Xenobiotica the fate of foreign compounds in biological systems
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Peroxidase/hydrogen peroxide—or bone marrow homogenate/hydrogen peroxide—mediated activation of phenol and binding to protein V. V. Subrahmanyam, L. G. McGirr & P. J. O'brien To cite this article: V. V. Subrahmanyam, L. G. McGirr & P. J. O'brien (1990) Peroxidase/ hydrogen peroxide—or bone marrow homogenate/hydrogen peroxide—mediated activation of phenol and binding to protein, Xenobiotica, 20:12, 1369-1378 To link to this article: http://dx.doi.org/10.3109/00498259009046635
Published online: 27 Aug 2009.
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Date: 09 November 2015, At: 06:22
XENOBIOTICA,
1990, VOL. 20,
NO.
12, 1369-1378
Peroxidase/hydrogen peroxide-or bone marrow homogenate/hydrogen peroxide-mediated activation of phenol and binding to protein V. V. SUBRAHMANYAMT, L. G. McGIRR and P. J. O'BRIENS Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 1Al
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Received 17 October 1989; accepted 5 April 1990
1. "C-Phenol was metabolized by rat bone marrow homogenate and H,O,. The homogenate catalyst, however, was inactivated by preincubation with H,O,, presumably due to inactivation of the enzyme(s) involved in phenol metabolism. 2. The majority of the metabolized I4C-phenol was bound to bone marrow proteins. o,o'-Biphenol and p,p'-biphenol were the principal non-protein-bound products. Ascorbate was unable to remove phenol oxidation products bound to protein, although o,o'biphenol recovery from the reaction mixture was markedly enhanced. Prior alkylation of protein thiols with N-ethylmaleimide decreased the binding of '*C-phenol oxidation products to bone marrow proteins by only 10-20%.
3. '*C-Phenol (200pM) metabolism by horseradish peroxidase (1Opg) and H,O, (200p ~also ) resulted in extensive binding to externally added bovine serum albumin. The absorption spectrum of "C-phenol oxidation products bound to bovine serum albumin was similar to that of bound oxidation products of o,d-biphenol but not of p,p'-biphenol.
4. Protease digestion of bovine serum albumin bound 14C-phenol oxidation products, followed by ethyl acetate extraction, extracted 75% of the '*C, indicating that most of the binding is probably non-covalent. Up to 32% of the '4C-phenol oxidation products binding to bovine serum albumin may be covalent, since derivation with dinitrofluorobenzene and extraction under acid, but not alkaline, conditions extracted the I4C. The percentage of metabolites covalently bound to bovine serum albumin was increased to 59% when horseradish peroxidase concentration was decreased to 0 2pg.
5. The thiol groups of bovine serum albumin were unaffected by o,o'-biphenol oxidation products, slightly decreased by phenol oxidation products, but were completely depleted by p, p'-biphenol oxidation products. 6. These results indicate that o,o'-biphenol oxidation products are responsible for much of the 14C-phenol binding to protein.
Introduction Peroxidases are well known to oxidize a wide variety of phenols (Saunders 1964). Recently, peroxidase activities in bone marrow and zymbal gland have been implicated in the activation of phenol to reactive metabolites which may have some role in benzene-induced carcinogenicity or toxicity (O'Brien et al. 1985, Smith et al. 1989). We have previously shown that phenol can be activated by myeloperoxidase in intact polymorphonuclear leucocytes in the presence of phorbol-myristate-acetate (a tumour promoter), to reactive metabolites that bind to nuclear DNA in the leucocyte (O'Brien et al. 1985). We and others have also shown that phenol metabolism by purified horseradish peroxidase and myeloperoxidase results in the formation of o,o'-biphenol and p,p'-biphenol (Danner et al. 1973, Eastmond et al. 1986, Sawahata and Neal 1982, Subrahmanyam and O'Brien 1985 a, b), the former
t Present address: Department of Biomedical and Environmental Health Sciences, School of Public Health, 322 Warren Hall, University of California, Berkeley, CA 94720,USA. $ T o whom correspondence should be addressed. 0049-8254/90 $ 3 0 0
01990 Taylor & Francis Ltd.
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being the major metabolite (Subrahmanyam and O’Brien 1985 a, b). Further oxidation of o,o’-biphenol but not p, p‘-biphenol leads to extensive binding to calf thymus DNA. p,p’-Biphenol can be further oxidized top, p’-biphenoquinone which forms a covalent conjugate with glutathione to form 3-S(-glutathion-yl)-p,pbiphenol (Eastmond et al. 1986, McGirr et al. 1986). Recently, Kariya et al. (1987) found that rat bone marrow contains at least two different peroxidases, namely myeloperoxidase and eosinophil peroxidase. Sawahata and colleagues (1982, 1985) showed that phenol was activated by bone marrow homogenate and hydrogen peroxide to products of which o,o‘-biphenol and p, p‘biphenol represented about 5%, indicating that peroxidatic metabolism of phenol indeed occurs in bone marrow. No other metabolites were found, but extensive binding to bone marrow protein was observed. They suggested that p,p’biphenoquinone may be the major reactive metabolite binding to protein, but the detailed mechanisms of phenol activation by bone marrow peroxidases are not clear. This has prompted us to further investigate phenol activation by bone marrow homogenate in order to find out the nature of the products involved in protein binding.
Materials and methods Materials Phenol, o,o’-biphenol, p, p’-biphenol, glutathione (GSH), oxidized glutathione (GSSG), horseradish peroxidase (HRP) type VI, ascorbic acid, protease (type XI), N-ethylmaleimide, dinitrofluorobenzene (DNFB) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Hydrogen peroxide (30% w/v) was obtained from Fisher Chemical Company (Toronto, O N T , Canada). Ur14C1Phen~l(71.1 mCilmmo1~was Durchased from Amersham Chemical Co. (Oakville. ONT. Canada) with a staied radiochemical puriiy of 99%. L
A
Preparation of bone marrow homogenate Bone marrow cells were isolated from male Sprague-Dawley rats (200-300 g) and a homogenate was prepared as described previously (McGirr et al. 1986). Analysis of ‘‘C-phenol metabolism by bone marrow homogenate 14C-Phenol (0.2 mM) was incubated with bone marrow homogenate (&200pg) and HzOz (&lo mM) in 3 ml of 0.1 M Tris-HC1 buffer pH 7.4 at 37°C. Reactions were started by the addition of H,Oz. After appropriate times of incubation the reactions were terminated by extraction with ethyl acetate (3 ml x 3). The ethyl acetate extracts were pooled and concentrated to 0 5 ml under N,. The product analysis was carried out either by t.1.c. or by h.p.1.c. as described previously (Subrahmanyam and O’Brien 1985 a, b). Quantitative determination of irreversible binding to protein Quantitative determination of irreversible binding to protein was performed as described by Sivarajah et al. (1978). The protein in the aqueous layer was precipitated with 100% trichloroacetic acid (TCA, final concn lo%),washed with 10% T C A (1ml) and the protein, after centrifugation, was extracted twice with methanol-water (4 : 1 v/v) and chloroform-methanol (2 : 1 v/v). The washed protein was solubilized in 1 ml of 0.1 M NaOH, radioactivity was then determined by scintillation counting and the amount of protein was determined by the method of Lowry et al. (1951). Approximately 7 5 4 5 % of protein was always recovered. The nature of protein binding The protein samples obtained after methanol extractions, as described above, were further examined to determine how strongly the products are bound to protein, using the method described by Jollow et al. (1973). The protein samples were suspended in 3 ml of 0 1 M Tris-HC1 buffer p H 7.4 and digested with protease for 12 h at 37°C. Maximal digestion occurred within 12 h, after which further incubation had no effect on the amount digested. The digest was extracted with ethyl acetate ( 3 ml x 2) and the organic layers were removed. T h e aqueous layers were bubbled with N, for a few minutes (to evaporate excess ethyl acetate), and an aliquot was removed for measuring the radioactivity. Further aliquots which were previously extracted with methanol and digested with protease, were mixed with DNFB (5% w/v) in ethanol and left for 4 h at 25°C in the dark, with occasional stirring. The
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pH was adjusted to 9.0 by addition of NaHCO, and the reaction mixtures were extracted with 3 ml ethyl acetate (twice). An aliquot of the aqueous layer was measured for radioactivity. The remaining aqueous ~ and the reaction mixtures were further extracted with ethyl layers were acidified to pH 1.0 with 6 0 HCI acetate (3 ml x 2). The radioactivity in the ethyl acetate extract was determined.
