Carcinogenic polycyclic aromatic hydrocarbons induce CYP1A1 in human cells via a p53-dependent mechanism.

The tumour suppressor gene TP53 is mutated in more than 50 % of human tumours, making it one of the most important cancer genes. We have investigated the role of TP53 in cytochrome P450 (CYP)-mediated metabolic activation of three polycyclic aromatic hydrocarbons (PAHs) in a panel of isogenic colorectal HCT116 cells with differing TP53 status. Cells that were TP53(+/+), TP53(+/-), TP53(-/-), TP53(R248W/+) or TP53(R248W/-) were treated with benzo[a]pyrene (BaP), dibenz[a,h]anthracene and dibenzo[a,l]pyrene, and the formation of DNA adducts was measured by (32)P-postlabelling analysis. Each PAH formed significantly higher DNA adduct levels in TP53(+/+) cells than in the other cell lines. There were also significantly lower levels of PAH metabolites in the culture media of these other cell lines. Bypass of the need for metabolic activation by treating cells with the corresponding reactive PAH-diol-epoxide metabolites resulted in similar adduct levels in all cell lines, which confirms that the influence of p53 is on the metabolism of the parent PAHs. Western blotting showed that CYP1A1 protein expression was induced to much greater extent in TP53(+/+) cells than in the other cell lines. CYP1A1 is inducible via the aryl hydrocarbon receptor (AHR), but we did not find that expression of AHR was dependent on p53; rather, we found that BaP-induced CYP1A1 expression was regulated through p53 binding to a p53 response element in the CYP1A1 promoter region, thereby enhancing its transcription. This study demonstrates a new pathway for CYP1A1 induction by environmental PAHs and reveals an emerging role for p53 in xenobiotic metabolism.

Protective action of n-3 fatty acids on benzo[a]pyrene-induced apoptosis through the plasma membrane remodeling-dependent NHE1 pathway.

Plasma membrane is an early target of polycyclic aromatic hydrocarbons (PAH). We previously showed that the PAH prototype, benzo[a]pyrene (B[a]P), triggers apoptosis via DNA damage-induced p53 activation (genotoxic pathway) and via remodeling of the membrane cholesterol-rich microdomains called lipid rafts, leading to changes in pH homeostasis (non-genotoxic pathway). As omega-3 (n-3) fatty acids can affect membrane composition and function or hamper in vivo PAH genotoxicity, we hypothesized that addition of physiologically relevant levels of polyunsaturated n-3 fatty acids (PUFAs) might interfere with B[a]P-induced toxicity. The effects of two major PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), were tested on B[a]P cytotoxicity in the liver epithelial cell line F258. Both PUFAs reduced B[a]P-induced apoptosis. Surprisingly, pre-treatment with DHA increased the formation of reactive B[a]P metabolites, resulting in higher levels of B[a]P-DNA adducts. EPA had no apparent effect on B[a]P metabolism or related DNA damage. EPA and DHA prevented B[a]P-induced apoptotic alkalinization by affecting Na(+)/H(+) exchanger 1 activity. Thus, the inhibitory effects of omega-3 fatty acids on B[a]P-induced apoptosis involve a non-genotoxic pathway associated with plasma membrane remodeling. Our results suggest that dietary omega-3 fatty acids may have marked effects on the biological consequences of PAH exposure.

Proposal on limits for chemical exposure in saturation divers' working atmosphere: the case of benzene.

