Molecular cloning and functions of rat liver hydroxysteroid sulfotransferases catalysing covalent binding of carcinogenic polycyclic arylmethanols to DNA

Chemico-Biological Interactions ELSEVIER

Chemico-Biological Interactions 92 (1994) 87-105

Molecular cloning and functions of rat liver hydroxysteroid sulfotransferases catalysing covalent binding of carcinogenic polycyclic arylmethanols to DNA Tadashi Watabe*, Kenichiro Ogura, Masahiro Satsukawa, Haruhiro Okuda, Akira Hiratsuka Laboratory of Drug Metabolism and Toxicology, Department of Hygienic Chemistry, Tokyo College of Pharmacy, 1432-1 Horinouchi. Hachioji-shi, Tokyo 192-03, Japan Received 16 August 1993; revision received 23 February 1994; accepted 24 February 1994

Abstract

Three sulfotransferases (STs) catalysing the metabolic activation of potent carcinogenic polycyclic arylmethanols were purified from female Sprague-Dawley (SD) rat liver cytosol without loss of their enzyme activities in the presence of Tween 20 used for preventing the enzymes from aggregation during purification and identified as hydroxysteroid sulfotransferases (HSTs). All the purified HSTs, STa, STb, and STc, with different electric charges had an apparently equal size of subunit (30.5 kDa) and cross-reacted with polyclonal antibody raised against STa. Our study on molecular cloning of cDNA libraries from two female SD rat livers indicated that both contained cDNA inserts coding for 5 different HST subunits, consisting of 284-285 amino acid residues (Mr, 33 084-33 535) and sharing strong amino acid sequence identity ( > 83%). Of the 5 HST subunits, two had an identical amino acid sequence except for only one amino acid residue, and the other two contained only 6 amino acid substitutions in their sequences.

Keywords: Carcinogen; Polycyclic arylmethanoi; Metabolic activation; Rat liver cytosol; Hydroxysteroid sulfotransferase; Molecular cloning

* Corresponding author. 0009-2797/94/$07.00 © 1994 Elsevier Science ireland Ltd. All rights reserved SSDI 0009-2797(94)03298-M

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1. Introduction

Despite their weak or little carcinogenicity, a number of polycyclic aromatic hydrocarbons, such as benz[a]anthracene (BA), and chrysene (CR), are known to become potent or extremely potent carcinogens by the introduction of a methyl group or groups to their aromatic nuclei [1]. 7- or 12-monomethyl- and 7,12-dimethyl-BAs and 5-methyl-CR (5-MCR) are typical instances of which 7,12-dimethyl-BA (DMBA) has been demonstrated to be the most potent carcinogen to rodents among known polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BP) [1]. Major oxidative metabolites of these carcinogenic methylarenes in untreated rat liver are the corresponding arylmethanols with potent carcinogenicity. 7-Hydroxymethyl-BA (7-HBA), 7-hydroxymethyl-12-methyl-BA (7-HMBA), 12hydroxymethyl-7-methyl-BA (12-HMBA), 7,12-dihydroxymethyl-BA (DHBA), 5hydroxymethyl-CR (5-HCR), and 6-hydroxymethyl-BP (6-HBP) have been demonstrated to have carcinogenicity comparable to the corresponding methylarenes [2-81. 2. Metabolic activation of carcinogenic arylmethanois by rat liver ST

In the presence of dialysed cytosol from rat liver and 3'-phosphoadenosine 5'phosphosulfate (PAPS), the arylmethanols, 7-HBA [9], 7-HMBA [10,11], 12-HMBA [11], DHBA [12,13], 5-HCR [14], 1-hydroxymethylpyrene (1-HP) [10,15,16], and 6-HBP [17] showed potent mutagenicity toward Salmonella typhimurium TA98. Reactive sulfate esters were isolated from incubation mixtures, containing the arylmethanols except 1-HP and 6-HBP, rat liver cytosol, and PAPS in phosphate buffer, by ion-pair extraction using tetra-n-butylammonium bromide and identified by coelution with the corresponding synthetic specimens on an octadecylsilica column by high-performance liquid chromatography (HPLC) [9-14]. The sulfate esters of the hydroxymethyl-BAs decomposed to the corresponding benzylalcohols with very short half-lives in water [13], but were stable enough to remain unchanged as sodium salts and as ion-pair complexes in aprotic solvents such as dimethyl sulfoxide and ethyl acetate. The latter solvent was used for extraction of the metabolically formed sulfate esters of the arylmethanols as their ion-pair complexes with tetra-n-butyl cation. These sulfate esters reacted with various nucleophiles in a concerted manner with concomitant loss of the sulfate anion. 7-HMBA sulfate, an intrinsic mutagen metabolically formed from 7-HMBA, reacted in phosphate buffer, pH 7.4, at a significant rate not only with the exocyclic amino groups of adenine and guanine residues of calf thymus DNA, but also with the nucleophilic functional groups of amino acid residues, Cys, Met, and Lys, of rat liver cytosolic protein [11,18,19]. 7-HMBA also showed the formation of the same nucleic acid base adducts in rat liver cytosol containing PAPS and calf thymus DNA in phosphate buffer [11,18]. Similar evidence was provided by Surh et al. [20] with hepatic chromosomal DNA in infant rats given 7-HMBA. The carcinogenic bifunctional alcohol, DHBA, was regioselectively conjugated by rat liver cytosolic ST at the 7-hydroxymethyl group to form the reactive sulfate ester,

T. Watabe et al. ~Chem.-Biol. Interact. 92 (1994) 87-105

89

DHBA 7-sulfate, which also reacted with the exocyclic amino groups of adenine and guanine residues of calf thymus DNA [12,13]. That the regioselectivity in enzymatic sulfation of DHBA is attributable to a steric hindrance effect of the 1,2-benzo ring carbons on the 12-hydroxymethyl group was supported by the higher regioselectivity in formation of the 7-monosulfate with chlorosulfonic acid in alkylpyridines than in pyridine [12]. Our unpublished data obtained with preweanling rats given DHBA and its 7-sulfate indicated that the same type of covalent binding of the carcinogen to hepatic chromosomal DNA took place in the same adenine to guanine adducts ratio as shown with calf thymus DNA in vitro. The hepatic cytosolic formation of the 7-sulfate from DHBA was strongly inhibited by the HST inhibitor, dehydroepiandrosterone (DHA) sulfate, but little affected by phenol ST (PST) inhibitors, pentachlorophenol and 2,6-dichloro-4-nitrophenol [131. Rat liver cytosolic HSTs also converted the potent carcinogen, 5-HCR, to the reactive and mutagenic conjugate, 5-HCR sulfate, with a marked sex difference, females >> males [14,21]. 5-HCR sulfate had a much longer half-life (11 h) than the sulfate esters of hydroxymethyl-BAs (< 1-8 min) at 37°C in water. In spite of its low reactivity with nucleophiles, 5-HCR sulfate reacted at a significant rate with the exocyclic amino groups of adenine and guanine residues of calf thymus DNA [22] (Fig. 1). The facile covalent binding of 5-HCR sulfate to the nucleic acid bases might be

