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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 μM) or phosphate plus Ca2+. Swelling was determined as maximal rate of absorption decrease at 540 nm (ΔA/Δt. Surprisingly, ΔA/Δt-values of mitochondria from AAF-fed rats were significantly lower (16.3 ± 4.8 x 103/min) than of mitochondria from control animals (32.7 ± 4.1 x 103/min; P < 0.02). In the presence of phosphate (15 mM), ΔA/Δt-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 μM) at a Ca2+ concentration of 80 μM 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. [ABSTRACT FROM PUBLISHER]

The self-templating nature of prions plays a central role in prion pathogenesis and is associated with infectivity and transmissibility. Since propagation of proteopathic seeds has now been acknowledged a principal pathogenic process in many types of dementia, more insight into the molecular mechanism of prion replication is vital to delineate specific and common disease pathways. By employing highly discriminatory anti-PrP antibodies and conversion-tolerant PrP chimera, we here report that de novo PrP conversion and formation of fibril-like PrP aggregates are distinct in mechanistic and kinetic terms. De novo PrP conversion occurs within minutes after infection at two subcellular locations, while fibril-like PrP aggregates are formed exclusively at the plasma membrane, hours after infection. Phenotypically distinct pools of abnormal PrP at perinuclear sites and the plasma membrane show differences in N-terminal processing, aggregation state and fibril formation and are linked by exocytic transport via synaptic and large-dense core vesicles., (© 2023. Crown.)

To dissect the N-terminal residues within the cellular prion protein (PrP C ) that are critical for efficient prion propagation, we generated a library of point, double, or triple alanine replacements within residues 23-111 of PrP, stably expressed them in cells silenced for endogenous mouse PrP C and challenged the reconstituted cells with four common but biologically diverse mouse prion strains. Amino acids (aa) 105-111 of Charge Cluster 2 (CC2), which is disordered in PrP C , were found to be required for propagation of all four prion strains; other residues had no effect or exhibited strain-specific effects. Replacements in CC2, including aa105-111, dominantly inhibited prion propagation in the presence of endogenous wild type PrP C whilst other changes were not inhibitory. Single alanine replacements within aa105-111 identified leucine 108 and valine 111 or the cluster of lysine 105, threonine 106 and asparagine 107 as critical for prion propagation. These residues mediate specific ordering of unstructured CC2 into β-sheets in the infectious prion fibrils from Rocky Mountain Laboratory (RML) and ME7 mouse prion strains., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Prion diseases are fatal neurodegenerative diseases that affect humans and animals. Prion strains, conformational variants of misfolded prion proteins, are associated with distinct clinical and pathological phenotypes. Host-strain interactions result in the selective damage of distinct brain areas and they are responsible for strain selection and/or adaptation, but the underlying molecular mechanisms are unknown. Prion strains can be distinguished by their cell tropism in vivo and in vitro , which suggests that susceptibility to distinct prion strains is determined by cellular factors. The neuroblastoma cell line PK1 is refractory to the prion strain Me7, but highly susceptible to RML. We challenged a large number of clonal PK1 lines with Me7 and successfully selected highly Me7-susceptible subclones (PME) to investigate whether the prion strain repertoire of PK1 can be expanded. Notably, the Me7-infected PME clones were more protease-resistant when compared to RML-infected PME clones, which suggested that cell-adapted Me7 and RML are distinct prion strains. Strikingly, Me7-refractory cells, including PK1 and astrocytes in cortico-hippocampal cultures, are highly susceptible to prions, being derived from homogenates of Me7-infected PME cells, suggesting that the passage of Me7 in PME cells leads to an extended host range. Thus, PME clones represent a compelling cell model for strain selection and adaptation.

The mouse-adapted scrapie prion strain RML is one of the most widely used in prion research. The introduction of a cell culture-based assay of RML prions, the scrapie cell assay (SCA) allows more rapid and precise prion titration. A semi-automated version of this assay (ASCA) was applied to explore a range of conditions that might influence the infectivity and properties of RML prions. These include resistance to freeze-thaw procedures; stability to endogenous proteases in brain homogenate despite prolonged exposure to varying temperatures; distribution of infective material between pellet and supernatant after centrifugation, the effect of reducing agents and the influence of detergent additives on the efficiency of infection. Apparent infectivity is increased significantly by interaction with cationic detergents. Importantly, we have also elucidated the relationship between the duration of exposure of cells to RML prions and the transmission of infection. We established that the infection process following contact of cells with RML prions is rapid and followed an exponential time course, implying a single rate-limiting process.