Saturation diving is performed under extreme environmental conditions. The divers are confined to a limited space for several weeks under high environmental pressure and elevated oxygen partial pressure. At present, divers are protected against chemical exposure by standard exposure limits only adjusted for the increased exposure length, i.e. from 8 to 24 hours a day and from 5 to 7 days a week. The objective of the present study was to indicate a procedure for derivation of occupational exposure limits for saturation diving, termed hyperbaric exposure limits (HEL). Using benzene as an example, a procedure is described that includes identification of the latest key documents, extensive literature search with defined exclusion criteria for the literature retrieved. Hematotoxicity and leukemia were defined as the critical effects, and exposure limits based upon concentration and cumulative exposure data and corresponding risks of leukemia were calculated. Possible interactions of high pressure, elevated pO2, and continuous exposure have been assessed, and incorporated in a final suggestion of a HEL for benzene. The procedure should be applicable for other relevant chemicals in the divers' breathing atmosphere. It is emphasized that the lack of interactions from pressure and oxygen indicated for benzene may be completely different for other chemicals.

Studies on ethylene glycol poisoning: one patient - 154 admissions.

OBJECTIVE: Fomepizole is the antidote of choice in toxic alcohol poisonings. Potential side effects from frequent use of fomepizole were studied in a patient admitted 154 times with ethylene glycol (EG) poisoning. The intra-individual correlation between the serum-ethylene glycol (serum-EG) and the osmolal gap (OG) EG-kinetics, and other laboratory parameters were also studied. METHODS: Combined pro- and retrospective collection of material from three different hospitals, and results from autopsy. RESULTS: A 26-year-old female with a dissociative disorder was admitted with EG poisoning a total of 154 times. Her admission data revealed a median pH of 7.31 (range 6.87-7.49), pCO(2): 4.2 kPa (1.2-6.7) (32 mmHg [9-50]), HCO-3: 15 mmol/L (4-26) (15 mEq/L [4-26]), base deficit (BD): 10 mmol/L (- 4 to 27) (10 mEq/L [-4 to 27]), serum-creatinine 65 µmol/L (40-133) (0.74 mg/dL [0.45-1.51]), OG 81 mOsm/kgH(2)O (25-132), and serum-EG 44 mmol/L (4-112) (250 mg/dL [25-700]). She was treated with fomepizole 99 times, ethanol 60 times (with a combination of both six times), and dialysis 73 times. The correlation between serum-EG and OG was good (r(2) = 0.76). She was finally found dead outside hospital with an EG blood concentration of 81 mmol/L (506 mg/dL). An autopsy revealed calcium oxalate crystals in the kidneys, slight liver steatosis, and slight edema of the lungs. DISCUSSION: The frequent use of fomepizole in this young patient was not associated with any detectable side effects; neither on clinical examination and lab screening, nor on the later autopsy. Regarding the sequelae from the repetitive EG-poisoning episodes, her kidney function seemed to normalize after each overdose. She was treated with buffer and antidote without hemodialysis 81 times without complications, supporting the safety of this approach in selected cases.

Importance of CYP1A1 and CYP1B1 in bioactivation of benzo[a]pyrene in human lung cell lines.

Polycyclic aromatic hydrocarbons are ubiquitous environmental pollutants classified as carcinogens in humans and rodents. The cytochromes P4501A1 and 1B1 have both shown capacity to carry out bioactivation of the prototype PAH, benzo[a]pyrene (B[a]P) to its ultimate carcinogenic B[a]P-diol-epoxide-I-1 form. The part played by each enzyme in human lung cells, however, has not been clarified. To get further insight into their individual role in the metabolic activation of B[a]P, RNA-interference was used to down-regulate CYP1A1 and/or CYP1B1 gene expression in the human lung cell lines BEP2D and NCIH2009. Fluorescence-HPLC analysis revealed that formation of B[a]P-tetrol-I-1 (hydrolyzed form of the corresponding diol-epoxide) was dependent primarily on CYP1A1. In cells without down-regulation of CYP1A1, the B[a]P-tetrol-I-1 was the major tested isomer formed. In contrast, the B[a]P-cis- and trans-7,8-dihydrodiol isomers were readily formed in cells expressing high levels of either CYP-gene. Simultaneous down-regulation of CYP1A1 and CYP1B1 mRNA resulted in low levels of metabolites overall. Residual unmetabolized B[a]P levels followed the expression of CYP1A1 in an inverse manner. In conclusion, these results indicate a major role of CYP1A1 in the bioactivation of B[a]P to carcinogenic B[a]P-diol-epoxides and in overall metabolism of B[a]P in human lung cell lines. In contrast, both CYP1A1 and CYP1B1 contribute significantly to the formation of the B[a]P-cis- and trans-7,8-dihydrodiol isomers.

