Cylindrospermopsin enhances the conjugative transfer of plasmid-mediated multi-antibiotic resistance genes through glutathione biosynthesis inhibition.

Cylindrospermopsin (CYN), a cyanobacterial toxin, has been detected in the global water environment. However, information concerning the potential environmental risk of CYN is limited, since the majority of previous studies have mainly focused on the adverse health effects of CYN through contaminated drinking water. The present study reported that CYN at environmentally relevant levels (0.1-100 µg/L) can significantly enhance the conjugative transfer of RP4 plasmid in Escherichia coli genera, wherein application of 10 µg/L of CYN led to maximum fold change of ∼6.5- fold at 16 h of exposure. Meanwhile, evaluation of underlying mechanisms revealed that environmental concentration of CYN exposure could increase oxidative stress in the bacterial cells, resulting in ROS overproduction. In turn, this led to an upregulation of antioxidant enzyme-related genes to avoid ROS attack. Further, inhibition of the synthesis of glutathione (GSH) was also detected, which led to the rapid depletion of GSH in cells and thus triggered the SOS response and promoted the conjugative transfer process. Increase in cell membrane permeability, upregulation of expression of genes related to pilus generation, ATP synthesis, and RP4 gene expression were also observed. These results highlight the potential impact on the spread of antimicrobial resistance in water environments.

Cylindrospermopsin exposure promotes redox unbalance and tissue damage in the liver of Poecilia reticulata, a neotropical fish species.

There is a growing concern regarding the adverse risks exposure to cylindrospermopsin (CYN) might exert on animals and humans. However, data regarding the toxicity of this cyanotoxin to neotropical fish species are scarce. Using the fish species Poecilia reticulata, the influence of CYN concentrations equal to and above the tolerable for drinking water may produce on liver was determined by assessing biomarkers of antioxidant defense mechanisms and correlated to qualitative and semiquantitative histopathological observations. Adult females were exposed to 0.0 (Control); 0.5, 1 and 1.5 µg/L pure CYN for 24 or 96 hr, in triplicate. Subsequently the livers were extracted for biochemical assays and histopathological evaluation. Catalase (CAT) activity was significantly increased only by 1.5 µg/L CYN-treatment, at both exposure times. Glutathione -S-transferase (GST) activity presented a biphasic response for both exposure times. It was markedly decreased after exposure by 0.5 µg/L CYN treatment but significantly elevated by 1.5 µg/L CYN treatment. All CYN treatments produced histopathological alterations, as evidenced by hepatocyte cords degeneration, steatosis, inflammatory infiltration, melanomacrophage centers, vessel congestion, and areas with necrosis. Further, an IORG >35 was achieved for all treatments, indicative of the presence of severe histological alterations in P. reticulata hepatic parenchyma and stroma. Taken together, data demonstrated evidence that CYN-induced hepatotoxicity in P. reticulata appears to be associated with an imbalance of antioxidant defense mechanisms accompanied by histopathological liver alterations. It is worthy to note that exposure to low environmentally-relevant CYN concentrations might constitute a significant risk to health of aquatic organisms.

Cytotoxic Effects and Oxidative Stress Produced by a Cyanobacterial Cylindrospermopsin Producer Extract versus a Cylindrospermopsin Non-Producing Extract on the Neuroblastoma SH-SY5Y Cell Line.

The incidence and interest of cyanobacteria are increasing nowadays because they are able to produce some toxic secondary metabolites known as cyanotoxins. Among them, the presence of cylindrospermopsin (CYN) is especially relevant, as it seems to cause damage at different levels in the organisms: the nervous system being the one most recently reported. Usually, the effects of the cyanotoxins are studied, but not those exerted by cyanobacterial biomass. The aim of the present study was to assess the cytotoxicity and oxidative stress generation of one cyanobacterial extract of R. raciborskii non-containing CYN (CYN-), and compare its effects with those exerted by a cyanobacterial extract of C. ovalisporum containing CYN (CYN+) in the human neuroblastoma SH-SY5Y cell line. Moreover, the analytical characterization of potential cyanotoxins and their metabolites that are present in both extracts of these cultures was also carried out using Ultrahigh Performance Liquid Chromatography-Mass Spectrometry, in tandem (UHPLC-MS/MS). The results show a reduction of cell viability concentration- and time-dependently after 24 and 48 h of exposure with CYN+ being five times more toxic than CYN-. Furthermore, the reactive oxygen species (ROS) increased with time (0-24 h) and CYN concentration (0-1.11 µg/mL). However, this rise was only obtained after the highest concentrations and times of exposure to CYN-, while this extract also caused a decrease in reduced glutathione (GSH) levels, which might be an indication of the compensation of the oxidative stress response. This study is the first one performed in vitro comparing the effects of CYN+ and CYN-, which highlights the importance of studying toxic features in their natural scenario.

