Johanna Michl

Interplay between Fanconi anemia and homologous recombination pathways in genome integrity

The Fanconi anemia (FA) pathway plays a central role in the repair of DNA interstrand crosslinks (ICLs) and regulates cellular responses to replication stress. Homologous recombination (HR), the error‐free pathway for double‐strand break (DSB) repair, is required during physiological cell cycle progression for the repair of replication‐associated DNA damage and protection of stalled replication forks. Substantial crosstalk between the two pathways has recently been unravelled, in that key HR proteins such as the RAD51 recombinase and the tumour suppressors BRCA1 and BRCA2 also play important roles in ICL repair. Consistent with this, rare patient mutations in these HR genes cause FA pathologies and have been assigned FA complementation groups. Here, we focus on the clinical and mechanistic implications of the connection between these two cancer susceptibility syndromes and on how these two molecular pathways of DNA replication and repair interact functionally to prevent genomic instability.

Is aristolochic acid nephropathy a widespread problem in developing countries? A case study of Aristolochia indica L. in Bangladesh using an ethnobotanical-phytochemical approach.

ETHNOPHARMACOLOGICAL RELEVANCE Species of Aristolochia are associated with aristolochic acid nephropathy (AAN), a renal interstitial fibrosis and upper urinary tract cancer (UUC). Aristolochic acid nephropathy has been reported in ten countries but its true incidence is unknown and most likely underestimated. By combining an ethnobotanical and phytochemical approach we provide evidence for the risk of AAN occurring in Bangladesh. More specifically, we assess the intra-specific variation of aristolochic acid analogues in medicinally used Aristolochia indica samples from Bangladesh. MATERIALS AND METHODS Ethnobotanical information was collected from 16 kavirajes (traditional healers) in different study locations in Bangladesh. Plant samples were obtained from native habitats, botanical gardens, herbal markets and pharmaceutical companies. The samples were extracted using 70% methanol and were analysed using LC-DAD-MS and (1)H-NMR. RESULTS Roots as well as leaves are commonly used for symptoms such as snake bites and sexual problems. Among the informants knowledge about toxicity or side effects is very limited and Aristolochia indica is often administered in very high doses. Replacement of Aristolochia indica with other medicinal plants such as Rauvolfia serpentina (L.) Benth. ex Kurz was common. Aristolochia indica samples contained a variety of aristolochic acid analogues such as aristolochic acid I, aristolochic acid II, cepharadione A and related compounds. CONCLUSIONS AAN cases are likely to occur in Bangladesh and more awareness needs to be raised about the health risks associated with the use of Aristolochia indica and other species of Aristolochia as herbal medicines.

FANCD2 limits replication stress and genome instability in cells lacking BRCA2

The tumor suppressor BRCA2 plays a key role in genome integrity by promoting replication-fork stability and homologous recombination (HR) DNA repair. Here we report that human cancer cells lacking BRCA2 rely on the Fanconi anemia protein FANCD2 to limit replication-fork progression and genomic instability. Our results identify a new role of FANCD2 in limiting constitutive replication stress in BRCA2-deficient cells, thereby affecting cell survival and treatment responses.

LC-MS- and 1 H NMR-Based Metabolomic Analysis and in Vitro Toxicological Assessment of 43 Aristolochia Species

Species of Aristolochia are used as herbal medicines worldwide. They cause aristolochic acid nephropathy (AAN), a devastating disease associated with kidney failure and renal cancer. Aristolochic acids I and II (1 and 2) are considered to be responsible for these nephrotoxic and carcinogenic effects. A wide range of other aristolochic acid analogues (AAAs) exist, and their implication in AAN may have been overlooked. An LC-MS- and (1)H NMR-based metabolomic analysis was carried out on 43 medicinally used Aristolochia species. The cytotoxicity and genotoxicity of 28 Aristolochia extracts were measured in human kidney (HK-2) cells. Compounds 1 and 2 were found to be the most common AAAs. However, AA IV (3), aristolactam I (4), and aristolactam BI (5) were also widespread. No correlation was found between the amounts of 1 or 2 and extract cytotoxicity against HK-2 cells. The genotoxicity and cytotoxicity of the extracts could be linked to their contents of 5, AA D (8), and AA IIIa (10). These results undermine the assumption that 1 and 2 are exclusively responsible for the toxicity of Aristolochia species. Other analogues are likely to contribute to their toxicity and need to be considered as nephrotoxic agents. These findings facilitate understanding of the nephrotoxic mechanisms of Aristolochia and have significance for the regulation of herbal medicines.

