Image analysis of mechanistic protein biomarkers for the characterization of genotoxicants: Aneugens, clastogens, and reactive oxygen species inducers.

The early detection of genotoxicity contributes to cutting-edge drug discovery and development, requiring effective identification of genotoxic hazards posed by drugs while providing mode of action (MoA) information in a high throughput manner. In other words, there is a need to complement standard genotoxicity testing according to the test battery given in ICH S2(R1) with new in vitro tools, thereby contributing to a more in-depth analysis of genotoxic effects. Here, we report on a proof-of-concept MoA approach based on post-translational modifications of proteins (PTMs) indicative of clastogenic and aneugenic effects in TK6 cells using imaging technology (with automated analysis). Cells were exposed in a 96-well plate format with a panel of reference (geno)toxic compounds and subsequently analyzed at 4 and 24 hr to detect dose-dependent changes in PTMs, relevant for mechanistic analysis. All tested compounds that interfere with the spindle apparatus yielded a BubR1 (S640) (3/3) and phospho-histone H3 (S28) (7/9) positive dose-response reflecting aneugenicity, whereas compounds inducing DNA double-strand-breaks were associated with positive FANCD2 (S1404) and 53BP1 (S1778) responses pointing to clastogenicity (2/3). The biomarker p53 (K373) was able to distinguish genotoxicants from non-genotoxicants (2/4), while the induction of reactive oxygen species (ROS), potentially causing DNA damage, was associated with a positive Nrf2 (S40) response (2/2). This work demonstrates that genotoxicants and non-genotoxicants induce different biomarker responses in TK6 cells which can be used for reliable classification into MoA groups (aneugens/clastogens/non-genotoxicants/ROS inducers), supporting a more in-depth safety assessment of drug candidates.

Differentiation of Aneugens and Clastogens in the In Vitro Micronucleus Test by Kinetochore Scoring Using Automated Image Analysis.

The in vitro micronucleus test according to OECD Test Guideline 487 (TG 487) is widely used to investigate the genotoxic potential of drugs. Besides the identification of in vitro genotoxicants, the assay can be complemented with kinetochore staining for the differentiation between clastogens and aneugens. This differentiation constitutes a major contribution to risk assessment as especially aneugens show a threshold response. Thus, a novel method for automated MN plus kinetochore (k+) scoring by image analysis was developed based on the OECD TG 487. Compound-induced increases in MN frequency can be detected using the cytokinesis-block (cytochalasin B) method in V79 cells after 24 h in a 96-well format. Nuclei, MN, and kinetochores were labeled with nuclear counterstain and anti-kinetochore antibodies, respectively, to score MN in binuclear or multinuclear cells and to differentiate compound-induced MN by the presence of kinetochores. First, a reference data set was created by manual scoring using two clastogens and aneugens. After developing the automated scoring process, a set of 14 reference genotoxicants were studied. The automated image analysis yielded the expected results: 5/5 clastogens and 6/6 aneugens (sensitivity: 100%) as well as 3/3 non-genotoxicants (specificity: 100%) were correctly identified. Further, a threshold was determined for identifying aneugens. Based on the data for our internally characterized reference compounds, unknown compounds that induce ≥53.8% k+ MN are classified as aneugens. The current data demonstrate excellent specificity and sensitivity and the methodology is superior to manual microscopic analysis in terms of speed and throughput as well as the absence of human bias. Environ. Mol. Mutagen. 60:227-242, 2019. © 2018 Wiley Periodicals, Inc.

The rat bone marrow micronucleus test: Statistical considerations on historical negative control data.

