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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.

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.

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.

Abstract: 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. [Copyright &y& Elsevier]