Biotransformation of benzo[a]pyrene in Ahr knockout mice is dependent on time and route of exposure.

Benzo[a]pyrene (BP) is an ubiquitous environmental pollutant with potent mutagenic and carcinogenic properties. The Ah receptor (Ahr) is important in the metabolic activation of BP and is therefore central to BP-induced carcinogenesis. Although Ahr(-/-) mice are refractory to BP-induced carcinogenesis, higher levels of BP-DNA and -protein adducts were formed in them than in wild-type mice. These results indicated the presence of an Ahr-independent and/or a slower biotransformation of BP in Ahr knockout mice. To address this issue further, we have now performed a time-course experiment, with mice receiving a single oral dose of BP (100 mg/kg). Wild-type mice have an effective clearance of BP metabolites, mainly through 3-hydroxybenzo[a]pyrene and 9-hydroxybenzo[a]pyrene in the feces with reduced levels of DNA and protein adducts in the examined tissues. On the other hand, the Ahr(-/-) mice appear to have a lower metabolic clearance of BP resulting in increased levels of DNA and protein adducts and of unmetabolized BP. In addition, we have performed an administration route experiment and found that skin-exposed Ahr(-/-) mice showed lower levels of protein adducts along with markedly reduced P450 1B1 expression, but only in the exposed area, as compared with the wild-type mice. In addition, the systemic uptake of BP is increased in the Ahr(-/-) mice as compared with the wild-type mice. Hence, the lack of a functional Ah receptor results in an Ahr-independent biotransformation of BP with a slower clearance of BP and higher levels of DNA and protein adducts, but the distribution and levels of BP and BP-protein adducts are clearly dependent on the route of exposure.

Background, approaches and recent trends for setting health-based occupational exposure limits: a minireview.

The setting of occupational exposure limits (OELs) are founded in occupational medicine and the predictive toxicological testing, resulting in exposure-response relationships. For compounds where a No-Observed-Adverse-Effect-Level (NOAEL) can be established, health-based OELs are set by dividing the NOAEL of the critical effect by an overall uncertainty factor. Possibly, the approach may also be used for carcinogens if the mechanism is epigenetic or the genetic effect is secondary to effect from reactions with proteins such as topoisomerase inhibitors, and mitotic and meiotic spindle poisons. Additionally, the NOAEL approach may also be used for compounds with weak genotoxic effect, playing no or only a minor role in the development of tumours. No health-based OEL can be set for direct-acting genotoxic compounds where the life-time risks may be estimated from the low-dose linear non-threshold extrapolation, allowing a politically based exposure level to be set. OELs are set by several agencies in the US and Europe, but also in-house in major chemical and pharmaceutical companies. The benchmark dose approach may in the future be used where it has advantage over the NOAEL approach. Also, more attention should be devoted to sensitive groups, toxicological mechanisms and interactions as most workplace exposures are mixtures.

Sensitive determination of a glyoxal-DNA adduct biomarker candidate by column switching capillary liquid chromatography electrospray ionization mass spectrometry.