Protein thiols Quantitative determination of protein thiols was performed according to the method of Sedlak and Lindsay (1 968) with slight modifications.Two-millilitre aliquots of reaction mixtures were added to tubes containing 1OOpl of 70% perchloric acid to precipitate proteins. The samples were then centrifuged at 2500 rpm for 5 min, and the supernatant discarded. The pellet was then washed twice with 3 ml of 5% perchloric acid, mixing well, centrifuging at 2500rpm for 5min and discarding the supernatant. Three millilitres of 0 5 M Tris buffer, containing 3% sodium dodecyl sulphate (SDS) at pH 8.0, was then added to the pellet and mixed well until all the protein was solubilized. One millilitre of this solution was processed as blank, while another 1ml aliquot was processed as the sample. For the blank, l00pl of N-ethylmaleimide (1 mM in Tris/SDS buffer) was added, mixed well and allowed to react for 15 min. This N-ethylmaleimide treatment alkylated all sulphydryl groups, thus acid (200p ~in) serving as an internal blank for each sample. Then 100pl of 5,5’-dithiobis-2-nitrobenzoic Tris/SDS buffer) was added to each sample and blank pair, and left in the dark for 15 min to allow it to react with SH groups. One millilitre of distilled water was then added to both the blank and the sample, and the absorbance of each sample was read against the appropriate blank at 412 nm. Protein thiol values were compared with GSH standards, which were prepared and treated as described above.
Results Preliminary studies showed that phenol metabolism was dependent on bone marrow protein concentration, the rate of phenol metabolism increased with increasing protein concentration. With 200 nmol phenol, 25 pg bone marrow protein and 10mM H,02, only 15f4nmol of phenol equivalents were bound per mg protein. Increasing the protein concentration to 200 pg, the binding of phenol equivalents to bone marrow protein increased to 57 f6 nmollmg protein. Figure 1 shows that the amount of 14C-phenol bound to protein is also proportional to the concentration of H 2 0 2(up to 10 mM). The need for a large excess of H , 0 2 indicates that most of the H , 0 2 may be decomposed by catalase in the bone marrow homogenate. Figure 1 also shows that preincubation, for Smin, of bone marrow homogenate with H20, results in inhibition of protein binding, presumably by inactivating the enzyme(s) involved in phenol activation. Table 1 shows the time-course for the metabolism and protein binding of 14Cphenol activated by bone marrow homogenate and H,O,. As shown in the table, 100%of the phenol was metabolized within 5 min. Approx. 7 5 4 5 % of the phenol was bound to bone marrow protein. Both o,o’-biphenol and p , p’-biphenol were formed from phenol. However, they accounted for less than 20% of the total phenol oxidized. It is possible that both the biphenols are further oxidized to products which bind to bone marrow protein. No hydroquinone or catechol formation was detected under these conditions. Addition of ascorbate at the end of the incubation period just before ethyl acetate extraction resulted in increased amounts of extractable o,o’-biphenol (table 1). A maximum of a four-fold increase in o,o’-biphenol formation was observed. Although small increases in p,p’-biphenol formation were also observed, the total p, p’biphenol formed was at least four-fold lower than the total o,o‘-biphenol formed, when compared at the 5 min time point where maximal yields of o,o‘-biphenol were obtained. These results therefore indicate that o,o‘-biphenol is the major metabolite formed from phenol during activation by bone marrow homogenate and H 2 0 z .The results in table 1 also show no decrease in protein-bound radioactivity in the presence of ascorbate, indicating that ascorbate is unable to remove the products already bound to the protein. Ascorbate therefore presumably acts by reducing the
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150
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100
50
0
0
1
3
5
10
15
20
H202 concn. (rnM)
Figure 1.
Effect of different concentrations of hydrogen peroxide on protein-binding of I4C-phenol catalysed by bone marrow homogenate and hydrogen peroxide. ’‘C-Phenol (200 nmol) was incubated with bone marrow homogenate (200pg), H 2 0 2 (concentrationsas indicated) in 1 ml of 0.1 M Tris-HC1 buffer pH 7.4 at 37°C. 0, as described above; A , preincubated for 5 min with 1 mM H 2 0 2 ; 0, preincubated for 5 min with 10 mM H 2 0 2before the addition of phenol and H 2 0 , .
o,o’-biphenol quinone product and p, p’-biphenoquinone back to o,o’-biphenol and p, p’-biphenol respectively. A minor unknown product with an R, = 0.00 was detected (both in the presence and in the absence of ascorbate), only at 5min of incubation. However, the amount formed appears to be unchanged compared to the amount formed in the absence of ascorbate. This compound was not eluted from the silica gel with ethyl acetate. A compound with similar properties, presumably a polymer, was formed in previous studies when horseradish peroxidase (HRP) was used instead of bone marrow homogenate (Subrahmanyam and O’Brien 1985 a, b). Preincubation of bone marrow homogenate with N-ethylmaleimide (1 mM), and then dialysing to remove excess N-ethylmaleimide, decreased protein thiols by 90%. However, protein binding of 14C-phenol oxidation products was decreased by only 10-20%, indicating that the majority of the binding of phenol oxidation products to protein may not involve sulphydryl groups. T o further investigate how strongly phenol oxidation products are bound to protein, oxidation of phenol by H R P and H 2 0 2 in the presence of BSA was investigated. Figure 2 shows the spectral properties of phenol oxidation products bound to BSA. In the absence of BSA, phenol oxidation product(s) had absorbance maxima at 210 and 399 nm and a shoulder in the 2 4 6 2 5 0 nm region. In the presence of BSA, phenol oxidation product(s) had absorbance maxima at 242 and 294nm. T h e reaction mixtures were extracted with ethyl acetate, after which the protein in the aqueous layer was precipitated with T C A (final concentration 10%);the protein pellet was redissolved in 3 ml of 0.1 M Tris-HC1 buffer p H 7.4 and re-scanned. T h e products bound to protein were not removed during extraction and precipitation, as evidenced by the presence of absorbance maxima at 242 and 294 nm. Figure 2 also
2f0
1 f0 0 0
2+0
1+ o 0 0
6+2
o,o’-Biphenol
p, p‘-Biphenol Unknown 1 Protein bound
Phenol oxidized
15+3
1+o 0 4f2
2+0
None
15+3
1 +o 0 4+ 1
3+0
Ascorbate
30 s
27+3
1 +o 0 22*4
2+0
None
26f4
1 +o 0 20f4
420
Ascorbate
1 min
6825
1 f0 0 61f6
3+0
None
67+5
2fO 0 5826
6+0
Ascorbate
2 min
100
2+0 1f O . 5 84+8
4+0
None
100
4+0 1 fO.5 78+7
16+0
Ascorbate
5 min
o,o’-Biphenol and p,p’-biphenol were quantified by h.p.1.c. and t.l.c., as described in Materials and methods. Unknown 1 was quantified from its radioactivity on t.1.c. Protein binding was determined as described in Materials and methods. Incubation conditions: phenol ( 0 2 m ~was ) incubated with 200pg bone marrow homogenate and 1OmM H,O, at 37°C in 1 ml of 0 1 M Tris-HCI buffer pH7.4. Note: ascorbic acid (1 mM) was added at the end of various incubation times, before extraction with ethyl acetate. Mean values fSEM for three experiments are given. The experiments were performed in triplicate on the same day from one preparation of bone marrow homogenate, in order to reduce the statistical variation due to experimental errors.
