Christina Ziemann

Evaluation of immunohistochemical markers to detect the genotoxic mode of action of fine and ultrafine dusts in rat lungs

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.

Toxicity profile of the GATA-3-specific DNAzyme hgd40 after inhalation exposure

DNAzymes are single-stranded catalytic DNA molecules that bind and cleave specific sequences in a target mRNA molecule. Their potential as novel therapeutic agents has been demonstrated in a variety of disease models. However, no studies have yet addressed their toxicology and safety pharmacology profiles in detail. Here we describe a detailed toxicological analysis of inhaled hgd40, a GATA-3-specific DNAzyme designed for the treatment of allergic bronchial asthma. Subacute toxicity, immunotoxicity, and respiratory, cardiovascular, and CNS safety pharmacology were analyzed in rodents and non-rodents, and genotoxicity was assessed in human peripheral blood. Overall, hgd40 was very well tolerated when delivered by aerosol inhalation or slow intravenous infusion. Only marginal reversible histopathological changes were observed in the lungs of rats receiving the highest dose of inhaled hgd40. The changes consisted of slight mononuclear cell infiltration and alveolar histiocytosis, and moderate hyperplasia of bronchus-associated lymphoid tissue. No local or systemic adverse effects were observed in dogs. No compound-related respiratory, cardiovascular, or CNS adverse events were observed. The only relevant immunological findings were very slight dose-dependent changes in interleukin-10 and interferon-γ levels in bronchoalveolar lavage fluid. Taken together, these results support direct delivery of a DNAzyme via inhalation for the treatment of respiratory disease.

Absence of genotoxic potential of 902 MHz (GSM) and 1747 MHz (DCS) wireless communication signals: In vivo two-year bioassay in B6C3F1 mice

Purpose: The aim of the present investigation was to determine the incidence of micronuclei in peripheral blood erythrocytes of B6C3F1 mice that had been chronically exposed to radiofrequencies (RF) used for mobile communication. Materials and methods: ‘Ferris wheels’ were used to expose tube-restrained male and female mice to simulated environmental RF signals of the Global System for Mobile Communications (GSM, 902 MHz) or Digital Cellular System (DCS, 1747 MHz). RF signals were applied to the mice for 2 hours/day on 5 days/week for two years, at maximal whole-body-averaged specific absorption rates of 0.4, 1.3, and 4.0 W/kg body weight. Concurrent sham-exposed mice, cage controls, and positive controls injected with mitomycin C were included in this investigation. At necropsy, peripheral blood smears were prepared, and coded slides were stained using May-Grünwald-Giemsa or acridine orange. The incidence of micronuclei was recorded for each mouse in 2000 polychromatic and 2000 normochromatic erythrocytes. Results: There were no significant differences in the frequency of micronuclei between RF-exposed, sham-exposed, and cage control mice, irrespective of the staining/counting method used. Micronuclei were, however, significantly increased in polychromatic erythrocytes of the positive control mice. Conclusions: In conclusion, the data did not indicate RF-induced genotoxicity in mice after two years of exposure.

New investigations into the genotoxicity of cobalt compounds and their impact on overall assessment of genotoxic risk.

The genotoxicity of cobalt metal and cobalt compounds has been widely studied. Several publications show induction of chromosomal aberrations, micronuclei or DNA damage in mammalian cells in vitro in the absence of S9. Mixed results were seen in gene mutation studies in bacteria and mammalian cells in vitro, and in chromosomal aberration or micronucleus assays in vivo. To resolve these inconsistencies, new studies were performed with soluble and poorly soluble cobalt compounds according to OECD-recommended protocols. Induction of chromosomal damage was confirmed in vitro, but data suggest this may be due to oxidative stress. No biologically significant mutagenic responses were obtained in bacteria, Tk(+/-) or Hprt mutation tests. Negative results were also obtained for chromosomal aberrations (in bone marrow and spermatogonia) and micronuclei at maximum tolerated doses in vivo. Poorly soluble cobalt compounds do not appear to be genotoxic. Soluble compounds do induce some DNA and chromosomal damage in vitro, probably due to reactive oxygen. The absence of chromosome damage in robust GLP studies in vivo suggests that effective protective processes are sufficient to prevent oxidative DNA damage in whole mammals. Overall, there is no evidence of genetic toxicity with relevance for humans of cobalt substances and cobalt metal.