A method based on column switching packed capillary liquid chromatography electrospray mass spectrometry has been developed for the determination of the adduct glyoxal-deoxyguanosine, a biomarker candidate for the assessment of glyoxal exposure, in DNA hydrolysate solutions. Microgram amounts of DNA were isolated and enzymatically hydrolyzed to deoxyribonucleosides, prior to ultrafiltration and subsequent dilution to a sample solution consisting of water-acetonitrile-formic acid (98 : 2 : 0.2, v/v). The sample solution was loaded onto a 1 mm I.D. x 5 mm Hypercarb (5 mum) porous graphitic carbon trap column for analyte enrichment using an injection volume of 200 mul, and was subsequently back-flushed onto a 0.30 mm I.D. x 150 mm Lichrospher diol (5 mum) analytical column. The samples were loaded with a flow rate of 40 mul min(-1) and glyoxal-deoxyguanosine was desorbed from the trap column and eluted with an isocratic mobile phase consisting of water-acetonitrile-formic acid (50 : 50 : 0.2, v/v) at a flow rate of 5 mul min(-1). Mass spectrometric determination of glyoxal-deoxyguanosine was obtained by multiple reaction monitoring of the transition [M + H](+)m/z 326 --> m/z 210. The method was evaluated over the concentration range 0.25-50 ng ml(-1) of glyoxal-deoxyguanosine in the hydrolysate of 5 mug DNA. The method was linear with a correlation coefficient of 0.9998 in this range. The within-day (n = 6) and between-day (n = 6) precisions were determined as 1.2-11% and 1.4-11% RSD, respectively, and the recovery was close to 100%. The mass limit of detection was 15 pg, corresponding to a concentration limit of detection of 75 fg mul(-1) DNA hydrolysate solution, corresponding to 48 adducts per 10(6) normal nucleosides. The method was applied for the determination of glyoxal-deoxyguanosine in DNA hydrolysate solutions of calf thymus DNA and cell cultures after reaction or incubation with glyoxal.

Testing REACH draft technical guidance notes for conducting chemical safety assessments-the experience of a downstream user of a preparation.

The goal of REACH is the safe use of chemicals. This study examines the efficiency and usefulness of two draft technical guidance notes in the REACH Interim Project 3.2-2 for the development of the chemical safety report and exposure scenarios. A case study was carried out for a paint system for protection of structural steel. The focuses of the study were risk assessment of preparations based on Derived No Effect Level (DNEL) and Predicted No Effect Concentrations (PNEC) and on effective and accurate communication in the supply chain. Exposure scenarios and generic descriptions of uses, risk management measures, and exposure determinants were developed. The study showed that communication formats, software tools, and guidelines for chemical risk assessment need further adjustment to preparations and real-life situations. Web platforms may simplify such communication. The downstream formulator needs basic substance data from the substance manufacturer during the pre-registration phase to develop exposure scenarios for preparations. Default values need to be communicated in the supply chain because these were critical for the derivation of applicable risk management demands. The current guidelines which rely on the available toxicological knowledge are insufficient to advise downstream users on how to develop exposure scenarios for preparations.

Determination of the dialdehyde glyoxal in workroom air-development of personal sampling methodology.

The dialdehyde glyoxal (ethanedial) is an increasingly used industrial chemical with potential occupational health risks. This study describes the development of a personal sampling methodology for the determination of glyoxal in workroom air. Among the compounds evaluated as derivatizing agents; N-methyl-4-hydrazino-7-nitrobenzofurazan (MNBDH), 1,2-phenylenediamine (OPDA), 1-dimethylaminonaphthalene-5-sulfonylhydrazine (dansylhydrazine, DNSH) and 2,4-dinitrophenylhydrazine (DNPH), DNPH was the only reagent that was suitable. Several different samplers were evaluated for sampling efficiency of glyoxal in workroom air using DNPH as derivatizing agent; in-house DNPH coated silica particles packed in two different types of glass tubes, impingers containing acidified DNPH solution, filter cassettes containing glass fibre filters coated with DNPH, a commercially available solid phase cartridge sampler originally developed for formaldehyde sampling (Waters Sep-Pak DNPH-silica cartridge), and the commercially available SKC UMEx 100 passive sampler originally developed for formaldehyde sampling. Aldehyde atmospheres for sampler evaluation were generated with an in-house made vapour atmosphere generator coupled to a sampling unit, with the possibility of parallel sampling. The resulting glyoxal-DNPH derivative was determined using both LC-UV and LC-APCI-MS with negative ionization. By far, the highest recovery of glyoxal was obtained employing one of the in-house DNPH coated silica samplers (93%, RSD = 3.6%, n = 12).