Recent updates of the OECD Guidelines for the Testing of Chemicals (Section 4: Health Effects) on genotoxicity testing emphasize the use of appropriate statistical methods for data analysis and proficiency proof. Updates also concern the mammalian erythrocyte micronucleus test (OECD 474), as the currently most often performed regulatory in vivo test. As the updated guideline gives high importance to adequate statistical assessment of historical negative control data to estimate validity of experiments and judge results, the present study evaluated statistical methodologies for handling of historical negative control data sets, and comes forward with respective proposals and reference data. Therefore, the working group "Statistics" within the German-speaking "Gesellschaft für Umwelt-Mutationsforschung e.V." (GUM) compiled a data set of 891 negative control rats from valid OECD 474-studies of four laboratories. Based on these data, Analysis-of-Variance (ANOVA) identified "laboratory" and "strain", but not "gender" as relevant stratification parameters, and argued for approximately normally distributed micronucleus frequencies in polychromatic erythrocytes per animal. This assumption provided the basis for further specifying one-sided parametric tolerance intervals for determination of corresponding upper historical negative control limits. Finally, the stability of such limits was investigated as a function of the number of experiments performed, using a simulation-based statistical strategy.

A tripartite mode of action approach for investigating the impact of aneugens on tubulin polymerization.

Chemical-induced disruption of the cellular microtubule network is one key mechanism of aneugenicity. Since recent data indicate that genotoxic effects of aneugens show nonlinear dose-response relationships, margins of safety can be derived with the ultimate goal to perform a risk assessment for the support of drug development. Furthermore, microtubule-interacting compounds are widely used for cancer treatment. While there is a need to support the risk assessment of tubulin-interacting chemicals using reliable mechanistic assays, no standard assays exist to date in regulatory genotoxicity testing for the distinction of aneugenic mechanisms. Recently reported methods exclusively rely on either biochemical, morphological, or cytometric endpoints. Since data requirements for the diverse fields of application of those assays differ strongly, the use of multiple assays for a correct classification of aneugens is ideal. We here report a tripartite mode of action approach comprising a cell-free biochemical polymerization assay and the cell-based methods cellular imaging and flow cytometry. The biochemical assay measures tubulin polymerization over time whereas the two cell-based assays quantify tubulin polymer mass. We herein show that the flow cytometric method yielded IC50 values for tubulin destabilizers and EC50 values for tubulin stabilizers as well as cell cycle information. In contrast, cellular imaging complemented these findings with characteristic morphological patterns. Biochemical analysis yielded kinetic information on tubulin polymerization. This multiplex approach is able to create holistic effect profiles which can be individually customized to the research question with regard to quality, quantity, usability, and economy. Environ. Mol. Mutagen. 59:188-201, 2018. © 2017 Wiley Periodicals, Inc.

Classification of in vitro genotoxicants using a novel multiplexed biomarker assay compared to the flow cytometric micronucleus test.

Regulatory in vitro genotoxicity testing exhibits shortcomings in specificity and mode of action (MoA) information. Thus, the aim of this work was to evaluate the performance of the novel MultiFlow® assay composed of mechanistic biomarkers quantified in TK6 cells after treatment (4 and 24 hr): γH2AX (DNA double strand breaks), phosphorylated H3 (mitotic cells), translocated p53 (genotoxicity), and cleaved PARP1 (apoptosis). A reference dataset of 31 compounds with well-established MoA was studied using the MicroFlow® micronucleus assay. A positive call was raised following the earlier published criteria from Litron Laboratories. In the light of our data, these evaluation criteria should probably be adjusted since only 8/11 (73%) nongenotoxicants and 18/20 (90%) genotoxicants were correctly identified. Moreover, there is a need for new in vitro tools to delineate the predominant MoA as in the MicroFlow® assay only 5/9 (56%) aneugens and 4/11 (36%) clastogens were correctly classified. In contrast, the MultiFlow® assay provides more in-depth information about the MoA and therefore reliably discriminates clastogens, aneugens, and nongenotoxicants. By using a lab-specific, practical threshold for the aforementioned biomarkers, 10/11 (91%) nongenotoxicants and 19/20 genotoxicants (95%), 9/11 (82%) clastogens, and 8/9 (89%) aneugens were correctly categorized, suggesting a clear improvement over the MicroFlow® . Furthermore, the MultiFlow markers were benchmarked against established methods to assess the validity of the data. Altogether, these findings demonstrated good agreement between the MultiFlow® assay and the benchmarking methods. Finally, p21 may improve class discrimination given the correct identification of 4/4 (100%) aneugens and 2/5 (40%) clastogens. Environ. Mol. Mutagen. 58:662-677, 2017. © 2017 Wiley Periodicals, Inc.

