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In order to explore the possible role of sulfation in the inactivation of environmental estrogens at gastrointestinal sites and their subsequent removal, we investigated the effects of phenolic environmental estrogens on sulfotransferase (ST) activity. The mouse intestine and a human colon carcinoma cell line, Caco-2, were studied. ST enzymes were found to have a high affinity for diethylstilbestrol (DES) and bisphenol A (BPA), whereas phenol ST (PST) activity was strongly inhibited by nonylphenol and genistein in both mice and humans. Kinetic analysis showed that this inhibition was competitive. These observations suggest that nonylphenol and genistein compounds might inhibit PST activity in the human intestine and that they might escape detoxification by sulfation.

Previously we demonstrated the presence of phenol sulphotransferase (P-ST) in mouse nasal cytosols and identified its zonal location in mouse nasal cavity by staining with an antiserum raised against a rat liver P-ST isoenzyme, PSTg. In the present study a cDNA was isolated from a mouse olfactory cDNA library by immunological screening with the antiserum. The isolated cDNA consisted of 1347 bp with a 912 bp open reading frame encoding a 304-residue polypeptide. Both the nucleotide and deduced amino acid sequences of the cDNA were 94% identical with those of a rat liver P-ST isoenzyme, ST1C1. The expressed enzyme in Escherichia coli displayed high P-ST activity towards phenolic odorants such as eugenol and guaiacol, and it showed a high N-hydroxy-2-acetylaminofluorene sulphation activity in comparison with the rat ST1C1 enzyme. These results indicate that the olfactory P-ST encoded by the cDNA is a mouse orthologue of rat ST1C1; however, expression of the olfactory P-ST mRNA is specific for nasal tissues as revealed by reverse transcriptase-mediated PCR (RT–PCR).

By means of peptide sequence information, several cDNA clones encoding a 42 kDa choline/ethanolamine kinase were isolated from a rat kidney cDNA library. Eight clones were sequenced with all of them resulting in identical overlapping nucleotide sequences. Four of them possessed entire open reading frame which could encode 394 amino acids with a calculated molecular size of 45 100. The predicted amino acid sequence contained all of the internal peptide fragment sequences derived from the purified 42 kDa enzyme. When the open reading frame was introduced into pGEX-2T vector and transfected into E. coli cells, a significant choline/ethanolamine kinase activity did appear in the cell lysate. A homology comparison with the previously reported choline kinase cDNAs (CKR1 and CKR2) from rat liver showed 66%-68% in entire nucleotide sequences and 57%-59% in amino acid sequences, indicating that the cloned cDNA here must be a novel CK/EK gene product. (c) 1998 Elsevier Science B. V.

Estradiol benzoate (EB) and testosterone propionate (TP) were administered to male and female Wistar rats, and their effects on the zonal expression of hepatic phenol sulfotransferase (P-ST) activities were determined at pH 5.5 and 7.4. Cytosolic fractions from periportal (PP) and perivenous (PV) hepatocytes were prepared by the dual-digitonin-pulse perfusion technique. In control rats, P-ST activities assayed at pH 5.5 and 7.4 were higher in males than in females, and were higher in the PV fraction than in the PP fraction in both sexes. P-ST activities were increased by the administration of TP in the PP and PV fractions of females, whereas the same treatment diminished the enzyme activities in both fractions of the males. EB administration gave reduced P-ST activity at pH 5.5 of both fractions, irrespective of sex, but not a marked difference at pH 7.4. Chromatofocusing of PP and PV fractions revealed the presence of P-ST isoforms eluted at approx. pH 8.0 (peak 1), 7.5 (peak 2), 7.0 (peak 3 ) and 6.0 (peak 4). In male rats, peak 3, which showed high enzyme activity at pH 5.5 in the PP and PV fractions, was markedly decreased by EB treatment, whereas in females, peak 3 was present only in the PV fraction and was not affected by EB administration. TP treatment did not show remarkable changes in P-ST peaks in the males, while peaks 2 and 3 were increased in the females. Immunoblot analysis revealed the presence of multiple P-ST isoforms which showed different immunoreactivity and electrochemical properties.

Mouse nasal cytosols show high sulfotransferase (ST) activities toward phenolic aromatic odorants, but have little activities for most alcoholic aromatic odorants. Most ST activities toward the phenolic odorants preferred slightly acidic pH (6.4) and were sensitive to 2,6-dichloro-4-nitrophenol, a specific inhibitor for phenol ST (P-ST) but were not inhibited by triethylamine and tetra-n-butylammonium chloride, which are specific inhibitors for hydroxysteroid ST (HS-ST). These results suggested that P-ST activities are responsible for sulfation of the phenolic odorants. The spectra of the ST activities for these odorants were similar in mouse nasal and liver cytosols, however, nasal cytosols showed much higher ST activity toward cinnamyl alcohol than liver cytosols. This activity preferred higher pH (7.4) compared to the phenolic odorant-ST activities and was inhibited by both types of inhibitors, specific for P-ST and HS-ST. These results appear to indicate the participation of multiple ST isoforms for the sulfation of odorants in mouse nasal cytosols. The existence of P-ST(s) active for the phenolic odorants in olfactory cytosols suggests a role in odorant perception, in particular, in the signal termination process.