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20,000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved., (© 2015 The Authors.)

Prion diseases (transmissible spongiform encephalopathies) are fatal neurodegenerative diseases, including Creutzfeldt-Jakob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. While genome-wide association studies in human and quantitative trait loci mapping in mice have provided evidence for multiple susceptibility genes, few of these have been confirmed functionally. Phenotyping mouse models is generally the method of choice. However, this is not a feasible option where many novel genes, without pre-existing models, would need to be tested. We have therefore developed and applied an in-vitro screen to triage and prioritize candidate modifier genes for more detailed future studies which is faster, far more cost effective and ethical relative to mouse bioassay models. An in vitro prion bioassay, the scrapie cell assay, uses a neuroblastoma-derived cell line (PK1) that is susceptible to RML prions and able to propagate prions at high levels. In this study, we have generated stable gene silencing and/or overexpressing PK1-derived cell lines to test whether perturbation of 14 candidate genes affects prion susceptibility. While no consistent differences were determined for seven genes, highly significant changes were detected for Zbtb38, Sorcs1, Stmn2, Hspa13, Fkbp9, Actr10 and Plg, suggesting that they play key roles in the fundamental processes of prion propagation or clearance. Many neurodegenerative diseases involve the accumulation of misfolded protein aggregates and 'prion-like' seeding and spread has been implicated in their pathogenesis. It is therefore expected that some of these prion-modifier genes may be of wider relevance in neurodegeneration., (© The Author 2014. Published by Oxford University Press.)

Prions consist of aggregates of abnormal conformers of the cellular prion protein (PrP(C)). They propagate by recruiting host-encoded PrP(C) although the critical interacting proteins and the reasons for the differences in susceptibility of distinct cell lines and populations are unknown. We derived a lineage of cell lines with markedly differing susceptibilities, unexplained by PrP(C) expression differences, to identify such factors. Transcriptome analysis of prion-resistant revertants, isolated from highly susceptible cells, revealed a gene expression signature associated with susceptibility and modulated by differentiation. Several of these genes encode proteins with a role in extracellular matrix (ECM) remodelling, a compartment in which disease-related PrP is deposited. Silencing nine of these genes significantly increased susceptibility. Silencing of Papss2 led to undersulphated heparan sulphate and increased PrP(C) deposition at the ECM, concomitantly with increased prion propagation. Moreover, inhibition of fibronectin 1 binding to integrin α8 by RGD peptide inhibited metalloproteinases (MMP)-2/9 whilst increasing prion propagation. In summary, we have identified a gene regulatory network associated with prion propagation at the ECM and governed by the cellular differentiation state., (© 2014 The Authors. Published under the terms of the CC BY 4.0 license.)

Prion diseases are incurable transmissible neurological disorders. In many natural and experimental prion diseases, infectious prions can be detected in the lymphoreticular system (LRS) long before they reach the brain where they cause a fatal rapidly progressive degeneration. Although major cell types that contribute to prion accumulation have been identified, the mode of prion dissemination in the LRS remains elusive. Recent evidence of a remarkably fast splenic prion accumulation after peripheral infection of mice, resulting in high prion titers in dendritic cells (DCs) and a release of prions from infected DCs via exosomes suggest that intercellular dissemination may contribute to rapid prion colonization in the LRS. A vast body of evidence from retroviral infections shows that DCs and other antigen-presenting cells (APCs) share viral antigens by intercellular transfer to warrant immunity against viruses if APCs remain uninfected. Evolved to adapt the immune response to evading pathogens, these pathways may constitute a portal for unimpeded prion dissemination owing to the tolerance of the immune system against host-encoded prion protein. In this review we summarize current paradigms for antigen-sharing pathways which may be relevant to better understand dissemination of rogue neurotoxic proteins., (Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles.

Intraperitoneal administration of ICSM18 and 35, monoclonal antibodies against prion protein (PrP), has been shown to significantly delay the onset of prion disease in mice, and humanized versions are candidate therapeutics for prion and Alzheimer's diseases. However, a previous report of severe and widespread apoptosis after intracerebral injection of anti-PrP monoclonal antibodies raised concerns about such therapy and led to an influential model of prion neurotoxicity via cross-linking of cell surface PrP by disease-related PrP aggregates. In extensive studies including ICSM18 and 35, fully humanized ICSM18, and the previously reported proapoptotic antibodies, we found no evidence of apoptosis, thereby questioning this model of prion neurotoxicity.