6f2
Ascorbate
None
Product formed
15s
Table 1. Time-course for 14C-phenol metabolism by bone marrow homogenate (percentage of 14C incorporated into products).
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h
ir
< b
t? s* 3
3
a
Q
0
a g
3
b
8
B
3
5 z
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1.0
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0.5
3g9
.-..
..: ..: : -.. ...
200
300
400
!
Wavelength (nm) Figure 2.
Spectral properties of phenol or o,o’-biphenol oxidation products bound to bovine serum albumin (BSA).
......, Phenol ( 1 0 0 p ~ ) l H R P(10pg)/H,02 ( 1 0 0 ~ (absorbance ~) scale 0-1.0); -, phenol ( 1 0 0 p ~ ) / H R P(lOpg)/H,O, ( 1 0 0 p ~ ) / B S A(5 mg) (absorbance scale 0-20); ---, o,o’-biphenol ( 1 0 0 p ~ ) / H R P(l0pg) H 2 0 2 ( 1 0 0 p ~ ) / B S A(5 mg) (absorbance scale G20). Reactions were performed in 3ml of 0.1 M Tris-HCI buffer pH7.4 at room temp. After incubation for 30min, reaction mixtures containing BSA were extracted with ethyl acetate (twice) and the BSA was precipitated with T C A (10% final). BSA was redissolved in 3 ml of 0.1 M Tris-HC1 buffer pH 7.4 and scanned using a Shimadzu UV-240 spectrophotometer (reference cuvettes contained equal amount of BSA). Reaction mixtures containing no BSA, were also scanned after 30min of incubation.
shows the spectrum for o,o’-biphenol oxidation products bound to BSA. The spectrum shows absorbance maxima at 242 and 294 nm, indicating that the products binding to protein during o,o’-biphenol oxidation are similar to that found when phenol oxidation binds to protein. On the other hand, p,p’-biphenol oxidation products bound to BSA have an absorbance maximum at 259nm (McGirr et al. 1986). Table 2 shows the effects of protease digestion and DNFB derivatization of the resulting amino acids, on the I4C-phenol oxidation products bound to BSA.Before protease digestion (column A), approximately 75% of the I4C remained in the aqueous layer after ethyl acetate extractions, if phenol was incubated with 10 pg HRP and equimolar H,O,. To decrease the rate of phenol polymerization we used 0.2 pg HRP and determined whether this would increase the covalently bound metabolites to protein. Only 30% of the 14C remained in the aqueous layer, after ethyl acetate
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Bone marrow peroxidase activation of phenol
Table 2. Effects of protease treatment and protease/DNFB treatment on bovine serum albumin containing bound phenol oxidation products. Percentage 14C' remaining in aqueous phase after extractions with ethyl acetateb
Incubation condition
Undigested Pronase Pronase digested end DNFB treated reaction digested pH 1.0 mixture reaction mixture pH 9 0
Phenol + 10pg HRP + 0 . 2 ~HRP+H,O, +lOpg HRP+HzOz
0.3 f 0.2 0.3*0.2 295525 741f5.8
0.3 f 0 2 0.3 50.2 176f2.4 242f3.4
0.3k0.2 03f02 166 3.2 24.4 & 6.0
0.3f0.2 03k0.2 1.2k0.6 21.k0.8
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~ ~ _ _ _
Phenol ( 2 0 0 ~ was ~ ) incubated with horseradish peroxidase (HRP) ( 0 . 2 ~or 1Opg) and H,O, (200p ~ in)3 ml of Tris-HC1 buffer pH 7 4 for 30 min. Controlsin the absenceof H,O, or in the absence of HRP and H,O, were included. 100% '*C represents the total '*C added to the incubation mixture. bThe rest of the '*C was recovered in the ethyl acetate. For detailed experimental procedures see Materials and methods. Mean values+ SEM for three experiments are given.
extractions, if phenol was incubated with 02pg HRP and equimolar HzOz. The remaining 14C was recovered in the ethyl acetate extracts. Digestion of the BSAbound phenol oxidation products with protease (column B) and extraction with ethyl acetate removed approximately 41% and 68% of the 14C bound to BSA into the ethyl acetate layer, with 0.2 pg HRP and 10pg HRP, respectively. This indicated that most of the products bound to BSA may be non-covalent in nature, and that decreasing the HRP concentration decreases the percentage of non-covalently bound metabolites to protein. We further investigated whether the 14Cremaining in the aqueous layers is due to covalent binding or phenol oxidation products to the amino acids. Amino acids from an aqueous solution can be extracted into an organic solvent following derivatization with DNFB and acidification. Thus, amino acids covalently bound to drug metabolites can be effectively isolated by this procedure (Jollow et al. 1973). Column C in table 2 shows that more than 95% of the 14Cwas extracted from the acidified reaction mixtures containing BSA bound with phenol oxidation products, after digestion with protease and derivatization with DNFB. However, DNFB treatment and extraction under alkaline conditions did not increase the amount of 14Cextracted from the aqueous layers, compared to that extracted from the reaction mixtures treated with DNFB. This indicates that the binding of some fraction of the phenol oxidation products to BSA may be covalent. Thus, when phenol was oxidized with 10pg and 0 2 pg HRP respectively, approximately 32% and 59% of the products bound to BSA were covalent in nature. The presence of GSH in the reaction mixture prevented the binding of 14Cphenol oxidation products to protein (data not shown). We have, however, previously shown that GSH also prevents peroxidase-catalysed phenol oxidation to dimers, presumably by reducing the phenoxy radicals back to phenol (Subrahmanyam and O'Brien 1985~).In the absence of phenol, little GSH oxidation occurred, indicating that inhibition of peroxidase-mediated oxidation of phenol by GSH was not due to its ability to act as a competitive peroxidase substrate. However, all the oxidized GSH in the presence of phenol was recovered as GSSC, indicating that GSH does not covalently interact with phenoxy radicals. Table 3 compares the effectiveness of phenol, o,o'-biphenol or p,p'-biphenol oxidation
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Table 3. Reaction of phenol, o,o’-biphenoland p,p’-biphenol oxidation products with thiol groups of bovine serum albumin. Treatment
nmol-SH/mg protein
None Phenol hydrogen peroxide o,o’-Biphenol hydrogen peroxide +p,p’-Biphenol +hydrogen peroxide +Hydrogen peroxide’ +N-Ethylmaleimideb
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+ +
+
+
98f05 7.4f0.3 8 8 f0 5 1.4f0.3 9.5 & 0.6 0 3 f0 2
Reaction mixtures of 3 ml of 0.1 M Tris-HC1 buffer pH7.4 contained: 5 O p ~phenol (or PM p,p‘-biphenol or 2 5 p o,o’-biphenol), ~ 1 equiv. of H,O, and lOyg HRP. The reaction mixtures were incubated for 30min at 22°C. ‘H,O, ( 5 0 ~ ~ ) . 3 ml of 0 1 M Tris-HC1 buffer pH 7.4, BSA (5mg) and N-ethylmaleimide (20 p ~ were ) incubated as above. Mean valuesf SEM for three experiments are given.
products in reacting with thiol groups on BSA. As shown, the thiol group of BSA reacted with p, p’-biphenol oxidation products as effectively as with Nethylmaleimide. However, the o,o’-biphenol oxidation products or H,O, were ineffective in decreasing the number of thiol groups. The small decrease in thiol groups, observed with phenol oxidation products, therefore, may best be attributed to the reaction with p, p’-biphenoquinone formed from phenol oxidation.
