Department of Toxicology, German Institute of Human Nutrition (DIfE), 14558 Bergholz-Rehbrücke, Germany
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: BSA, bovine serum albumin; h1B1, human sulfotransferase 1B1; HMBP, 6-hydroxymethylbenzo[a]pyrene; KCP, phosphate-buffered KCl solution; LC50, lethal concentration 50 (leading to a 50% decrease in cell number compared with control cultures); OH-CPC, 4H-cyclopenta[def]chrysen-4-ol; PAH, polycyclic aromatic hydrocarbon; PAPS, 3'-phosphoadenosine-5'-phosphosulfate; PBS, phosphate-buffered saline; PCR, polymerase-chain reaction; r1B1, rat sulfotransferase 1B1; SULT, member of the superfamily of soluble sulfotransferases.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Unlike other conjugation reactions, sulfonation involves a relatively high risk of the formation of reactive, potentially mutagenic and carcinogenic, metabolites (4,5). This activation pathway is not detected in standard in vitro mutagenicity test systems, since the target cells do not express SULTs and conventional external activating systems lack the cofactor PAPS (6). Even if PAPS is supplemented, reactive sulfo conjugates may not efficiently penetrate the target cell since they are charged at physiological pH (7). For this reason, we expressed directly various SULTs in Ames's Salmonella typhimurium strains (5,8). We have found nearly 100 chemicals that are activated to mutagens in bacterial strains expressing mammalian SULTs. In general, pharmaceuticals and industrial chemicals have to be tested for mutagenicity not only in bacterial systems but also in mammalian cells. Some V79 cell lines expressing various individual rat and human SULTs have already been constructed and used for metabolism and mutagenicity studies (916). Here we describe the construction and characterization of cell lines expressing rat SULT1B1 [subsequently abbreviated r1B1, or if the species is clear from the context, 1B1; analogous abbreviations are used for other rat and human (h) SULT forms].
r1B1 is one of at least nine SULT forms detected in the rat (2). It was originally identified as an enzyme with activity towards dopa and thyroid hormones. Some other small phenols also are good substrates for r1B1 (17). Interestingly, two relatively large, non-phenolic compounds, the benzylic alcohols 6-hydroxymethylbenzo[a]pyrene (HMBP) and 4H-cyclopenta[def]chrysen-4-ol (OH-CPC), were efficiently activated to mutagens by r1B1 [and human sulfotransferase 1B1 (h1B1)] expressed in S.typhimurium strains, whereas they were inactive or much less active in 20 strains expressing other SULTs (15).
IARC has classified coal-tar pitches, coal-tars, mineral oils, soots, tobacco smoke, aluminum production, coke production and coal gasification into group I (`recognized to be carcinogenic to humans') (18). Using animal models, polycyclic aromatic hydrocarbons (PAHs) have been identified as important carcinogens in these mixtures and exposure circumstances. However, as humans are exposed to complex mixtures of many PAHs, it is difficult to assess the carcinogenic risk of individual congeners to humans. A few PAHs that are carcinogenic to animals and have been investigated thoroughly in humans in vivo and/or in human cells in culture (e.g. with regard to metabolism and DNA adduct formation) have been classified into groups IIa and IIb (`probably' and `possibly' carcinogenic to humans, respectively). Many other PAHs are in group III (`not classifiable as to carcinogenicity in humans') or have not yet been subjected to the classification procedure, as the data available are insufficient. It is important to better understand the mechanism of action of these compounds, in particular the bioactivation pathways, in animal models and humans, before risk assessments can be made.
Methylated PAHs are formed geochemically from terpenoid compounds and are present in large quantities in mineral oils and coal (19). They have also been detected in exhausts of petrol and diesel motors (20) and cigarette smoke (21). Benzylic hydroxylation is a major metabolic pathway of some methylated PAHs (22). Bay-region methylene-bridged PAHs, including 4H-cyclopenta[def]chrysene, were detected after pyrolysis of methylated PAHs at temperatures in excess of 700°C (23). In addition to 4H-cyclopenta[def]chrysene, an oxygenated derivative, 4H-cyclopenta[def]chrysen-4-one, has been detected in the environment (24). This compound is reduced to OH-CPC by human intestinal bacteria (H.Schneider and H.R.Glatt, unpublished data).
