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Quantitative determination of the hydrolysis products from proteins and DNA gives valuable information regarding the reactive metabolite that forms the protein and DNA adduct. Quantification of protein−benzo[a]pyrene (BP) adducts represents a more sensitive method than quantification of BP−DNA adducts. The aim of the present study was to identify two hydrolysis products from BP-derived protein adducts found in vitro and in vivo in a previous study. Male Wistar rats were injected i.p. with BP, and serum albumin was isolated and subjected to acid hydrolysis at 70 °C for 3 h. The hydrolysate was subjected to LC separation, and fractions of the two unknown compounds were collected. The molecular masses of the two unknown compounds were in accordance with being tetrols as judged by LC electrospray mass spectrometry. The fragmentation patterns were characteristic of tetrols with formation of the molecular ion and the loss of water molecules. In addition, the compounds were subjected to acid hydrolysis at 70 °C with 0.1 M HCl for 3 h. We observed that two of the known tetrols epimerized to the two unknown tetrols and vice versa. This is probably a characteristic epimerization involving not only position C10−OH but also another site like position C7−OH. The in vivo findings of the two unknown adducts are probably the result of the formation of BPDE III in the metabolism of BP. These two tetrols must then have the C7−OH and C8−OH groups in a cis position.

The present work displays capillary liquid chromatographic column switching methodology tailored for determination of benzoapyrene tetrol isomers in biological matrices using on-line fluorescence and μ-electrospray ionization mass spectrometric detection. A well-established off-line crude solid phase extraction procedure was used in order to make the method compatible with several biological matrices. The solid phase extraction eluates were evaporated to dryness, redissolved in 1.0 ml methanol : water 10 : 90, vv, loaded onto a 0.32 mm I.D. × 40 mm 5 µm Kromasil C18pre-column for analyte enrichment and back-flushed elution onto a 0.30 mm I.D. × 150 mm 3.5 µm Kromasil C18analytical column. The samples were loaded with a flow rate of 50 µl min−1and the tetrols were separated at a flow rate of 4 µl min−1with an acetonitrile : 10 mM ammonium acetate gradient from 10 to 90. A sample loading flow rate up to 50 µl min−1was allowed. The fluorescence excitation and emission were set to 342 and 385 nm, respectively, while mass spectrometric detection of the benzoapyrene tetrols was obtained by monitoring their M − H−molecular ions at mz319. The method was validated over the concentration range 0.1–50 ng ml−1benzoapyrene tetrols in a cell culture medium with 100 µl injection volume, fluorescence detection and the first eluting tetrol isomer as model compound, resulting in a correlation coefficient of 0.993. The within-assay n 6 and between-assay n 6 precisions were determined to 2.6–8.6 and 3.8–9.6, respectively, and the recoveries were determined to 97.9–102.4 within the investigated concentration range. The mass limit of detection by fluorescence was 3 pg for all the tetrol isomers, corresponding to a concentration limit of detection of 30 pg ml−1cell culture medium. The corresponding mass spectrometric mass limits of detection were 4–10 pg, corresponding to concentration limits of detection of 40–100 pg ml−1cell culture medium.

Studies suggest that resveratrol (trans-3,4',5-trihydroxystilbene), which is a diphenolic antioxidant found in plants and foods, has cancer chemopreventive and chemotherapeutic potential. A lower risk of lung cancer among consumers of wine compared with consumers of other beverages has been observed, which may be partly attributed to the high content of resveratrol particularly in red wine. We have studied the effect of resveratrol on the expression of genes involved in the metabolism of polycyclic aromatic hydrocarbons in the human bronchial epithelial cell line BEP2D. Expression of the cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1), microsomal epoxide hydrolase (mEH), and glutathione S-transferase P1 (GSTP1) genes was measured by quantitative reverse transcriptase-polymerase chain reaction. The cells were treated either with benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin in the presence or absence of resveratrol. Resveratrol inhibited both the constitutive and the induced expression of CYP1A1 and CYP1B1 in a dose-dependent manner. In contrast, the expression of the mEH gene was increased in response to resveratrol and no change in the expression of GSTP1 was found. The altered gene expression in response to resveratrol was reflected in a reduced overall level of benzo[a]pyrene metabolism. These data indicate that resveratrol may exert lung cancer chemopreventive activity through altering the expression of genes involved in the metabolism of polycyclic aromatic hydrocarbons, resulting in altered formation of carcinogenic benzo[a]pyrene metabolites in human bronchial epithelial cells. © 2001 Wiley-Liss, Inc.