By employing dual-digitonin-pulse perfusion technique, the cytosolic fractions were prepared from periportal (PP) and perivenous (PV) hepatocytes of male and female rat livers. Sulfotransferase (ST) activities toward 2-naphthol (2NAP) (at pH 5.5 and 7.4), 4-nitrophenol, dehydroepiandrosterone (DHEA), and cortisol were measured in each fraction. DHEA-ST activity was mainly localized in PP fraction in males, while in females it was slightly higher in PP fraction than in PV samples. In contrast, phenol ST activities were higher in PV fraction in both sexes. With anti-HS-ST and anti-P-ST antisera, the levels of immunoreactive ST polypeptides were compatible with the levels of ST activities except that in female PP fraction the level of immunoreactive P-ST polypeptide was low in spite of comparatively high levels of P-ST activities. Chromatofocusing of PP and PV fractions separated P-ST activities into three major fractions (I–III), which have distinct catalytic and electrochemical properties. Fraction I was localized only in the PP samples in both sexes and revealed ST activities toward 2NAP at pH 5.5 and 7.4, while fraction II was localized in the PP and PV samples in both sexes with 2NAP-ST activity only at pH 7.4. Fraction III which had ST activities at pH 5.5 and 7.4 was present only in the PV samples in female rats, whereas in male rats it was present in both PP and PV samples. With anti-P-ST antiserum, the immunoreactive polypeptide was present in fraction III, but no immunoreactive band was detected in fractions I and II, suggesting the presence of immunochemically and electrochemically different P-ST(s) in these fractions.

Sulfation is an important conjugation reaction in the metabolism of various xenobiotics and endogenous compounds and is catalyzed by sulfotransferase (ST) present in cytosols. The cloning studies on STs have provided the basis for the understanding of the ST multigene family. STs are classified into hydroxysteroid (or alcohol), aryl (or phenol), estrogen, flavonol and polysaccharide STs and recent developments in the molecular characterization of these isoforms are reviewed. Regulation and localization of ST isoforms in various tissues are characterized at the molecular level by virtue of the specific antibodies and the corresponding cDNA probes. The recent developments are summarized. ST inhibitors are potent tools for the study on ST multiplicity and for the characterization of the enzyme structure. It also appears to be important to understand exogenous and endogenous ST inhibitors in clinical environment. The recent developments are reviewed.

Rat hepatic hydroxysteroid sulfotransferase with sulfoconjugates androsterone (androsterone-sulfating sulfotransferase) is an oligomer consisting of several subunits with distinct pI values but with the same molecular mass (pI variants). N-terminal amino acid sequences of the pI variants are all identical. The enzyme is exclusively present in the liver, in which its lobular localization is sex-dependent. The localization of the enzyme is markedly different from that of an isoenzyme of phenol sulfotransferase. In weanling and adult female rats, the relative abundance of the pI variants is different. During development from weanling stage to adulthood, the amounts of acidic variants increase, whereas the relative levels of alkaline variants remain constant.

We have regionally assigned the androsterone UDP- glucuronosyltransferase(UGT2B2) gene to rat chromosome 14 at the band p21.2-p22 by fluorescence in situ hybridization. LA Wistar rats have a deficiency of UGT2B2 due to large deletion of the gene. Fischer rats and Nagase analbuminemia rats also show very low UGT2B2 activity similar to LA Wistar rats. It was clarified that the enzyme protein was absent in these strains, however, the molecular mechanism of the defect of UGT2B2 are to be elucidated.

Sulfation is an important conjugation reaction in the metabolism of a diversity of xenobiotics and endogenous compounds such as drugs, food additives, carcinogens, steroids and neurotransmitters. Sulfate conjugation is catalyzed by various kinds of sulfotransferase (ST) such as hydroxysteroid ST (HS-ST), phenol ST (P-ST) and estrogen ST (E-ST) present in cytosols. Our laboratory has been studying the multiplicity of rat and mouse STs. We found that tertiary amines such as triethylamine selectively inhibited rat hepatic HS-ST. Developmental changes and zonal distributions of rat liver HS-ST and P-ST isoenzymes provided evidence for their complex regulatory mechanisms. Studies on site-directed mutagenesis and chimeras of rat HS-ST cDNAs, ST-40 and ST-20, revealed the importance of the C-terminal region for the substrate specificity and involvement of multiple regions for the enzyme activities. These two cDNAs have been mapped to the same chromosomal region 1q21.3-->q22.1 by fluorescence in situ hybridization. Mouse olfactory P-ST was present in the cytoplasm of olfactory sustentacular cells and its cloning study revealed that it is 94% identical with rat ST1C1 in amino acid sequences.