Tumor-initiating activity has been demonstrated on mouse skin for 6-methylbenzo[a]pyrene (25,26), HMBP (26), 4H-cyclopenta[def]chrysene and 4H-cyclopenta[def]chrysen-4-one (27). HMBP induced liver tumors in the newborn mouse (28) and produced sarcomas in the rat after subcutaneous injection (29). OH-CPC and HMBP are extremely weak direct mutagens to conventional S.typhimurium strains (3032). Their chemically synthesized sulfo conjugates are moderately potent mutagens, whose activity is strongly affected in the presence of small nucleophilic molecules (such as halogen ions and amino acids), suggesting the formation of secondary, membrane-penetrating reactive species (3335). Likewise, the corresponding alcohols were activated to mutagens in the presence of rat hepatic cytosolic fraction and PAPS (30,36). HMBP was a good substrate for the female-specific rat hydroxysteroid sulfotransferase a (37), whereas OH-CPC was efficiently activated by r1A1 (32), a male-dominant form in liver of adult rats (38). In these experiments, a fixed, high concentration (50 µM) of the benzylic alcohols was used in combination with varying amounts of enzyme preparation. However, when varying concentrations of the substrates were used in a Salmonella strain expressing r1B1, mutagenicity was detected even at very low concentrations of HMBP and OH-CPC (<50 nM) (15).
Fujita et al. (39) and Araki et al. (40) reported that r1B1 protein expression in liver is independent of gender, which is in contrast to all other SULT forms of the rat analyzed for sex-dependent expression (41). r1B1 mRNA was detected in liver, kidney and intestine (41) and its protein in liver and kidney (40). The orthologous human enzyme is highly expressed in intestinal mucosa (42,43). Therefore, we have compared the r1B1 protein level in the recombinant cell lines with those observed in liver and colon mucosa of the rat.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals
Wistar rats were purchased from Tierzucht Schönwalde (Schönwalde, Germany).
Preparation of r1B1 cDNA and insertion into a eucaryotic expression vector
Total RNA was prepared from 50 mg of liver from a 9-week-old male and a 2-year-old female rat, which were employed primarily for another study, using the RNeasy Kit from Qiagen (Hilden, Germany). r1B1 cDNA was obtained by reverse transcription using the following reverse primer from InViTek (Berlin, Germany) priming at position 1034 according to the published sequence (17) (GenBank accession number U38419): 5' CTC CCT CAC AAA TCC ATC TGA TTT CCC 3' and Superscript II reverse transcriptase from Gibco BRL (Gaithersburg, MD). Subsequent polymerase chain reaction (PCR) was carried out using 5' CCT GCT ACA AAA ATG GGT ACT GCA G 3' (InViTek) priming at position 79 according to the published sequence and the same reverse primer as used for reverse transcription. After an initial denaturing step for 5 min at 94°C, PCR with Pfu polymerase (Stratagene GmbH, Heidelberg, Germany) was carried out for 30 cycles of 1 min at 94°C, followed by 1 min at 60°C and 1 min at 72°C and a final elongation step for 10 min at 72°C using the thermocycler Cyclogene (Techne, Duxford Cambridge, UK). The identity of the PCR product was confirmed by XbaI (MBI Fermentas, Vilnius, Lithuania) restriction fragment length analysis after separation on a 1% agarose gel. PCR products and plasmids were purified using kits from Qiagen. The cDNA was inserted by blunt-end ligation into pCR Script (Stratagene) and the pCR-r1B1 plasmid was used to transform competent Escherichia coli XL-1 Blue (Stratagene). After verification of the cDNA sequence using an A.L.F. DNA sequencer (Amersham Pharmacia Biotech, Freiburg, Germany), r1B1 cDNA was subcloned into pT7 Blue (Calbiochem-Novabiochem, Bad Soden, Germany) using the 5' HindIII and 3' SacI restriction sites in the pCR-Script polylinker. Using this procedure, 5'- and 3'-flanking EcoRI sites were obtained that could be used for inserting the cDNA into the EcoRI restriction site of the eukaryotic expression vector pMPSV (44). The correct orientation of the cDNA was checked by digestion with XbaI.
Cell culture and transfection
As Chinese hamster V79 is a very old, rapidly growing cell line (45), cells kept in different laboratories may differ in some properties. The subline used here, now termed V79-MZ, has been thoroughly studied for xenobiotic-metabolizing activities (46,47). It is deficient in cytochromes P450, UDP-glucuronosyltransferases and sulfotransferases, and has been widely used for the heterologous expression of these enzyme classes (48). The construction of the control cell line V79p, which only expresses the selection marker puromycin acetyltransferase, was described in a previous study (9). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin (all from Gibco BRL) and maintained at 37°C in a humidified atmosphere containing 5% CO2 in air.