1Osmolal and anion gaps are helpful in the diagnosis and evaluation of intoxications with methanol and ethylene glycol. Reported reference values for osmolal gap and anion gap are -1 (± 6) mosm kg-1H2O and 16 (± 2) mmol I -1, respectively. However, we have repeatedly found unexplained increased gaps in patients admitted to our department, and the relevance of the established reference values has been questioned.2Osmolal and anion gaps were determined in an unselected population of patients consecutively admitted to an emergency medical department. In the case of unexplained gaps, the blood samples were analysed with respect to the presence of alcohols and organic acids.3We included all accessible patients admitted during 14 days. Appropriate blood samples were obtained in 177 patients (88 male, 89 female), with a mean age of 65 years (range 17-94).4The mean and (standard deviation) for osmolal and anion gaps in our material were 5.2 mosm kg-1H2O (7.0) and 12.9 mmol/l (4.2). Neither methanol nor ethylene-glycol was detected in serum from any patients. Small amounts of ethanol were found in 5 patients, and high lactate levels explained in part the most extensively increased anion gaps. However, the calculated analytical standard deviation accounted entirely for the variation in our material, and we suggest that the present reference values be adjusted.

We have examined DNA restriction fragment length polymorphisms (RFLP) of the Ha-ras-1 gene in DNA from 118 lung cancer patients and 123 unaffected controls. When DNA samples were digested with MspI/ HpaII restriction endonucleases. Southern blot analysis demonstrated 4 common, 4 intermediate and 7 different rare alleles in the combined population after hybridization to the pGDa1 probe. Six of the rare alleles were unique for the lung cancer group and 1 rare allele for the control group. The frequency of rare alleles in lung cancer patients (10/236) was significantly different (P<0.01) from the control group (1/246). The lung cancer group also had a significantly lower frequency of the common 2.57 kb fragment than the controls (P<0.02). The results thus indicate that Ha-ras genotyping may be of value in lung cancer risk assessment.

The CYP1A1, CYP2D6and GSTM1genes encode biotransforming enzymes involved in activation and detoxification of xenobiotics. Metabolically activated chemical compounds may interact with DNA and form adducts. In this study, the effect of the GSTM1, CYP1A1exon 7 and CYP2D6polymorphisms on DNA adduct levels was studied in 170 healthy volunteers. DNA adducts levels were measured by 32P-postlabelling in mononuclear white blood cells (WBC, lymphocytes and monocytes) and granulocytes collected in summer and winter. The influence of the genotype on the level of DNA adducts in both types of WBCs was observed only in summer samples. Individuals with GSTM1deficient (null) genotype had significantly elevated level of adducts in mononuclear WBCs (p=0.045) and granulocytes (p=0.031) compared to GSTM1positives. Higher adduct levels in carriers of combined GSTM1(null)/CYP1A1-Ile/Valgenotype were found in both types of WBCs when compared to GSTM1(+)/CYP1A1-Ile/Ilegenotype carriers (p=0.046 in granulocytes, p=0.092 in mononuclear WBCs). CYP2D6wild-type homozygotes (EMs) and heterozygotes (HEMs) were shown to have significantly higher mononuclear WBC DNA adduct levels than mutant homozygotes (PMs) (p=0.037 and p=0.014). When confounding factors associated with PAH exposure were taken into account a statistically significant effect of CYP1A1exon 7 polymorphism on DNA adduct levels was found (p=0.012 in mononuclear WBCs, p=0.043 in granulocytes). In a subgroup of current smokers (n=95) high DNA adduct levels in granulocytes were associated with GSTM1-(null)genotype, and increased adduct levels in mononuclear WBCs correlated with CYP2D6EM and HEM genotypes. In winter samples the association between the genotype and DNA adduct levels was not observed.

