title: Evaluation of immunohistochemical markers to detect the genotoxic mode of action of fine and ultrafine dusts in rat lungs.
abstract: Data on local genotoxicity after particle exposure are crucial to resolve mechanistic aspects such as the impact of chronic inflammation, types of DNA damage, and their role in lung carcinogenesis. We established immunohistochemical methods to quantify the DNA damage markers poly(ADP-ribose) (PAR), phosphorylated H2AX (γ-H2AX), 8-hydroxyguanosine (8-OH-dG), and 8-oxoguanine DNA glycosylase (OGG1) in paraffin-embedded tissue from particle-exposed rats. The study was based on lungs from a subchronic study that was part of an already published carcinogenicity study where rats had been intratracheally instilled with saline, quartz DQ12, amorphous silica (Aerosil(®) 150), or carbon black (Printex(®) 90) at monthly intervals for 3 months. Lung sections were stained immunohistochemically and markers were quantified in alveolar lining cells. Local genotoxicity was then correlated with already defined endpoints, i.e. mean inflammation score, bronchoalveolar lavage parameters, and carcinogenicity. Genotoxicity was most pronounced in quartz DQ12-treated rats, where all genotoxicity markers gave statistically significant positive results, indicating considerable genotoxic stress such as occurrence of DNA double-strand breaks (DSB), and oxidative damage with subsequent repair activity. Genotoxicity was less pronounced for Printex(®) 90, but significant increases in γ-H2AX- and 8-OH-dG-positive nuclei and OGG1-positive cytoplasm were nevertheless detected. In contrast, Aerosil(®) 150 significantly enhanced only 8-OH-dG-positive nuclei and oxidative damage-related repair activity (OGG1) in cytoplasm. In the present study, γ-H2AX was the most sensitive genotoxicity marker, differentiating best between the three types of particles. The mean number of 8-OH-dG-positive nuclei, however, correlated best with the mean inflammation score at the same time point. This methodological approach enables integration of local genotoxicity testing in subchronic inhalation studies and makes immunohistochemical detection, in particular of γ-H2AX and 8-hydroxyguanine, a very promising approach for local genotoxicity testing in lungs, with prognostic value for the long-term outcome of particle exposure.
authors.affiliation: Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover, Germany. susanne.rittinghausen@item.fraunhofer.de
authors.forename: Susanne
authors.initials: S
authors.lastname: Rittinghausen
authors.forename: Bernd
authors.initials: B
authors.lastname: Bellmann
authors.forename: Otto
authors.initials: O
authors.lastname: Creutzenberg
authors.forename: Heinrich
authors.initials: H
authors.lastname: Ernst
authors.forename: Angelika
authors.initials: A
authors.lastname: Kolling
authors.forename: Inge
authors.initials: I
authors.lastname: Mangelsdorf
authors.forename: Rupert
authors.initials: R
authors.lastname: Kellner
authors.forename: Sascha
authors.initials: S
authors.lastname: Beneke
authors.forename: Christina
authors.initials: C
authors.lastname: Ziemann
mesh_terms: D000818:Animals; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects; D045647:DNA Glycosylases / Q000378:metabolism; D005260:Female; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D007150:Immunohistochemistry; D008168:Lung / Q000187:drug effects* / Q000473:pathology; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity*; D010316:Particle Size; D010750:Phosphoproteins / Q000378:metabolism; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D011791:Quartz / Q000633:toxicity*; D051381:Rats; D017208:Rats, Wistar; D012822:Silicon Dioxide / Q000633:toxicity*; D053260:Soot / Q000633:toxicity*
chemical_list: D006657:Histones; D009153:Mutagens; D010750:Phosphoproteins; D053260:Soot; C577575:gamma-H2AX protein, rat; D006151:Guanosine; D011791:Quartz; D011064:Poly Adenosine Diphosphate Ribose; C046215:8-hydroxyguanosine; D012822:Silicon Dioxide; D045647:DNA Glycosylases; C483741:OGG1 protein, rat