UDP-glucuronosyltransferase (UGT) catalyzes a transfer of glucuronic acid from UDP-glucuronic acid to various endogenous and exogenous compounds and is located in the endoplasmic reticulum of liver and other tissues. Glucuronidation is a major detoxication reaction and provides great effects on the metabolism, disposition and excretion of pharmacologically and toxicologically active compounds. There are several UGT isoenzymes with different and overlapping substrate specificities and under different regulation. During the last decade, there has been a dramatical increase in the understanding of the structure and function of UGT isoenzymes especially through the recombinant DNA technology. This review concentrates on the recent developments in the research on glucuronidation and deals with purification, cloning, gene structure, deficiency, chromosomal location and function of UGT isoenzymes and glucuronides as active metabolites.

Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC50=0.61 microM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50% of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3'-phosphoadenosine 5'-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [35S]-3'-phosphoadenosine 5'-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina.

We investigated the expression of sulfotransferases (STs) in the human colon carcinoma cell line, Caco-2, which is widely used as a human intestine model for orally administered drugs. By reverse transcription-polymerase chain reaction (RT-PCR), we could detect mRNA expressions of phenol STs (P-ST), hydroxysteroid ST (HS-ST) and estrogen ST (E-ST). Treatment with 1,25-dihydroxyvitamine D3, which induces CYP3A4 expression in Caco-2 cells, did not affect the ST expression of the cells. We detected significant P-ST activity toward 2-naphthol and dopamine in the cell extract. We also observed detectable HS-ST activity toward dehydroepiandrosterone (DHEA), but no E-ST activity toward beta-estradiol was observed in the extract. Analyses by RT-PCR and separation of P-ST activities on ion-exchange column chromatography showed that the thermolabile monoamine phenol sulfotransferase (M-PST) is dominant in Caco-2 cells.

Tea and fruit juices are beverages consumed daily all over the world. The present study reports the inhibitory effects of these beverages on the activity of mammalian intestinal phenol sulfotransferases (P-STs). Green tea strongly inhibited the E. coli-expressed mouse intestinal P-ST activity in vitro. (-)-Epigallocatechin gallate (EGCG) was found to be the most potent inhibitor among the catechins tested (IC50=0.93 microM). (-)EGCG also inhibited the P-ST activity of the human colon carcinoma cell line, Caco-2. Kinetic analysis showed that the inhibition was competitive. Among fruit juices examined (apple, grape, grapefruit and orange), grape juice exhibited the most potent inhibitory action on the P-ST activity of mouse intestines and human colon carcinoma cells. The inhibitory activity of grape juice was located mainly in the skin and seeds. Flavonols, such as quercetin and kaempferol, inhibited the P-ST activity at low concentrations. These observations suggest the possible inhibition of P-ST activity in human intestines by green tea or grape juice.

A sulfotransferase (ST) cDNA was isolated from a mouse intestinal cDNA library using a probe which was generated by reverse transcription (RT)-PCR based on the conserved amino acid sequences of the ST molecules. The isolated cDNA (1.1 kb) contained an 858 bp open reading frame encoding a 286 amino acid polypeptide with molecular weight of 33439. The deduced amino acid sequence shares 55.1% and 40.2% identity with mouse liver aryl/phenol (mSTp1) and alcohol (mSTa1 or mSTa2) STs, respectively, and it is highly similar to those of rat and human liver phenol ST (P-ST) isozymes, ST1B1 (87.8%) and ST1B2 (71.0%), respectively. RT-PCR analyses showed abundant expression of the P-ST mRNA in the intestine as well as in the liver in the mouse tissues examined (brain, heart, intestine, kidney, liver and lung) of both sexes. E. coli-expressed enzyme is capable of catalyzing the sulfation of 2-naphthol at Km = 3.3 microM and Vmax = 3.33 nmol/min/mg protein and also showed sulfation activity for L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine. Among food constituents tested, tannic acid and epigallocatechin gallate strongly inhibited the P-ST activity in vitro.