Like the other SULTs that have been expressed in V79 cells (9,11,12,16), the r1B1 cDNA was inserted into the mammalian expression vector pMPSV to yield pMPSV-r1B1. In this construct, the LTR promoter of the myeloproliferative sarcoma virus controls the expression (44). The protocol for transfection has been described elsewhere (9). Briefly, pMPSV-r1B1 and pBSpacp, an expression vector conferring puromycin resistance, were co-transfected into V79-MZ cells using the calcium phosphate precipitation method. Crystals of calcium phosphate and 20 µg of plasmid-DNA consisting of 19 µg pMPSV-r1B1 and 1 µg pBSpac
p were distributed on a 24 h old culture of initially 7.5 x 105 V79-MZ cells. The following day, the cells were harvested by treatment with 0.05% trypsin and 0.5 mM EDTA in phosphate-buffered saline (150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.4; PBS) for 5 min and seeded onto 15 150 cm2 dishes. They were allowed to grow in standard medium for another day, then the medium was exchanged with the selection medium containing 5 mg/l puromycin. Dead cells were removed by renewing the selection medium after five additional days. Puromycin-resistant colonies were transferred 23 weeks after transfection onto separate plates and expanded. These cultures were given the passage number 1. For the subsequent passages, cultures were normally diluted by a factor of 2050.
Confirmation of the sequence of r1B1 DNA inserted into the genome of V79-r1B1 cells
Genomic DNA from V79p and V79-r1B1 cells (of passage 9) was used as template in a PCR designed for amplification of any DNA inserted into the pMPSV cloning site. The primers pMPSV-S (5' GTT AAC TGG TAA GTT TAG TC 3') and pMPSV-R (5' GTG GTT TGT CCA AAC TCA TC 3') primed at positions 675 and 805, respectively, of pMPSV. PCR was performed as described above using CombiPol polymerase (InViTek) with an annealing temperature of 55°C. The PCR products were purified and sent to InViTek for sequencing.
Cell population doubling time
Cells were seeded at an initial density of 1.5 x 106 cells/150 cm2 dish. At varying time points, 2249 h after the initiation, cells from two cultures were harvested and counted using a Casy 1 cell counter (Schärfe, Reutlingen, Germany). Cell population doubling times were calculated from three experiments performed with cells of different stem cultures.
Preparation of cytosolic fraction from cell lines and rat tissues
Cells were grown for 6872 h at an initial density of 1.5 x 106 cells/150 cm2 dish, harvested by treatment with trypsin/EDTA (5 min, 37°C), diluted with 10 ml of 10% fetal bovine serum in PBS, centrifuged, washed with KCP (150 mM KCl, 10 mM sodium phosphate buffer pH 7.4), resuspended in ice-cold KCP (100 µl/plate), sonicated and centrifuged at 100000 g for 1h (4°C). The resulting supernatant is termed cytosolic fraction.
Cytosolic fractions of tissues were prepared from male and female 11-week-old rats. Intestines were opened, placed on an ice-cooled glass plate, rinsed with ice-cold PBS and the mucosa was scraped off with a rubber scalpel. Mucosa and liver were homogenized in 35 ml KCP containing 1 mM EDTA and Complete Protease Inhibitor Cocktail (Boehringer Mannheim, Mannheim, Germany) per gram tissue using a Potter-Elvehjem tissue grinder. Cytosolic fraction was prepared by ultracentrifugation as described for V79 cells.
Protein concentrations were determined according to the method of Lowry using bovine serum albumin (BSA) as a standard.
Immunodetection of SULTs in cytosolic fractions
Cytosolic fractions of cells and tissues were separated on an 11% sodium dodecylsulfate-polyacrylamide gel. Cytosolic fractions of S.typhimurium expressing various rat SULTs (8) were included as standards. After electrophoresis, proteins were transferred to a Hybond ECL membrane (Amersham) and probed with polyclonal antibodies raised in a rabbit against h1B1 at a dilution of 1:10 000. Goat anti-rabbit IgGperoxidase conjugate (Sigma), at a dilution of 1:2000, was used as the secondary antibody. Visualization of the immunoreactive bands was achieved using the enhanced-chemoluminescence system together with Hyperfilm ECL (Amersham).