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Fig. 1. Metabolic activation of the carcinogen 5-MCR by cytochrome P-450/HST, inactivation by GST Yrs-Yrs and covalent binding to DNA of the reactive metabolite 5-HCR sulfate. Details are described by Okuda and coworkers [14,22,23] and as to GST Yrs-Yrs, by Hiratsuka et al. [241.

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T, Watabe et al. ~Chem.-Biol. Interact. 92 (1994) 87-105

attributable not only to the intercalation of its aryl moiety between base pairs of DNA, but also to the ion-pair complex formation between its sulfate moiety and basic nitrogens of nucleic acid bases. Unlike the sulfate esters of 7-HMBA [18] and DHBA [13], both of which bound to the guanine residue in preference to the adenine residue of calf thymus DNA, the adenine to guanine adducts ratio was 27:1 in covalent binding of 5-HCR sulfate to the nucleic acid [22]. The higher adenine adduct formation strongly blocked the DNA synthesis from 5-HCR sulfate-pretreated single-stranded phage DNA by DNA polymerase [25]. Later, it was suggested that highly reactive sulfate esters formed by rat liver cytosolic HST from polycyclic arylmethanols such as 1-HP [15,16], 7-HMBA [26], and 9-hydroxymethyl-10-methylanthracene [16], were partly transformed in the presence of chloride anion to the corresponding chloromethylarenes as more reactive species of putative intrinsic mutagens. As to synthetic l-HP sulfate, Glatt et al. [15] have demonstrated that it reacts with chloride anion in an aqueous medium containing potassium chloride to form 1-chloromethylpyrene which was shown to be more reactive with salmon sperm DNA and more mutagenic to bacteria than the sulfate ester. However, Surh et al. have shown only a little difference in covalent binding to calf thymus DNA of the two species of reactive intermediates from 1-HP [16]. The bacterial mutagenicity of synthetic 7-HMBA sulfate is also enhanced in the presence of chloride anion [26], and covalent binding to calf thymus DNA of the sulfate ester metabolically formed from 7-HMBA by a purified rat liver HST is accelerated by chloride anion [27]. The polycyclic chloromethylarenes are more unstable than the corresponding sulfate esters at pH 7.4 in aqueous media, so that 6-HBP sulfate could exert more bacterial mutagenicity in the absence than in the presence of chloride anion [17]. Surh et al. have demonstrated that 1-HP sulfate administered to rats binds covalently to liver DNA in spite of little binding of 1-chloromethylpyrene to the DNA in vivo [16]. All the sulfate esters metabolically formed from 7-HMBA, DHBA, and 5-HCR in rat liver cytosol were rapidly and completely scavenged in the presence of glutathione (GSH), so that the carcinogens could not show any appreciable mutagenicity to S. typhimurium TA98 or bind covalently to calf thymus DNA added to the cytosolic incubation mixtures, containing GSH and PAPS [1 l, 13,23,28,29]. Synthetic sulfate esters were also rapidly scavenged by GSH in the cytosol. Stable GSH conjugates Ar-CH2-SG formed were isolated and identified with the corresponding synthetic specimens. The GSH conjugation reactions, in most part, proceeded enzymatically and were demonstrated to be mediated by the new class (theta) GSH Stransferase (GST) Yrs-Yrs, a homodimeric Y protein scavenging reactive sulfate esters [24]. GST Yrs-Yrs is unique in view of the fact that it is unretainable on the S-hexyl-GSH- and GSH-affinity columns, used for isolation of alpha, mu, and pi classes of GSTs from liver cytosol, and has little activity toward typical GST substrates, 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene. The class theta GST, existing at a concentration of approximately 0.1% of the total cytosolic protein in Sprague-Dawley (SD) rat liver, is most likely to play an important role in preventing carcinogenesis induced by the polycyclic arylmethanols in rat liver as all the other classes of rat GSTs have little activity toward the reactive sulfate esters

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T. Watabe et al. ~Chem.-Biol. Interact. 92 (1994) 87-105

[24]. In the SD rat, the G S T Y r s - Y r s activity is highest in the liver relative to the other tissues, including the skin which is one o f the target tissues for these carcinogens and contains little G S T Y r s - Y r s compared with HSTs [24]. Purification and molecular cloning o f G S T Y r s - Y r s have been achieved by Hiratsuka et al. [24] and Ogura et al. [30], respectively.

3. Rat liver HSTs catalysing the formation of reactive sulfate esters from carcinogenic arylmethanols HSTs play an important role not only in the transformation of a variety o f endogenous steroidal and xenobiotic alcohols to the corresponding hydrophilic sulfate esters in hepatic and extrahepatic tissues for their excretion [31-33], but also in the production o f neurosteroids, such as the sulfate esters o f D H A and pregnenolone, in brain [34,35]. Participation o f HSTs in the metabolic activation o f the carcinogenic arylmethanols in rat liver cytosol was strongly suggested by using the inhibitors for HSTs and PSTs [13] and by the marked sex difference (females >> males) in enzyme activity [21] as mentioned above. Previous workers have demonstrated female SD rat liver cytosol to contain three HSTs which are separable on an anion-exchange column and have different sizes o f

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4 Fraction number Fig. 2. Separation of the HSTs, STa, STb, and STc, activating the carcinogen 5-HCR in female SD rat liver cytosol by DEAE-Sephadex A-50 column chromatography. A pooled rat liver cytosolic fraction (26 ml, 1074mg protein) from livers of three 9-week-old rats was directly applied to the anion-exchange column (3.5 x 60 cm) which was pre-equilibrated with 5 mM Tris-HCl buffer, pH 7.5, containing sucrose (250 mM) and 2-mercaptoethanol (3 mM). Elution of HSTs was carried out with the buffer (90 ml) and then with a 0-0.3 M KCI linear gradient in the buffer (1800 ml) at a flow rate of 90 ml/h. The eluate was collected in 12-ml fractions. Enzyme activities were monitored with 5-HCR (D) and DHA (0) for HSTs and 4-NP (A) for PST. Protein (broken line) was monitored at 280 nm by absorptiometry. Details are described by Ogura et al. [36].