Rat hydroxysteroid sulfotransferase (HS-SULT) cDNAs, ST-40 and ST-20 are 90% identical in amino acid sequences and show different substrate specificities toward dehydroepiandrosterone (DHEA), androsterone (AD) and cortisol (CS). ST-40 enzyme is active toward the three substrates, whereas ST-20 enzyme is preferentially active for CS. First we prepared mutants of well conserved histidine, lysine and asparagine by site-directed mutagenesis. Secondly we constructed 20 chimeric HS-SULTs by reciprocal exchange of five protein domains between ST-20 and ST-40 enzymes. The studies on the expressed mutant and chimeric enzymes indicate the importance of the C-terminal region for the substrate specificity and the involvement of multiple regions for the enzyme activities. Next we determined the genetic loci of ST-40 and ST-20 by fluorescence in situ hybridization. Biotinylated ST-20 and ST-40 probes gave a pair of fluorescent spots on the same region of rat chromosome 1 and the loci of these genes were localized to the same chromosomal region of 1q21.3→q22.1. Finally we studied mouse olfactory phenol SULT (P-SULT). It was immunolocalized in the cytoplasm of mouse olfactory sustentacular cells and mouse nasal cytosols show high SULT activities toward phenolic aromatic odorants. We subsequently isolated a mouse P-SULT cDNA from mouse olfactory cDNA library. It encodes 304 amino acid polypeptide and is 94% identical with rat ST1C1 in amino acid sequences.

Isolation of several hydroxysteroid sulfotransferase (HS-ST) cDNAs from the rat liver cDNA library has demonstrated the possible expression of these HS-ST isoforms in rat liver. We devised a method to detect unequivocally ST-20 and ST-40 mRNAs by a reverse transcription-polymerase chain reaction, using specific primers. ST-40 mRNA was expressed only in liver, but ST-20 mRNA was present predominantly in the liver and slightly in extrahepatic tissues. On chromatofocusing, expressed ST-20 or ST-40 enzymes were eluted at approx. pH 8.2 and 5.7-4.7 or at pH 6.4-5.4, respectively. Chromatofocusing of adult female rat liver cytosols resolved HS-ST isoenzymes in a broad range of pH, and ST fractions A, B, C, D and E were eluted at approx. pH 8.2, 7.6, 7.5-6.8, 6.2 and 6.1-5,5, respectively. After PAP-agarose affinity column chromatography and SDS-polyacrylamide gel electrophoresis (PAGE), their N-terminal amino acid sequences were determined. ST isoenzymes present in fractions B and E showed identical N-terminal amino acid sequences with those of ST-21 and ST-20, respectively, whereas the ST isoenzymes present in fractions C and D had the same N-terminal amino acid sequence as those of ST-40 (and/or ST-41). The results demonstrated the presence of at least three HS-ST isoenzymes in adult female rat liver.

Sulfation is an important conjugation reaction in the metabolism of a diversity of xenobiotics and endogenous compounds such as drugs, food additives, carcinogens, steroids and neurotransmitters. Sulfate conjugation is catalyzed by various kinds of sulfotransferase (ST) such as hydroxysteroid ST (HS-ST), phenol ST (P-ST) and estrogen ST (E-ST) present in cytosols. Our laboratory has been studying the multiplicity of rat and mouse STs. We found that tertiary amines such as triethylamine selectively inhibited rat hepatic HS-ST. Developmental changes and zonal distributions of rat liver HS-ST and P-ST isoenzymes provided evidence for their complex regulatory mechanisms. Studies on site-directed mutagenesis and chimeras of rat HS-ST cDNAs, ST-40 and ST-20, revealed the importance of the C-terminal region for the substrate specificity and involvement of multiple regions for the enzyme activities. These two cDNAs have been mapped to the same chromosomal region 1q21.3-->q22.1 by fluorescence in situ hybridization. Mouse olfactory P-ST was present in the cytoplasm of olfactory sustentacular cells and its cloning study revealed that it is 94% identical with rat ST1C1 in amino acid sequences.

Two cDNA clones of rat hepatic hydroxysteroid sulfotransferase (ST) (ST-40 and ST-20) were isolated and expressed in Escherichia coli cells. Several histidine residues in their coding regions are highly conserved in the ST superfamily, and histidine mutants were constructed by site-directed mutagenesis. The substitution of alanine or lysine for the histidine at position 98 in the ST-40 enzyme resulted in a loss of ST activities toward dehydroepiandrosterone (DHEA), androsterone (AD) and cortisol (CS). The mutation of histidine 98 into alanine abolished the specific binding to 3'-phosphoadenosine 5'-phosphate agarose, suggesting that the residue is located at a critical position in the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding site. In the ST-20 enzyme, the replacement of histidine 98 with alanine also resulted in the loss of ST activity toward its preferential substrate, CS. In the ST-40 enzyme, the mutation at histidine 256 into alanine markedly reduced CS-ST activity, but DHEA-ST activity was not changed. Furthermore, selective decrease in CS-ST activity was also observed in the alanine mutant at lysine 254 or at asparagine 255 of the ST-40 enzyme. Kinetic analysis on the ST-40 and its mutant at asparagine 255 indicated that the Km value for CS was significantly increased in the mutant without any change in the Km values for 3'-phosphoadenosine 5'-phosphosulfate and DHEA. Inhibition studies demonstrated that DHEA-ST activity was competitively inhibited by AD, but not by CS in the ST-40 enzyme, whereas triethylamine, a noncompetitive inhibitor of hydroxysteroid ST, inhibited DHEA-ST activity in the ST-40 enzyme but did not inhibit CS-ST activity in either ST-40 or ST-20 enzymes. These data provide evidence that DHEA and CS bind to different sites, which probably function in a different manner in the ST-40 enzyme.