Prion infections induce severe disruption of the central nervous system with neuronal vacuolation and extensive glial reactions, and invariably lead to death of affected individuals. The molecular underpinnings of these events are not well understood. To better define the molecular consequences of prion infections, we analyzed the transcriptional response to persistent prion infection in a panel of three murine neural cell lines in vitro. Colony spot immunochemistry assays indicated that 65-100% of cells were infected in each line. Only the Nav1 gene was marginally modulated in one cell line, whereas transcripts previously reported to be derailed in prion-infected cells were not confirmed in the present study. We attribute these discrepancies to the experimental stringency of the current study, which was performed under conditions designed to minimize potential genetic drifts. These findings are at striking variance with gene expression studies performed on whole brains upon prion infections in vivo, suggesting that many of the latter changes represent secondary reactions to infection. We conclude that, surprisingly, there are no universal transcriptional changes induced by prion infection of neural cells in vitro.

The molecular basis for neuronal death in prion disease is not established, but putative pathogenic roles for both disease-related prion protein (PrP(Sc)) and accumulated cytosolic PrP(C) have been proposed. Here we report that only prion-infected neuronal cells become apoptotic after mild inhibition of the proteasome, and this is strictly dependent upon sustained propagation of PrP(Sc). Whereas cells overexpressing PrP(C) developed cytosolic PrP(C) aggregates, this did not cause cell death. In contrast, only in prion-infected cells, mild proteasome impairment resulted in the formation of large cytosolic perinuclear aggresomes that contained PrP(Sc), heat shock chaperone 70, ubiquitin, proteasome subunits, and vimentin. Similar structures were found in the brains of prion-infected mice. PrP(Sc) aggresome formation was directly associated with activation of caspase 3 and 8, resulting in apoptosis. These data suggest that neuronal propagation of prions invokes a neurotoxic mechanism involving intracellular formation of PrP(Sc) aggresomes. This, in turn, triggers caspase-dependent apoptosis and further implicates proteasome dysfunction in the pathogenesis of prion diseases.

Prions typically accumulate in nervous and lymphoid tissues. Because proinflammatory cytokines and immune cells are required for lymphoid prion replication, we tested whether inflammatory conditions affect prion pathogenesis. We administered prions to mice with five inflammatory diseases of the kidney, pancreas, or liver. In all cases, chronic lymphocytic inflammation enabled prion accumulation in otherwise prion-free organs. Inflammatory foci consistently correlated with lymphotoxin up-regulation and ectopic induction of FDC-M1+ cells expressing the normal cellular prion protein PrPC. By contrast, inflamed organs of mice lacking lymphotoxin-alpha or its receptor did not accumulate the abnormal isoform PrPSc, nor did they display infectivity upon prion inoculation. By expanding the tissue distribution of prions, chronic inflammatory conditions may act as modifiers of natural and iatrogenic prion transmission.

The mechanisms involved in prion neurotoxicity are unclear, and therapies preventing accumulation of PrPSc, the disease-associated form of prion protein (PrP), do not significantly prolong survival in mice with central nervous system prion infection. We found that depleting endogenous neuronal PrPc in mice with established neuroinvasive prion infection reversed early spongiform change and prevented neuronal loss and progression to clinical disease. This occurred despite the accumulation of extraneuronal PrPSc to levels seen in terminally ill wild-type animals. Thus, the propagation of nonneuronal PrPSc is not pathogenic, but arresting the continued conversion of PrPc to PrPSc within neurons during scrapie infection prevents prion neurotoxicity.

Prions are usually quantified by bioassays based on intracerebral inoculation of mice that are slow, imprecise, and costly. We have isolated neuroblastoma N2a sublines highly susceptible to mouse prions, as evidenced by accumulation of infectivity and the scrapie form of prion protein (PrPSc), and developed quantitative in vitro assays for prion infectivity. In the scrapie cell (SC) assay, susceptible N2a cells are exposed to prion-containing samples for 3 days, grown to confluence, and split 1:10 three times, and the proportion of PrPSc-containing cells is determined with automated counting equipment. In a log/log plot, the dose-response is linear over two logs of prion concentrations. The SC assay is about as sensitive as the mouse bioassay, 10 times faster, >2 orders of magnitude less expensive, and suitable for robotization. SC assays performed in a more time-consuming end point titration format extend the sensitivity and show that infectivity titers measured in tissue culture and in the mouse are similar.