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subunits without i m m u n o - h o m o l o g y , e.g. ST 1 (28 kDa, homooligomer), ST 2 (32 kDa, homooligomer), and ST 3 (60 kDa, homodimer) by J a k o b y et al. [31] and ST I (28 kDa, homooligomer), ST II (not well characterized), and ST III (30 kDa, homodimer) by Singer [32]. However, except for the amino acid compositions o f some HSTs, neither of the two groups o f pioneers provided further evidence for molecular data, such as the N-terminal amino acid sequences of the enzyme subunits and/or peptides obtained thereof, nor did they show any co-electrophoretic patterns of their purified enzyme forms on the same gel slabs. During the course o f their investigation, Ogura et al. found that there existed at least three HST enzyme forms, designated STa, STb, and STc, bioactivating carcinogenic polycyclic arylmethanols in female SD rat liver cytosol, which were separable on a D E A E - S e p h a d e x column [36]. The anion-exchange column chromatograms, monitored by the carcinogen 5 - H C R and the hydroxysteroid D H A as substrates, were almost superimposable on each other (Fig. 2). The more abundant enzyme form STa was purified to homogeneity by three steps of column c h r o m a t o g r a p h y in which 3 ' - p h o s p h o a d e n o s i n e 5 ' - p h o s p h a t e (PAP)-affinity column c h r o m a t o g r a p h y played a central part (Table 1). The enzyme purification study indicated that the sulfating activities of STa toward 5 - H C R and D H A were inseparable throughout the purification procedures used. Actually, a kinetic experiment showed D H A to inhibit the sulfation o f 5 - H C R by purified STa in a competitive manner [36]. Purified STa had no activity toward 4-nitrophenol (4-NP) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF). A study on the expression of the STa subunits in E s c h e r i c h i a c o l i provided crucial evidence that STa did catalyse both sulfations of 5 - H C R and D H A (see Ogura et al. [68]). As previously pointed out by a number of workers, rat liver HSTs readily aggregate to become insoluble during purification and concentration o f the purified or

Table 1 Isolation and purification of STa from female SD rat liver cytosola Step

Specific activitiesb (nmol/mg protein/min) 5-HCR

Cytosol 0.15 DEAE-Sephadex 1.11 A-50 PAP-agarose 16.2 Sephadex G-100 14.7

DHA 0.58 3.2 64.3 60.3

4-NP

N-OH-AAF

0.4 0.1

---

ND ND

-ND

Purification folds

Yields(%)

5-HCR

5-HCR

DHA

100 38.9

100 28.1

16.6 14.3

16.8 15.0

1 7.6 110 100

DHA 1 5.6 112 105

aThe table data [36] were obtained before Tween 20 was found to be very effective for preventing the enzyme from aggregating leading to a marked loss of the enzyme activity. In the presence of 0.5% (w/v) detergent, the recovery of the enzyme activity from the pooled DEAE-Sephadex column chromatographic fraction for STa (Fig. 2) was quantitative by successivechromatography on the PAP-agarose affinity and Sephadex G-100 gel filtration columns. However, addition of the detergent directly to the cytosolic fraction decreases the separation of HST enzyme forms, STa, STb, and STc, on the anion-exchange column. bND, not detectable (< 0.08 nmol/mg protein/rain). Details are described in Ogura et al. [36].

T. Watabe et al./ Chem.-Biol. Interact. 92 (1994) 87-105

93

partially purified enzymes, resulting in a marked loss of their enzyme activities [31,36-39]. The aggregation takes place first on the PAP-agarose affinity column which concentrates the enzyme protein selectively with concomitant removal of most cytosolic proteins playing an important role as stabilizers for HSTs in rat liver cytosol and also on the DEAE-gel column. No effective device has been made to prevent the aggregation of the partially purified and purified enzymes. All of STa, STb, and STc also readily aggregated when concentrated on the affinity column or on the membrane filter. The enzyme activities toward 5-HCR and DHA of a very diluted solution of STa (2/~g/ml) used for enzyme assay were found to decrease during incubations. A 60-min preincubation of the diluted STa solution in the presence or in the absence of PAPS prior to the addition of the substrate decreased its activity to lower than 35%, strongly suggesting that an invisible aggregation of STa took place even at the very low concentration (Fig. 3). Tween 20 was the best stabilizing agent for STa among eight neutral detergents examined. In the presence of 0.05% (w/v) Tween 20, no appreciable decrease in enzyme activity of STa took place even after the 60-min preincubation carried out under the above conditions (Fig. 3). The lost enzyme activity of STa during the preincubation in the absence of Tween 20 could not be restored by the addition of the detergent. The detergent used at a concentration higher than 0.5% (w/v) strongly inhibited the sulfation of 5-HCR by STa without any influence on the enzymatic sulfation of DHA. However, by diluting the detergent in the STa solution to 0.1-0.05% (w/v) with detergent-free buffer, the

A

100 t

6o

4o

20

0

20 4 Preincubation time (rain)

60

Fig. 3. Stabilizing effect of Tween 20 on the enzyme activities of purified STa toward 5-HCR and DHA. STa (2/zg/ml) purified in the absence of Tween 20 as described in Table 1 was preincubated in the presence (triangles) and in the absence (circles) of 0.05% (w/v) Tween 20 at 37°C for 0-60 min in the mixture containing PAPS (120 ~,M) in a final volume of 250 ~1 of 0.1 M phosphate buffer, pH 7.4 for 5-HCR (closed symbols) and pH 6.8 for DHA (open symbols). The reaction was started by the addition of the substrate (0.1 mM) dissolved in dimethyl sulfoxide (5% (w/v)) and terminated by immersing the reaction vessels into boiling water for 3 min. Enzymatic sulfation of 5-HCR and DHA were determined as previously reported [36]. Data are expressed as mean values of at least three experiments.