Novel Class of Potent and Cellularly Active Inhibitors Devalidates MTH1 as Broad-Spectrum Cancer Target.

MTH1 is a hydrolase responsible for sanitization of oxidized purine nucleoside triphosphates to prevent their incorporation into replicating DNA. Early tool compounds published in the literature inhibited the enzymatic activity of MTH1 and subsequently induced cancer cell death; however recent studies have questioned the reported link between these two events. Therefore, it is important to validate MTH1 as a cancer dependency with high quality chemical probes. Here, we present BAY-707, a substrate-competitive, highly potent and selective inhibitor of MTH1, chemically distinct compared to those previously published. Despite superior cellular target engagement and pharmacokinetic properties, inhibition of MTH1 with BAY-707 resulted in a clear lack of in vitro or in vivo anticancer efficacy either in mono- or in combination therapies. Therefore, we conclude that MTH1 is dispensable for cancer cell survival.

Interlaboratory evaluation of a multiplexed high information content in vitro genotoxicity assay.

We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent-liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho-histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow® DNA Damage Kit-p53, γH2AX, Phospho-Histone H3. For these experiments, seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and nongenotoxicants. TK6 cells were exposed to chemicals in 96-well plates over a range of concentrations for 24 hr. At 4 and 24 hr, cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96-well plate via a robotic walk-away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all interlaboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or nongenotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals' predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay. Environ. Mol. Mutagen. 58:146-161, 2017. © 2017 Wiley Periodicals, Inc.

Aldosterone activates the oncogenic signals ERK1/2 and STAT3 via redox-regulated mechanisms.

Epidemiological studies found an increased risk for kidney cancer in hypertensive patients, of which a subgroup has high aldosterone (Ald) levels. We recently showed that Ald is genotoxic both in kidney tubular cells and in rats with mineralocorticoid-mediated hypertension. The present work investigated in vitro and in vivo, if the oxidative stress-mediated activation of the ERK1/2 pathway, and its downstream target STAT3, could be one mechanism involved in the potential oncogenic capability of excess Ald exposure. The effects of excess Ald were investigated in LLC-PK1 cells and in Ald-induced hypertensive rats. Ald caused cRaf, MEK1/2, and ERK1/2 phosphorylation both in LLC-PK1 cells and in rat kidneys. ERK1/2 activation led to an increased phosphorylation of MSK1, p90RSK, and STAT3. The involvement of ERK1/2 in the activation of STAT3 was evidenced by the capacity of the MEK inhibitor U0126 to prevent Ald-mediated ERK1/2 and STAT3 phosphorylation. Both in vitro and in vivo, the activation of ERK1/2 and STAT3 by Ald was dependent on the mineralocorticoid receptor and was triggered by an increase in cellular oxidants. Ald-mediated oxidant increase was in part due to the activation of the enzymes NADPH oxidase and NO synthase. Proliferation was significantly enhanced and apoptosis decreased in Ald-treated rat kidneys and/or LLC-PK1 cells. Results support the concept that the oxidant-mediated long-term activation of ERK1/2/STAT3 by persistently high Ald levels could trigger proliferative and prosurvival events. Ald-mediated promotion of cell survival and DNA damage could result in kidney cell transformation and initiation of cancer in hypertensive patients with hyperaldosteronism.

Aldosterone induces fibrosis, oxidative stress and DNA damage in livers of male rats independent of blood pressure changes.

