ARTICLE pubs.acs.org/crt
DNA Adducts Formed from p-Benzoquinone, an Electrophilic Metabolite of Benzene, Are Extensively Metabolized in Vivo Igor Linhart,*,† Petr Mikes,‡ Emil Frantík,§ and Jaroslav Mraz§ †
Department of Organic Chemistry, Faculty of Chemical Technology, Institute of Chemical Technology, Prague, Technicka 1905, CZ-166 28 Prague, Czech Republic ‡ Apigenex, Ltd., Podebradska 56, CZ-180 66 Prague, Czech Republic § National Institute of Public Health, Srobarova 48, CZ-100 42 Prague, Czech Republic ABSTRACT: Benzetheno adducts derived from p-benzoquinone (p-BQ), a reactive metabolite of benzene, were reported to be formed by the reaction of p-BQ with DNA in vitro but have never been detected either in vivo or in experiments with living cells. Two of them, 3-hydroxy-3,N4-benzetheno-20 -deoxycytidine (DCBQ) and 7-hydroxy-1,N2-benzetheno-20 -deoxyguanosine (DGBQ), were administered to rats by single ip injections at the doses of 2 mg/kg each. The excretion of unchanged compounds DCBQ and DGBQ within 2 days amounted to 8.2 ( 1.9 and 4.5 ( 1.2% (mean ( SE) of the dose, respectively. Additionally, deribosylated metabolites of DCBQ and DGBQ, 3-hydroxy-3,N4-benzethenocytosine (CBQ) and 7-hydroxy-1,N2-benzethenoguanine (GBQ), were found amounting to 45.7 ( 10.2 and 2.9 ( 2.1% of the dose, respectively. An additional portion of CBQ and GBQ was liberated from their corresponding conjugates by acidic hydrolysis. Therefore, total recoveries of CBQ and GBQ in urine were 82.1 ( 13.5 and 11.6 ( 5.1% of the dose. To identify conjugated metabolites, DCBQ and DGBQ were administered intraperitoneally at the doses 10.5 and 11.0 mg/kg, respectively, to one animal each. Glucuronides of DCBQ, DGBQ, and GBQ as well as sulfates of DGBQ, CBQ, and GBQ were identified by ESI-LC-MS according to (M - H)- ions and their fragmentation. In addition, two oxygenated metabolites and their corresponding conjugates were detected for DGBQ and GBQ. One of these metabolites was identified as 2,7-dihydroxy-1,N2-benzethenoguanine OGBQ1. It coeluted with the product obtained by the reaction of HQ and p-BQ mixture with 8-hydroxy-20 -deoxyguanosine followed by acid hydrolysis. These findings suggest that both DCBQ and DGBQ undergo extensive biotransformation in vivo. CBQ appears to be the only p-BQ derived DNA adduct, which can be efficiently recovered from its conjugates and might be therefore useful in molecular dosimetry of benzene.
’ INTRODUCTION Benzene is a widely used industrial chemical produced on a mass scale. It is emitted into the atmosphere as a vapor from many industrial processes and from automobile exhausts, the latter being the main source of environmental exposure. Benzene is a proven human and animal carcinogen. In humans, longterm exposure has been associated with bone marrow toxicity and leukemia.1-3 Metabolic activation of benzene is shown in Scheme 1. It proceeds via benzene oxide, formed mainly in the liver. This reactive intermediate is spontaneously rearranged to phenol, which is further hydroxylated by cytochrome P450 2E1 to hydroquinone (HQ) and catechol (CAT). Both HQ and CAT are transported to the bone marrow where they are further oxidized by myeloperoxidases to corresponding benzoquinones (BQ), which are electrophilic enough to attack nucleophilic sites in the DNA.4-6 Oxidation of HQ and CAT to p-BQ and o-BQ, respectively, proceeds via stabilized semiquinone radicals. This process has been linked to important toxic events in the bone marrow such as mutagenicity,6 induction of sister chromatid exchanges,7 and micronuclei.8 Altogether, four benzetheno adducts, two arising from in vitro reactions r 2011 American Chemical Society
of p-BQ with dC9,10 and dA10,11 and another two with dG,12-14 were hitherto identified. These adducts were also found when reacting p-BQ with DNA in vitro.9,11,13,14 The most abundant benzetheno adduct was that with dC followed by that with dA and two adducts with dG.14 Surprisingly, none of these adducts has been detected in vivo following exposure to benzene.4,15 Bodell et al. could not detect any of them in the DNA isolated from HL-60 cells treated with HQ or BQ by 32P-postlabeling,16,17 and to our knowledge, there is no report on the excretion of benzetheno adducts in urine. Possible explanation of these negative findings may lie in efficient biotransformation. To test this hypothesis, we decided to perform a biotransformation study on two synthesized DNA adducts in rats in vivo and to determine their recovery in urine.
’ EXPERIMENTAL PROCEDURES General. Acetonitril for LC/MS Chromasolv was from Riedel de Ha€en, methanol supragradient HPLC grade was from Scharlau, formic Received: October 1, 2010 Published: January 19, 2011 383
dx.doi.org/10.1021/tx1003408 | Chem. Res. Toxicol. 2011, 24, 383–391
Chemical Research in Toxicology
ARTICLE
318 f 202 f 184 (M þ H - deoxyribosyl - H2O, 174 (M þ H deoxyribosyl - CO)þ, 147 (M þ H - deoxyribosyl - C3H3O)þ 316 f 200 f 172 (M - H - deoxyribosyl - CO)-, 145 (M - H deoxyribosyl - C3H3O)-. 3-Hydroxy-3,N4-benzethenocytosine, (3-Hydroxypyrimido[1,6-a]benzimidazol-1(2H)-one, CBQ). DCBQ (10 mg, 31.5 μmol) was dissolved in 2 mL of 0.1 M HCl, and the solution was heated for 1 h at 95 °C. After cooling to room temperature, the reaction mixture was neutralized by diluted ammonia and evaporated to dryness in a vacuum. Column chromatography on silica gel using 17:3:3:2 ethyl acetateethanol-acetone-water as an eluent yielded 5 mg (25 μmol, 81%) of a greyish powder identified as CBQ. Rf = 0.67. UV absorption: λmax = 331 and 237 nm (pH 6). 1H NMR (DMSO-d6): δ = 6.49 (dd, J = 7.7 and 0.9 Hz, 1H, C10-H); 6.91 (ddd, J = 8.7, 2.6, and 0.9 Hz, 1H, C4-H); 7.3 (dd, J = 7.7 and 0.9 Hz, 1H, C9-H); 7.50 (d, 8.8 Hz, 1H, C5-H); 7.71 (d, J = 2.4 Hz, 1H, C2-H). ESI-MS (m/z): 202 (M þ H)þ, 200 (M - H)-; MS2: 202 f 184 (M þ H - H2O, 174 (M þ H - CO)þ, 147 (M þ H - C3H3O)þ, 200 f 172 (M - H - CO)-, 145 (M - H - C3H3O)-. 7-Hydroxy-1,N2-benzetheno-20 -deoxyguanosine (7-Hydroxy-3(20 -deoxyribosyl)-benzimidazo[1,2-a]purin-11-one, DGBQ). 20 -Deoxyguanosine monohydrate (500 mg, 1.75 mmol) and HQ (191 mg, 1.75 mmol) were dissolved in 24 mL of DMF, potassium carbonate (242 mg, 1.75 mmol) was added, and the resulting mixture was stirred at room temperature for 24 h. The solvent was distilled off in a vacuum (