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selectively inhibited enzyme activity toward 5 - H C R was completely restored. That may be due to the much higher K m value o f 23.2 # M for 5 - H C R than for D H A (Kin, 2.0/zM) toward" STa. Use o f Tween 20 (0.5%, w/v) in the eluants applied to the PAP-agarose and gel filtration columns allowed the quantitative recovery o f the STa and STb activities from the anion-exchange column (Fig. 2). The PAP-agarose column used for the quantitative recovery o f the H S T activities o f STa and STb retained 80% o f the H S T activity o f the pooled fraction for STc in the presence o f 0.5% (w/v) Tween 20. The column-retained H S T activity o f STc was also quantitatively recovered from the affinity column by the elution with a buffer solution containing 0.5% Tween 20 (w/v) and A D P in gradient manner as used for the elution o f STa and STb. All the pooled fractions, A, B, and C for STa, STb, and STc, from the PAPagarose affinity column contained 30.5-kDa proteins appearing on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which were all intensely immuno-stained with polyclonal antibody raised against purified STa (Fig. 4). STa and STb, both appearing at 30.5 k D a on S D S - P A G E , were purified to homogeneity

M

STa

A

B

C

STa

A

B

C

kDa 66

--~

45 36

--~

29 24

"-~ -="

kDa .......

20.1 ~

.....

SDS-PAGE

Western-blot

Fig. 4. SDS-PAGE and Western blot analyses of partially purified STa, STb, and STc. The pooled fractions for STa, STb, and STc in the DEAE-Sephadex column chromatogram (Fig. 2) were applied to a PAP-agarose column (1.5 x l0 cm) pre-equilibrated with 50 mM Tris-HC1, pH 8.0, containing sucrose (250 mM), 2-mercaptoethanol (3 mM), and 0.5% (w/v) Tween 20 (buffer I). After the column was washed with the same buffer (50 ml), it was eluted with a 0-18 mM ADP linear gradient in buffer I (200 ml) at a flow rate of 40 ml/h in 8-ml fractions. Pooled fractions containing partially purified STa (A), STb (B), and STc (C), eluting as single activity peaks from the PAP-agarose column, were used for SDS-PAGE and Western blot analyses. Protein samples resolved by SDS-PAGE [40] were stained with Coomassie Brilliant Blue R-250. For Western blot analysis, proteins resolved by SDS-PAGE were transferred to a nitrocellulose membrane according to the method of Towbin et al. [41], and the membrane was immunostained with the anti-STa-antibody as previously described [36]. Lane STa, 0.5 #g of purified STa; lanes A, B, and C, pooled fractions (2/zg protein each) from the PAP-agarose column after separation by DEAE-Scphadex A-50 column chromatography; and lane M, Mr markers used: bovine serum albumin (Mr, 66 000), ovalbumin (Mr, 45 000), rabbit muscle glyceraldehyde 3-phosphate dehydrogenasc (Mr, 36 000), bovine erythrocyte carbonic anhydrase (Mr, 29 000), bovine pancreas trypsinogen (Mr, 24 000), and soybean trypsin inhibitor (Mr, 20 100).

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T. Watabe et al./ Chem.-Biol. Interact. 92 (1994) 87-105

Cytosols i

M

STa

STb

STc

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i Male

STa

STb

STc

kDa 66

~

45

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36

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....

29 24

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Fig. 5. SDS-PAGE and Western blot analyses of purified STa, STb, STc and female and male rat liver cytosols. Further purification of STa and STb from the pooled fractions A and B shown in Fig. 4 was performed as follows: the fractions containing STa and STb from the PAP-agarose affinity column were separately applied to a Sephadex G-100 column (2 x 100 cm) that was eluted with buffer I (200 ml, see Fig. 4) at a flow rate of 30 ml/h in 5-ml fractions. The gel filtration chromatographic fractions eluting as single enzyme activity peaks, containing homogeneous enzyme proteins, STa and STb, active toward DHA and 5-HCR, were subjected to SDS-PAGE and Western blot analyses. Purification of STc from the pooled fraction C shown in Fig. 4 was carried out as follows: the pooled fraction containing STc from the affinity column was dialysed against 10 mM K2HPOa-KH2PO4 buffer, pH 6.8, containing sucrose (250 mM), 2-mercaptoethanol (3 mM) and 0.1% (w/v) Tween 20 (buffer II). After dialysis, the fraction was applied to a hydroxyapatite column (1.8 x 10 cm) pre-equilibrated with buffer II. The column was eluted by a step gradient with 30 ml each of 0.1, 0.3, 0.5, and 0.7 M K2HPO4-KH2PO4 buffer, pH 6.8, containing sucrose (250 mM), 2-mercaptoethanol (3 mM), and 0.1% (w/v) Tween 20, at a flow rate of 15 ml/h in 2-ml fractions. The fractions for homogeneous STc, eluting in the 0.5 M phosphate buffer, were combined and used for subsequent analyses. Purified enzyme proteins, STa, STb, and STc, resolved by SDS-PAGE were stained with Coomassie Brilliant Blue R-250. For Western blot analysis,proteins resolved by SDS-PAGE were immuno-stained with the anti-STa-antibody. Lanes STa, STb, and STc, 0.5/~g of purified STa, STb, and STc; lanes cytosols female and male, cytosols (10/~g protein each) from female and male SD rat (9 weeks of age) livers; and lane M, the same Mr markers as used in Fig. 4.

from the pooled fractions A a n d B by subsequent gel filtration c h r o m a t o g r a p h y o n Sephadex G-100 (Fig. 5) a n d proved to be the only c o m p o n e n t s for the H S T activities in the pooled fractions. A n extensive survey by the electrophoretic apparatus, PhastSystem TM,of the affinity a n d gel filtration c o l u m n c h r o m a t o g r a p h i c fractions before pooling d e m o n s t r a t e d that the other proteins appearing at different molecular masses o n S D S - P A G E o f the pooled fractions A a n d B had n o H S T activity toward D H A a n d 5-HCR. The pooled H S T fraction C had a potent sulfating activity toward 4-NP. The PST activity in the fraction C was separated from the H S T activity by hydroxyapatite c o l u m n c h r o m a t o g r a p h y a n d f o u n d by S D S - P A G E / e n z y m e assay to be due to the protein appearing at 32 k D a o n S D S - P A G E , which was weakly i m m u n o - s t a i n e d by the a n t i - S T a - a n t i b o d y c o m p a r e d with the 30.5-kDa protein (Fig.