A hallmark of tumorigenesis is resistance to apoptosis. To explore whether resistance to cell death precedes tumor formation, we have studied the short-term effects of the hepatocarcinogen 2-acetylaminofluorene (AAF) on liver mitochondria, on hepatocytes, and on the response to bacterial endotoxin lipopolysaccharide (LPS) in albino Wistar rats. We show that after as early as two weeks of AAF feeding liver mitochondria developed an increased resistance to opening of the permeability transition pore (PTP), an inner membrane channel that is involved in various forms of cell death. Consistent with a mitochondrial adaptive response in vivo, (i) AAF feeding increased the expression of BCL-2 in mitochondria, and (ii) hepatocytes isolated from AAF-fed rats became resistant to PTP-dependent depolarization, cytochrome c release, and cell death, which were instead observed in hepatocytes from rats fed a control diet. AAF-fed rats were fully protected from the hepatotoxic effects of the injection of 20-30 microg of LPS plus 700 mg of d-galactosamine (d-GalN) x kg-1 of body weight, a treatment that in control rats readily caused a large increase of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in liver cryosections and release of alanine and aspartate aminotransferase into the bloodstream. Treatment with LPS and d-GalN triggered cleavage of BID, a BCL-2 family member, in the livers of both control- and AAF-fed animals, whereas caspase 3 was cleaved only in control-fed animals, indicating that the mitochondrial proapoptotic pathway had been selectively suppressed during AAF feeding. Phenotypic reversion was observed after stopping the carcinogenic diet. These results underscore a key role of mitochondria in apoptosis and demonstrate that regulation of the mitochondrial PTP is altered early during AAF carcinogenesis, which matches, and possibly causes, the increased resistance of hepatocytes to death stimuli in vivo. Both events precede tumor formation, suggesting that suppression of apoptosis may contribute to the selection of a resistant phenotype, eventually increasing the probability of cell progression to the transformed state.

The "protein only" hypothesis states that the infectious agent causing transmissible spongiform encephalopathies is a conformational isomer of PrP, a host protein predominantly expressed in brain, and is strongly supported by many lines of evidence. Prion diseases are so far unique among conformational diseases in that they are transmissible, not only experimentally but also by natural routes, mainly by ingestion. A striking feature of prions is their extraordinary resistance to conventional sterilization procedures, and their capacity to bind to surfaces of metal and plastic without losing infectivity. This property, first observed in a clinical setting, is now being investigated in experimental settings, both in animals and in cell culture.

The "protein only" hypothesis holds that the infectious agent causing transmissible spongiform encephalopathies is a conformational isomer of PrP, a host protein predominantly expressed in brain and is strongly supported by many lines of evidence. Prion diseases are so far unique among conformational diseases in that they are transmissible, not only experimentally but also by natural routes, mainly by ingestion. The pathway of prions to the brain has been elucidated in outline. A striking feature of prions is their extraordinary resistance to conventional sterilisation procedures, and their capacity to bind to surfaces of metal and plastic without losing infectivity. This property, first observed in a clinical setting, is now being investigated in experimental settings, both in animals and in cell culture.

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.

2-Acetylaminofluorene (AAF) is a complete carcinogen in rat liver. The genotoxic effects of reactive metabolites are considered necessary but not sufficient to explain tumor formation. An overview is given of an AAF-feeding experiment designed to demonstrate early effects, preceding the development of enzyme-altered foci to support the hypothesis that toxic effects lead to a cirrhosis-like transformation as a prerequisite for the expansion of initiated foci and how those effects influence the dose-time-response relationship of tumor formation. Male Wistar rats were fed 0.005, 0.01, 0.02, 0.04 and 0.08% AAF in the diet for 2, 4, 8, and 16 weeks. GST-P-positive foci developed more than proportionately only at 16 weeks. As a first sign of morphological alterations the number of apoptoses increased (2 weeks), the proliferation rate followed with some delay and was maximal at 4 weeks. The most sensitive parameter for adaptive responses was the inhibition of the mitochondrial permeability transition, studied ex vivo. All parameters increased dose-dependently at low doses. A threshold could not be detected, but effects developed much more gradually with the lowest, non-toxic dose. The situation of massive development of foci observed with the higher doses at 16 weeks was not reached. Apoptosis and proliferation rate reach a plateau between 4 and 8 weeks with some of the doses indicating a period in which some balance between adaptation and stress response exists.