Immunomodulatory Effects of Cylindrospermopsin in Human T Cells and Monocytes.

Cylindrospermopsin (CYN) is a cyanotoxin with an increasing occurrence, and therefore it is important to elucidate its toxicity profile. CYN has been classified as a cytotoxin, although the scientific literature has already revealed that it affects a wide range of organs and systems. However, research on its potential immunotoxicity is still limited. Thus, this study aimed to evaluate the impact of CYN on two human cell lines representative of the immune system: THP-1 (monocytes) and Jurkat (lymphocytes). CYN reduced cell viability, leading to mean effective concentrations (EC50 24 h) of 6.00 ± 1.04 µM and 5.20 ± 1.20 µM for THP-1 and Jurkat cells, respectively, and induced cell death mainly by apoptosis in both experimental models. Moreover, CYN decreased the differentiation of monocytes to macrophages after 48 h of exposure. In addition, an up-regulation of the mRNA expression of different cytokines, such as interleukin (IL) 2, IL-8, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (INF-γ), was also observed mainly after 24 h exposure in both cell lines. However, only an increase in TNF-α in THP-1 supernatants was observed by ELISA. Overall, these results suggest the immunomodulatory activity of CYN in vitro. Therefore, further research is required to evaluate the impact of CYN on the human immune system.

Potential Endocrine Disruption of Cyanobacterial Toxins, Microcystins and Cylindrospermopsin: A Review.

Microcystins (MCs) and cylindrospermopsin (CYN), although classified as hepatotoxins and cytotoxins, respectively, have been shown to also induce toxic effects in many other systems and organs. Among them, their potential endocrine disruption (ED) activity has been scarcely investigated. Considering the increasing relevance of ED on humans, mammals, and aquatic organisms, this work aimed to review the state-of-the-art regarding the toxic effects of MCs and CYN at this level. It has been evidenced that MCs have been more extensively investigated than CYN. Reported results are contradictory, with the presence or absence of effects, but experimental conditions also vary to a great extent. In general, both toxins have shown ED activity mediated by very different mechanisms, such as estrogenic responses via a binding estrogen receptor (ER), pathological changes in several organs and cells (testis, ovarian cells), and a decreased gonad-somatic index. Moreover, toxic effects mediated by reactive oxygen species (ROS), changes in transcriptional responses on several endocrine axes and steroidogenesis-related genes, and changes in hormone levels have also been reported. Further research is required in a risk assessment frame because official protocols for assessment of endocrine disrupters have not been used. Moreover, the use of advanced techniques would aid in deciphering cyanotoxins dose-response relationships in relation to their ED potential.

Cylindrospermopsin Disrupts Estrous Cycle and Increases Spermatogenesis in Mice.

Cylindrospermopsin (CYN) is a water-soluble cyanotoxin that has been linked to several cases of poisoning in the world. In vitro studies have shown that CYN acts as an endocrine disruptor by inhibiting progesterone synthesis in primary cell cultures of women, showing estrogenic activity. However, in vivo assessment of CYN in the female and male reproductive systems remains unknown. We thus aimed to evaluate the in vivo effects of CYN in both the female and male reproductive systems of mice. A single intraperitoneal exposure to 64 µg of CYN/kg body weight was performed in females. Estrous cycle was evaluated daily by vaginal cytology, and serum progesterone and estradiol levels were measured after 50 days. We showed an impairment in the estrous cycle as well as a decrease in circulating plasma progesterone levels. In males, weekly intraperitoneal doses of 20 µg of CYN/kg body weight were given and groups were killed after one, two, or four doses. CYN increased the testosterone levels in the groups that received one or two doses of CYN. Additionally, CYN induced a transient increase in spermatozoa in males after four doses. Our results highlight that CYN interferes with both male and female reproductive systems and may lead to infertility. As far as we know, this is the first report showing the impacts of CYN on the mammalian reproductive system, suggesting a threat from this cyanotoxin to human and environmental health.

