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The genotoxic effects of 2-acetylaminofluorene (AAF) alone cannot explain the formation of rat liver tumors. It has been proposed that mitochondrial effects are associated with its tumor-promoting properties. These mitochondrial effects are thought to trigger apoptosis and regenerative proliferation, which alters the liver lobule in a cirrhosis-like manner. A situation is generated which favors the growth of initiated cells. To test this sequence of events, the dose dependence of early effects with time was studied. Male Wistar rats received 50, 100, 200, 400, or 800 ppm AAF in the diet and the following endpoints were analyzed at 2, 4, 8, and 16 weeks of feeding: apoptotic cell death, cell proliferation, GST-P-positive foci (placental form of glutathione S-transferase), and morphological alterations. Hydrolyzable hemoglobin adducts were used as a dosimeter for metabolic activation after 2 weeks of feeding. The hemoglobin adduct levels increased linearly with dose. With the conventional carcinogenic concentration of 200-ppm AAF in the diet, the number of apoptoses increased first, predominantly in the periportal area (2 weeks). Cell proliferation followed with some delay (4 weeks). This reflects regenerative tissue response and not the growth of initiated cells, because the number of enzyme-altered foci was still extremely low at that time. Foci developed only later when the morphology had changed. With 50 ppm AAF in the diet, a no-effect level had not been reached for any of the endpoints, but foci developed much more gradually than with higher doses. Unexpectedly, the proliferative response stabilized at 8 weeks with a labeling index of 12-17, with all AAF concentrations. The observed sequence of events supports the hypothesis. It is concluded that (1) The proliferation of initiated cells-defined as promotable cells-is largely determined by the cellular environment, such as morphologically altered liver. (2) The morphological alterations in rat liver result from imperfect regeneration from toxic effects. (3) Imperfect regeneration results from limitations in the possibilities to adapt to chemical stress. (4) If these limits are overwhelmed and morphology has changed to a certain extent, preneoplastic foci develop; this occurs much faster, at least, than without these changes.

In the present study we investigated c-fos expression in rat livers, that was initiated with the three arylamines, 2-acetylaminofluorene, 2-acetylaminophenanthrene and trans-4-acetylaminostilbene. The tumor promoter phenobarbital was applied chronically for 26, 52 and 100 weeks. Gene expression, determined by the mRNA level, and FOS protein were increased after 52 weeks of treatment in arylamine initiated as well as in phenobarbital only treated animals. Expression of c-fos seems to be a phenobarbital induced effect that is independent of additional initiator treatments. This finding was supported by immunohistochemical studies demonstrating increased FOS levels to be localized around the central vein. The results indicate that phenobarbital, a widely used tumor promoter, induces c-fos expression. In addition, we demonstrated enhanced FOS in GST-P-positive foci and in tumors.

The tumour promoting properties of carcinogenic 2-acetylaminofluorene (AAF) in rat liver are essentially unknown. We proposed that mitochondria are a target for the cytotoxic effects of 2-nitrosofluorene (NOF), a metabolite of AAF, since NOF induces a redox-cycle at complex I and complex III of the respiratory chain, and impairs respiration and oxidative phosphorylation. We now demonstrate that NOF is a potent inducer of the mitochondrial permeability transition pore (PTP) in isolated mitochondria. In the presence of Ca2+, NOF induced rapid swelling of mitochondria in a dose-dependent manner and depolarized the mitochondrial membrane. Permeability transition as well as depolarization were abolished completely by pre-incubation with the PTP inhibitor cyclosporin A. To study whether the PTP is involved in in vivo toxicity, rats were fed a diet containing AAF (0.04%) for 2 weeks. After isolation of mitochondria, permeability transition was induced by high Ca2+ concentrations (150-400 microM) or phosphate plus Ca2+. Swelling was determined as maximal rate of absorption decrease at 540 nm (delta A/delta t). Surprisingly, delta A/delta t-values of mitochondria from AAF-fed rats were significantly lower (16.3 +/- 4.8 x 10(3)/min) than of mitochondria from control animals (32.7 +/- 4.1 x 10(3)/min; P < 0.02). In the presence of phosphate (15 mM), delta A/delta t-values of mitochondria from AAF-fed rats were even lower (10% of control). Moreover, the membrane potential which was dissipated rapidly by the PTP-inducer NOF (30 microM) at a Ca2+ concentration of 80 microM in mitochondria from control animals, remained constant in mitochondria of AAF-treated rats. We therefore propose that the regulation of the PTP is altered on chronic AAF-feeding. The increased resistance of mitochondria against permeability transition may alter the threshold for apoptosis and thus suppress apoptosis. We also discuss the role of epigenetic modifications in early stages of carcinogenesis.