Lack of marked cyto- and genotoxicity of cristobalite in devitrified (heated) alkaline earth silicate wools in short-term assays with cultured primary rat alveolar macrophages

Abstract Alkaline earth silicate (AES) wools are low-biopersistence high-temperature insulation wools. Following prolonged periods at high temperatures they may devitrify, producing crystalline silica (CS) polymorphs, including cristobalite, classified as carcinogenic to humans. Here we investigated the cytotoxic and genotoxic significance of cristobalite present in heated AES wools. Primary rat alveolar macrophages were incubated in vitro for 2 h with 200 µg/cm2 unheated/heated calcium magnesium silicate wools (CMS1, CMS2, CMS3; heat-treated for 1 week at, or 4 weeks 150 °C below, their respective classification temperatures) or magnesium silicate wool (MS; heated for 24 h at 1260 °C). Types and quantities of CS formed, and fiber size distribution and shape were determined by X-ray diffraction and electron microscopy. Lactate dehydrogenase release and alkaline and hOGG1-modified comet assays were used, ± aluminum lactate (known to quench CS effects), for cytotoxicity/genotoxicity screening. Cristobalite content of wools increased with heating temperature and duration, paralleled by decreases in fiber length and changes in fiber shape. No marked cytotoxicity, and nearly no (CMS) or only slight (MS) DNA-strand break induction was observed, compared to the CS-negative control Al2O3, whereas DQ12 as CS-positive control was highly active. Some samples induced slight oxidative DNA damage, but no biological endpoint significantly correlated with free CS, quartz, or cristobalite. In conclusion, heating of AES wools mediates changes in CS content and fiber length/shape. While changes in fiber morphology can impact biological activity, cristobalite content appears minor or of no relevance to the intrinsic toxicity of heated AES wools in short-term assays with rat alveolar macrophages.

Update on COX-2 inhibitor patents with a focus on optimised formulation and therapeutic scope of drug combinations making use of COX-2 inhibitors

Selective cyclooxygenase-2 (COX-2) inhibitors, like coxibs, demonstrate efficacy in the treatment of pain and inflammation, comparable to unselective non-steroidal anti-inflammatory drugs (NSAIDs). COX-2 expression was, for a long time, linked only to pathophysiology, but ongoing research has also demonstrated constitutive COX-2 expression within normal tissues such as brain, kidney, pancreas, intestine and blood vessels. Irrespective of their tendency to reduce severe gastrointestinal (GI) events, strong debates have been ignited regarding new adverse effects of specific COX-2 inhibitors, most notably those of a cardiovascular (CV) nature, such as blood pressure elevation, myocardial infarction and stroke. This review evaluates the recent patent literature concerning new therapeutic options offered by selective COX-2 inhibitors, with emphasis on technological improvements and COX-2 inhibitors as components of drug combinations.

Importance of DNA-Adduct Formation and Gene Expression Profiling of Disease Candidate Genes in Rats Exposed to Bitumen Fumes

The equivocal experimental and epidemiological evidence of bitumen fumes and the possible mechanisms of toxicity remain uncertain. This study therefore aimed at investigating the genotoxicity of bitumen fumes, the biotransformation and urinary excretion of PAHs, and altered expression of a selected number of genes in lung, nasal epithelium, and white blood cells of rats. Animals were exposed to three different concentrations (low: 4 mg/m 3 ; medium: 20 mg/m 3 ; high: 100 mg/m 3 ) of bitumen fume condensate for 5 days, 30 days, and 12 months (6 hours per day) or ambient air. Notably, no dose-related signs of intolerance were observed throughout the inhalation period but dose dependent uptake of bitumen fumes was observed based on urinary excretion of PAHs and metabolites. At best, measurements of naphthols enabled an estimate of dose-dependent body burden. Excretion of 1-hydroxy- and 2-hydroxyphenanthrene was dose dependent and their production is catalyzed by the CYP1A1 monooxygenase which we found to be strongly induced upon exposure to bitumen fumes. Furthermore, pyrene, a minor component in bitumen fumes, produced hydroxypyrene levels close to the detection limit in rat urine. We additionally determined DNA adduct formation by the 32 P-postlabelling assay and observed a dose and time dependent increase of 3 to 4 stable DNA adducts in lung, nasal, and alveolar epithelium. DNA adduct levels were highest in nasal epithelium, the relative adduct level (RAL) being 450 adducts per 10 9 nucleotides. For lung and alveolar epithelium the RAL was 114 and 76 adducts per 10 9 nucleotides, respectively. However, we did not observe micronucleated red blood cells of the peripheral blood or with polychromatic erythrocytes of the bone marrow (after 12 months). It is important to note that erythrocyte cell count in bone marrow smears was reduced in four out of six animals after 12 months of exposure, clearly demonstrating that components of bitumen fume had reached the bone marrow. Finally, we investigated by reverse transcription polymerase chain reaction regulation of genes with known functions in inflammation, asthma and other pulmonary diseases. Gene expression changed during the time of exposure. With the monooxygenases CYP1A1 and CYP2G1 we observed dose dependent regulation in nasal and lung tissue. We also observed significant, but dose independent, regulation of cathepsin K and D, cadherin 22, platelet activating factor acetylhydrolase isoform 1b alpha subunit and the regulator of G-protein signalling in nasal epithelium of male rats after exposure to bitumen fumes. We found bitumen fumes to be genotoxic in target tissue of exposure and observed altered regulation of genes involved in the metabolic activation of polycyclic aromatic hydrocarbons and cellular inflammatory processes. These findings are consistent with the histopathology observed in the respiratory tract of rats chronically exposed to bitumen fume. An understanding of the regulation of suspected disease candidate genes in target tissues of exposure will be an interesting objective for further research into the mechanisms of toxicity.