Four decades of progress in cylindrospermopsin research: The ins and outs of a potent cyanotoxin.

The cyanotoxin cylindrospermopsin (CYN), a toxic metabolite from cyanobacteria, is of particular concern due to its cosmopolitan occurrence, aquatic bioaccumulation, and multi-organ toxicity. CYN is the second most often recorded cyanotoxin worldwide, and cases of human morbidity and animal mortality are associated with ingestion of CYN contaminated water. The toxin poses a great challenge for drinking water treatment plants and public health authorities. CYN, with the major toxicity manifested in the liver, is cytotoxic, genotoxic, immunotoxic, neurotoxic and may be carcinogenic. Adverse effects are also reported for endocrine and developmental processes. We present a comprehensive review of CYN over the past four decades since its first reported poisoning event, highlighting its global occurrence, biosynthesis, toxicology, removal, and monitoring. In addition, current data gaps are identified, and future directions for CYN research are outlined. This review is beneficial for understanding the ins and outs of this environmental pollutant, and for robustly assessing health hazards posed by CYN exposure to humans and other organisms.

In vitro assessment of the combination of cylindrospermopsin and the organophosphate chlorpyrifos on the human neuroblastoma SH-SY5Y cell line.

Cylindrospermopsin (CYN) is a cyanotoxicant which occurrence is increasing due to climate change. Cylindrospermopsin is able to exert damage in the organism at several levels, among them, in the nervous system. Moreover, it is important to take into account that it is not usually present isolated in nature, but in combination with some other pollutants, being the case of the pesticide chlorpyrifos (CPF). Thus, the aim of the present work was to assess the effects of the interaction of CYN in combination with CPF in the human neuroblastoma cell line SH-SY5Y by evaluating cytotoxicity and mechanistic endpoints. The mixtures 0.25 + 21, 0.5 + 42, 1 + 84 µg/mL of CYN + CPF based on cytotoxicity results, were evaluated, and the isobologram method detected an antagonistic effect after 24 and 48 h of exposure. Moreover, although no alterations of reactive oxygen species were detected, a significant decrease of glutathione levels was observed after exposure to both, CPF alone and the combination, at all the concentrations and times of exposure assayed. In addition, CYN + CPF caused a marked decrease in the acetylcholinesterase activity, providing similar values to CPF alone. However, these effects were less severe than expected. All these findings, together with the morphological study results, point out that it is important to take into account the interaction of CYN with other pollutants. Further research is required to contribute to the risk assessment of CYN and other contaminants considering more realistic exposure scenarios.

In vivo genotoxicity evaluation of cylindrospermopsin in rats using a combined micronucleus and comet assay.

Cylindrospermopsin (CYN) is a potent cyanotoxin recognized as an emerging human threat due to its cytotoxicity and potential carcinogenicity. Although the genotoxicity of CYN has been extensively studied in vitro, limited data are available on its in vivo genotoxicity. The aim of this study was to evaluate the in vivo genotoxicity of pure CYN (7.5-75 µg/kg body weight) after oral exposure of rats through a combined assay of the micronucleus test (MN) in bone marrow, and the standard and modified comet assay in stomach, liver and blood. Also, histopathological changes in stomach and liver were evaluated. Positive results in the MN test were observed in bone marrow in the exposed rats at all the tested concentrations. However, the comet assay revealed that CYN did not induce DNA strand breaks nor oxidative DNA damage in any of the tissues investigated. Finally, histopathological changes were observed in stomach and liver (7.5-75 µg/kg) in intoxicated rats. These results could indicate that CYN is able to induce irritation in stomach before its biotransformation in rats orally exposed, and genotoxicity in bone marrow.