We have immunolocalized phenol sulfotransferase (PST)G, an isoform of PST in sustentacular cells which reside in the dorso-medial portion of the nasal cavity of the mouse. The same topographical pattern of gene expression has been reported for some olfactory neuron-specific genes. When several established (phenol-containing) odorants were used as substrates, mouse nasal tissue cytosol showed a significant level of PST activity, as does mouse liver cytosol. This study is the first to demonstrate that gene expression in the olfactory sustentacular cells is also organized zonally, and indicates the involvement of sulfo-conjugation in olfactory perireceptor processes, such as odorant clearance and xenobiotic detoxification.

Tetraalkylammonium salts having n-propyl to n-amyl side chains inhibited rat liver sulfotransferase (ST) activities toward dehydroepiandrosterone and cortisol, but not ST activity toward 2-naphthol, whereas trialkylamines having ethyl to n-amyl side chains inhibited ST activity toward dehydroepiandrosterone, but not ST activities toward cortisol and 2-naphthol. A comparison of I50 values, which represent inhibitor concentration resulting in 50% inhibition of dehydroepiandrosterone ST activity, revealed that the values for the tetraalkylammonium salts were 0.015 to 0.017 mM, whereas the values for the trialkylamines were 0.20 to 0.33 mM. Introduction of hydrophilic groups such as hydroxyl, thiol, nitrile and acetamide groups or substitution by methyl and allyl groups in the alkyl side chains markedly diminished the inhibitory effect of triethylamine. These data indicate that ethyl to n-amyl side chains are a prerequisite for the alkylamine-type inhibitor. Tertiary amine drugs such as imipramine, dimenhydrinate, cyclizine, chlorpromazine and promethazine inhibited ST activities toward dehydroepiandrosterone and cortisol similar to the tetraalkylammonium salts, although the drugs were weaker inhibitors of hydroxysteroid ST activities. These results imply that in addition to trialkylamine side chains, the other portion of the drugs may participate in the inhibition of hydroxysteroid ST activities.

Triethylamine, which was used as an elution solvent for column chromatography to purify chemically synthesized 3'-phosphoadenosine 5'-phosphosulfate (PAPS), was a potent inhibitor of rat liver sulfotransferase (ST) activities toward androsterone and dehydroepiandrosterone, but not ST activities toward cortisol and 2-naphthol. Examination of fourteen primary, secondary and tertiary amines revealed that a secondary amine, di-n-butylamine, and three tertiary amines, triethylamine, tri-n-propylamine and tri-n-butylamine, specifically inhibited ST activities toward androgen.

Rat androsterone UDP-glucuronosyltransferase gene (UGT2B2) was regionally assigned to chromosome band 14p21.2-p22 by fluorescence in situ hybridization using a biotinylated cDNA (1.8-kb insert) probe. The present mapping is the first report on the precise localization of the rat UGT2B2 on high resolution banded metaphase chromosomes.

A major isoenzyme of hepatic androsterone-sulfating sulfotransferase (AD-ST) was purified from adult female rats. The activity was purified 122-fold over that found in the cytosol and showed a single protein band with a subunit molecular mass of 30 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme exhibited four isoelectric variants of subunits on denaturing isoelectrofocusing gels (pI = 5.8, 6.1, 6.7 and 7.2). Rabbit antiserum raised against the enzyme specifically detected AD-ST polypeptide in rat liver cytosol. Immunoblot analysis of liver cytosol from female and male rats at various ages showed good correlation between the levels of AD-ST activity and AD-ST polypeptide. Significant levels of AD-ST activity and polypeptide were detected in senescent male rats, though normal adult male rats have very low levels of AD-ST activity and protein. The relative content of the isoelectric variants of AD-ST were different in liver cytosol of weanling and adult females, indicating that age- and gender-related alterations of hepatic AD-ST activity are primarily determined by the levels of AD-ST polypeptide and the relative amounts of the four isoelectric variants of the enzyme.