96

T. Watabe et al. ~Chem.-Biol. Interact. 92 (1994) 87-105

4). Purified STa, STb, and STc (Fig. 5) had no activity toward 4-NP at pH 5.5-7.4. In young adult SD rats, the concentration of hepatic cytosolic HST protein immunostained by the anti-STa-antibody was much higher in females than in males (Fig. 5). That is in good accordance with our previous data on the metabolic activation of 5-HCR [21] and with those obtained by Surh et al. with DNA binding of 7-HMBA [421 and 6-HBP [43] in vitro. The weakly immuno-stained band appearing at 32 kDa in female rat liver cytosol and the rather intensely stained band compared with the 30.5-kDa band in male rat liver cytosol (Fig. 5) corresponded to the PST found in the pooled fraction C of the PAP-affinity column chromatogram for the female HSTs (Fig. 4). The major HSTs, STa and STb, had sulfating activities toward all four of the carcinogens, 5-HCR, 7-HMBA, 7-HBA, and DHBA, DHA, cortisol, and n-butanol with different substrate specificities (Fig. 6). The enzyme activities in this experiment were determined by measuring PAP formed from PAPS by HPLC. As to STa, the purified enzyme was eluted from a TSKgel G3000SW gel filtration column as a dimer in the presence of 0.1% (w/v) Tween 20 and as an oligomer larger than a tetramer in the absence of the detergent. The dimeric form of STa was also confirmed by native PAGE carried out in the presence of the detergent. As mentioned above, we could not detect any HST protein smaller or larger than 30.5 kDa as a subunit of HST in SD rat liver cytosol although previous workers had

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Fig. 6. Sulfations of carcinogenic polycyclic arylmethanols, steroids, and n-butanol by purified STa and STb. A reaction mixture consisted of purified enzyme (4 t~g/ml), substrate (100 #M) dissolved in dimethyl sulfoxide (5% (w/v)), PAPS (120 ~M), and 0.05% (w/v) Tween 20 in Na2HPO4-KH2PO 4 buffer, pH 6.8. Reaction was started by addition of the substrate solution after preincubation of the mixture at 37°C for 3 min. After 20 min, the reaction was terminated by immersing reaction vessels into ice-cold water, followed by rapid removal of the substrate by extraction of the incubation mixture with ethyl acetate. Enzymatic sulfation was determined by measuring PAP formed from PAPS by HPLC on an anion-exchange column as previously reported [36]. Data are expressed as mean values of at least three experiments.

T. Watabe et al. ~Chem.-Biol. Interact. 92 (1994) 87-105

97

reported that the cytosol contained HSTs which were separable on the anionexchange column and consisted of subunits with different sizes and without immuno-homology [31,32]. Our result on the equality of the subunit size would be in good accordance with that obtained by Homma and et al. [44,45] who have very recently demonstrated a purified and denatured Wistar rat liver HST to consist of at least four pI variants with the equal subunit size of 30 kDa. Despite current advances in technology, the isolation and characterization of the major rat liver HST enzyme forms remains difficult. Actually, molecular cloning studies indicate that at least seven different subunits, including our five ones with a very narrow range of M r values (Mr, 33 084-33 535), may exist in the female SD rat liver cytosol as described below. 4. Molecular cloning of SD rat liver HST subunits The first success in molecular cloning of the ST catalysing the sulfation of xenobiotics has been achieved with the major rat liver HST, STa, by Ogura et al. [46,47] who cloned two cDNAs (ST-20 and ST-40) encoding the corresponding HST subunits, ST-20P and ST-40P, from a female SD rat liver Xgtll c D N A library by

ST-20P

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P ..............

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T ........................................

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............

P ....

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M-S--N

hHSTI

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....

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....

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.........

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......

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....

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....

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.........

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......

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....

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....

P-ISYQR-I-EDIR

......

101

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..................

I ...................

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i01

..................

I ...............

KT ...................

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i01

..................

I ...............

KT .....................

ST-60P

102

..................

I .........

F ......

A---M

.....

v .................................

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99

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102

IQ--P--F

..........

M ...........

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hHST2

102

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..........

M ...........

F--K-MKFI---K-WEE-F---CQ-T-V

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102

IQ--P--F

..........

M ...........

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ST-20P

ST-40P

202 201

.........................

N ....

N-ME-EL-LP-FTF

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201

.........................

N ....

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202

..........

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199

---N-G

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202

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202

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202

......

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..................

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.....

I

F ....

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...........................

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I ......

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.......

.....

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.........................

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A .........

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....

R ......

V ....................

T ......

....... ..... .......

I .....

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.............. P ..............

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.....

S .......

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.....

S--EL-Q--GR--E---Q-

D--RG-14P-R-EK

.....

S--EL-Q--GR--E---Q-

A .......................

D

A .......................

D

284 284 284

......

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A ........................

285

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A ........................

282

L .......

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....

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Fig. 7. Alignment of the amino acid sequences deduced from c D N A s encoding rat and human liver HSTs. Amino acid sequences are given in the conventional single letter code. Dashes in the sequences represent amino acid residues identical with those of ST-20P. The nucleotide sequence of the ST-60 cDNA is in the GenBankrM/EMBL Data Bank with the accession number D14989. Three gaps are introduced into the sequence of SMP-2 to maximize pairing of identical amino acid residues as indicated by asterisks. Dots are placed at 10-amino acid residue intervals. Amino acid positions are shown on the left and right sides of the sequences. The amino acid sequences of hHSTI, hHST2, and hHST3 were reported by Otterness et al. [48], Kong et al. [49], and Comer et al. [50], respectively. The proposed PAPS-binding site (see the text) is shown in white upon black.

98

T. Watabe et al, ~Chem.-Biol, Interact. 92 ( 1 9 9 4 ) 8 7 - 1 0 5

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T. Watabe et al. / Chem.-Biol. Interact. 92 (1994) 87-105

screening with the polyclonal anti-STa-antibody, sequenced them, and identified ST40P with STa by matching the deduced amino acid sequence with the chemically determined N-terminal and partial amino acid sequences of the rat liver enzyme. The first 20 N-terminal amino acid sequence of STa differed from that of ST-20P by two amino acid residues (Fig. 7). The second success in molecular cloning of the ST for xenobiotic metabolism was reported with rat PST-1 by Ozawa et al. [51] who used polyclonal antibody raised against a PST purified from male SD rat liver cytosol for screening their SD rat liver c D N A library. The identification of the polypeptide deduced from PST-1 c D N A with a rat liver PST has been achieved by Hirshey et al. [52]. Apart from STs sulfating xenobiotics, Nash et al. [53], earlier than Ogura et al. [46], reported the structural identification of the bovine placenta estrogen ST (EST) with the polypeptide deduced from a c D N A cloned from the bovine placenta cDNA library. Bovine placenta EST, however, is likely to play little role in metabolism of xenobiotics as the enzyme has been demonstrated to show no activity toward 4-NP and D H A [54]. The HST subunit ST-40P shares weak sequence identity with those of PST-1 (37.8%) and EST (41.4%) (Table 2). Thus, belatedly compared with other fields of enzyme research, the door to further studies on ST structure and function was opened. Increasing information has accumulated in the last half decade about the primary structures of STs in various species of living organisms, human to plant (Table 2). cDNAs encoding five different HST subunits, including ST-20P [46] and ST-40P [47], have been cloned by Ogura et al. from both of the c D N A libraries 1 and 2 from two female SD rat livers by screening with the anti-STa-antibody or a synthetic oligonucleotide probe. The three new HST subunits were designated ST-21P, ST-41P,

