We optimized and validated a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of six metabolites of homocysteine metabolism: homocysteine, methionine, cysteine, S-adenosylmethionine, S-adenosylhomocysteine and betaine. The detection limits for these metabolites were in the nanomolar range, and the intra- and inter-day precisions were lower than 20% of the relative standard deviations. The method was specifically designed for the determination of the intracellular concentrations of the metabolites in cultured cells. To study the role of betaine-homocysteine S-methyltransferase (BHMT), HepG2 cells and HepG2 cells that were stably transfected with BHMT ((BHMT) HepG2) were treated with homocysteine or with a specific inhibitor of BHMT, and metabolite levels were subsequently measured. Severely compromised methyl group metabolism in the HepG2 cells, which is typical of cancer-derived cells, prevented clear evaluation of the changes caused by the external manipulations of homocysteine metabolism. However, the ease of handling these cells and the almost unlimited source of experimental material supplied by cells in permanent culture allowed us to develop a reliable methodology. The precautions concerning intracellular metabolite determinations using LC-MS/MS in cultured cells that are expressed in this work will have global validity for future metabolomics studies.

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,N(4)-benzetheno-2'-deoxycytidine (DCBQ) and 7-hydroxy-1,N(2)-benzetheno-2'-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,N(4)-benzethenocytosine (CBQ) and 7-hydroxy-1,N(2)-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,N(2)-benzethenoguanine OGBQ1. It coeluted with the product obtained by the reaction of HQ and p-BQ mixture with 8-hydroxy-2'-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.

Metabolism of benzene, an important environmental and industrial carcinogen, produces three electrophilic intermediates, namely, benzene oxide and 1,2- and 1,4-benzoquinone, capable of reacting with the DNA. Numerous DNA adducts formed by these metabolites in vitro have been reported in the literature, but only one of them was hitherto identified in vivo. In a search for urinary DNA adducts, specific LC-ESI-MS methods have been developed for the determination in urine of six nucleobase adducts, namely, 7-phenylguanine, 3-phenyladenine, 3-hydroxy-3,N(4) -benzethenocytosine, N(2) -(4-hydroxyphenyl)guanine, 7-(3,4-dihydroxyphenyl)guanine and 3-(3,4-dihydroxyphenyl)-adenine (DHPA), with detection limits of 200, 10, 260, 50, 400 and 200 pg ml(-1) , respectively. Mice were exposed to benzene vapors at concentrations of 900 and 1800 mg m(-3) , 6 h per day for 15 consecutive days. The only adduct detected in their urine was DHPA. It was found in eight out of 30 urine samples from the high-exposure group at concentrations of 352 ± 146 pg ml(-1) (mean ± SD; n = 8), whereas urines from the low-exposure group were negative. Assuming the DHPA concentration in the negative samples to be half of the detection limit, conversion of benzene to DHPA was estimated to 2.2 × 10(-6) % of the absorbed dose. Thus, despite the known high mutagenic and carcinogenic potential of benzene, only traces of a single DNA adduct in urine were detected. In conclusion, DHPA is an easily depurinating adduct, thus allowing indication of only high recent exposure to benzene, but not long-term damage to DNA in tissues.

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