Liver sections prepared from male and female rats were immunohistochemically stained with the antisera against phenol sulfotransferase G (P-STG), an isoenzyme of phenol ST as well as androsterone-sulfating ST (AD-ST), an isoform of hydroxysteroid ST. Localization of these isoenzymes in liver is sex-dependent and is markedly different between the two. P-STG is preferentially localized in the hepatocytes proximal to the central vein in females, whereas it is present in all the hepatocytes throughout the liver in males. On the other hand, AD-ST is evident in the hepatocytes proximal to the portal triad in males, while in females it is synthesized and localized in all the hepatocytes. The polarized sex-related localization of these ST isoenzymes appears to correlate with differential hormonal regulation of the enzymes.

LA Wistar rats have a deficiency of androsterone UDP-glucuronosyltransferase (UDPGT) and are present in Wistar rat colonies around the world. In order to clarify the molecular mechanism of the deficiency, androsterone UDPGT cDNA clone, pGT2 was isolated from rat liver cDNA library and was digested with restriction enzymes to afford three probes for Northern and Southern blot analyses in HA (normal), heterozygous LA and LA Wistar rats. In Northern blot analysis, androsterone UDPGT mRNA was totally absent in LA Wistar rat liver. Southern blot analysis suggested a large deletion of androsterone UDPGT gene in the rats. Genomic DNA amplifications with synthetic primers which have nucleotide sequences corresponding to the 5′-region of androsterone UDPGT cDNA, suggested that androsterone UDPGT gene has exon 1 with a length of some 700 bp and that this exon is deleted in LA Wistar rats. Based on these lines of evidence, it is concluded that the large portion of androsterone UDPGT gene is deleted in LA Wistar rats, which results in the absence of androsterone UDPGT mRNA and consequently the corresponding enzyme protein.

Some properties of androsterone (AD)-sulfating sulfotransferase (ST) present in female rat livers were characterized. Based on the substrate specificities of the enzyme preparation obtained by anion exchange chromatography and 3'-phosphoadenosine 5'-phosphate (PAP)-agarose affinity chromatography, AD-ST was supposed to be among isoenzymes of hydroxysteroid STs. The identity of the AD-ST with the isoenzymes of hydroxysteroid ST, however, remains unclear at present. The enzyme preparation revealed a wide range of native molecular weight with a major Mr of some 600000. The AD-ST did not appear to have a homogeneous isoelectric point, because the enzymatic activity was spread over a wide range of the pH gradient, centering around pH 6.6 on chromatofocusing. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the AD-ST showed a subunit with Mr of 30000, which was similar to the hydroxysteroid STs purified previously. Under denaturing conditions the subunit was demonstrated to be composed of three protein species containing distinct pI values (pI 6.1, 6.7 and 7.2). The AD-ST was thus supposed to be an oligomer with high molecular weight, in which the subunits of different pI values are assembled in various association numbers.

Bilirubin and phenol UDP-glucuronosyltransferases (UGTs) are located on the same chromosome and comprise the UGT1 gene complex. A 1,763-bp cDNA probe (UGT1*0) specific for rat liver bilirubin UGT was used to localize the UGT1 complex locus (Ugt1a1) to chromosome region 9q35→q36 by fluorescence in situ hybridization. This assignment is the first report on the location of a gene of the rat UGT1 complex using high-resolution banded metaphase chromosomes.

Male and female Wistar rats with high and low hepatic microsomal UDP-glucuronosyltransferase activity towards androsterone were classified by partial hepatectomy. The breeding experiments between the classified high-activity and low-activity rats show that the genetic expression of the high transferase activity is inherited in an autosomal dominant fashion.

Twenty-one cases of esophageal foreign bodies in children were treated at the authors' clinic during the last 9 years (1970-1978). In the present paper, 4 cases out of the 21 who presented troublesome clinical courses were reported.Case 1: A 16-month-old girl misswallowed a piece of washer. The initial symptoms of vomiting and odynophagia disappeared gradually, while dysphagia continued. The foreign body was removed endoscopically 20 days later.Case 2: A 10-month-old boy suffered from a hypopharyngeal foreign body (pin). The main symptom was stridor and, for this reason, the definite diagnosis was delayed. It was found that the pin penetrated through the hypopharyngeal wall to the laryngeal cavity.Case 3: A 3-year and 8-month-old boy, who underwent a surgery for congenital atresia of the esophagus at age 1, misswallowed an iron ball. The foreign body stuck at the stricture of the site of anastmosis.Case 4: A 5-year and 11-month-old boy misswallowed his dental prosthesis. This was a rare foreign body in childhood.The incidence of such foreign bodies in children as observed in Cases 3 and 4 may increase in future. In order to prevent foreign bodies of these types, a good knowledge of the parents and child himself seems mandatory.