Table 3 Microheterogeneity in nucleotide sequences of ST-20 and ST-21 cDNAs found in both cDNA libraries from two female SD rat livers Base position

18b 136 244 392 453 555 760 762 785

Nucleotide basesa

Amino acid position

ST-20

ST-21

C

T

GAC__ ACC G TC GCG CA A GGT ACT G TT

GAA ACT A TC GT_G CGA GGA ATT A'l~

Amino acid substitution --

33 69 ll9 139 173 241 242 250

Asp -- Glu Thr (silent) Val - Ile Ala -- Val Gln - Arg Gly (silent) Thr -- lie Val - Ile

aSubstituted bases in the codons are underlined. The ST-21 cDNA consists of two heterogeneous cDNAs, ST-21a and ST-21b, that differ only in the length of their 3'-non-coding regions before the poly(A). Their complete nucleotide sequences are in the GenBankTM/EMBLData Bank with accession numbers, M31363, D14987, and D14988 for ST-20, ST-21a, and ST-21b cDNAs, respectively. bThis position is in the 5'-non-coding regions of ST-20 and ST-21 cDNAs.

100

T. Watabe et al. ~Chem.-Biol. Interact. 92 (1994) 87-105

and ST-60P related to the corresponding cDNAs. The five HST subunits consist of 284 or 285 amino acid residues (Mr, 33 084-33 535) and share extremely strong sequence identity with ST-40P like ST-20P (89.8%): ST-21P (90.5%), ST-41P (99.6%), and ST-60P (86.3%) (Table 2). The very strong sequence identity and the very narrow range of Mr values of these HST subunits are in good accordance with the aforementioned facts that all the HSTs collected on the PAP-agarose affinity column from the rat liver cytosol show an intense immuno-cross-reactivity with the antiSTa-antibody by Western blot analysis and have an apparently equal subunit size (30.5 kDa) on SDS-PAGE (Figs. 4 and 5). The ST-20 c D N A differs from the ST-21 c D N A only by 8 nucleotide residues in nucleotide sequence of their coding-regions, leading to 6 amino acid substitutions (Table 3). There exists a difference only by three nucleotide residues between the coding regions of ST-40 and ST-41 cDNAs, leading to the only amino acid substitution (Table 4). Numbers of these full length cDNAs cloned from the two female SD rat liver c D N A libraries were 6 and 3 for ST-20, 2 and 2 for ST-21, 2 and 2 for ST-40, and 3 and 3 for ST-41 from the libraries 1 and 2, respectively. Extensive studies on molecular cloning of HSTs suggest that microheterogeneous ST-40 and ST-41 cDNAs may the be result of reverse transcription of native m R N A s in the livers of the two female rats used to prepare the expression libraries, and not the result of cloning artifact. Human liver HST has been recognized to be a single homodimer appearing at 35 kDa on SDS-PAGE [63] and was demonstrated to consist of three microheterogeneous subunits differing in only one or two amino acid residues [48-50]. However, the microheterogeneity in the human liver HST subunit has not been demonstrated with cDNAs cloned from a single c D N A library but from three different libraries, strongly suggesting it to be due to the individual differences in expression. The microheterogeneous subunit proteins, ST-40P and ST-41P, were separately expressed in E. coli which had been transformed with an expression plasmid containing the coding region of the ST-40 or ST-41 cDNA. Homodimers of the recombinant proteins, ST-40P and ST-41P, purified by the same method as used for the rat liver HST, STa, were chromatographically, electrophoretically, and enzyme-functionally

Table 4 Extreme microheterogeneityin nucleotide sequences of ST-40 and ST-41 cDNAs found in both cDNA libraries from two female SD rat livers Base position

175 503 640

Nucleotide basesa ST-40

ST-41

ACA TAT CTA

ACG A AT CTG

Amino acid position

Amino acid substitution

46 156 201

Thr (silent) Try -- Asn Leu (silent)

aSubstituted bases in the codons are underlined. Complete nucleotide sequences are in the GenBankTM/EMBL Data Bank with accession numbers, M33329 and X63410, for the ST-40 and ST-41 cDNAs, respectively. See Fig. 1 in Ogura et al. [68].