In Vitro Mutagenic and Genotoxic Assessment of a Mixture of the Cyanotoxins Microcystin-LR and Cylindrospermopsin.

The co-occurrence of various cyanobacterial toxins can potentially induce toxic effects different than those observed for single cyanotoxins, as interaction phenomena cannot be discarded. Moreover, mixtures are a more probable exposure scenario. However, toxicological information on the topic is still scarce. Taking into account the important role of mutagenicity and genotoxicity in the risk evaluation framework, the objective of this study was to assess the mutagenic and genotoxic potential of mixtures of two of the most relevant cyanotoxins, Microcystin-LR (MC-LR) and Cylindrospermopsin (CYN), using the battery of in vitro tests recommended by the European Food Safety Authority (EFSA) for food contaminants. Mixtures of 1:10 CYN/MC-LR (CYN concentration in the range 0.04-2.5 µg/mL) were used to perform the bacterial reverse-mutation assay (Ames test) in Salmonella typhimurium, the mammalian cell micronucleus (MN) test and the mouse lymphoma thymidine-kinase assay (MLA) on L5178YTk± cells, while Caco-2 cells were used for the standard and enzyme-modified comet assays. The exposure periods ranged between 4 and 72 h depending on the assay. The genotoxicity of the mixture was observed only in the MN test with S9 metabolic fraction, similar to the results previously reported for CYN individually. These results indicate that cyanobacterial mixtures require a specific (geno)toxicity evaluation as their effects cannot be extrapolated from those of the individual cyanotoxins.

Cylindrospermopsin induces cellular stress and activation of ERK1/2 and p38 MAPK pathways in adult human liver stem cells.

Cyanobacterial toxin cylindrospermopsin (CYN) is an emerging freshwater contaminant, whose expanding environmental occurrence might result into increased human health risks. CYN is potent hepatotoxin, with cytotoxicity and genotoxicity documented in primary hepatocytes or hepatoma cell lines. However, there is only limited information about CYN effects on adult human liver stem cells (LSCs), which play an important role in liver tissue development, regeneration and repair. In our study with human liver cell line HL1-hT1 which expresses characteristics of LSCs, CYN was found to be cytotoxic and increasing cell death after 24-48 h exposure to concentrations >1 µM. Subcytotoxic 1 µM concentration did not induce cell death or membrane damage, but inhibited cellular processes related to energy production, leading to a growth stagnation after >72 h. Interestingly, these effects were not associated with increased DNA damage, reactive oxygen species production, or endoplasmic reticulum stress. However, CYN induced a sustained (24-48 h) activation of mitogen-activated protein kinases ERK1/2 and p38, and increased expression of stress-related transcription factor ATF3. Thus, LSCs were not primarily affected by CYN-induced genotoxicity and oxidative stress, but via activation of signaling and transcriptional pathways critical for regulation of cell proliferation, stress responses, cell survival and inflammation. Alterations of LSCs during CYN-induced liver injury, including the role of nongenotoxic mechanisms, should be therefore considered in mechanistic assessments of chronic CYN hepatotoxicity and hepatocarcinogenicity.

Identification of key pathways involved in the toxic response of the cyanobacterial toxin cylindrospermopsin in human hepatic HepaRG cells.

The hepatotoxin cylindrospermopsin (CYN) has been involved in cases of poisoning in humans following ingestion. As its liver toxicity process is complex, we studied the transcriptomic profile of HepaRG cells exposed to CYN. The affected pathways were confirmed through the expression of key genes and the investigation of toxicity markers. In addition, CYP450 activities and cell redox homeostasis were investigated following acute and repeated exposure. CYN induced the down-regulation of genes involved in xenobiotic metabolism and cell cycle progression. There was cell cycle disturbance characterised by an accumulation of G1/S and G2/M cells and an increase in phospho-H3-positive cells. This was linked to the induction of DNA damage demonstrated by an increase in γH2AX-positive cells as well as an accumulation of sub-G1 cells indicating apoptosis but not involving caspase-3. While glutathione (GSH) content sharply decreased following acute exposure to CYN, it increased following repeated exposure, reflecting an adaptive response of cell redox homeostasis. However, our data also suggested that CYN induced the down-regulation of phase I and II metabolism gene products, and CYP450 activities were affected following both acute and repeated exposure to CYN. Our study indicated that repeated exposure of liver cells to low concentrations of CYN may affect their detoxification capacities.