Discussion Sawahata and colleagues (1985) have proposed that p , 9’-biphenoquinone may be the major reactive product binding to protein when phenol is activated with bone marrow homogenate and hydrogen peroxide. However, the present results indicate that althoughp, p‘-biphenoquinone may be one of the reactive metabolites binding to protein it is not the major reactive metabolite. The three-fold increase in o,o‘biphenol formation, in the presence of ascorbate, indicates that the majority of the binding may be due to o,o‘-biphenol-derived reactive products. The possibility that phenoxy radicals may be directly involved in protein binding appears to be unlikely, because if the phenoxy radicals are directly binding to proteins, there should have been an inhibition or lag period in the formation of biphenols. However, both o,o’biphenol and p,p‘-biphenol were detected in the first 15 s of the reaction, at which time no protein binding was observed. Sawahata et al. (1985) also reported that p, p’biphenol was formed when phenol was incubated with hydrogen peroxide and partially purified myeloperoxidase, or with bone marrow homogenatehydrogen peroxide. They also found that p, p’-biphenoquinone was formed with myeloperoxidase but not with bone marrow, although the results shown in table 1 indicate that p, p’-biphenol and o,o’-biphenol are further oxidized by bone marrow homogenate. N-Ethylmaleimide (1 mM), a sulphydryl reagent, appears to decrease the binding of phenol oxidation product(s) to proteins in bone marrow homogenate by approximately lo%, indicating that groups other than thiols are involved, or that the binding might not be covalent. In order to understand the nature of interaction between phenol metabolites and proteins in more detail, the oxidation of phenol by peroxidase and hydrogen peroxide in the presence of BSA was investigated (tables 2 and 3). In support of non-covalent interactions it was found that most of the product (s) bound to BSA was released when BSA (containing bound phenol oxidation
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products) was digested with protease, and the released product(s) were extracted into ethyl acetate. This implies that the majority of the binding of phenol oxidation products to BSA may be through non-covalent interactions. It was proposed earlier (Subrahmanyam and O’Brien 1985 b) that binding of phenol oxidation products to DNA may be non-covalent and that the binding of phenol oxidation products may occur via charge-transfer complexes. The product(s) released by the enzymic hydrolysis of DNA, however, were not extracted into butanol (or ethyl acetate) but stayed at the interphase between the organic and aqueous layers. Interestingly, the protease digestion of BSA (with bound 14C-phenol oxidation products) released products which were extracted into ethyl acetate. If BSA was absent from the reaction mixture, the phenol was oxidized to insoluble ‘polymeric’ products which also were not extracted into ethyl acetate but stayed at the interphase of the organic and aqueous layers. This indicates that BSA prevents the precipitation of the phenol ‘polymeric’ oxidation products or binds the soluble ‘polymeric’ products. Some fraction of the bound products was not released from BSA when BSA was digested with protease. However, bound products were extracted into ethyl acetate when the digested BSA was treated with DNFB and acidified. This implies that these metabolites were covalently linked to amino acid residues. The fraction of covalently bound products increased with decreasing HRP concentrations. The nature of the covalently bound metabolites is not clear, although o,o‘-biphenol oxidation products may represent a major part of this fraction. However, our results also indicated that o,o’-biphenol oxidation products do not appear to bind covalently to sulphydryl groups. Whether other functional groups such as amino groups on proteins can covalently interact with o,o’-biphenol oxidation products requires further investigation. Table 3 shows that p,p’-biphenol oxidation product(s) react readily with thiol groups on BSA. However, o,o‘-biphenol oxidation product(s) react poorly with thiol groups on BSA, although phenol oxidation product(s) reacted more readily with BSA thiols. It is therefore tempting to postulate that p,p’-biphenoquinone (the further oxidation product of p, p’-biphenol) formed from phenol could be reacting with thiol groups on BSA. A similar reaction may explain why p,p‘-biphenoquinone was not detected when phenol was oxidized by bone marrow homogenate, but was detected when oxidized by partially purified myeloperoxidase (Sawahata et al. 1985). Furthermore, we recently reported that the peroxidase-catalysed oxidation of p, p’biphenol in the presence of GSH results in the formation of covalent conjugates with the thiol group (McGirr et al. 1986). The major conjugate formed was identified as 3S-(glutathion-y1)-p, p’-biphenol. Furthermore, the aqueous layer after ethyl acetate extraction of the protease digested BSA bound with oxidized phenol products, showed an absorption peak at 257 nm, which is characteristic of a p,p‘-biphenolGSH conjugate (McGirr et al. 1986). This indicates that the covalent interactions with BSA may involve reactions of p,p’-biphenoquinone with a cysteine residue. In conclusion, the results presented in this communication indicate that o,o‘biphenol is the major product formed when phenol is oxidized by bone marrow homogenate and hydrogen peroxide. The data are also consistent with the suggestion that further oxidation product(s) of o,o’-biphenol may account for most of the products binding non-covalently to protein. Some o,o’-biphenol oxidation products may also covalently interact with proteins. Sulphydryl groups appear not to be involved in the protein-binding by o,o’-biphenol oxidation products. p, p’-Biphenol oxidation, however, resulted in the depletion of protein thiols, presumably due to
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covalent interaction withp, p‘-biphenoquinone. Phenol metabolism by bone marrow peroxidases to toxic metabolites may play an important role in benzene-induced myelotoxicity and leukemogenesis.