Immunostaining of V79 cultures
Cells were seeded onto glass slides in Quadriperm culture dishes (In vitro Systems & Services, Osterode, Germany) at an initial density of 5x 104/20 cm2 slide and cultivated for 72 h. Cells were then washed with PBS (37°C), fixed in methanol (2°C) for 7 min, and dried at room temperature. Endogenous peroxidases were inhibited by treatment with H2O2 (0.3%) for 10 min. The slides were then rinsed with PBS-Triton (PBS containing 0.1% Triton X-100), incubated with 1% BSA in PBS-Triton for 1 h to inhibit non-specific binding of antiserum, and treated with anti-h1B1 antiserum diluted 1:1500 in 1% BSA (for 12 h at 4°C). Bound h1B1-antibodies were detected using Vectastain ABC kit (Vector Laboratories, Burlingame, CA) in combination with the diaminobenzidine chromogen system (Immunotech, Marseille, France). These procedures were carried out on the Sequenza Immunostaining Center (Shandon, Pittsburgh, PE). Nuclei were stained with methylgreen (Sigma), 0.8% in 20% ethanol, for microscopical visualization of SULT-negative cells.
Determination of sulfotransferase activity
Sulfotransferase activity was determined using 1-naphthol as the substrate and [35S]PAPS (adjusted to 14 GBq/mmol) as the co-substrate. The reaction mixture (150 µl) contained [35S]PAPS (333 nM), KCP, cytosolic fraction (0.55µg protein, supplemented with V79p cytosolic fraction to 5µg) and 1-naphthol (1 µM). After incubation for 10 min at 37°C, PAPS was precipitated with barium (49), and the radioactivity in the supernatant was determined in a scintillation counter. Values from control incubations (no 1-naphthol) were subtracted. Preliminary experiments with varying amounts of protein were performed for each cytosolic fraction to determine the linear range. Incubations were performed in duplicate. Activities are expressed as pmol 1-naphthylsulfate formed per min and mg cytosolic protein.
Mutagenicity and cytotoxicity assay
Acquisition of resistance to 6-thioguanine was used as a marker of mutagenic activity. It involves inactivation of the X-chromosomal hypoxanthine phosporibosyl transferase (hprt) locus. A detailed protocol for the mutagenicity assay has been described elsewhere (50). Briefly, 1.5 x 106 cells were added to 30 ml medium in a 15 cm Petri dish. After 18 h, the test compound (dissolved in 60 µl dimethylsulfoxide), or the solvent alone, was added. The exposure was terminated after 24 h by a change of medium. Two days later, the cells were detached by treatment with trypsin. Their number, expressed as a percent of the corresponding value of the solvent control cultures, was used as a measure of the cytotoxicity of the treatment. The cells were subcultured in normal medium for 3 days, and then subcultured again using 6-thioguanine-supplemented (7 µg/ml) medium for the selection of mutants (106 cells/15 cm Petri dish, six dishes) and normal medium for determining the total number of colony-forming cells (100 cells/6 cm Petri dish, three dishes). After 12 days, the cultures were fixed and stained, the colonies were counted and mutant frequencies were calculated.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
r1B1 cDNA was inserted into the expression vector pMPSV. The resulting plasmid, pMPSV-r1B1, was co-transfected together with plasmid pBSpacp, encoding a puromycin-resistance marker, into V79-MZ cells. Ten puromycin-resistant cell colonies were expanded and analyzed for 1-naphthol sulfotransferase activity. Two clones were void of any activity (as were V79p control cells) and six clones had an enzyme activity between 0.1 and 6 pmol/mg/min. The remaining two clones exhibited activities of >10 pmol/mg/min, and were selected for further investigations. They were named V79-rSULT1B1-A and -B (abbreviated to V79-r1B1-A and -B in the present paper).
A pMPSV-r1B1 fragment including the entire r1B1 cDNA was amplified by PCR from genomic DNA of both V79-r1B1 cell lines at passage 9. Sequencing proved the correctness of the r1B1 cDNA integrated into the recombinant cell lines.