Neurotoxicity induced by microcystins and cylindrospermopsin: A review.

Microcystins (MCs) and cylindrospermopsin (CYN) are among the most frequent toxins produced by cyanobacteria. These toxic secondary metabolites are classified as hepatotoxins and cytotoxin, respectively. Furthermore, both may present the ability to induce damage to the nervous system. In this sense, there are many studies manifesting the potential of MCs to cause neurotoxicity both in vitro and in vivo, due to their probable capacity to cross the blood-brain-barrier through organic anion transporting polypeptides. Moreover, the presence of MCs has been detected in brain of several experimental models. Among the neurological effects, histopathological brain changes, deregulation of biochemical parameters in brain (production of oxidative stress and inhibition of protein phosphatases) and behavioral alterations have been described. It is noteworthy that minority variants such as MC-LF and -LW have demonstrated to exert higher neurotoxic effects compared to the most studied congener, MC-LR. By contrast, the available studies concerning CYN-neurotoxic effects are very scarce, mostly showing inflammation and apoptosis in neural murine cell lines, oxidative stress, and alteration of the acetylcholinesterase activity in vivo. However, more studies are required in order to clarify the neurotoxic potential of both toxins, as well as their possible contribution to neurodegenerative diseases.

Neurotoxic assessment of Microcystin-LR, cylindrospermopsin and their combination on the human neuroblastoma SH-SY5Y cell line.

Microcystin-LR (MC-LR) and Cylindrospermopsin (CYN) are produced by cyanobacteria. Although being considered as a hepatotoxin and a cytotoxin, respectively, different studies have revealed neurotoxic properties for both of them. The aim of the present work was to study their cytotoxic effects, alone and in combination, in the SH-SY5Y cell line. In addition, toxicity mechanisms such as oxidative stress and acetylcholinesterase (AChE) activity, and morphological studies were carried out. Results showed a cytotoxic response of the cells after their exposure to 0-100 µg/mL of MC-LR or 0-10 µg/mL CYN in both differentiated and undifferentiated cells. Thus, CYN resulted to be more toxic than MC-LR. Respect to their combination, a higher cytotoxic effect than the toxins alone in the case of undifferentiated cells, and almost a similar response to the presented by MC-LR in differentiated cells were observed. However, after analyzing this data with the isobolograms method, an antagonistic effect was mainly obtained. The oxidative stress study only showed an affectation of glutathione levels at the highest concentrations assayed of MC-LR and the combination in the undifferentiated cells. A significant increase in the AChE activity was observed after exposure to MC-LR in undifferentiated cells, and after exposure to the combination of both cyanotoxins on differentiated cells. However, CYN decreased the AChE activity only on differentiated cultures. Finally, the morphological study revealed different signs of cellular affectation, with apoptotic processes at all the concentrations assayed. Therefore, both cyanotoxins isolated and in combination, have demonstrated to cause neurotoxic effects in the SH-SY5Y cell line.

Identification of novel uracil derivatives incorporating benzoic acid moieties as highly potent Dipeptidyl Peptidase-IV inhibitors.

Dipeptidyl Peptidase-IV (DPP-4) is a validated therapeutic target for type 2 diabetes. Aiming to interact with both residues Try629 and Lys554 in S2' site, a series of novel uracil derivatives 1a-l and 2a-i incorporating benzoic acid moieties at the N3 position were designed and evaluated for their DPP-4 inhibitory activity. Structure-activity relationships (SAR) study led to the identification of the optimal compound 2b as a potent and selective DPP-4 inhibitor (IC50 = 1.7 nM). Docking study revealed the additional salt bridge formed between the carboxylic acid and primary amine of Lys554 has a key role in the enhancement of the activity. Furthermore, compound 2b exhibited no cytotoxicity in human hepatocyte LO2 cells up to 50 µM. Subsequent in vivo evaluations revealed that the ester of 2b robustly improves the glucose tolerance in normal mice. The overall results have shown that compound 2b has the potential to a safe and efficacious treatment for T2DM.