Mineralocorticoid receptor blockers show antifibrotic potential in hepatic fibrosis. The mechanism of this protective effect is not known yet, although reactive oxygen species seem to play an important role. Here, we investigated the effects of elevated levels of aldosterone (Ald), the primary ligand of the mineralocorticoid receptor, on livers of rats in a hyperaldosteronism model: aldosterone-induced hypertension. Male Sprague-Dawley rats were treated for 4 weeks with aldosterone. To distinguish if damage caused in the liver depended on increased blood pressure or on increased Ald levels, the mineralocorticoid receptor antagonist spironolactone was given in a subtherapeutic dose, not normalizing blood pressure. To investigate the impact of oxidative stress, the antioxidant tempol was administered. Aldosterone induced fibrosis, detected histopathologically, and by expression analysis of the fibrosis marker, α-smooth muscle actin. Further, the mRNA amount of the profibrotic cytokine TGF-ß was increased significantly. Fibrosis could be reduced by scavenging reactive oxygen species, and also by blocking the mineralocorticoid receptor. Furthermore, aldosterone treatment caused oxidative stress and DNA double strand breaks in livers, as well as the elevation of DNA repair activity. An increase of the transcription factor Nrf2, the main regulator of the antioxidative response could be observed, and of its target genes heme oxygenase-1 and γ-glutamylcysteine synthetase. All these effects of aldosterone were prevented by spironolactone and tempol. Already after 4 weeks of treatment, aldosteroneinfusion induced fibrosis in the liver. This effect was independent of elevated blood pressure. DNA damage caused by aldosterone might contribute to fibrosis progression when aldosterone is chronically increased.

Aldosterone activates transcription factor Nrf2 in kidney cells both in vitro and in vivo.

AIMS: An increased kidney cancer risk was found in hypertensive patients, who frequently exhibit hyperaldosteronism, known to contribute to kidney injury, with oxidative stress playing an important role. The capacity of kidney cells to up-regulate transcription factor nuclear factor-erythroid-2-related factor 2 (Nrf2), a key regulator of the cellular antioxidative defense, as a prevention of aldosterone-induced oxidative damage was investigated both in vitro and in vivo. RESULTS: Aldosterone activated Nrf2 and increased the expression of enzymes involved in glutathione (GSH) synthesis and detoxification. This activation depended on the mineralocorticoid receptor (MR) and oxidative stress. In vitro, Nrf2 activation, GSH amounts, and target gene levels decreased after 24 h, while oxidant levels remained high. Nrf2 activation could not protect cells against oxidative DNA damage, as aldosterone-induced double-strand breaks and 7,8-dihydro-8-oxo-guanine (8-oxodG) lesions steadily rose. The Nrf2 activator sulforaphane enhanced the Nrf2 response both in vitro and in vivo, thereby preventing aldosterone-induced DNA damage. In vivo, Nrf2 activation further had beneficial effects on the aldosterone-caused blood pressure increase and loss of kidney function. INNOVATION: This is the first study showing the activation of Nrf2 by aldosterone. Moreover, the results identify sulforaphane as a substance that is capable of preventing aldosterone-induced damage both in vivo and in vitro. CONCLUSION: Aldosterone-induced Nrf2 adaptive response cannot neutralize oxidative actions of chronically increased aldosterone, which, therefore could be causally involved in the increased cancer incidence of hypertensive individuals. Enhancing the cellular antioxidative defense with sulforaphane might exhibit beneficial effects.

Effect of gum arabic on oxidative stress and inflammation in adenine-induced chronic renal failure in rats.

Inflammation and oxidative stress are known to be involved in the pathogenesis of chronic kidney disease in humans, and in chronic renal failure (CRF) in rats. The aim of this work was to study the role of inflammation and oxidative stress in adenine-induced CRF and the effect thereon of the purported nephroprotective agent gum arabic (GA). Rats were divided into four groups and treated for 4 weeks as follows: control, adenine in feed (0.75%, w/w), GA in drinking water (15%, w/v) and adenine+GA, as before. Urine, blood and kidneys were collected from the rats at the end of the treatment for analysis of conventional renal function tests (plasma creatinine and urea concentration). In addition, the concentrations of the pro-inflammatory cytokine TNF-α and the oxidative stress markers glutathione and superoxide dismutase, renal apoptosis, superoxide formation and DNA double strand break frequency, detected by immunohistochemistry for γ-H2AX, were measured. Adenine significantly increased the concentrations of urea and creatinine in plasma, significantly decreased the creatinine clearance and induced significant increases in the concentration of the measured inflammatory mediators. Further, it caused oxidative stress and DNA damage. Treatment with GA significantly ameliorated these actions. The mechanism of the reported salutary effect of GA in adenine-induced CRF is associated with mitigation of the adenine-induced inflammation and generation of free radicals.