Stability and homogeneity of 1B1 expression
Cytosolic fraction was prepared and enzyme activity was measured every second or third passage over ~20 passages (100 cell population doublings) using several separate starting cultures of V79-r1B1-A and -B cells (data not shown). Activity was maintained over the entire observation period, although with some variation and possibly a trend to decreased activity in some subcultures with increasing passage number (especially in cell line V79-r1B1-A after about passage 16). Therefore, we decided to normally use cells of passages 616 for biochemical and toxicological studies. As each passage corresponds to an expansion of the cell population by a factor of 20100, this implies no practical limitations by the number of cells available. V79-r1B1-A and -B cells from passages 6 to 16 showed mean sulfotransferase activities (±SD) of 1020 ± 220 and of 57 ± 9 pmol/mg/min, respectively.
It was also studied whether cell density affects the expression of 1B1. For this purpose, cells were seeded at two different densities (1.5 and 3 x 104/cm2) and harvested after three different culture periods (24, 48 and 72 h). The activities were similar under all six conditions (2-fold variation for each cell line).
An immunoblot analysis using an anti-h1B1 antibody demonstrated the presence of a single immunoreactive band in cytosol from V79-r1B1-A and -B cells and its absence in V79p cytosol (Figure 1). The electrophoretic mobility of that band was identical to that of r1B1 expressed in S.typhimurium and an immunoreactive protein detected in rat liver and colon. Signals of similar intensity were obtained with 3 µg cytosolic protein of V79-r1B1-A cells and 100 µg of V79-r1B1-B cells. The enzyme activities in the actual cytosolic fractions used for the immunoblot differed by a factor of 24, indicating a good correlation between the amount of immunoreactive protein and the sulfotransferase enzyme activity.
|
|
|
Mutagenicity and cytotoxicity of OH-CPC and HMBP in V79-r1B1 and V79p cells
Mutagenicity was determined at the hprt locus, and cytotoxicity was measured within the mutagenicity experiment. The mutant frequencies in control cultures were similar for all cell lines. They amounted to 2 and 6 x 10-6 for V79p cells, 2 and 3 x 10-6 for V79-r1B1-A cells and 1 and 2 x 10-6 for V79-r1B1-B cells in the final experiments (Figures 3 and 4), and were also similar in the preceding concentration-finding experiments (data not shown). The concentrations of OH-CPC and HMBP that decreased the cell number to 50% compared with solvent control cultures (LC50) were ~0.15 µM in V79-r1B1-A cells for both compounds, but 150- and 30-fold higher in SULT-deficient control cells (V79p), respectively (Figures 3A and 4A
). The cytotoxicity of these compounds was only slightly higher in the cell line with the lower expression level for r1B1 (V79-r1B1-B) than in the SULT-deficient cell line V79p (Figures 3A and 4A
, and other data).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cloned r1B1 sequence was expressed in cells that are deficient in endogenous SULTs. The transfection procedure used can lead to the integration of one or several plasmid copies in varying chromosomal sites and, therefore, various expression levels and stabilities in the recombinant clones. Two clones were selected for further analysis. The r1B1 protein level in clone V79-r1B1-B was similar to that found in liver, whereas expression in clone V79-r1B1-A was nearly 20-fold higher. However, it is not known whether r1B1 is evenly expressed in liver tissue. For other SULT forms and activities, large differences in expression have been observed between parenchymal, endothelial and Kupffer cells (51) and between different zones of the liver lobule (5254). Moreover, 1B1 is inducible by dexamethasone (55). Therefore, the r1B1 level may be higher in some cells and/or physiological states than the mean constitutive level in liver.
The correctness of the r1B1 cDNA integrated into V79-r1B1-A and -B cells was verified by re-amplifying its coding part from the cell lines and sequencing. 1B1 enzyme activity was expressed in these cell lines for at least 20 culture passages (100 population doubling times). The activity was substantially higher in V79-r1B1-A cells than V79-r1B1-B cells throughout this period, although marked variations were observed among individual cultures of the cell lines. The expression did not depend on the cell density or the age of the culture within the range investigated. Expression in V79-r1B1-A, but not V79-r1B1-B, cells was sufficiently high to allow immunostaining of cultures. Staining was rather homogeneous within colonies but more variable between colonies. It is not probable that this variation was due to a staining artifact, since some neighboring colonies were stained differentially. Initially we considered the possibility that the original V79-r1B1-A colony was not homogeneous (e.g. because the 1B1 sequence was only integrated after one or several cell divisions). Therefore, we subcloned the V79-r1B1-A line. However, all 10 sublines showed the same expression level (as determined by immunoblotting and activity measurements) and the same colony-dependent heterogeneity of immunostaining as the original V79-r1B1-A line. We have not elucidated the reason for the heterogeneity between colonies. Perhaps, cells within colonies are synchronized and expression is cell cycle-dependent.