Freshwater Cyanotoxin Cylindrospermopsin Has Detrimental Stage-specific Effects on Hepatic Differentiation From Human Embryonic Stem Cells.

Cylindrospermopsin (CYN) has been recognized as a potent waterborne hepatotoxin with an increasing environmental occurrence. However, CYN effects on the specific populations of hepatic cells involved in liver tissue development, renewal, and regeneration, have not been characterized yet. We used human embryonic stem cells to analyze the hepatic differentiation stage-specific effect of CYN. Our results strongly suggest that CYN might contribute to the development of chronic adverse outcomes by disrupting liver tissue homeostasis in terms of (1) cellular stress and damage induced in the mature differentiated hepatocytes, which was associated with a necrotic cell death and thus possibly also inflammatory responses; (2) selective elimination of HNF4α+ cells from populations of progenitor cells and immature hepatocytes during hepatic differentiation, which could possibly lead to an impaired liver renewal and regeneration; (3) impaired hepatic functions of immature hepatocytes, such as decreased albumin secretion or increased lipid accumulation, which could contribute to the development of liver steatosis; and (4) survival of the immature and AFP-expressing cells with the limited ability to further differentiate, which could represent a tumor-promoting condition.

Cytotoxic and morphological effects of microcystin-LR, cylindrospermopsin, and their combinations on the human hepatic cell line HepG2.

Cylindrospermopsin (CYN) and Microcystin-LR (MC-LR) are toxins produced by different cyanobacterial species, which are found mainly in freshwater reservoirs. Both of them can induce, separately, toxic effects in humans and wildlife. However, little is known about the toxic effects of the combined exposure, which could likely happen, taking into account the concomitant occurrence of the producers. As both cyanotoxins are well known to induce hepatic damage, the human hepatocellular HepG2 cell line was selected for the present study. Thus, the cytotoxicity of both pure cyanotoxins alone (0-5 µg/mL CYN and 0-120 µg/mL MC-LR) and in combination for 24 and 48 h was assayed, as long as the cytotoxicity of extracts from CYN-producing and nonproducing cyanobacterial species. The potential interaction of the combination was evaluated by the isobologram or Chou-Talalay's method, which provides a combination index as a quantitative measure of the two cyanotoxins interaction's degree. Moreover, a morphological study of the individual pure toxins and their combinations was also performed. Results showed that CYN was the most toxic pure cyanotoxin, being the mean effective concentrations obtained ≈4 and 90 µg/mL for CYN and MC-LR, respectively after 24 h. However, the simultaneous exposure showed an antagonistic effect. Morphologically, autophagy, at low concentrations, and apoptosis, at high concentrations were observed, with affectation of the rough endoplasmic reticulum and mitochondria. These effects were more pronounced with the combination. Therefore, it is important to assess the toxicological profile of cyanotoxins combinations in order to perform more realistic risk evaluations.

Mutagenic and genotoxic potential of pure Cylindrospermopsin by a battery of in vitro tests.

Cylindrospermopsin (CYN) is a cyanobacterial toxin with an increasing world-wide occurrence. The main route of human exposure is through the ingestion of contaminated food and water. The European Food Safety Authority has identified the need to further characterize the toxicological profile of cyanotoxins and in this regard the genotoxicity is a key toxicological effect. The data available in the scientific literature show contradictory results. Thus, the aim of this study was to investigate the mutagenic and genotoxic effects of pure CYN using a battery of different in vitro assays including: the bacterial reverse-mutation assay in Salmonella typhimurium (Ames test) (0-10 µg/mL), the mammalian cell micronucleus (MN) test (0-1.35 µg/mL and 0-2 µg/mL in absence or presence of S9 fraction, respectively) and the mouse lymphoma thymidine-kinase assay (MLA)(0-0.675 µg/mL) on L5178YTk ±â€¯cells, and the standard and enzyme-modified comet assays (0-2.5 µg/mL) on Caco-2 cells. Positive results were obtained only when the metabolic fraction S9 was employed in the MN test, suggesting pro-genotoxic properties of CYN. Also, DNA damage was not mediated by oxidative stress as CYN did not induced changes in the modified comet assay. These data could contribute to a better risk assessment of this cyanotoxin.