Characterization of adducts formed in the reaction of glutaraldehyde with 2'-deoxyadenosine.

Glutaraldehyde (1,5-pentanedial) is a widely used industrial chemical that has been found to be mutagenic in bacteria and mammalian cells. In this study, we examined the reaction of glutaraldehyde with 2'-deoxyadenosine and calf thymus DNA in aqueous buffered solutions. The 2'-deoxyadenosine adducts were isolated by reversed phase HPLC and characterized by their UV absorbance and 1H and 13C NMR spectroscopic and mass spectrometric features. The reaction produced three major adducts. The adduct dA567 was derived from two 2'-deoxyadenosine units bound together with a piperidine unit, and its yield was 10.4%. The carbons of the piperidine ring originated from glutaraldehyde, whereas the nitrogen of the ring originated from the exocyclic amino group of one of the 2'-deoxyadenosine units. The adduct dA451d (yield 0.6%) was similar in structure to dA567, but one of the deoxyribose moieties from 2'-deoxyadenosine was missing. The third adduct, dA334, consisted of a hydroxy-tetrahydropyridine moiety derived from glutaraldehyde and N6 of 2'-deoxyadenosine (yield 4.0%). Furthermore, LC-ESI-MS/MS analysis of the reaction mixture revealed the formation of compounds with ion peaks of m/z = 352. None of these compounds were sufficiently stable for preparative isolation. They were tentatively identified as a pair of diastereomers of 2,6-dihydroxypiperidine derivatives, which are likely precursors to dA334. Plausible mechanisms for the formation of the adducts are presented. In the reaction of glutaraldehyde with single and double stranded calf thymus DNA, the dA334 adduct was formed.

Different mechanisms involved in apoptosis following exposure to benzo[a]pyrene in F258 and Hepa1c1c7 cells.

The present study compares and elucidates possible mechanisms why B[a]P induces different cell signals and triggers apparently different apoptotic pathways in two rather similar cell lines (hepatic epithelial cells of rodents). The rate and maximal capacity of metabolic activation, as measured by the formation of B[a]P-tetrols and B[a]P-DNA adducts, was much higher in mouse hepatoma Hepa1c1c7 cells than in rat liver epithelial F258 cells due to a higher induced level of cyp1a1. B[a]P increased intracellular pH in both cell lines, but this change modulated the apoptotic process only in F258 cells. In Hepa1c1c7 cells reactive oxygen species (ROS) production appeared to be a consequence of toxicity, unlike F258 cells in which it was an initial event. The increased mitochondrial membrane potential found in F258 cells was not observed in Hepa1c1c7 cells. Surprisingly, F258 cells cultured at low cell density were somewhat more sensitive to low (50nM) B[a]P concentrations than Hepa1c1c7 cells. This could be explained partly by metabolic differences at low B[a]P concentrations. In contrast to the Hepa1c1c7 model, no activation of cell survival signals including p-Akt, p-ERK1/2 and no clear inactivation of pro-apoptotic Bad was observed in the F258 model following exposure to B[a]P. Another important difference between the two cell lines was related to the role of Bax and cytochrome c. In Hepa1c1c7 cells, B[a]P exposure resulted in a "classical" translocation of Bax to the mitochondria and release of cytochrome c, whereas in F258 cells no intracellular translocation of these two proteins was seen. These results suggest that the rate of metabolism of B[a]P and type of reactive metabolites formed influence the resulting balance of pro-apoptotic and anti-apoptotic cell signaling, and hence the mechanisms involved in cell death and the chances of more permanent genetic damage.

Quantitative analysis of benzo[a]pyrene biotransformation and adduct formation in Ahr knockout mice.