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References DANNER, D. J., BRIGNAC, JR,P. J., and PATEL, V., 1973, The oxidation of phenol and its reaction product by horseradish peroxidase. Archives of Biochemistry and Biophysics, 156, 759-763. EASTMOND, D. A,, SMITH,M. T., Ruzo, L. O., and Ross, D., 1986, Metabolic activation of phenol by human myeloperoxidase and horseradish peroxidase. Molecular Pharmacology, 30, 67M79. JOLLOW, D. J., MITCHELL, J. R., POTTER, W. Z., DAVIS,D. C., GILLETTE, J. R., and BRODIE, B. B., 1973, Acetaminophen-induced hepatic necrosis. II-Role of covalent binding in wivo. Journal of Pharmacology and Experimental Therapy, 187, 195-202. KARIYA, K., LEE, E., HIROUCHI, M., HOSOKAWA, M., and SAYO,H., 1987, Purification and some properties of peroxidases of rat bone marrow. Biochimica et Biophysica Acta, 99, 95-101. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J., 1951,Protein measurement with the folin-phenol reagent. Journal of Biological Chemistry, 193, 265-275. MCGIRR,L. G., SUBRAHMANYAM, V. V., MOORE, G. R., and O’BRIEN, P. J., 1986, Peroxidase-catalyzed3S-(glutathion-y1)-p,p’-biphenolformation. Chemico-Biological Interactions, 60,85-99. O’BRIEN, P. J., SUBRAHMANYAM, V. V., GREGORY, B., FOURNEY, R., DAVIDSON, W. S., RAHIMTULA, A. D., and TSURUTA, Y., 1985, One-electron oxidation in cell toxicity. In Microsomes and Drug Oxidations, edited by A. R. Boobis, J. Caldwell, F. Dematties and C. R. Elcombe (London: Taylor & Francis), pp. 284-295. SAUNDERS, B. C., HOLMER-SIEDLE, A. G., and STARK, B. P. (eds), 1964, Peroxidase (Washington, DC: Butterworth). SAWAHATA, T., and NEAL, R. A,, 1982, Horseradish peroxidase-mediated oxidation of phenol. Biochemical and biophysical Research Communications, 109. 988-994. SAWAHATA, T., RICKERT, D. E., and GREENLEE, W. F., 1985, Metabolism ofbenzene and its metabolites in bone marrow. In Toxicology of the Blood and Bone Marrow, edited by R. D. Irons (New York: Raven Press), pp. 141-148. SEDLAK, J., and LINDSAY, R. H., 1968, Estimation of total, protein-bound and non-protein sulfydryl groups in tissues with Ellman’s reagent. Analytical Biochemistry, 25, 192-205. SIVARAJAH, K., ANDERSON, M. W., and ELING,T . E., 1978, Metabolism of benzo(a)pyrene to reactive intermediates via prostaglandin biosynthesis. Life Sciences, 25, 2571-2578. SMITH,M. T., YAGER, J. W., STEINMETZ, K. L., and EASTMOND, D. A., 1989, Peroxidase-dependent metabolism of benzene’s phenolic metabolites and its potential role in benzene toxicity and carcinogenicity. Environmental Health Perspectives, 82, 23-29. SUBRAHMANYAM, V. V., and O’BRIEN,P. f . , 1985 a, Peroxidase-catalysed phenol binding to DNA. Xenobiotica, 15, 859-871. SUBRAHMANYAM, V. V., and O’BRIEN,P. J., 1985b, Phenol oxidation product (s), formed by a peroxidase reaction, that bind to DNA. Xenobiotica, 15, 873-885. SUBRAHMANYAM, V. V., and OIBRIEN, P. J., 1985c, Peroxidase-catalyzed oxygen activation by arylamine carcinogens and phenol. Chemico-Biological Interactions, 56, 185-1 99.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Peroxidase/hydrogen peroxide—or bone marrow homogenate/hydrogen peroxide—mediated activation of phenol and binding to protein V. V. Subrahmanyam, L. G. McGirr & P. J. O'brien To cite this article: V. V. Subrahmanyam, L. G. McGirr & P. J. O'brien (1990) Peroxidase/ hydrogen peroxide—or bone marrow homogenate/hydrogen peroxide—mediated activation of phenol and binding to protein, Xenobiotica, 20:12, 1369-1378 To link to this article: http://dx.doi.org/10.3109/00498259009046635
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Date: 09 November 2015, At: 06:22
XENOBIOTICA,
1990, VOL. 20,
NO.
12, 1369-1378
Peroxidase/hydrogen peroxide-or bone marrow homogenate/hydrogen peroxide-mediated activation of phenol and binding to protein V. V. SUBRAHMANYAMT, L. G. McGIRR and P. J. O'BRIENS Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 1Al
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Received 17 October 1989; accepted 5 April 1990
1. "C-Phenol was metabolized by rat bone marrow homogenate and H,O,. The homogenate catalyst, however, was inactivated by preincubation with H,O,, presumably due to inactivation of the enzyme(s) involved in phenol metabolism. 2. The majority of the metabolized I4C-phenol was bound to bone marrow proteins. o,o'-Biphenol and p,p'-biphenol were the principal non-protein-bound products. Ascorbate was unable to remove phenol oxidation products bound to protein, although o,o'biphenol recovery from the reaction mixture was markedly enhanced. Prior alkylation of protein thiols with N-ethylmaleimide decreased the binding of '*C-phenol oxidation products to bone marrow proteins by only 10-20%.
3. '*C-Phenol (200pM) metabolism by horseradish peroxidase (1Opg) and H,O, (200p ~also ) resulted in extensive binding to externally added bovine serum albumin. The absorption spectrum of "C-phenol oxidation products bound to bovine serum albumin was similar to that of bound oxidation products of o,d-biphenol but not of p,p'-biphenol.
4. Protease digestion of bovine serum albumin bound 14C-phenol oxidation products, followed by ethyl acetate extraction, extracted 75% of the '*C, indicating that most of the binding is probably non-covalent. Up to 32% of the '4C-phenol oxidation products binding to bovine serum albumin may be covalent, since derivation with dinitrofluorobenzene and extraction under acid, but not alkaline, conditions extracted the I4C. The percentage of metabolites covalently bound to bovine serum albumin was increased to 59% when horseradish peroxidase concentration was decreased to 0 2pg.
5. The thiol groups of bovine serum albumin were unaffected by o,o'-biphenol oxidation products, slightly decreased by phenol oxidation products, but were completely depleted by p, p'-biphenol oxidation products. 6. These results indicate that o,o'-biphenol oxidation products are responsible for much of the 14C-phenol binding to protein.
Introduction Peroxidases are well known to oxidize a wide variety of phenols (Saunders 1964). Recently, peroxidase activities in bone marrow and zymbal gland have been implicated in the activation of phenol to reactive metabolites which may have some role in benzene-induced carcinogenicity or toxicity (O'Brien et al. 1985, Smith et al. 1989). We have previously shown that phenol can be activated by myeloperoxidase in intact polymorphonuclear leucocytes in the presence of phorbol-myristate-acetate (a tumour promoter), to reactive metabolites that bind to nuclear DNA in the leucocyte (O'Brien et al. 1985). We and others have also shown that phenol metabolism by purified horseradish peroxidase and myeloperoxidase results in the formation of o,o'-biphenol and p,p'-biphenol (Danner et al. 1973, Eastmond et al. 1986, Sawahata and Neal 1982, Subrahmanyam and O'Brien 1985 a, b), the former
t Present address: Department of Biomedical and Environmental Health Sciences, School of Public Health, 322 Warren Hall, University of California, Berkeley, CA 94720,USA. $ T o whom correspondence should be addressed. 0049-8254/90 $ 3 0 0
01990 Taylor & Francis Ltd.
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being the major metabolite (Subrahmanyam and O’Brien 1985 a, b). Further oxidation of o,o’-biphenol but not p, p‘-biphenol leads to extensive binding to calf thymus DNA. p,p’-Biphenol can be further oxidized top, p’-biphenoquinone which forms a covalent conjugate with glutathione to form 3-S(-glutathion-yl)-p,pbiphenol (Eastmond et al. 1986, McGirr et al. 1986). Recently, Kariya et al. (1987) found that rat bone marrow contains at least two different peroxidases, namely myeloperoxidase and eosinophil peroxidase. Sawahata and colleagues (1982, 1985) showed that phenol was activated by bone marrow homogenate and hydrogen peroxide to products of which o,o‘-biphenol and p, p‘biphenol represented about 5%, indicating that peroxidatic metabolism of phenol indeed occurs in bone marrow. No other metabolites were found, but extensive binding to bone marrow protein was observed. They suggested that p,p’biphenoquinone may be the major reactive metabolite binding to protein, but the detailed mechanisms of phenol activation by bone marrow peroxidases are not clear. This has prompted us to further investigate phenol activation by bone marrow homogenate in order to find out the nature of the products involved in protein binding.
