• Effects of nitrated-polycyclic aromatic hydrocarbons and diesel exhaust particle extracts on cell signalling related to apoptosis: possible implications for their mutagenic and carcinogenic effects.

      Landvik, Nina E.; Gorria, Morgane; Arlt, Volker M.; Asare, Nana; Solhaug, Anita; Lagadic-Gossmann, Dominique; Holme, Jorn A. (2007-03-07)
      Nitrated-polycyclic aromatic hydrocarbons (nitro-PAHs) and diesel exhaust particle extracts (DEPE) induced apoptosis in Hepa1c1c7 cells with the following potency: 1,3-dinitropyrene (1,3-DNP)>1-nitropyrene (1-NP) >> DEPE >> 1,8-dinitropyrene (1,8-DNP). The compounds induced cyp1a1, and activated various intracellular signalling pathways related to apoptosis. The CYP inhibitor alpha-naphthoflavone strongly reduced 1,3-DNP-induced cell death, whereas cell death induced by 1-NP was rather increased. Toxic 1,3-DNP and 1-NP were found to induce a concentration-dependent lipid peroxidation. 1,3-DNP caused pro-apoptotic events, including increased phosphorylation and accumulation of p53 in the nucleus, cleavage of bid and of caspases 8 and 3, down-regulation of bcl-x(L) and phosphorylation of p38 and JNK MAPK. Furthermore, 1,3-DNP increased the activation of survival signals including phosphorylation of Akt and inactivation (phosphorylation) of pro-apoptotic bad. Although less potent, rather similar effects were observed following exposure to DEPE, compared to 1-NP. The most important finding was that the most mutagenic and carcinogenic compound tested, 1,8-DNP, induced little (if any) cell death, despite the fact that this compound seemed to give the most DNA damage as judged by DNA adduct formation, increased phosphorylation of p53 and accumulation of cells in S-phase. Immunocytochemical studies revealed that the p53 protein did not accumulate into the nucleus suggesting that 1,8-DNP inactivated the pro-apoptotic function of the p53 protein by a non-mutagenic event. These results suggest that after exposure to 1,8-DNP more cells may survive with DNA damage, thereby increasing its mutagenic and carcinogenic potential.
    • The environmental pollutant and carcinogen 3-nitrobenzanthrone induces cytochrome P450 1A1 and NAD(P)H:quinone oxidoreductase in rat lung and kidney, thereby enhancing its own genotoxicity.

      Stiborova, Marie; Dracinska, Helena; Mizerovska, Jana; Frei, Eva; Schmeiser, Heinz H.; Hudecek, Jiri; Hodek, Petr; Phillips, David H.; Arlt, Volker M. (2008-05-02)
      3-Nitrobenzanthrone (3-NBA) is a carcinogen occurring in diesel exhaust and air pollution. Using the (32)P-postlabelling method, we found that 3-NBA and its human metabolite, 3-aminobenzanthrone (3-ABA), are activated to species forming DNA adducts by cytosols and/or microsomes isolated from rat lung, the target organ for 3-NBA carcinogenicity, and kidney. Each compound generated identical five DNA adducts. We have demonstrated the importance of pulmonary and renal NAD(P)H:quinone oxidoreductase (NQO1) to reduce 3-NBA to species that are further activated by N,O-acetyltransferases and sulfotransferases. Cytochrome P450 (CYP) 1A1 is the essential enzyme for oxidative activation of 3-ABA in microsomes of both organs, while cyclooxygenase plays a minor role. 3-NBA was also investigated for its ability to induce NQO1 and CYP1A1 in lungs and kidneys, and for the influence of such induction on DNA adduct formation by 3-NBA and 3-ABA. When cytosols from rats treated i.p. with 40mg/kg bw of 3-NBA were incubated with 3-NBA, DNA adduct formation was up to 2.1-fold higher than in incubations with cytosols from control animals. This increase corresponded to an increase in protein level and enzymatic activity of NQO1. Incubations of 3-ABA with microsomes of 3-NBA-treated rats led to up to a fivefold increase in DNA adduct formation relative to controls. The stimulation of DNA adduct formation correlated with the potential of 3-NBA to induce protein expression and activity of CYP1A1. These results demonstrate that 3-NBA is capable to induce NQO1 and CYP1A1 in lungs and kidney of rats thereby enhancing its own genotoxic and carcinogenic potential.
    • Influence of aryl hydrocarbon- (Ah) receptor and genotoxins on DNA repair gene expression and cell survival of mouse hepatoma cells.