Many polycyclic aromatic amines are mutagenic, and their genotoxic properties are held responsible for their biological effects. The genotoxic effects are explained by the formation of metabolites that react with macromolecules by forming adducts (Kriek 1969; Franz et al. 1986). DNA adducts may cause mutations which are responsible for tumorigenic effects. Results from comparative studies, however, indicate that tissue-specific and species-specific tumor formation caused by arylamines cannot be explained readily by the extent of DNA modifications. Long-term feeding of 2-acetylaminofluorene (AAF) typically produces liver tumors; 2-acetylaminophenanthrene (AAP), mammary tumors; andtrans-4-acetylaminostilbene (AAS), Zymbal’s gland tumors in rats (Neumann et al. 1970). All three arylamines are genotoxic. They generate comparable DNA adduct levels in rat liver, and in liver more extensively than in any other tissue (Neumann 1983; Ruthsatz and Neumann 1988; Gupta et al. 1989). But only AAF is a complete carcinogen in rat liver. AAS and AAP are able to produce liver tumors in rats only, if initiation is followed by some promotion treatment (Hammerl 1989). Therefore, DNA binding may reflect the formation of some critical lesions related to tumor initiation but is not sufficient to explain carcinogenic effects. This raises two questions: (1) Do differences in genotoxic initiating effects contribute to tissue specificity? and (2) What are the additional promoting properties of AAF that distinguish it from the incomplete liver carcinogens?

2-Acetylaminofluorene (2-AAF) is a complete carcinogen in rat liver. To investigate the specific properties, that distinguish 2-AAF from incomplete carcinogens, rats were fed 0.02% AAF in the diet for 6, 12, 16 weeks and some indicators of genotoxic and chronic toxic effects were studied immunohistochemically. GST-P, a marker for single initiated cells and preneoplastic foci, was induced in response to 2-AAF exposure. The effects were slight after 6 weeks of feeding, after 12 weeks GST-P-positive preneoplastic foci were present. The proto-oncogenes c-fos and c-jun are induced by several tumor promoters. In the present study c-FOS protein levels were increased in all 2-AAF treated animals at early stages not only in preneoplastic foci. However, all GST-P-positive foci were also c-FOS-positive. Surprisingly c-JUN was not enhanced in GST-P positive foci. It was comparatively expressed in hepatocytes and bile duct cells in all animals. We did not observe any immunolabeling for p53, either in preneoplastic foci or in hepatocytes from treated animals. A significant increase of apoptoses was noted in the whole liver lobule but also gathered in groups in the periportal area. The results support our proposal that oxidative stress and energy impairment in the mitochondria of periportal hepatocytes trigger morphological alterations in the rat liver.

Many polycyclic aromatic amines are mutagenic. Their genotoxic properties are explained by the formation of metabolites that react with DNA by forming adducts (Kriek 1969, Franz et al. 1986). These DNA adducts may cause mutations which are responsible for tumorigenic effects. The carcinogenesis induced by aromatic amines is very often characterized by high tissue specificity. Chronic feeding of 2-acetylaminofluorene (AAF) typically produces liver tumors in rodents, 2-acetylaminophenanthrene (AAP) mammary tumors and trans-4-acetylaminostilbene (AAS) Zymbal’s gland tumors (Neumann et al. 1970). Many attempts have been made to correlate DNA binding or the resulting lesions with tissue specific tumor formation. However, this phenomenon cannot be readily explained by the extent of DNA modifications. All three amines generate comparable DNA adduct levels in rat liver and moreover in liver more extensively than in any other tissue (Neumann 1983, Ruthsatz and Neumann 1988, Gupta et al. 1989). Therefore, DNA binding may reflect the formation of some critical lesions related to tumor initiation but is not sufficient to explain carcinogenic effects. AAS and AAP are able to produce liver tumors in rats, but only if initiation is followed by some promotion treatment (Hammerl 1989). AAF in contrast is a complete carcinogen for this tissue.

Liver tumors were generated in Wistar rats in an initiation-promotion experiment. 2-Acetylaminofluorene (AAF), 2-acetylaminophenanthrene (AAP), and trans-4-acetylaminostilbene (AAS) were administered to newborn animals as initiators, and phenobarbital as a promoter was added to the drinking water after weaning. Livers were examined after 26, 52, 78, and 104 weeks. Tumors were present in all groups except for at the first time point. The potency of the initiators decreased in the order AAS > AAP > AAF. DNA from tumors of all groups and of control livers was analyzed for mutations in the H-ras gene, but no mutations could be found. The sequence of almost the entire H-ras gene was determined and was compared to other H-ras genes. There are some differences with the sequence in other rat strains, particularly in intron D containing the alternative splicing site. The expression of the H-ras gene has also been studied by various methods in enzyme altered foci and tumors, but no alterations could be found. It is, therefore, concluded that structural of functional alterations of this proto-oncogene are not involved in the generation of liver tumors in Wistar rats by the three genotoxic arylamines.