T. Watabe et aL ~Chem.-Biol. Interact. 92 (1994) 87-105

101

identical with STa (see Ogura et al. [68]). Therefore, STa in rat liver cytosol may consist of these inseparable microheterogeneous subunits, ST-40P and ST-41P. There may exist at least two other HST subunits in SD rat liver cytosol; one is senescence marker protein 2 (SMP-2) [551, and the other bile acid STI (BAST I) [39]. SMP-2 is estrogen-dependently expressed and androgen-dependently suppressed, so that its hepatic level increases at the prepubertal and senescent stages, decreases between these stages in male rats, and is kept at a high level after the pubertal to the presenescent stages in the female animals. Ogura et al. have demonstrated SMP-2 to be a subunit of HST by sequence alignment of ST-20P and ST-40P with SMP-2 and pointed out that the reported coding region of the cDNA for SMP-2 contained erroneous deletions of 9 nucleotide residues [47]. Introducing gaps for the deleted three amino acid residues into the reported amino acid sequence, SMP-2 shares strong sequence identity (>74%) with those of ST-20P and ST-40P (Table 2). Limiting the amino acid sequence to four common domains covering 62% of their total amino acid residues, the sequence identity of ST-40P with SMP-2 increased up to 91.5% [47]. However, we failed to isolate the cDNA insert for SMP-2 from our two rat liver cDNA libraries. The other cDNA that we failed to isolate was that coding for rat liver BAST I whose primary structure had not been elucidated by molecular cloning. However, the reported first 24 N-terminal amino acid residues of purified BAST I shares very strong sequence identity (92%) with that of STa. Amino acid sequence alignment of rat and human HSTs with other reported mammalian and plant STs strongly suggests the existence of the consensus sequence RKGXXGDWKXXFT as a putative PAPS-binding site, in their sequences, which consists of 13 amino acid residues located at positions corresponding to 246-258 for ST-20P or ST-40P (shown in white upon black in Fig. 7). In connection with this, Satishchandran et al. [64] have proposed the sequence KG/AXXGXXXNXFT for the putative cofactor-binding site existing in the deduced amino acid sequence of adenosine 5 '-phosphosulfate kinase in E. coli, based on the result of its sequence alignment with STs, including our ST-20P and ST-40P. Similarly, Hashimoto et al. [65] have also proposed the sequence, GXXGXXK, for the putative PAPS-binding site of rat liver N-heparan sulfate ST. In our proposed PAPS-binding site, the two lysines may play an important role in simultaneous ionic interactions with the sulfate and 3 '-phosphate anions of PAPS, and the two glycines in capturing the adenine moiety by conforming a loop or pocket in the higher structure of the enzymes as previously suggested with E. coli H+-ATPase [66] and pig muscle adenylate kinase [67]. As mentioned above, all of the HST activities in the rat liver were eluted from the DEAE-Sephadex column in three major chromatogram peaks containing STa, STb, and STc activities (Fig. 2). However, molecular cloning of the rat liver HST subunits suggested that these three peaks may contain more than several HST subunits in miscellaneous forms probably as homo- or hetero-dimers or/and oligomers. These subunits have an almost equal molecular mass, share strong sequence identity in amino acid sequence, and are, consequently, likely to be cross-reactive with a polyclonal antibody raised against an HST purified from liver cytosol. Purification of the rat liver HSTs as homogeneous proteins, therefore, may be predicted to be almost impossible.

102

T. Watabe et al. ~Chem.-Biol. Interact. 92 (1994) 87-105

Is t h e r e a n y w a y for us to solve the p r o b l e m o n the s t r u c t u r e a n d f u n c t i o n o f the rat liver HST?. O n e o f the m o s t d i r e c t a n s w e r s to this q u e s t i o n c o u l d be to use r e c o m b i n a n t H S T s e x p r e s s e d in p r o k a r y o t i c o r e u k a r y o t i c cells as d e m o n s t r a t e d by O g u r a et al. [68]. H o w e v e r , t h a t w o u l d n o t give us an a n s w e r to a n o t h e r i m p o r t a n t q u e s tion, such as, to w h a t e x t e n t t h e y are e x p r e s s e d d u r i n g d e v e l o p m e n t o f m a l e a n d fem a l e a n i m a l s ? M o n o c l o n a i a n t i b o d i e s h i g h l y specific to e a c h o f the H S T subunits, if possible to p r e p a r e , are e x p e c t e d to b e c o m e a v a i l a b l e for this p u r p o s e . H o w e v e r , c o u l d a m o n o c l o n a l a n t i b o d y be p r e p a r e d w h i c h w o u l d d i s t i n g u i s h b e t w e e n the ext r e m e l y similar m i c r o h e t e r o g e n e o u s s u b u n i t s S T - 4 0 P a n d S T - 4 1 P ?

5. References 1 J.K. Selkirk, Chemical carcinogenesis: a brief overview of the mechanism of action of polycyclic hydrocarbons, aromatic amines, nitrosamines, and aflatoxins, in: T.J. Slaga (Ed.), Carcinogenesis, Vol. 5, Raven Press, New York, 1980, pp. 1-31. 2 E. Boyland and P. Sims, The carcinogenic activities in mice of compounds related to benz[a]anthracene, Int. J. Cancer, 2 (1967) 500-504. 3 A. Dipple, Polynuclear aromatic carcinogens, in: C.E. Searle (Ed.), Chemical Carcinogens, ACS Monograph 173, American Chemical Society, Washington DC, 1976, pp. 245-314. 4 E. Cavalieri, R. Roth and E. Rogan, Hydroxylation and conjugation at the benzylic carbon atom: a possible mechanism of carcinogenic activation for some methyl-substituted aromatic hydrocarbons, in: P.W. Jones and P. Leber (Eds.), Polynuclear Aromatic Hydrocarbons, Third International Symposium on Chemistry and Biology, Carcinogenesis and Mutagenesis, Ann Arbor Science, Michigan, 1979, pp. 517-529. 5 E. Boyland, P. Sims, and C. Huggins, Induction of adrenal damage and cancer with metabolites of 7,12-dimethylbenz[a]anth racene, Nature (London), 207 (1965) 816-817. 6 J.W. Flesher and K.L. Sydnor, Carcinogenicity of derivatives of 7,12-dimethylbenz[a]anthracene, Cancer Res., 31 (1971) 1951-1954. 7 E. Cavalieri, R. Roth, C. Grandjean, J. Althoff, K. Patil, S. Liakus and S. Marsh, Carcinogenicity and metabolic profiles of 6-substituted benzo[a]pyrene derivatives on mouse skin, Chem.-Biol. Interact., 22 (1978) 53-67. 8 S. Amin, A. Juchatz, K. Furuya and S.S. Hecht, Effects of fluorine substitution on the tumor initiating activity and metabolism of 5-hydroxymethylchrysene, a tumorigenic metabolite of 5methylchrysene, Carcinogenesis, 2 (1981) 1027-1032. 9 T. Watabe, Y. Hakamata, A. Hiratsuka and K. Ogura, A 7-hydroxymethyl sulphate ester as an active metabolite of the carcinogen, 7-hydroxymethylbenz[a]anthracene, Carcinogenesis, 7 (1986) 207-214. 10 T. Watabe, T. Ishizuka, M. Isobe and N. Ozawa, A 7-hydroxymethyl sulfate ester as an active metabolite of 7,12-dimethylbenz[a]anthracene, Science, 215 (1982) 403-405. 11 T. Watabe, T. Ishizuka, T. Fujieda, A. Hiratsuka and K. Ogura, Sulfate esters of hydroxymethyl-' methyl-benz[a]anthracenes as active metabolites of 7,12-dimethylbenz[a]anthracene, Jpn. J. Cancer Res. (Gann), 76 (1985) 684-698. 12 T. Watabe, A. Hiratsuka, K. Ogura and K. Endoh, A reactive hydroxymethyl sulfate ester formed regioselectively from the carcinogen, 7,12-dihydroxymethylbenz[a]anthracene, by rat liver sulfotransferase, Biochem. Biophys. Res. Commun., 131 (1985)694-698. 13 T. Watabe, A. Hiratsuka and K. Ogura, Sulphotransferase-mediated covalent binding of the carcinogen 7,12-dihydroxymethylbenzla]anthracene to calf thymus DNA and its inhibition by glutathione transferase, Carcinogenesis, 8 (1987) 445-453. 14 H. Okuda, A. Hiratsuka, H. Nojima and T. Watabe, A hydroxymethyl sulphate ester as an active metabolite of the carcinogen, 5-hydroxymethylchrysene, Biochem. Pharmacol., 35 (1986) 535-538. 15 H. Glatt, R. Henschler, D.H. Phillips, J.W. Blake, P. Steinberg, A. Seidel and F. Oesch, Sulfotransferase-mediated chlorination of l-hydroxymethylpyrene to a mutagen capable of penetrating indicator cells, Environ. Health Perspect., 88 (1990) 43-48.