Blood pressure has only minor influence on aldosterone-induced oxidative stress and DNA damage in vivo.

Epidemiological studies found an increased kidney cancer risk in hypertensive patients. These patients frequently present an increase in the mineralocorticoid aldosterone (Ald) due to a stimulated renin angiotensin aldosterone system (RAAS). Recently, we showed pro-oxidative and genotoxic effects of Ald in vitro. Here, we investigated the influence of blood pressure on aldosterone-induced oxidative damage. To distinguish whether effects in Sprague-Dawley rats treated with Ald were caused by Ald or by increased blood pressure, the mineralocorticoid receptor (MR) antagonist spironolactone was administered in a subtherapeutical dose, not lowering the blood pressure, and hydralazine, a RAAS-independent vasodilator, was given to normalize the pressure. With the antioxidant tempol, oxidative stress-dependent effects were demonstrated. Ald treatment caused kidney damage and oxidative and nitrative stress. Structural DNA damage and the mutagenic oxidative base modification 7,8-dihydro-8-oxoguanine were increased, as well as DNA repair activity and nuclear NF-κB translocation. Spironolactone and tempol decreased all markers significantly, whereas hydralazine had just slight effects. These data comprise the first report of essentially blood pressure-independent tissue- and DNA-damaging effects of Ald. A fully activated MR and the production of reactive oxygen and nitrogen species were crucial for these effects.

Aldosterone, oxidative stress, and NF-κB activation in hypertension-related cardiovascular and renal diseases.

The mineralocorticoid aldosterone regulates electrolyte and fluid balance and is involved in blood pressure homoeostasis. Classically, it binds to its intracellular mineralocorticoid receptor to induce expression of proteins influencing the reabsorption of sodium and water in the distal nephron. Aldosterone gained special attention when large clinical studies showed that blocking its receptor in patients with cardiovascular diseases reduced their mortality. These patients present increased plasma aldosterone levels. The exact mechanisms of the potential toxic effects of aldosterone leading to cardiovascular damage are not known yet. The observation of reduced nitric oxide bioavailability in hyperaldosteronism implied the generation of oxidative stress by aldosterone. Subsequent studies confirmed the increase of oxidative stress markers in patients with chronic heart failure and in animal models of hyperaldosteronism. The effects of reactive oxygen species have been related to the activation of transcription factors, such as NF-κB. This review summarizes the present-day knowledge of aldosterone-induced oxidative stress and NF-κB activation in humans and different experimental models.

Aldosterone increases kidney tubule cell oxidants through calcium-mediated activation of NADPH oxidase and nitric oxide synthase.

Chronic hyperaldosteronism has been associated with an increased cancer risk. We recently showed that aldosterone causes an increase in cell oxidants, DNA damage, and NF-κB activation. This study investigated the mechanisms underlying aldosterone-induced increase in cell oxidants in kidney tubule cells. Aldosterone caused an increase in both reactive oxygen and reactive nitrogen (RNS) species. The involvement of the activation of NADPH oxidase in the increase in cellular oxidants was demonstrated by the inhibitory action of the NADPH oxidase inhibitors DPI, apocynin, and VAS2870 and by the migration of the p47 subunit to the membrane. NADPH oxidase activation occurred as a consequence of an increase in cellular calcium levels and was mediated by protein kinase C. The prevention of RNS increase by BAPTA-AM, W-7, and L-NAME indicates a calcium-calmodulin activation of NOS. A similar pattern of effects of the NADPH oxidase and NOS inhibitors was observed for aldosterone-induced DNA damage and NF-κB activation, both central to the pathogenesis of chronic aldosteronism. In summary, this paper demonstrates that aldosterone, via the mineralocorticoid receptor, causes an increase in kidney cell oxidants, DNA damage, and NF-κB activation through a calcium-mediated activation of NADPH oxidase and NOS. Therapies targeting calcium, NOS, and NADPH oxidase could prevent the adverse effects of hyperaldosteronism on kidney function as well as its potential oncogenic action.