A multivariate approach linking reported side effects of clinical antidepressant and antipsychotic trials to in vitro binding affinities

The vast majority of approved antidepressants and antipsychotics exhibit a complex pharmacology. The mechanistic understanding of how these psychotropic medications are related to adverse drug reactions (ADRs) is crucial for the development of novel drug candidates and patient adherence. This study aims to associate in vitro assessed binding affinity profiles (39 compounds, 24 molecular drug targets) and ADRs (n=22) reported in clinical trials of antidepressants and antipsychotics (n>59.000 patients) by the use of robust multivariate statistics. Orthogonal projection to latent structures (O-PLS) regression models with reasonable predictability were found for several frequent ADRs such as nausea, diarrhea, hypotension, dizziness, headache, insomnia, sedation, sleepiness, increased sweating, and weight gain. Results of the present study support many well-known pharmacological principles such as the association of hypotension and dizziness with α1-receptor or sedation with H1-receptor antagonism. Moreover, the analyses revealed novel or hardly investigated mechanisms for common ADRs including the potential involvement of 5-HT6-antagonism in weight gain, muscarinic receptor antagonism in dizziness, or 5-HT7-antagonism in sedation. To summarize, the presented study underlines the feasibility and value of a multivariate data mining approach in psychopharmacological development of antidepressants and antipsychotics.

BRCA1 and BRCA2 tumor suppressors protect against endogenous acetaldehyde toxicity

Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR‐compromised cells are sensitive to acetaldehyde, similarly to FANCD2‐deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2‐deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR‐deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication‐associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde‐arrested replication forks require BRCA2 and FANCD2 for protection against MRE11‐dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2‐deficient tumors and ex vivo in patient‐derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP‐ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2‐deficient cells and tumors.

Medicinally Used Asarum Species: High-Resolution LC-MS Analysis of Aristolochic Acid Analogs and In vitro Toxicity Screening in HK-2 Cells

Species of Asarum are used in traditional Chinese medicine and, similar to members of the genus Aristolochia, they contain aristolochic acid analogs (AAAs). These compounds are known for their nephrotoxic and carcinogenic effects. So far, the phytochemistry and nephrotoxicity of species of Asarum is not well studied. A high-resolution LC-MS-based metabolomic approach was used to study the phytochemical variation in medicinally used Asarum species. The cytotoxicity of the samples was assessed using human kidney (HK-2) cells. The majority of samples contained potentially nephrotoxic AAAs, including 9-methoxy aristolactam (AL) IV, AL I, and AL IV. These compounds were present in methanol as well as water extracts. AAAs were detected in all parts of the plant. The majority of the extracts were not cytotoxic to HK-2 cells at the doses tested. However, other mechanisms relating to aristolochic acid nephropathy and cancer development, such as DNA adduct formation may occur. The results of this study provide a model for assessing lesser-known plant species for toxicity.

Toxicological risk assessment of Aristolochia species

Aristolochia species are toxic plants used as herbal medicines worldwide. They are known to cause aristolochic acid nephropathy (AAN), a disease associated with kidney failure and kidney cancer. Aristolochic acid (AA) I and AA II are considered to be responsible for AAN. However, a wide range of aristolochic acid analogues exists and their implication in AAN may have been overlooked. The aims of this project are to assess the health risks associated with different Aristolochia species and to elucidate the principles behind their nephrotoxic effects. 44 medicinally used Aristolochia species were analyzed using LC-MS and 1H- NMR. The cytotoxicity and genotoxicity of 28 Aristolochia extracts was measured in human kidney (HK-2) cells. Several AAAs were isolated from natural sources. Their molecular mechanisms were analysed in hepatoma G2 (Hep G2) cells. Furthermore, we studied ethnobotanical uses of Aristolochia indica L. in Bangladesh. AA I and AA II are the most common AAAs. However, aristolactam (AL) I, AA IV and AL BI are widespread as well. Several of the extracts caused cytotoxic effects and micronuclei induction in HK-2 cells. No correlation was found between the amounts of AA I or AA II and the extractsʼ toxicity. While both AA I and AA II formed DNA adducts in Hep G2 cells, only AA I was cytotoxic and caused oxidative stress. AL I and AA IIIa caused oxidative stress to a lesser extent. Other components exhibited no toxic effects. Furthermore we demonstrated that Aristolocha indica is widely available in Bangladesh and more awareness needs to be raised about the health risks associated its use. By linking a metabolomic analysis with in vitro studies, we were able to show that the assumption that the toxicity of Aristolochia species is caused by AA I and AA II alone is incorrect.