A rapid method for the separation and purification of uridine diphosphate-glucuronosyltransferases (GT) was developed with the use of chromatofocusing on a high-performance liquid chromatograph. GT isoenzymes solubilized from hepatic microsomes of Wistar rats were separated on a Mono P column, a pre-packed column for chromatofocusing. Using 4-nitrophenol, testosterone and androsterone as substrates, four fractions with different GT activities were separated in a pH gradient from 9.5 to 7.0. Two isoenzymes, testosterone GT and androsterone GT were purified to apparent homogeneity. They were eluted at pH 8.9 and 8.0 and had subunit molecular weight values of 50000 and 52000, respectively. Approximately 10 mg of solubilized microsomal proteins was applied and the elution was completed within 2 h. Addition of N-nitrodiethylamine, an in vitro activator of GT activity, enhanced the GT activity toward 4-nitrophenol in the three fractions. This chromatographic analysis confirmed the absence of androsterone GT isoenzyme in LA Wistar rats, a mutant strain in terms of androsterone glucuronidation.

[3H]androsterone was administered intraperitoneally into male Wistar rats with biliary fistulas. The major portion of the radioactivity was recovered in the bile within 24 hr. There was marked diversity in androsterone metabolism. Half of the rats excreted large amounts of steroid monoglucuronides rapidly into bile, whereas the remaining rats eliminated various steroid conjugates at a much slower rate. In both groups, androsterone was metabolized to 2- and 16-oxygenated C19O3 and C19O4 steroids and probably poly oxygenated steroids. In contrast, the injected [3H]androsterone glucuronide was recovered rapidly and quantitatively in the bile. These results indicate the extensive metabolism of androsterone prior to glucuronidation and probably the existence of variations of UDP-glucuronyltransferase toward the oxygenated metabolites of androsterone.

Sulfotransferases (STs)active on androsterone (AD), cortisol (CS) and 4-nitrophenol (NP) were separated by diethylaminoethyl-cellulose chromatography from cytosolic fractions of female rat liver and were divided into five ST fractions (peaks I-V) with different activities toward three substrates. The precipitates obtained in the 68% of saturation of ammonium sulfate were passed through a Sephadex G-100 column and purified by agarose-hexane adenosine 3',5'-bisphosphate affinity chromatography. AD-ST isoenzyme (peak I) was purified 85-fold, had low CS-ST activity, was devoid of NP-ST activity and appeared to correspond to hydroxysteroid ST 1. Peaks II and V appeared to consist mainly of hydroxysteroid ST and aryl ST, respectively.

Seven cases of retropharyngeal abscess in children were treated at the author's clinic during the last 9 years (1970-1978). In the present paper, the clinical findings in these cases and their treatment are discussed.Ages ranged from 4 months to 5 years with four cases under 1 year old. There were 3 males and 4 females. The common symptoms were fever, swelling of the neck and respiratory distress. The soft tissue X-ray film of the neck was helpful in the diagnosis. The common pathogens were β-streptococcus hemolytics A and staphylococcus aureus.As soon as the diagnosis is established, prompt surgical incision and drainage are indicated since the increasing danger of grave respiratory distress will be expected in due time among these children.

N-Nitrosodiethylamine (NEN) and N-nitrodiethylamine (NEA) are carcinogens and in vitro activators of hepatic UDP-glucuronosyltransferase (GT) toward 2-aminophenol (AP) and 4-nitrophenol (NP). In this communication, they were intraperitoneally administered to male Wistar rats for 7 days and GT activities were determined towards AP, NP, phenolphthalein (PH) and testosterone (TS). Administration of 30 or 20 mg/kg dose of NEN caused marked decrease of liver and body weights, and did not affect hepatic GT activities. Injection of 10 mg/kg dose of NEN did not diminish liver and body weights, and increased the maximally activated GT activities toward AP and NP. In contrast, 30 mg/kg dose of NEA, did not affect either liver and body weights or GT activities. N-Nitrosodimethylamine (NMN), which is a carcinogen and a weak in vitro AP GT activator, was more toxic than NEN, and 3.6 mg/kg dose of NMN appears to induce GT toward NP and AP. Administration of 46.5 mg/kg N-nitrosodibutylamine (NBN), which is a carcinogen but not a GT activator, did not affect GT activities or liver body weights.

Male and female Wistar rats showed discontinuous variation in hepatic microsomal UDP-glucuronyltransferase activities toward androsterone but not toward testosterone. In contrast, Sprague-Dawley rats. Hartley guinea pigs and CF#1 mice did not exhibit such diversity. Microsomes with a high level of transferase activity toward androsterone (HG) and microsomes with a low level of transferase activity (LG) were found approximately in the ratio 5:4 in male and female Wistar rats, the HG to LG specific activity ratio being about 9. In the presence of Triton X-100, the enzyme activity was stimulated maximally (5-fold) and the HG to LG specific activity ratio increased to 16. Kinetic studies revealed that the transferase had a much lower affinity toward androsterone in the LG microsomes than in the HG micro-somes. These results confirm the view that hepatic UDP-glucuronyltransferase is principally responsible for the large variations in the metabolism in vivo of androsterone and testosterone in female Wistar rats.