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16

17

18

19

20

21

22

23

24

25 26

27

28

29

30

31

32

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Y.-J. Surh, J.C. Blomquist, A. Liem and J.A. Miller, Metabolic activation of 9-hydroxymethyl-10methylanthracene and l-hydroxymethylpyrene to electrophilic, mutagenic and tumorigenic sulfuric acid esters by rat hepatic sulfotransferase activity, Carcinogenesis, 11 (1990) 1451-1460. Y.-J. Surh, A. Liem, E.C. Miller and J.A. Miller, The strong hepatocarcinogenicity of the electrophilic and mutagenic metabolite 6-sulfooxymethylbenzo[a]pyrene and its formation of benzylic DNA adducts in the livers of infant male B6C3FI mice, Biochem. Biophys. Res. Commun., 172 (1990) 85-91. T. Watabe, T. Fujieda, A. Hiratsuka, T. lshizuka, Y. Hakamata and K. Ogura, The carcinogen, 7hydroxymethyl-12-methylbenz[a]anthracene, is activated and covalently binds to DNA via a sulphate ester, Biochem. Pharmacol., 34 (1985) 3002-3005. T. Watabe, T. Ishizuka, Y. Hakamata, T. Aizawa and M. lsobe, Covalent binding of the proximate carcinogen, 7-hydroxymethyl-12-methylbenz[a]anthracene (7-HMBA) to rat liver cytosolic protein via 7-HMBA sulphate, Biochem. Pharmacol., 32 (1983) 2120-2122. Y.-J. Surh, C.-C. Lai, J.A. Miller and E.C. Miller, Hepatic DNA and RNA adduct formation from the carcinogen 7-hydroxymethyl-12-methylbenz[a]anthracene and its electrophilic sulfuric acid ester metabolite in preweanling rats and mice, Biochem. Biophys. Res. Commun., 144 (1987) 576-582. H. Okuda, H. Nojima, N. Watanabe and T. Watabe, Sulphotransferase-mediated activation of the carcinogen 5-hydroxymethylchrysene: species and sex differences in tissue distribution of the enzyme activity and a possible participation of hydroxysteroid sulfotransferases, Biochem. Pharmacol., 38 (1989) 3003-3009. H. Okuda, H. Nojima, K. Miwa, N. Watanabe and T. Watabe, Selective covalent binding of the active sulfate ester of the carcinogen 5-(hydroxymethyl)chrysene to the adenine residue of calf thymus DNA, Chem. Res. Toxicol., 2 (1989) 15-22. H. Okuda, K. Miwa, H. Nojima and T. Watabe, Inactivation of the carcinogen, 5-hydroxymethylchrysene, by glutathione conjugation via a sulphate ester in hepatic cytosol, Biochem. Pharmacol., 35 (1986) 4573-4576. A. Hiratsuka, N. Sebata, K. Kawashima, H. Okuda, K. Ogura, T. Watabe, K. Satoh, I. Hatayama, S. Tsuchida, T. lshikawa and K. Sato, A new class of rat glutathione S-transferase Yrs-Yrs inactivating reactive sulfate esters as metabolites of carcinogenic arylmethanols, J. Biol. Chem., 265 (1990) 11973-11981. M. Suzuki, M. Kojima, H. Okuda, T. Watabe and M. Tada, DNA polymerase action blocked by adenine adducts induced 5-hydroxymethylchrysene sulfate, Biochem. Int., 25 (1991) 19-27. Y.-J. Surh, A. Liem, E.C. Miller and J.A. Miller, 7-Sulfooxymethyl-12-methylbenz[a]anthracene is an electrophilic mutagen, but does not appear to play a role in carcinogenesis by 7,12dimethylbenz[a]anthracene or 7-hydroxymethyl-12-methyl-benz[a]anthracene, Carcinogenesis, 12 (1991) 339-347. C.N. Falany, J. Wheeler, L. Coward, D. Keehan, J.L. Falany and S. Barnes, Bioactivation of 7hydroxymethyl-12-methylbenz[a]anthracene by rat liver bile acid sulfotransferase I. J. Biochem. Toxicol., 7 (1992) 241-248. T. Watabe, T. Ishizuka, N. Ozawa and M. Isobe, Conjugation of 7-hydroxymethyl-12methylbenz[a]anthracene (7-HMBA) with glutathione via a sulphate ester in hepatic cytosol, Biochem. Pharmacol., 31 (1982) 2542-2544. T. Watabe, A. Hiratsuka and K. Ogura, Regioselective glutathione conjugation of the carcinogen, 7,12-dihydroxymethylbenz[a]anthracene, via reactive 7-hydroxymethyl sulfate ester in rat liver cytosol, Biochem. Biophys. Res. Commun., 134 (1986) 100-105. K. Ogura, T. Nishiyama, T. Okada, J. Kajita, H. Narihata, T. Watabe, A. Hiratsuka and T. Watabe, Molecular cloning and amino acid sequencing of rat liver class theta glutathione S-transferase Yrs-Yrs inactivating reactive sulfate esters of carcinogenic arylmethanols, Biochem. Biophys. Res. Commun., 181 (1991) 1294-1300. W.B. Jakoby, M.W. Duffel, E.S. Lyon, and S. Ramaswamy, Sulfotransferase active with xenobiotics --comments on mechanism, in: J.W. Bridges and L.F. Chasseaud (Eds.), Progress in Drug Metabolism, Vol. 8, Taylor & Francis, London, 1984, pp. 11-33. S.S. Singer, Preparation and characterization of the different kinds of sulfotransferases, in: D. Zakim and D.A. Vessay (Eds.), Biochemical Pharmacology and Toxicology, Vol. 1, WileyInterscience, New York, 1985, pp. 95-159.

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