The present study was designed to investigate the effects of 2-nitrosofluorene (NOF), a metabolite of carcinogenic 2-acetylaminofluorene, on mitochondrial respiration and oxidative phosphorylation. NOF reacts with the NADH:ubiquinone oxidoreductase (complex I) and consumes oxygen in a rotenone-insensitive manner. Unlike menadione, which is able to bypass the rotenone-block and to restore ATP-formation, NOF-induced electron flow was almost completely uncoupled. In normal respiration both redox-cyclers decreased the respiratory control and P/O ratios at low concentrations (2-20 nmol/mg) in NADH-dependent oxidation. With succinate as substrate, only NOF was significantly active. In contrast to NOF, the hydroxamic acid N-hydroxy-2-acetylaminofluorene (N-OH-AAF) impaired mitochondrial energy conversion only at much higher concentrations (80 nmol/mg). At concentrations > 10 nmol/mg, NOF inhibited electron flow through the respiratory chain in NADH- and succinate-dependent oxidation, as determined by dinitrophenolate-uncoupled respiration. The small protective effect of L-cysteine indicates that covalent binding of the nitroso-compound to SH-groups may not explain sufficiently the inhibitory effect of NOF. The results support the notion that redox cyclers impair oxidative phosphorylation by establishing alternative pathways for electron transport in the respiratory chain.

2-Acetylaminofluorene (AAF) is one of the most widely studied model carcinogens. It produces liver tumors in rats. Comparison with other arylamides shows that promutagenic DNA lesions are necessary but not sufficient to explain this tissue-specific effect. Mutagenicity of AAF was studied in AS52 cells and compared with that of 2-acetylaminophenanthrene and trans-4-acetylaminostilbene which are incomplete carcinogens in rat liver. The major mutations were G to T transversions in all cases. All three acetamides acted as initiators in an initiation-promotion experiment with phenobarbital as a promoter. Chronic toxic effects of AAF were attributed to specific effects of AAF metabolites on mitochondrial respiration. Electron drainage by 2-nitrosofluorene causes an uncoupling effect on oxidative phosphorylation in vitro. Corresponding compensatory effects were observed in vivo. Initiating as well as promoting properties of AAF are therefore considered responsible for the generation of rat liver tumors. The results support the hypothesis that genotoxic effects generate initiated cells which begin to proliferate only when microcirculation is disturbed due to cirrhotic alterations. These are triggered by non-genotoxic interference with mitochondrial respiration and oxidative phosphorylation.

We determined the sites of artificial electron transfer onto 2-nitrosofluorene (NOF), a metabolite of carcinogenic 2- acetylaminofluorene in mitochondria and isolated cytochrome bc1 complex. NOF-induced O2 consumption in mitochondria was sensitive to antimycin A, but insensitive to myxothiazol. In the isolated cytochrome bc1 complex, NOF induced rapid MOA-stilbene-insensitive reoxidation of cytochrome b, whereas in the presence of antimycin A, reoxidation was very slow. The corresponding hydroxylamine, N-hydroxy-2-aminofluorene (N-OH-AF), reduced cytochrome b specifically through center N of the cytochrome bc1 complex. We conclude that NOF and N-OH-AF bind to center N of the cytochrome bc1 complex and act as electron acceptor and donor, respectively. The N-OH-AF/NOF interconversion is considered to be involved in the cytotoxicity of 2-acetylaminofluorene in vivo.

The present study was designed to confirm the recent proposal that 2-nitrosofluorene (2-NOF) as well as N-hydroxy-2-aminofluorene (N-OH-AF) induce a redox-cycle in rat liver mitochondria as part of the chronic toxic effects of the carcinogen 2-acetylaminofluorene (2-AAF). The formation of O2.- was demonstrated in submitochondrial particles by the formation of adrenochrome with NADH and succinate as respiratory substrates. 2-NOF was as effective as paraquat, a known redox-cycler, the lowest effective concentration being 0.4 nmol 2-NOF/mg protein. Experiments with isolated mitochondria showed that 2-NOF, in contrast to N-OH-AF, induces cyanide-resistant O2 consumption only in the presence of respiratory substrates, indicating that the reduction, but not the reoxidation, depends on a continuous flow of electrons through the respiratory chain of the mitochondrial membrane. Lipid peroxidation was estimated by the formation of thiobarbituric-acid-reactive substances. In comparison to the well-known prooxidant tert-butylhydroperoxide, 2-NOF was not significantly active. The results support the notion that 2-NOF induces oxidative stress by mitochondrial redox-cycling in vivo. Effects other than lipid peroxidation seem to be important for the chronic toxicity of 2-AAF.