Evaluation of Uracil, Sodium Ascorbate, and Rosiglitazone as Promoters of Urinary Bladder Transitional Cell Carcinomas in Male Sprague-Dawley Rats.

The purpose of this study was to establish a 2-stage model of urinary bladder carcinogenesis in male Sprague-Dawley rats to identify tumor promoters. In phase 1 of the study, rats ( n = 170) were administered 100 mg/kg of the tumor initiator, N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), twice weekly by oral gavage (po) for a period of 6 weeks. Phase 2 consisted of dividing rats into 4 groups ( n = 40 per group) and administering one of the following for 26 weeks to identify putative tumor promoters: (1) vehicle po, (2) 25 mg/kg/day rosiglitazone po, (3) 5% dietary sodium l-ascorbate, and (4) 3% dietary uracil. Rats were necropsied after 7.5 months, and urinary bladders were evaluated by histopathology. BBN/vehicle treatments induced the development of urothelial hyperplasia (83%) and papillomas (15%) but no transitional cell carcinomas (TCCs). Rosiglitazone increased the incidence and severity of papillomas (93%) and resulted in TCC in 10% of treated rats. Uracil was the most effective tumor promoter in our study and increased the incidence of papillomas (90%) and TCC (74%). Sodium ascorbate decreased the incidence of urothelial hyperplasia (63%) and did not increase the incidence of urothelial papillomas or TCC. These data confirm the capacity of our 2-stage model to identify urinary bladder tumor promoters.

Estrogenic activity of cylindrospermopsin and anatoxin-a and their oxidative products by FeIII-B*/H2O2.

The cyanotoxins released into waters during cyanobacterial blooms can pose serious hazards to humans and animals. Apart from their toxicological mechanisms, cyanotoxins have been shown to be involved in estrogenic activity by in vivo and in vitro assays; however, there is limited information on the change in estrogenicity of cyanotoxins following chemical oxidation. In this study, the estrogenic activity of cylindrospermopsin (CYL) and anatoxin-a (ANA) at concentrations ranging from 2.4 × 10-7 M to 2.4 × 10-12 M (CYL) and 7.1 × 10-6 M to 7.1 × 10-11 M (ANA), and after treatment by the FeIII-B*/H2O2 catalyst system, was investigated by the yeast estrogen screen (YES) assay. The results indicate that CYL and ANA acted as agonists in the YES assay (CYL logEC50 = -8.901; ANA logEC50 = -6.789), their binding affinity to estrogen receptors is associated with their intrinsic properties, including ring structures and toxicant properties. CYL and ANA were shown to simulate endocrine disrupting chemicals (EDCs) to modulate the 17ß-estradiol-induced estrogenic activity, resulting in non-monotonic dose responses. The treated CYL showed a significantly altered estrogenicity compared to the untreated CYL (T(2) = 8.168, p ≤ .05), while the estrogenicity of the treated ANA was not significantly different to the untreated ANA (T(2) = 1.295, p > .05). Intermediate products generated from CYL and ANA oxidized by FeIII-B*/H2O2 were identified using Q-Exactive Tandem Mass Spectrometry (LC-MS/MS). Treatment with FeIII-B*/H2O2 yielded open-ring by-products which likely resulted in CYL's reduced binding affinity to estrogen receptors. The insignificant change in the estrogenicity of treated ANA was possibly a result of its multiple ring structure products, which were likely able to bind to estrogen receptors. The comparisons for the estrogenicity of these cyanotoxins before and after FeIII-B*/H2O2 treatment suggest that the reductions in estrogenicity achieved by oxidation were dependent on the levels of cyanotoxins removed, as well as the estrogenicity of the degradation products. This is the first study on the change in the estrogenicity of CYL and ANA upon oxidation by FeIII-B*/H2O2, a high activity catalyst system.