Benzo[a]pyrene (BP) is an ubiquitous environmental pollutant with potent mutagenic and carcinogenic properties. The Ah receptor (Ahr) is involved in the metabolic activation of BP and is therefore important in the induction of chemical carcinogenesis. In this study, the relationship between Ahr genotype and biotransformation of BP in internal organs was investigated in Ahr (+/+), Ahr (+/-) and Ahr (-/-) mice. The mice were treated with BP (100mg/kg) by gavage. Gene expression was measured after 24h by real-time RT-PCR and showed induction of Cyp1a1 in liver and lung, and Cyp1b1 in lung in both Ahr (+/+) and Ahr (+/-). No induction of the Cyp genes was observed in the Ahr (-/-). There was a significant basal expression of Cyp1b1 in the liver of all genotypes, and this expression was independent of the BP exposure. Analyzed by HPLC-fluorescence, there were increased levels of protein and DNA adducts, metabolites, conjugates and unmetabolized BP in the internal organs of Ahr (-/-) as compared to Ahr (+/+) and Ahr (+/-) mice. This may be partly explained by a delayed bioactivation of BP in the Ahr deficient mice. The BP metabolism observed in the Ahr (-/-) mice is also evidence of an Ahr independent biotransformation of BP.

Epimerization and stability of two new cis-benzo[a]pyrene tetrols by the use of liquid chromatography-fluorescence and mass spectrometry.

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 degrees 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 degrees 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 C(10)-OH but also another site like position C(7)-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 C(7)-OH and C(8)-OH groups in a cis position.

Survey of air pollution in Cotonou, Benin--air monitoring and biomarkers.

Exposure to genotoxic compounds present in ambient air has been studied in Cotonou, Benin, a city where two-stroke motorbikes are the major form of transportation and gasoline quality is poor. Personal monitoring and biomarkers were used to assess the exposure. Non-smoking taxi-moto drivers (city) and village residents were the study subjects. Benzene exposure was significantly higher in the city, as compared to the village (76.0+/-26.8 microg/m(3) versus 3.4+/-3.0, p=0.0004). Urinary excretion of benzene and S-phenylmercapturic acid (S-PMA) were also highest in subjects living in the city, whereas 1-hydroxypyrene was not different. The level of total polycyclic aromatic hydrocarbons (PAHs), associated with particles, ranged from 76.21 to 103.23 in Cotonou versus 1.55 ng/m(3) for the village. Determination of DNA damage in lymphocytes showed that subjects from the city had elevated number of lesions compared to subjects in the village in terms of bulky DNA adducts, 8-hydroxy-2'-deoxyguanosine and 5-methylcytosine, whereas DNA fragmentations analysed by alkaline gel electrophoresis was not different between the subjects. In conclusion, this study shows that air pollution is pronounced in Cotonou, Bénin and is associated with elevated levels of DNA damage in residents of the city compared to people living in a non-polluted rural village.

Chromosome aberrations in tunnel workers exposed to acrylamide and N-methylolacrylamide.

OBJECTIVES: The aim of this study was to examine chromosome aberrations in 25 tunnel workers exposed to acrylamide-containing grout in injection work. METHODS: Blood samples were collected from 25 exposed and 25 unexposed tunnel workers matched for age, gender, and smoking habits. Whole blood was cultured for 50-53 hours according to conventional methods. Chromosome damage was scored in 200 metaphases per person on coded slides. The distribution of glutathione S-transferase (GST) genotypes (M1 and T1) was examined for all the workers. Exposure assessment was performed with detailed interviews and questionnaires. RESULTS: The chromosome examinations showed no statistically significant differences between the 25 exposed and 25 unexposed workers for cells with chromosome aberrations or for chromatid breaks, chromosome breaks, and chromosome gaps. The exposed workers had a significantly higher number of chromatid gaps (mean 10.6, SD 5.6) than the unexposed workers (mean 6.4, SD 4.4, P=0.004), but there was no exposure-response relationship. The limited stratum-specific numbers showed that the exposed workers with the GSTM1-/GSTT1-genotype had nonsignificantly higher frequencies of all the effect parameters than the unexposed workers; this finding indicates that individual susceptibility related to the detoxification of acrylamide and N-methylolacrylamide may have played a role in the observed effect. CONCLUSIONS: No increase in chromosome breaks or aberrations was observed for 25 workers exposed to acrylamide-containing grout during tunnel work. The increased frequency of chromatid gaps in the exposed workers may indicate a slight genotoxic effect related to exposure to acrylamide or N-methylolacrylamide.