      Schreck, Ilona; Chudziak, Doreen; Schneider, Sandra; Seidel, Albrecht; Platt, Karl L.; Oesch, Franz; Weiss, Carsten (2009-05-17)
      The aryl hydrocarbon receptor (AhR) mediates toxicity of a variety of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and dioxins. However, the underlying mechanisms and genetic programmes regulated by AhR to cause adverse effects but also to counteract poisoning are still poorly understood. Here we analysed the effects of two AhR ligands, benzo[a]pyrene (B[a]P), a DNA damaging tumour initiator and promotor and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a pure tumour promoter, on cell survival and on nucleotide excision repair (NER) gene expression. NER deals with so called "bulky" DNA adducts including those generated by enzymatically activated B[a]P. Therefore, the hypothesis that AhR may enhance NER gene expression to trigger DNA repair in the presence of genotoxic AhR ligands was tested. Furthermore, we investigated a potential cytoprotective effect of AhR activation by the non-genotoxic ligand TCDD against cell death induced by various genotoxins. Finally, the actions of genotoxins themselves on NER gene expression were studied. As a cell culture model we used mouse hepatoma cells (Hepa-c7) proficient for AhR and its partner protein ARNT as well as subclones deficient in AhR (Hepa-c12) or ARNT (Hepa-c4) to study involvement of AhR and ARNT in response to B[a]P and TCDD. Indeed, the mRNA levels of the two NER genes XP-C and DNA polymerase kappa were increased by B[a]P and TCDD, however, this was not accompanied by an increase in the amount of the respective proteins. Pretreatment of cells with TCDD did not reduce cytotoxicity induced by various genotoxins. Thus, in Hepa-c7 cells AhR has no major effects on the expression of these crucial NER proteins and does not prevent genotoxin-provoked cell death. As expected, the genotoxins B[a]P and cis-platin led to p53 accumulation and induction of its target p21. Interestingly, however, NER gene expression was not enhanced but rather decreased. As two NER genes, XP-C and DNA damage binding protein ddb2, are up-regulated by p53 and ultraviolet radiation in human cells these findings suggest cell type, species or lesion specific actions of p53 on DNA repair gene expression. Importantly, in cells with damaged DNA up-regulation of p53 may not suffice to enhance DNA repair gene expression.
    • Molecular evidence for an involvement of organic anion transporters (OATs) in aristolochic acid nephropathy.

      Bakhiya, Nadiya; Arlt, Volker M.; Bahn, Andrew; Burckhardt, Gerhard; Phillips, David H.; Glatt, Hansruedi (2009-10-01)
      Aristolochic acid (AA), present in Aristolochia species, is the major causative agent in the development of severe renal failure and urothelial cancers in patients with AA nephropathy. It may also be a cause of Balkan endemic nephropathy. Epithelial cells of the proximal tubule are the primary cellular target of AA. To study whether organic anion transporters (OATs) expressed in proximal tubule cells are involved in uptake of AA, we used human epithelial kidney (HEK293) cells stably expressing human (h) OAT1, OAT3 or OAT4. AA potently inhibited the uptake of characteristic substrates, p-aminohippurate for hOAT1 and estrone sulfate for hOAT3 and hOAT4. Aristolochic acid I (AAI), the more cytotoxic and genotoxic AA congener, exhibited high affinity for hOAT1 (K(i)=0.6 microM) as well as hOAT3 (K(i)=0.5 microM), and lower affinity for hOAT4 (K(i)=20.6 microM). Subsequently, AAI-DNA adduct formation (investigated by (32)P-postlabelling) was used as a measure of AAI uptake. Significantly higher levels of adducts occurred in hOAT-expressing cells than in control cells: this effect was abolished in the presence of the OAT inhibitor probenecid. In Xenopus laevis oocytes hOAT-mediated efflux of p-aminohippurate was trans-stimulated by extracellular AA, providing further molecular evidence for AA translocation by hOATs. Our study indicates that OATs can mediate the uptake of AA into proximal tubule cells and thereby participate in kidney cell damage by this toxin.
    • Oxidatively damaged DNA and inflammation in the liver of dyslipidemic ApoE-/- mice exposed to diesel exhaust particles.

      Folkmann, Janne Kjaersgaard; Risom, Lotte; Hansen, Christian Stevns; Loft, Steffen; Moller, Peter (2007-07-31)
      Epidemiological studies have shown that exposure to air pollution particles is associated with cardiovascular diseases, whereas the role in the initiation of atherosclerosis is unresolved. Atherosclerosis is considered to be an inflammatory disease that also involves oxidative stress. Here we investigated effects of oxidative stress elicited by diesel exhaust particles (DEP) in the aorta, liver, and lung of dyslipidemic ApoE(-/-) mice at the age when visual plaques appear in the aorta (11-13 weeks). DEP was administrated by intraperitoneal injection (0, 50, 500 and 5,000 microg DEP/kg bodyweight) in order to omit vascular effects secondary to pulmonary inflammation. The mice were killed either 6 or 24h after the administration. Inflammation was measured as the expression of inducible nitric oxide synthase (iNOS) and serum nitric oxide and DNA damage was measured by the comet assay. The expression of iNOS mRNA was increased in the liver 6h after the administration. The level of oxidized purine bases, determined as formamidopyrimidine DNA glycosylase sites was increased by 67% (95% CI: 11-124%) in the liver after 24h in the mice administrated with only 50 microg/kg bodyweight. However, there was no indication of systemic inflammation determined as the serum concentration of nitric oxide and iNOS expression, and DNA damage was not increased in the aorta. These observations indicate that intraperitoneal DEP injection does not induce inflammation or oxidatively damaged DNA in the lung and aorta, whereas a direct effect in terms of inflammation and oxidized DNA was observed in the liver of dyslipidemic ApoE(-/-) mice.