Aldosterone induces oxidative stress, oxidative DNA damage and NF-κB-activation in kidney tubule cells.

An increase of the mineralocorticoid aldosterone is induced by a stimulated renin-angiotensin system in a subgroup of hypertensive patients. Epidemiological studies find higher cancer mortality in hypertensive patients and an increased risk to develop kidney cancer. This work investigated the involvement of oxidants in the genotoxicity of aldosterone and on a potential activation of transcription factor nuclear factor-κB (NF-κB) in kidney tubule cells. Aldosterone, at concentrations as low as 1 nM caused a significant increase of DNA damage, as assessed by comet assay and micronucleus frequency test. Aldosterone also led to a dose-dependent activation of NF-κB. Time courses of DNA damage and NF-κB-activation showed that these effects already occurred after 5 and 3 min of aldosterone exposure, respectively, suggesting non-genomic events of the hormone. Antioxidants prevented aldosterone-induced DNA damage and NF-κB-activation, indicating the involvement of oxidants. In fact, aldosterone caused an increase in intracellular oxidant levels, and in particular of superoxide anions. As a consequence, increased levels of the oxidized DNA modification 7,8-dihydro-8-oxo-guanine were observed in aldosterone-treated kidney cells. Aldosterone-induced DNA damage and NF-κB-activation was dependent on the involvement of the mineralocorticoid receptor. The induction of oxidant-mediated genotoxic effects, and of a long-term activation of the potentially oncogenic cell signal NF-κB by aldosterone could contribute to the increased kidney cancer incidence in hypertensive patients.

Mineralocorticoid receptor-mediated DNA damage in kidneys of DOCA-salt hypertensive rats.

Epidemiological studies exploring the connection between hypertension and cancer demonstrate a higher cancer incidence, especially of kidney cancer, and a higher cancer mortality in hypertensive patients. Hormones elevated in hypertension, i.e., aldosterone and angiotensin II, which exert genotoxic effects in vitro, could contribute to carcinogenesis in hypertension. The present study was conducted to investigate the possible DNA-damaging effect of aldosterone receptor activation in vivo. Crl:CD (Sprague-Dawley) rats were treated for 6 wk with desoxycorticosterone acetate (DOCA) and salt to induce a mineralocorticoid-dependent hypertension. DOCA-salt treatment caused increased blood pressure (+26 mmHg) compared to untreated rats, elevated markers of kidney failure (up to 62-fold for Kim-1), and the induction of several proinflammatory genes and proteins (up to 2.6-fold for tissue MCP-1). The mineralocorticoid receptor (MR) antagonist spironolactone (MR IC(50) 24 nM) and the novel nonsteroidal antagonist BR-4628 (MR IC(50) 28 nM) decreased these damage markers. DOCA-salt treatment also caused 8.8-fold increased structural DNA damage, determined with the comet assay, double-strand breaks (3.5-fold), detected immunohistochemically, and oxidative stress. Furthermore, the oxidatively modified mutagenic DNA base 7,8-dihydro-8-oxo-guanine (8-oxodG), quantified by LC-MS/MS, was almost 2-fold higher in DOCA-salt-treated kidneys. Our results suggest a mutagenic potential of high mineralocorticoid levels, frequent in hypertensive individuals.

Superoxide anion and hydrogen peroxide-induced signaling and damage in angiotensin II and aldosterone action.

The formation of reactive oxygen species (ROS) can be induced by xenobiotic substances, such as redox cycling molecules, but also by endogenous substances such as hormones and cytokines. Recent research shows the importance of ROS in cellular signaling. Here, the signaling pathways of the two blood pressure-regulating hormones angiotensin II and aldosterone are presented, focusing on both their physiological effects and the change of signaling owing to the action of increased concentrations or prolonged exposure. When present in high concentrations, both angiotensin II and aldosterone, as various other endogenous substances, activate NADPH oxidase, which produces superoxide. In this review the generation of superoxide anions and hydrogen peroxide in cells stimulated with angiotensin II or aldosterone, as well as the subsequently induced signaling processes and DNA damage is discussed.