Materials and methods Materials Phenol, o,o’-biphenol, p, p’-biphenol, glutathione (GSH), oxidized glutathione (GSSG), horseradish peroxidase (HRP) type VI, ascorbic acid, protease (type XI), N-ethylmaleimide, dinitrofluorobenzene (DNFB) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Hydrogen peroxide (30% w/v) was obtained from Fisher Chemical Company (Toronto, O N T , Canada). Ur14C1Phen~l(71.1 mCilmmo1~was Durchased from Amersham Chemical Co. (Oakville. ONT. Canada) with a staied radiochemical puriiy of 99%. L
A
Preparation of bone marrow homogenate Bone marrow cells were isolated from male Sprague-Dawley rats (200-300 g) and a homogenate was prepared as described previously (McGirr et al. 1986). Analysis of ‘‘C-phenol metabolism by bone marrow homogenate 14C-Phenol (0.2 mM) was incubated with bone marrow homogenate (&200pg) and HzOz (&lo mM) in 3 ml of 0.1 M Tris-HC1 buffer pH 7.4 at 37°C. Reactions were started by the addition of H,Oz. After appropriate times of incubation the reactions were terminated by extraction with ethyl acetate (3 ml x 3). The ethyl acetate extracts were pooled and concentrated to 0 5 ml under N,. The product analysis was carried out either by t.1.c. or by h.p.1.c. as described previously (Subrahmanyam and O’Brien 1985 a, b). Quantitative determination of irreversible binding to protein Quantitative determination of irreversible binding to protein was performed as described by Sivarajah et al. (1978). The protein in the aqueous layer was precipitated with 100% trichloroacetic acid (TCA, final concn lo%),washed with 10% T C A (1ml) and the protein, after centrifugation, was extracted twice with methanol-water (4 : 1 v/v) and chloroform-methanol (2 : 1 v/v). The washed protein was solubilized in 1 ml of 0.1 M NaOH, radioactivity was then determined by scintillation counting and the amount of protein was determined by the method of Lowry et al. (1951). Approximately 7 5 4 5 % of protein was always recovered. The nature of protein binding The protein samples obtained after methanol extractions, as described above, were further examined to determine how strongly the products are bound to protein, using the method described by Jollow et al. (1973). The protein samples were suspended in 3 ml of 0 1 M Tris-HC1 buffer p H 7.4 and digested with protease for 12 h at 37°C. Maximal digestion occurred within 12 h, after which further incubation had no effect on the amount digested. The digest was extracted with ethyl acetate ( 3 ml x 2) and the organic layers were removed. T h e aqueous layers were bubbled with N, for a few minutes (to evaporate excess ethyl acetate), and an aliquot was removed for measuring the radioactivity. Further aliquots which were previously extracted with methanol and digested with protease, were mixed with DNFB (5% w/v) in ethanol and left for 4 h at 25°C in the dark, with occasional stirring. The
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pH was adjusted to 9.0 by addition of NaHCO, and the reaction mixtures were extracted with 3 ml ethyl acetate (twice). An aliquot of the aqueous layer was measured for radioactivity. The remaining aqueous ~ and the reaction mixtures were further extracted with ethyl layers were acidified to pH 1.0 with 6 0 HCI acetate (3 ml x 2). The radioactivity in the ethyl acetate extract was determined.
Protein thiols Quantitative determination of protein thiols was performed according to the method of Sedlak and Lindsay (1 968) with slight modifications.Two-millilitre aliquots of reaction mixtures were added to tubes containing 1OOpl of 70% perchloric acid to precipitate proteins. The samples were then centrifuged at 2500 rpm for 5 min, and the supernatant discarded. The pellet was then washed twice with 3 ml of 5% perchloric acid, mixing well, centrifuging at 2500rpm for 5min and discarding the supernatant. Three millilitres of 0 5 M Tris buffer, containing 3% sodium dodecyl sulphate (SDS) at pH 8.0, was then added to the pellet and mixed well until all the protein was solubilized. One millilitre of this solution was processed as blank, while another 1ml aliquot was processed as the sample. For the blank, l00pl of N-ethylmaleimide (1 mM in Tris/SDS buffer) was added, mixed well and allowed to react for 15 min. This N-ethylmaleimide treatment alkylated all sulphydryl groups, thus acid (200p ~in) serving as an internal blank for each sample. Then 100pl of 5,5’-dithiobis-2-nitrobenzoic Tris/SDS buffer) was added to each sample and blank pair, and left in the dark for 15 min to allow it to react with SH groups. One millilitre of distilled water was then added to both the blank and the sample, and the absorbance of each sample was read against the appropriate blank at 412 nm. Protein thiol values were compared with GSH standards, which were prepared and treated as described above.
Results Preliminary studies showed that phenol metabolism was dependent on bone marrow protein concentration, the rate of phenol metabolism increased with increasing protein concentration. With 200 nmol phenol, 25 pg bone marrow protein and 10mM H,02, only 15f4nmol of phenol equivalents were bound per mg protein. Increasing the protein concentration to 200 pg, the binding of phenol equivalents to bone marrow protein increased to 57 f6 nmollmg protein. Figure 1 shows that the amount of 14C-phenol bound to protein is also proportional to the concentration of H 2 0 2(up to 10 mM). The need for a large excess of H , 0 2 indicates that most of the H , 0 2 may be decomposed by catalase in the bone marrow homogenate. Figure 1 also shows that preincubation, for Smin, of bone marrow homogenate with H20, results in inhibition of protein binding, presumably by inactivating the enzyme(s) involved in phenol activation. Table 1 shows the time-course for the metabolism and protein binding of 14Cphenol activated by bone marrow homogenate and H,O,. As shown in the table, 100%of the phenol was metabolized within 5 min. Approx. 7 5 4 5 % of the phenol was bound to bone marrow protein. Both o,o’-biphenol and p , p’-biphenol were formed from phenol. However, they accounted for less than 20% of the total phenol oxidized. It is possible that both the biphenols are further oxidized to products which bind to bone marrow protein. No hydroquinone or catechol formation was detected under these conditions. Addition of ascorbate at the end of the incubation period just before ethyl acetate extraction resulted in increased amounts of extractable o,o’-biphenol (table 1). A maximum of a four-fold increase in o,o’-biphenol formation was observed. Although small increases in p,p’-biphenol formation were also observed, the total p, p’biphenol formed was at least four-fold lower than the total o,o‘-biphenol formed, when compared at the 5 min time point where maximal yields of o,o‘-biphenol were obtained. These results therefore indicate that o,o‘-biphenol is the major metabolite formed from phenol during activation by bone marrow homogenate and H 2 0 z .The results in table 1 also show no decrease in protein-bound radioactivity in the presence of ascorbate, indicating that ascorbate is unable to remove the products already bound to the protein. Ascorbate therefore presumably acts by reducing the
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100
50
0
0
1
3
5
10
15
20
H202 concn. (rnM)
Figure 1.
Effect of different concentrations of hydrogen peroxide on protein-binding of I4C-phenol catalysed by bone marrow homogenate and hydrogen peroxide. ’‘C-Phenol (200 nmol) was incubated with bone marrow homogenate (200pg), H 2 0 2 (concentrationsas indicated) in 1 ml of 0.1 M Tris-HC1 buffer pH 7.4 at 37°C. 0, as described above; A , preincubated for 5 min with 1 mM H 2 0 2 ; 0, preincubated for 5 min with 10 mM H 2 0 2before the addition of phenol and H 2 0 , .