V79-r1B1 cell lines are a practically unlimited source for r1B1. Metabolism studies can be conducted in subcellular preparations or intact recombinant cells (10). The cells also can be used for toxicological studies, as illustrated here with HMBP and OH-CPC. These compounds may be used as r1B1-dependent positive controls in further toxicological studies.
The new cell lines take into account an activation mechanism that is ignored in conventional short-term tests for mutagenicity. Moreover, the expressed enzyme stands in a physiological environment, compared with studies using subcellular activating systems or recombinant bacteria. The fact that the cell lines constructed only express individual SULTs has some advantages and disadvantages. It requires that compounds with structural alerts for a SULT-mediated toxification may have to be studied as substrates of a battery of SULTs. As different SULTs differ in their regulation, e.g. their tissue distribution (1), identification of the critical forms allows substantiated prediction on potential target tissues and susceptible species, genotypes and physiological states. As only limited factors are used for this prediction, it has to be examined in more complex systems, using the results from the recombinant cell lines for guidance. Factors that might modulate the toxicological activities include enzymes that compete with SULTs for the substrate [e.g. UDP-glucuronosyltransferases and alcohol dehydrogenases with some benzylic alcohols (56)] or inactivate reactive sulfo conjugates [e.g. rat glutathione transferases from the theta class (57)]. Of course, such potential detoxifying systems are not uniformly present in the organism, but drastically vary between different cell types and physiological states. At present there is no way to mimic this complexity in vitro, although cell lines can be constructed that express a small number of defined heterologous enzymes to address specific questions. On the basis of such metabolic information, we had predicted that ethanol would enhance SULT-mediated toxification of 1-hydroxymethylpyrene via inhibition of the side-chain oxidation. Indeed, the formation of hepatic DNA adducts by this benzylic alcohol was increased 14-fold in animals receiving ethanol together with the test compound (56).
HMBP has been shown to be a substrate for the female-specific rat hydroxysteroid sulfotransferase a (37), but the form responsible for its activation in males was unknown. r1B1 is equally expressed in rat liver of both genders (40). As no other rat SULT has been identified that shows high activity towards HMBP, it is probable that 1B1 plays a major role in the activation of HMBP by male hepatic cytosol and contributes, in addition to hydroxysteroid sulfotransferase a, to its activation in female liver. r1A1, a male-dominant form, showed the highest activity towards OH-CPC among the rat SULT forms studied previously. The present finding that r1B1 is very active towards this substrate suggests a prominent role of 1B1 in the activation of OH-CPC by female hepatic cytosol, and a contribution, in addition to that of r1A1, to the activation in male liver. We suspect that the contribution of r1B1 to the activation of OH-CPC and HMBP is particularly high at low substrate concentrations.
Non-smokers are exposed to PAHs mainly via nutrition (5860). Enzymes of the gastro-intestinal tract and the liver may be particularly important for the toxification and detoxification of orally ingested xenobiotics. Benzylic alcohols may be formed from methylated PAHs and conjugated to glucuronic acids in liver, excreted via bile or intestinal mucosa into the gut lumen and deconjugated by bacterial glucuronidases in the large intestine to generate the free benzylic alcohols. Benzylic alcohols may also be formed by intestinal bacteria from oxygenated environmental PAHs, such as 4H-cyclopenta[def]chrysen-4-one. These benzylic alcohols may be absorbed and metabolized in the colon mucosa, and share this fate with other hydroxyl compounds released by bacteria from glycosylated phytochemicals and glucuronidated metabolites. Conjugation in colon mucosa would appear to be an appropriate pathway for the elimination of these compounds, except in the cases where the resulting conjugates are reactive and short-lived.
Araki et al. (40) did not detect any 1B1 mRNA or protein in rat intestine. However, Dunn and Klaassen (41) observed the mRNA in this tissue; its level was 25 and 15% of the hepatic level in males and females, respectively. In the present study we demonstrated the presence of 1B1 protein in rat colon mucosa. Its level was approximately one-fifth of the hepatic level. Interestingly, human colon mucosa expresses high levels of several SULTs (42,43). The level of h1B1 is several times higher in colon mucosa than in liver. A V79-derived cell line expressing h1B1 has been constructed and its characterization is currently under investigation.
![]() |
Notes |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|