We have compared several biomarkers for polycyclic aromatic hydrocarbon (PAH) exposure among electrode paste plant workers and workers not occupationally exposed to PAH. The PAH exposure was quantitated from samples collected with person-attached sampling devices. The mean particulate PAH exposure level in the plant was 14.4 µgm3. The level of pyrene was significantly correlated with both PAH level and the level of selected carcinogenic PAHs in this type of exposure. The mean concentration of the biomarker 1-hydroxypyrene in the PAH exposed workers' urine was 6.98 µimol of 1-hydroxypyrene per mole of creatinine compared with 0.08 and 0.14 µimol of 1-hydroxypyrene per mole of creatinine in the two reference groups. PAH-DNA adducts were measured in DNA from white blood cells by the ultrasensitive enzyme radioimmunoassay (USERIA) and the 32Ppostlabeling technique. Only urinary 1-hydroxypyrene was significantly increased in the PAH-exposed group.

The metabolism of thymine, thymidine, uracil, and uridine has been investigated in five different strains of Acinetobacter calcoaceticus. Attempts to isolate thymine and thymidine auxotrophic mutants were not successful. Consistent with this finding was the observation that uptake of radioactive thymine or thymidine could not be demonstrated. Search for enzymes capable of transforming thymine via thymidine to thymidine-5′-monophosphate in crude extracts was performed, and the following enzymes were absent judging from enzyme assays: thymidine phosphorylase (EC 2.4.2.4), trans-N-deoxyribosylase (EC 2.4.2.6), and thymidine kinase (EC 2.7.1.21). The enzymes responsible for the phosphorylation of thymidine-5′-monophosphate to thymidine-5′-triphosphate were present in crude extracts. Radioactive uracil was readily incorporated into both ribonucleic acid and deoxyribonucleic acid, the ratio being 6:1, and radioactivity was found only in pyrimidine bases. No uptake of uridine could be demonstrated. Uridine-5′-monophosphate pyrophosphorylase (EC 2.4.2.9) activity was detected in crude extracts, suggesting that uracil is converted directly to uridine-5′-monophosphate which is then phosphorylated to uridine-5′-triphosphate or transformed to other ribo- and deoxypyrimidine nucleotides.

An otherwise healthy methanol-poisoned male was treated with ethanol and forced diuresis. No acidosis or visual symptoms occurred. His blood ethanol concentrations varied between 10.7 and 30.0 mmoll (0.49–1.38 gl). His maximal blood methanol concentration was 14.4 mmoll (0.45 gl) and his plasma formate levels were within reference range (0.11–0.29 mmoll). In a 24 h period where methanol metabolism was blocked by ethanol, the total body clearance and renal clearance of methanol were 11.3 and 5.7 mlmin, respectively. The difference, 5.6 mlmin, to a large extent reflects the pulmonary clearance of methanol thus being low in man.

Intoxications with the antifreeze constituent ethylene glycol (EG) occur infrequently, but may be fatal if not recognized and treated promptly. The aim of the present work was to develop an analytical method for rapid diagnosis of EG poisoning and for monitoring EG removal by hemodialysis. EG was measured by gas chromatography upon direct injection of serum or urine samples (50 µL diluted in 200 µL of distilled water containing 2,3-butanediol as internal standard). A 2-m × 2-mm glass column with Chromosorb 101 (80/100 mesh) separates these glycols within four minutes at 200°C using nitrogen as the carrier gas. The glycols 1,2- and 1,3-propanediol were separated from EG and the internal standard. Acetone, methanol, and isopropanol did not interfere with the analysis. The limit of quantitation of EG was close to 0.5mM. Because no derivatization, extraction, or concentration procedures were necessary, EG may be determined quantitatively within 30 min, allowing for monitoring of hemodialysis, which should be performed for 15 h in severe cases. The diagnosis of ethylene glycol intoxication in a late stage may be secured by analysis of urine collected on admission.