FR-900098, an antimalarial development candidate that inhibits the non-mevalonate isoprenoid biosynthesis pathway, shows no evidence of acute toxicity and genotoxicity

ABSTRACT FR-900098 is an inhibitor of 1-deoxy-d-xylulose-5-phosphate (DXP) reductoisomerase, the second enzyme in the non-mevalonate isoprenoid biosynthesis pathway. In previous studies, FR-900098 was shown to possess potent antimalarial activity in vitro and in a murine malaria model. In order to provide a basis for further preclinical and clinical development, we studied the acute toxicity and genotoxicity of FR-900098. We observed no acute toxicity in rats, i.e. there were no clinical signs of toxicity and no substance-related deaths after the administration of a single dose of 3000 mg/kg body weight orally or 400 mg/kg body weight intravenously. No mutagenic potential was detected in the Salmonella typhimurium reverse mutation assay (Ames test) or an in vitro mammalian cell gene mutation test using mouse lymphoma L5178Y/TK+/− cells (clone 3.7.2C), both with and without metabolic activation. In addition, FR-900098 demonstrated no clastogenic or aneugenic capability or significant adverse effects on blood formation in an in vivo micronucleus test with bone marrow erythrocytes from NMRI mice. We conclude that FR-900098 lacks acute toxicity and genotoxicity, supporting its further development as an antimalarial drug.

Genotoxicity testing of sulfur dioxide (SO2) in a mouse bone marrow micronucleus test complemented with hematological endpoints

Sulfur dioxide (SO2) is a non-flammable, non-explosive, colorless gas. It is a ubiquitous environmental pollutant and an important chemical intermediate in several industrial processes. The toxicological properties of SO2, including its genotoxic potential, have been studied extensively. The majority of the available in vitro data indicate a lack of genotoxicity of SO2, while for sulfite salts some positive results have been reported. However, recent in vivo studies, using Kunming albino mice, have pointed to in vivo clastogenicity of SO2. To re-evaluate these positive findings, a bone-marrow micronucleus test according to OECD Guideline No. 474 was performed. NMRI mice (m/f) were exposed by inhalation via whole-body exposure to 0 (clean air), 2.7, 8, 27, or 80mg/m3 (0, 1, 3, 10, or 30ppm) SO2 for 4h/day on 7 consecutive days. Animals were sacrificed 24h after start of the last exposure, and blood samples (for complementing hematology) and bone marrow smears (for analysis of micronuclei) were prepared. Under the conditions used, exposure to SO2 caused no acute toxicity, mortality, or reduction in body weight. Compared with the clean-air controls, hematological parameters such as hematocrit, hemoglobin, erythrocyte/platelet/total leukocyte counts, differential white blood cell counts, and indicators of blood formation (reticulocyte counts, ratio of polychromatic to normochromatic erythrocytes in the bone marrow) remained unchanged by SO2 treatment. Unlike the previously reported studies on micronucleus formation, SO2 did not induce micronuclei in polychromatic erythrocytes of the bone marrow, whereas the positive control cyclophosphamide (60mg/kg body weight) was quite effective in this respect. Interestingly, SO2 treatment significantly enhanced malondialdehyde levels in erythrocyte lysates (TBARS method), indicating SO2-mediated oxidative stress, but also demonstrating systemic availability of the inhaled SO2. In conclusion, the present study could not reproduce the genotoxicity findings of the previously reported studies. SO2 is thus considered non-genotoxic in polychromatic erythrocytes in the bone marrow of NMRI mice under the conditions and in the concentrations used.

Quartz-Containing Ceramic Dusts: In vitro screening of the cytotoxic, genotoxic and pro-inflammatory potential of 5 factory samples

Inhalation of some respirable crystalline silica (MMAD RF>TG>Ti>BR>TC>Al2O3. DNA-damage was maximal for BR and TI followed by DQ12>TG>TC>RF>Al2O3. All dusts induced PGE2-liberation (DQ12>BR>TC>TG>Ti>RF>Al2O3) at 50μg/cm2 (4h), but TNF-a mRNA (10μg/cm2, 24h) was only increased by DQ12, TG (quartz-dependently), and TC. In conclusion, these in vitro tests were an adequate approach to screen the toxic potential of quartz-containing ceramic dusts, but the quartz-content was too low to differentiate the various quartz-varieties.