3α-Acetoxy-16α-bromo-5α-androstan-17-one (1) was converted into 16α-bromo-17,17-ethylenedioxy-5α-androstan-3α-ol (2), which was dehydrobrominated and hydrolysed to give 3α-hydroxy-5α-androst-15-en-17-one (5). Addition of benzyl alcohol to the Δ15-3α-ol (5) followed by hydrogenolysis yielded 3α, 15β-dihydroxy-5α-androstan-17-one (7). This was converted into 5α-androstane-3α, 15β, 17β-triol (8). Similarly 5α-androstane-3β, 15β, 17β-triol (10) was prepared from 3β-hydroxy-5α-androst-15-en-17-one (3). Hydroboration of 5α-androst-14-ene-3α, 17β-diol (12) afforded 5α-androstane-3α, 15α-17β-triol (13). The spectral properties of 5α-androstane-3,15,17β-triols are markedly affected by the configuration of the hydroxy-group at C-15.

Enzyme-catalyzed hydrogenation of Δ 4 -3-oxosteroids and their conjugates can be assayed without hydrolytic and chromatographic procedures. The assay medium is mixed with methanol, followed by centrifugation. The resultant supernatant is evaporated to dryness and subsequently condensed with the isonicotinic acid hydrazide reagent. The hydrazone of Δ 4 -3-oxosteroid thus formed is determined at 372 nm.

Hooded Gunn rats which have low-activity of 4-nitrophenol uridine diphosphate-glucuronosyltransferase (GT) and defect in bilirubin GT were crossed with albino LA Wistar rats with a defect in androsterone GT. From F2 and F3 progeny were selected Gunn-LA Wistar rats which have defective bilirubin and androsterone GTs and low-active 4-nitrophenol GT. These rats had either hooded or albino coat color. In order to establish a uniform genetic background, albino male Gunn-LA Wistar rats were crossed with heterozygous albino female Gunn-LA Wistar rats in terms of bilirubin GT. The offsprings were all albino and were classified to Gunn-LA Wistar rats and heterozygous Gunn-LA Wistar rats by assaying their hepatic GT activities or by the appearance of jaundice. The linkage relationships between GT and coat color genes are discussed.

In connection with the biochemical studies on D-glucaric acid, a normal constituent of human urine which is derived from D-glucuronolactone, we developed a colorimetric method for the quantitative determination of this acid. Thus, D-glucaric acid separated from mammalian urines using ion-exchange column chromatography was oxidized with periodic acid to give glyoxylic acid, which was further converted into the intensely colored 1, 5-diphenyl-formazan and determined colorimetrically. During the course of our studies on D-glucaric acid a much more highly sensitive method was required for the accurate estimation of normal serum level of D-glucaric acid in mammals. The present paper deals with the fluorometric determination of D-glucaric acid which is based on the condensation of glyoxylic acid derived from D-glucaric acid with 4'-hydrazino-2-stilbazole to yield a highly fluorescent product using a modification of the procedure for the determination of α-oxo acids originally reported by Mizutani, et al.(Fig. 1).

Metabolic hydrogenation of the ring A double bond in testosterone and its conjugates were studied by incubating 14C-testosterone, 14C-testosterone 17-N-acetylglucosaminide and 3H-testosterone 17-glucosiduronate with 20000×g or 105000×g supernatant fluids and microsomal fraction of male rat liver homogenate under carbon monoxide atmosphere or in air. It was demonstrated that in contrast to testosterone the conjugates were not good substrates for microsomal l4-5α-hydrogenase as well as for hydroxylases, while they were better substrates for soluble l4-5β-hydrogenase than testosterone. These findings are consistent with the observations on in vivo metabolism of testosterone and its conjugates in man.

Benzoyl migrations took place in the partical acid hydrolysis of 1, 2, 3, 5-tetra-O-benzoyl-4, 6-O-ethylidene-D-glucitol (III) to give 1, 2, 4, 6-tetra-O-benzoyl-D-glucitol (IV). This finding is principally based on the experimental result that chromium trioxide oxidation of IV in acetone-sulfuric acid afforded the following five compounds : 1, 3, 5, 6-tetra-O-benzoyl-L-sorbose (V), 1, 2, 4, 6-tetra-O-benzoyl-D-ribo-3-hexulose (IX), 2, 3, 5, 6-tetra-O-benzoyl-L-gulonolactone (VIII), di-O-benzoyl-L-glyceric acid (XI), and benzoylglycolic acid (XII). The formation of VIII from IV in this oxidation indicates that benzoyl migration occurred also during the oxidation.