Characterization of common CYP1B1 variants with different capacity for benzo[a]pyrene-7,8-dihydrodiol epoxide formation from benzo[a]pyrene.

Cytochrome P450 1B1 (CYP1B1), an extrahepatic enzyme inducible by smoking, is overexpressed in many tumors and catalyzes the metabolic activation of procarcinogens such as polycyclic aromatic hydrocarbons. In human, CYP1B1 is genetically polymorphic and five common missense mutations causing amino acid substitution have been identified. In this study, we have investigated CYP1B1 haplotypes present in a Spanish population and carried out functional analyses of the corresponding enzymes in yeast using benzo[a]pyrene as a substrate. CYP1B1*1, CYP1B1*2, CYP1B1*3, CYP1B1*4, CYP1B1*6, and CYP1B1*7, encoding combinations of the Arg48Gly, Ala119Ser, Leu432Val, Asn453Ser, and Ala443Gly amino acid substitutions, were present at frequencies of 14.3%, 25.5%, 38.8%, 18.1%, 0.4%, and 2.6%, respectively. The variant CYP1B1 forms were heterologously expressed with human reductase in Saccharomyces cerevisiae and kinetic analyses of benzo[a]pyrene metabolism were carried out. CYP1B1.7, having the amino acid substitutions Arg48Gly, Ala119Ser, Leu432Val, and Ala443Gly, exhibited a significantly decreased capacity (P < 0.001) for the formation of (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol from benzo[a]pyrene as indicated by lower intrinsic clearance (Vmax/Km). A somewhat decreased clearance was observed for CYP1B1.4, whereas no significant differences in kinetic properties among the remaining variant enzymes were observed as compared with CYP1B1.1. Thus, genetic polymorphism in the CYP1B1 gene, as defined by the haplotypes investigated, might cause interindividual differences in susceptibility (e.g., to lung cancer induced by smoking). The results indicate the necessity to make molecular epidemiologic investigations regarding the association of the specific CYP1B1 haplotypes and cancer risk.

Determination of benzo[a]pyrene tetrols by column-switching capillary liquid chromatography with fluorescence and micro-electrospray ionization mass spectrometric detection.

The present work displays capillary liquid chromatographic column switching methodology tailored for determination of benzo[a]pyrene tetrol isomers in biological matrices using on-line fluorescence and micro-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, v/v), loaded onto a 0.32 mm I.D. x 40 mm 5 microm Kromasil C(18) pre-column for analyte enrichment and back-flushed elution onto a 0.30 mm I.D. x 150 mm 3.5 microm Kromasil C(18) analytical column. The samples were loaded with a flow rate of 50 microl min(-1) and the tetrols were separated at a flow rate of 4 microl min(-1) with an acetonitrile:10 mM ammonium acetate gradient from 10 to 90%. A sample loading flow rate up to 50 microl min(-1) was allowed. The fluorescence excitation and emission were set to 342 and 385 nm, respectively, while mass spectrometric detection of the benzo[a]pyrene tetrols was obtained by monitoring their [M - H](-) molecular ions at m/z 319. The method was validated over the concentration range 0.1-50 ng ml(-1) benzo[a]pyrene tetrols in a cell culture medium with 100 microl 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(-1) cell 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(-1) cell culture medium.

