular characterization studies suggest a single form of DHEA-ST in human tissues. The properties of DHEA-ST expressed in either mammalian or bacterial cells are very similar to those of the native enzyme. DHEA-ST can also bioactivate a number of procarcinogens to reactive electrophilic forms. Hydroxymethyl PAHs are sulfated and bioactivated at a relatively rapid rate by DHEA-ST, whereas 1'-hydroxysafrole and N-hydroxy-2-acetylaminofluorene are bioactivated to a lesser extent.

Adrenal Glands↗
PubMed1995

Activation of benzylic alcohols to mutagens by rat and human sulfotransferases expressed in Escherichia coli.

Human hydroxysteroid sulfotransferase, human phenol-sulfating form of phenol sulfotransferase, rat hydroxysteroid sulfotransferase a and rat phenol sulfotransferase IV were expressed in Escherichia coli. Cytosol preparations of transformed bacteria were used as activating systems in mutagenicity tests with Salmonella typhimurium TA98. All test compounds, 1-hydroxymethylpyrene, 2-hydroxymethylpyrene, 1-(1-pyrenyl)ethanol, 9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene and 4H-cyclopenta[def]chrysen-4-ol, were activated by both hydroxysteroid sulfotransferases investigated. However, 1-(1-pyrenyl)ethanol was 67-fold more efficiently activated by the human enzyme, whereas 7-hydroxymethyl-12-methylbenz[a]anthracene was 27-fold more efficiently activated by the rat enzyme. The phenol sulfotransferases showed relatively low activities with the benzylic alcohols investigated. The only exception was 4H-cyclopenta[def]chrysen-4-ol, which was activated efficiently by rat phenol sulfotransferase IV. We had previously tested the ability of rat and human hepatic cytosol preparations to activate the same compounds. The results of a statistical analysis suggest that the activities of human hydroxysteroid sulfotransferase, rat hydroxysteroid sulfotransferase a and phenol sulfotransferase IV can account for a substantial portion of the activation of benzylic alcohols in human, female rat and male rat liver, respectively.

Animals↗
PubMed1995

Human platelet phenolsulfotransferases: cDNA cloning, stable expression in V79 cells and identification of a novel allelic variant of the phenol-sulfating form.

We have isolated cDNA clones encoding phenol- and monoamine-sulfating phenolsulfotransferases, using the reverse transcription-polymerase chain reaction method with human platelet mRNA as template. These cDNAs were stably expressed in the Chinese hamster fibroblast cell line V79 and their substrate specificities towards known sulfotransferase isoenzyme-selective compounds determined. The nucleotide and derived amino acid sequences of the monoamine-sulfating form were identical to those previously identified in human liver and brain, but the cDNA for the human platelet phenol-sulfating form we isolated contained 5 and 2 amino acid changes from the two previously published sequences from human liver and brain, suggesting that we have identified a new allelic variant of human phenol-sulfating phenolsulfotransferase.

Alleles↗
PubMed1995

Human fetal adrenal hydroxysteroid sulphotransferase: cDNA cloning, stable expression in V79 cells and functional characterisation of the expressed enzyme.

Dehydroepiandrosterone sulphate (DHEAS) is a major adrenal secretory product, particularly in the fetus where it serves as a substrate for oestrogen biosynthesis by the placenta. The enzyme in the adrenal responsible for synthesising DHEAS, hydroxysteroid sulphotransferase (HST), is therefore essential for human development. We have isolated a full-length cDNA clone, encoding human fetal adrenal HST, and constructed a stable cell line expressing it by transfection into V79 Chinese hamster lung fibroblast cells. This cDNA was essentially identical to that isolated from adult human liver, where the role of HST is less well understood. This recombinant cell line allowed determination of the substrate specificity and kinetic properties of this enzyme towards various steroid hormones, and by comparison of these activities with human liver cytosol we have shown that HST is the major sulphotransferase responsible for the sulphation of DHEA, androsterone and pregnenolone in man and that, functionally, the hepatic and adrenal enzymes are very similar. The expressed HST was also active with testosterone, cortisol (although at low levels) and the xenobiotic 17 alpha-ethinyloestradiol, but not with oestrone or 1-naphthol. We have therefore created a valuable resource for the study of this important enzyme.

Adrenal Glands↗