o,o’-biphenol quinone product and p, p’-biphenoquinone back to o,o’-biphenol and p, p’-biphenol respectively. A minor unknown product with an R, = 0.00 was detected (both in the presence and in the absence of ascorbate), only at 5min of incubation. However, the amount formed appears to be unchanged compared to the amount formed in the absence of ascorbate. This compound was not eluted from the silica gel with ethyl acetate. A compound with similar properties, presumably a polymer, was formed in previous studies when horseradish peroxidase (HRP) was used instead of bone marrow homogenate (Subrahmanyam and O’Brien 1985 a, b). Preincubation of bone marrow homogenate with N-ethylmaleimide (1 mM), and then dialysing to remove excess N-ethylmaleimide, decreased protein thiols by 90%. However, protein binding of 14C-phenol oxidation products was decreased by only 10-20%, indicating that the majority of the binding of phenol oxidation products to protein may not involve sulphydryl groups. T o further investigate how strongly phenol oxidation products are bound to protein, oxidation of phenol by H R P and H 2 0 2 in the presence of BSA was investigated. Figure 2 shows the spectral properties of phenol oxidation products bound to BSA. In the absence of BSA, phenol oxidation product(s) had absorbance maxima at 210 and 399 nm and a shoulder in the 2 4 6 2 5 0 nm region. In the presence of BSA, phenol oxidation product(s) had absorbance maxima at 242 and 294nm. T h e reaction mixtures were extracted with ethyl acetate, after which the protein in the aqueous layer was precipitated with T C A (final concentration 10%);the protein pellet was redissolved in 3 ml of 0.1 M Tris-HC1 buffer p H 7.4 and re-scanned. T h e products bound to protein were not removed during extraction and precipitation, as evidenced by the presence of absorbance maxima at 242 and 294 nm. Figure 2 also
2f0
1 f0 0 0
2+0
1+ o 0 0
6+2
o,o’-Biphenol
p, p‘-Biphenol Unknown 1 Protein bound
Phenol oxidized
15+3
1+o 0 4f2
2+0
None
15+3
1 +o 0 4+ 1
3+0
Ascorbate
30 s
27+3
1 +o 0 22*4
2+0
None
26f4
1 +o 0 20f4
420
Ascorbate
1 min
6825
1 f0 0 61f6
3+0
None
67+5
2fO 0 5826
6+0
Ascorbate
2 min
100
2+0 1f O . 5 84+8
4+0
None
100
4+0 1 fO.5 78+7
16+0
Ascorbate
5 min
o,o’-Biphenol and p,p’-biphenol were quantified by h.p.1.c. and t.l.c., as described in Materials and methods. Unknown 1 was quantified from its radioactivity on t.1.c. Protein binding was determined as described in Materials and methods. Incubation conditions: phenol ( 0 2 m ~was ) incubated with 200pg bone marrow homogenate and 1OmM H,O, at 37°C in 1 ml of 0 1 M Tris-HCI buffer pH7.4. Note: ascorbic acid (1 mM) was added at the end of various incubation times, before extraction with ethyl acetate. Mean values fSEM for three experiments are given. The experiments were performed in triplicate on the same day from one preparation of bone marrow homogenate, in order to reduce the statistical variation due to experimental errors.
6f2
Ascorbate
None
Product formed
15s
Table 1. Time-course for 14C-phenol metabolism by bone marrow homogenate (percentage of 14C incorporated into products).
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h
ir
< b
t? s* 3
3
a
Q
0
a g
3
b
8
B
3
5 z
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1.0
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0.5
3g9
.-..
..: ..: : -.. ...
200
300
400
!
Wavelength (nm) Figure 2.
Spectral properties of phenol or o,o’-biphenol oxidation products bound to bovine serum albumin (BSA).
......, Phenol ( 1 0 0 p ~ ) l H R P(10pg)/H,02 ( 1 0 0 ~ (absorbance ~) scale 0-1.0); -, phenol ( 1 0 0 p ~ ) / H R P(lOpg)/H,O, ( 1 0 0 p ~ ) / B S A(5 mg) (absorbance scale 0-20); ---, o,o’-biphenol ( 1 0 0 p ~ ) / H R P(l0pg) H 2 0 2 ( 1 0 0 p ~ ) / B S A(5 mg) (absorbance scale G20). Reactions were performed in 3ml of 0.1 M Tris-HCI buffer pH7.4 at room temp. After incubation for 30min, reaction mixtures containing BSA were extracted with ethyl acetate (twice) and the BSA was precipitated with T C A (10% final). BSA was redissolved in 3 ml of 0.1 M Tris-HC1 buffer pH 7.4 and scanned using a Shimadzu UV-240 spectrophotometer (reference cuvettes contained equal amount of BSA). Reaction mixtures containing no BSA, were also scanned after 30min of incubation.
shows the spectrum for o,o’-biphenol oxidation products bound to BSA. The spectrum shows absorbance maxima at 242 and 294 nm, indicating that the products binding to protein during o,o’-biphenol oxidation are similar to that found when phenol oxidation binds to protein. On the other hand, p,p’-biphenol oxidation products bound to BSA have an absorbance maximum at 259nm (McGirr et al. 1986). Table 2 shows the effects of protease digestion and DNFB derivatization of the resulting amino acids, on the I4C-phenol oxidation products bound to BSA.Before protease digestion (column A), approximately 75% of the I4C remained in the aqueous layer after ethyl acetate extractions, if phenol was incubated with 10 pg HRP and equimolar H,O,. To decrease the rate of phenol polymerization we used 0.2 pg HRP and determined whether this would increase the covalently bound metabolites to protein. Only 30% of the 14C remained in the aqueous layer, after ethyl acetate
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Table 2. Effects of protease treatment and protease/DNFB treatment on bovine serum albumin containing bound phenol oxidation products. Percentage 14C' remaining in aqueous phase after extractions with ethyl acetateb
Incubation condition
Undigested Pronase Pronase digested end DNFB treated reaction digested pH 1.0 mixture reaction mixture pH 9 0
Phenol + 10pg HRP + 0 . 2 ~HRP+H,O, +lOpg HRP+HzOz
0.3 f 0.2 0.3*0.2 295525 741f5.8
0.3 f 0 2 0.3 50.2 176f2.4 242f3.4
0.3k0.2 03f02 166 3.2 24.4 & 6.0
0.3f0.2 03k0.2 1.2k0.6 21.k0.8
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~ ~ _ _ _
Phenol ( 2 0 0 ~ was ~ ) incubated with horseradish peroxidase (HRP) ( 0 . 2 ~or 1Opg) and H,O, (200p ~ in)3 ml of Tris-HC1 buffer pH 7 4 for 30 min. Controlsin the absenceof H,O, or in the absence of HRP and H,O, were included. 100% '*C represents the total '*C added to the incubation mixture. bThe rest of the '*C was recovered in the ethyl acetate. For detailed experimental procedures see Materials and methods. Mean values+ SEM for three experiments are given.