Under the European Union (EU) Biocidal Products Directive 98/8/EC, comprehensive evaluations on substances of the Third Priority List were conducted until 31 July 2007. This list includes, among other categories, disinfectants for human hygiene (e.g., skin and surface disinfection). For environmental exposure assessment of biocides, the EU emission scenarios apply. Currently available default values for disinfectants are based on consumption data from not more than 8 hospitals and were originally assembled for other purposes. To revalidate these default values, a survey on annual consumption data was performed in 27 German hospitals. These data were analyzed to provide consumption data per bed and day and per nurse and day for particular categories of active ingredients and were compared with default values from the EU emission scenario documents. Although several deviations were detected, an overall acceptable correspondence between Emission Scenario Documents default values and the current survey data was found. Integr Environ Assess Manag 2010;6:273–280. © 2009 SETAC

The mouse spot test, an in vivo mutation assay, has been used to assess a number of chemicals. It is at present the only in vivo mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484). It is however rather insensitive, animal consuming and expensive type of test. More recently several assays using transgenic animals have been developed. From data in the literature, the present study compares the results of in vivo testing of over twenty chemicals using the mouse spot test and compares them with results from the two transgenic mouse models with the best data base available, the lacI model (commercially available as the Big Blue® mouse), and the lacZ model (commercially available as the Muta™ Mouse). There was agreement in the results from the majority of substances. No differences were found in the predictability of the transgenic animal assays and the mouse spot test for carcinogenicity. However, from the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo.

As part of a larger literature study on transgenic animals in mutagenicity testing, test results from the transgenic mutagenicity assays (lacI model; commercially available as the Big Blue® mouse, and the lacZ model; commercially available as the Muta™Mouse), were compared with the results on the same substances in the more traditional mouse bone marrow micronucleus test. 39 substances were found which had been tested in the micronucleus assay and in the above transgenic mouse systems. Although, the transgenic animal mutation assay is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation, the results for the majority of substances were in agreement. Both test systems, the transgenic mouse assay and the mouse bone marrow micronucleus test, have advantages and they complement each other. However, the transgenic animal assay has some distinct advantages over the micronucleus test: it is not restricted to one target organ and detects systemic as well as local mutagenic effects.

Applying labelled phenylalanine or tyrosine to purified intact spinach chloroplasts, only the corresponding phenylacetic acids but not the cinnamic acids could be detected. The addition of mercaptoethanol or dl-dithiothreitol and the variation of light conditions had only a slight effect. However, cinnamic acids could be found together with phenylacetic acids in leaf homogenates indicating the presence of phenylalanine and/or tyrosine ammonia lyase outside the spinach chloroplasts. Similar results were obtained with barley leaf homogenates, where cinnamic acids were the main products. Reviewing recent findings on amino acid synthesis in spinach leaves, it may be concluded that the synthesis of aromatic amino acids is restricted to the chloroplast, whereas the metabolism of secondary aromatic compounds is predominantly localized outside the chloroplasts.

In der Anwendung van Bioziden wird die Schaumversprühung zunehmend als sicherere Alternative zur Tropfenversprühung eingesetzt. Bislang fehlen jedoch Expositionsdaten sowie etablierte Ansätze zur Charakterisierung und Bewertung der potenziellen Aerosolfreisetzung bei der Schaumversprühung, sodass bei der Zulassung von Biozidwirkstoffen und -produkten Unsicherheiten bezüglich der Bewertung bestehen. Erste Untersuchungen im Rahmen der vorliegenden Studie zeigen, dass bei der Verwendung van Schaumen eine Aerosolbildung nicht ausgeschlossen werden kann, die Aerosolfreisetzung aber gegenüber der konventionellen Tropfenversprühung signifikant reduziert ist. Die derzeitige Bewertung auf der Basis van Daten für Sprühanwendungen kann jedoch ggf. zu einer Überschatzung der inhalativen Exposition führen. Weitere Untersuchungen sind notwendig zur Ermittlung von Korrelationen zwischen Aerosolfreisetzung und Produkt- und Prozessparametern. Diese können als Grundlage für die Bewertung des Expositionsrisikos sowie ggf. für die lmplementierung der Schaumversprühung in Expositionsmodellen dienen.