Genotoxicity of the neurotransmitter dopamine in vitro.

Alterations in dopamine levels play a role in several human pathological conditions and their pharmacological treatment. Here we describe an induction of genomic damage detected as micronucleus formation by concentrations in the low micromolar range (6.25-25 microM) in three cell lines in vitro. Rat neuronal PC12 cells exhibited a more pronounced induction (about 10-fold over control at 100 microM) than human lymphoblastoid TK6 cells and rat kidney NRK cells (about 2-fold over control at 100 microM). The role of transporters and receptors in the formation of genomic damage was investigated in PC12 cells, in which the effect of dopamine was reduced by addition of the antioxidants TEMPOL and dimethylthiourea, by inhibitors of the dopamine transporter (GBR 12909 and nomifensine) and by a D2 antagonist (sulpiride). Antioxidative effects of nomifensine and sulpiride, but not of GBR 12909, were excluded, since they did not protect oxidative stress sensitive HL-60 cells from hydrogen peroxide-induced damage in the comet assay. Thus, the transport of dopamine into the cell and the signalling upon binding to D2 receptors was required for the genotoxic effect of dopamine in PC12 cells, which was mediated by intracellular dopamine oxidation products and/or reactive oxygen species.

Angiotensin II induces DNA damage in the kidney.

Increased activity of the renin angiotensin system with enhanced levels of angiotensin II leads to oxidative stress with endothelial dysfunction, hypertension, and atherosclerosis. Epidemiologic studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. Because elevated angiotensin II levels might contribute to carcinogenesis, we tested whether angiotensin II induces DNA damage in the kidney. In isolated perfused mouse kidneys, as little as 1 nmol/L angiotensin II caused a significant increase in DNA strand breaks, measured with the comet assay. This damage was independent of the hemodynamic effect of angiotensin II and mediated by the angiotensin II type 1 receptor. Angiotensin II also caused double-strand breaks in the cells of the isolated perfused kidney, detected with an antibody against the double-strand break marker gamma-H2AX. Studies in cell culture allowed further characterization of the DNA damage induced by angiotensin II. Single- and double-strand breaks, abasic sites, and 7,8-dihydro-8-oxo-guanine, all types of oxidative DNA lesions, were detected in angiotensin II-treated renal cells. The majority of detected strand breaks was repaired within 1 hour, but double-strand breaks increased and persisted for at least 24 hours.

Curcumin induces cell-arrest and apoptosis in association with the inhibition of constitutively active NF-kappaB and STAT3 pathways in Hodgkin's lymphoma cells.

Although treatment of Hodgkin's lymphoma (HL) with a multi-drug approach has been very successful, its toxicity becomes evident after several years as secondary malignancies and cardiovascular disease. Therefore, the current goal in HL treatment is to find new therapies that specifically target the deregulated signaling cascades, such as NF-kappaB and STAT3, which cause Hodgkin and Reed-Sternberg (H-RS) cell proliferation and resistance of apoptosis. Based on the above information, we investigated the capacity of curcumin to inhibit NF-kappaB and STAT3 in H-RS cells, characterizing the functional consequences. Curcumin is incorporated into H-RS cells and acts inhibiting both NF-kappaB and STAT3 activation, leading to a decreased expression of proteins involved in cell proliferation and apoptosis, e.g. Bcl-2, Bcl-xL, cFLIP, XIAP, c-IAP1, survivin, c-myc and cyclin D1. Interestingly, curcumin caused cell cycle arrest in G2-M and a significant reduction (80-97%) in H-RS cell viability. Furthermore, curcumin triggered cell death by apoptosis, as evidenced by the activation of caspase-3 and caspase-9, changes in nuclear morphology and phosphatidylserine translocation. The above findings provide a mechanistic rationale for the potential use of curcumin as a therapeutic agent for patients with HL.