Reaction of glyoxal with 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, cytidine, thymidine, and calf thymus DNA: identification of DNA adducts.

Glyoxal (ethanedial) is an increasingly used industrial chemical that has been found to be mutagenic in bacteria and mammalian cells. In this study, the reactions of glyoxal with 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, cytidine, thymidine, and calf thymus DNA have been studied in aqueous buffered solutions. The nucleoside adducts were isolated by reversed-phase liquid chromatography and characterized by their UV absorbance and 1H and 13C NMR spectroscopic and mass spectrometric features. The reaction with 2'-deoxyguanosine gave one adduct, the previously known 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one adduct. The reaction of 2'-deoxyadenosine with glyoxal resulted in the formation of a previously not reported N6-(hydroxyacetyl)-2'-deoxyadenosine adduct. In the reaction of glyoxal with 2'-deoxycytidine and cytidine at neutral conditions and 37 degrees C, 5-hydroxyacetyl pyrimidine derivatives were obtained. When the cytidine reaction was performed at pH 4.5 and 50 degrees C, the 5-hydroxyacetyl derivative of uridine was formed through deamination of cytidine-glyoxal. Adducts in the thymidine reaction could not be detected. In the reaction of glyoxal with calf thymus DNA, the 2'-deoxyguanosine-glyoxal and 2'-deoxyadenosine-glyoxal adducts were obtained, the former being the major adduct.

Role of cell signaling in B[a]P-induced apoptosis: characterization of unspecific effects of cell signaling inhibitors and apoptotic effects of B[a]P metabolites.

Here we show that several cell signaling inhibitors have effect on cyp1a1 expression and the metabolism of benzo[a]pyrene (B[a]P) in Hepa1c1c7 cells. The CYP1A1 inhibitor alpha-naphthoflavone (alpha-NF), the p53 inhibitor pifithrin-alpha (PFT-alpha), the ERK inhibitors PD98059 and U0126, and the p38 MAPK inhibitors SB202190 and PD169316 induced the expression and level of cyp1a1 protein. On the other hand, during the first h the inhibitors appeared to reduce the metabolism of B[a]P as measured by the generation of tetrols and by covalent binding of B[a]P to macromolecules. In contrast, the phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin, had neither an effect on the cyp1a1 expression nor the B[a]P-metabolism. In order to avoid these unspecific effects, we characterized the mechanisms involved in the apoptotic effects of B[a]P-metabolites. B[a]P and the B[a]P-metabolites B[a]P-7,8-DHD and BPDE-I induced apoptosis, whereas B[a]P-4,5-DHD had no effect. B[a]P, B[a]P-7,8-DHD and BPDE-I induced an accumulation and phosphorylation of p53, while the Bcl-2 proteins Bcl-xl, Bad and Bid were down-regulated. Interestingly, the levels of anti-apoptotic phospho-Bad were up-regulated in response to B[a]P as well as to B[a]P-7,8-DHD and BPDE-I. Both p38 MAPK and JNK were activated, but the p38 MAPK inhibitors were not able to inhibit BPDE-I-induced apoptosis. PFT-alpha reduced the BPDE-I-induced apoptosis, while both the PI-3 kinase inhibitor and the ERK inhibitors increased the apoptosis in combination with BPDE-I. BPDE-I also triggered apoptosis in primary cultures of rat lung cells. In conclusion, often used cell signaling inhibitors both enhanced the expression and the level of cyp1a1 and more directly acted as inhibitors of cyp1a1 metabolism of B[a]P. However, studies with the B[a]P-metabolite BPDE-I supported the previous suggestion that p53 has a role in the pro-apoptotic signaling pathway induced by B[a]P. Furthermore, these studies also show that the reactive metabolites of B[a]P induce the anti-apoptotic signals, Akt and ERK. Neither the induction nor the activity of p38 MAPK and JNK seems to be of major importance for the B[a]P-induced apoptosis.