extractions, if phenol was incubated with 02pg HRP and equimolar HzOz. The remaining 14C was recovered in the ethyl acetate extracts. Digestion of the BSAbound phenol oxidation products with protease (column B) and extraction with ethyl acetate removed approximately 41% and 68% of the 14C bound to BSA into the ethyl acetate layer, with 0.2 pg HRP and 10pg HRP, respectively. This indicated that most of the products bound to BSA may be non-covalent in nature, and that decreasing the HRP concentration decreases the percentage of non-covalently bound metabolites to protein. We further investigated whether the 14Cremaining in the aqueous layers is due to covalent binding or phenol oxidation products to the amino acids. Amino acids from an aqueous solution can be extracted into an organic solvent following derivatization with DNFB and acidification. Thus, amino acids covalently bound to drug metabolites can be effectively isolated by this procedure (Jollow et al. 1973). Column C in table 2 shows that more than 95% of the 14Cwas extracted from the acidified reaction mixtures containing BSA bound with phenol oxidation products, after digestion with protease and derivatization with DNFB. However, DNFB treatment and extraction under alkaline conditions did not increase the amount of 14Cextracted from the aqueous layers, compared to that extracted from the reaction mixtures treated with DNFB. This indicates that the binding of some fraction of the phenol oxidation products to BSA may be covalent. Thus, when phenol was oxidized with 10pg and 0 2 pg HRP respectively, approximately 32% and 59% of the products bound to BSA were covalent in nature. The presence of GSH in the reaction mixture prevented the binding of 14Cphenol oxidation products to protein (data not shown). We have, however, previously shown that GSH also prevents peroxidase-catalysed phenol oxidation to dimers, presumably by reducing the phenoxy radicals back to phenol (Subrahmanyam and O'Brien 1985~).In the absence of phenol, little GSH oxidation occurred, indicating that inhibition of peroxidase-mediated oxidation of phenol by GSH was not due to its ability to act as a competitive peroxidase substrate. However, all the oxidized GSH in the presence of phenol was recovered as GSSC, indicating that GSH does not covalently interact with phenoxy radicals. Table 3 compares the effectiveness of phenol, o,o'-biphenol or p,p'-biphenol oxidation
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Table 3. Reaction of phenol, o,o’-biphenoland p,p’-biphenol oxidation products with thiol groups of bovine serum albumin. Treatment
nmol-SH/mg protein
None Phenol hydrogen peroxide o,o’-Biphenol hydrogen peroxide +p,p’-Biphenol +hydrogen peroxide +Hydrogen peroxide’ +N-Ethylmaleimideb
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+ +
+
+
98f05 7.4f0.3 8 8 f0 5 1.4f0.3 9.5 & 0.6 0 3 f0 2
Reaction mixtures of 3 ml of 0.1 M Tris-HC1 buffer pH7.4 contained: 5 O p ~phenol (or PM p,p‘-biphenol or 2 5 p o,o’-biphenol), ~ 1 equiv. of H,O, and lOyg HRP. The reaction mixtures were incubated for 30min at 22°C. ‘H,O, ( 5 0 ~ ~ ) . 3 ml of 0 1 M Tris-HC1 buffer pH 7.4, BSA (5mg) and N-ethylmaleimide (20 p ~ were ) incubated as above. Mean valuesf SEM for three experiments are given.
products in reacting with thiol groups on BSA. As shown, the thiol group of BSA reacted with p, p’-biphenol oxidation products as effectively as with Nethylmaleimide. However, the o,o’-biphenol oxidation products or H,O, were ineffective in decreasing the number of thiol groups. The small decrease in thiol groups, observed with phenol oxidation products, therefore, may best be attributed to the reaction with p, p’-biphenoquinone formed from phenol oxidation.
Discussion Sawahata and colleagues (1985) have proposed that p , 9’-biphenoquinone may be the major reactive product binding to protein when phenol is activated with bone marrow homogenate and hydrogen peroxide. However, the present results indicate that althoughp, p‘-biphenoquinone may be one of the reactive metabolites binding to protein it is not the major reactive metabolite. The three-fold increase in o,o‘biphenol formation, in the presence of ascorbate, indicates that the majority of the binding may be due to o,o‘-biphenol-derived reactive products. The possibility that phenoxy radicals may be directly involved in protein binding appears to be unlikely, because if the phenoxy radicals are directly binding to proteins, there should have been an inhibition or lag period in the formation of biphenols. However, both o,o’biphenol and p,p‘-biphenol were detected in the first 15 s of the reaction, at which time no protein binding was observed. Sawahata et al. (1985) also reported that p, p’biphenol was formed when phenol was incubated with hydrogen peroxide and partially purified myeloperoxidase, or with bone marrow homogenatehydrogen peroxide. They also found that p, p’-biphenoquinone was formed with myeloperoxidase but not with bone marrow, although the results shown in table 1 indicate that p, p’-biphenol and o,o’-biphenol are further oxidized by bone marrow homogenate. N-Ethylmaleimide (1 mM), a sulphydryl reagent, appears to decrease the binding of phenol oxidation product(s) to proteins in bone marrow homogenate by approximately lo%, indicating that groups other than thiols are involved, or that the binding might not be covalent. In order to understand the nature of interaction between phenol metabolites and proteins in more detail, the oxidation of phenol by peroxidase and hydrogen peroxide in the presence of BSA was investigated (tables 2 and 3). In support of non-covalent interactions it was found that most of the product (s) bound to BSA was released when BSA (containing bound phenol oxidation
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products) was digested with protease, and the released product(s) were extracted into ethyl acetate. This implies that the majority of the binding of phenol oxidation products to BSA may be through non-covalent interactions. It was proposed earlier (Subrahmanyam and O’Brien 1985 b) that binding of phenol oxidation products to DNA may be non-covalent and that the binding of phenol oxidation products may occur via charge-transfer complexes. The product(s) released by the enzymic hydrolysis of DNA, however, were not extracted into butanol (or ethyl acetate) but stayed at the interphase between the organic and aqueous layers. Interestingly, the protease digestion of BSA (with bound 14C-phenol oxidation products) released products which were extracted into ethyl acetate. If BSA was absent from the reaction mixture, the phenol was oxidized to insoluble ‘polymeric’ products which also were not extracted into ethyl acetate but stayed at the interphase of the organic and aqueous layers. This indicates that BSA prevents the precipitation of the phenol ‘polymeric’ oxidation products or binds the soluble ‘polymeric’ products. Some fraction of the bound products was not released from BSA when BSA was digested with protease. However, bound products were extracted into ethyl acetate when the digested BSA was treated with DNFB and acidified. This implies that these metabolites were covalently linked to amino acid residues. The fraction of covalently bound products increased with decreasing HRP concentrations. The nature of the covalently bound metabolites is not clear, although o,o‘-biphenol oxidation products may represent a major part of this fraction. However, our results also indicated that o,o’-biphenol oxidation products do not appear to bind covalently to sulphydryl groups. Whether other functional groups such as amino groups on proteins can covalently interact with o,o’-biphenol oxidation products requires further investigation. Table 3 shows that p,p’-biphenol oxidation product(s) react readily with thiol groups on BSA. However, o,o‘-biphenol oxidation product(s) react poorly with thiol groups on BSA, although phenol oxidation product(s) reacted more readily with BSA thiols. It is therefore tempting to postulate that p,p’-biphenoquinone (the further oxidation product of p, p’-biphenol) formed from phenol could be reacting with thiol groups on BSA. A similar reaction may explain why p,p‘-biphenoquinone was not detected when phenol was oxidized by bone marrow homogenate, but was detected when oxidized by partially purified myeloperoxidase (Sawahata et al. 1985). Furthermore, we recently reported that the peroxidase-catalysed oxidation of p, p’biphenol in the presence of GSH results in the formation of covalent conjugates with the thiol group (McGirr et al. 1986). The major conjugate formed was identified as 3S-(glutathion-y1)-p, p’-biphenol. Furthermore, the aqueous layer after ethyl acetate extraction of the protease digested BSA bound with oxidized phenol products, showed an absorption peak at 257 nm, which is characteristic of a p,p‘-biphenolGSH conjugate (McGirr et al. 1986). This indicates that the covalent interactions with BSA may involve reactions of p,p’-biphenoquinone with a cysteine residue. In conclusion, the results presented in this communication indicate that o,o‘biphenol is the major product formed when phenol is oxidized by bone marrow homogenate and hydrogen peroxide. The data are also consistent with the suggestion that further oxidation product(s) of o,o’-biphenol may account for most of the products binding non-covalently to protein. Some o,o’-biphenol oxidation products may also covalently interact with proteins. Sulphydryl groups appear not to be involved in the protein-binding by o,o’-biphenol oxidation products. p, p’-Biphenol oxidation, however, resulted in the depletion of protein thiols, presumably due to
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covalent interaction withp, p‘-biphenoquinone. Phenol metabolism by bone marrow peroxidases to toxic metabolites may play an important role in benzene-induced myelotoxicity and leukemogenesis.
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