The genus Artemia, the nanoplastics, the microplastics, and their toxic effects: a review.

Plastic pollution is a threat to the marine environment, the destination of mismanaged plastic. Due to reduced size, microplastics and nanoplastics (MNPs) can interact with a wide range of organisms. Non-selective filter feeder zooplanktonic microcrustaceans are potential targets for MNP accumulation. Zooplankton is a key group for the food web, linking primary producers to secondary consumers. The genus Artemia has been widely used to investigate the effects of plastic particles on the biota. The present work critically reviewed the ecotoxicological studies about plastic particles and Artemia, pointing out methodological aspects and effects caused by MNPs, highlighting their importance and limitations, and suggesting directions for future research. We analyzed twenty-one parameters into four categories: characteristics of plastic particles, general particularities of brine shrimp, methodologies of the cultures, and toxicological parameters. The principal gaps in the area are the lack of methodological standardization regarding the physicochemical parameters of the particles, the biology of the animals, and culture conditions. Even though few studies performed realistic exposure scenarios, results indicate MNPs as potential harmful contaminants to microcrustaceans. The main effects reported were particle ingestion and accumulation followed by reduced brine shrimp survival/mobility. The present review poses Artemia as suitable animals for investigations concerning the risks of MNP exposure at the individual level and to the ecosystems, although protocol standardization is still needed.

Effects of the toxic dinoflagellates Prorocentrum lima and Ostreopsis cf. ovata on immune responses of cultured oysters Crassostrea gasar.

Oyster production in Brazil has been highlighted as an important economic activity and is directly impacted by the quality of the environment, which is largely the result of human interference and climate change. Harmful algal blooms occur in aquatic ecosystems worldwide, including coastal marine environments which have been increasing over the last decades as a result of global change and anthropogenic activities. In this study, the native oysters Crassostrea gasar from Northeast of Brazil were exposed to two toxic benthic dinoflagellate species, Prorocentrum lima and Ostreopsis cf. ovata. Their respective effects on C. gasar physiology and defense mechanisms were investigated. Oyster hemocytes were first exposed in vitro to different concentrations of both dinoflagellate species to assess their effects on hemocyte functions, such as phagocytosis, production of reactive oxygen species, as well as mortality. Results highlighted an alteration of hemocyte phagocytosis and viability in presence of O. cf. ovata, whereas P. lima did not affect the measured hemocyte functions. In a second experiment, oysters were exposed for 4 days in vivo to toxic culture of O. cf. ovata to assess its effects on hemocyte parameters, tissues damages and pathogenic Perkinsus spp. infection. An increase in hemocyte mortality was also observed in vivo, associated with a decrease of ROS production. Histopathological analyses demonstrated a thinning of the epithelium of the digestive tubules of the digestive gland, inflammatory reaction and a significant increase in the level of infection by Perkinsus spp. in oysters exposed to O. cf. ovata. These results indicate that oysters C. gasar seem to be pretty resilient to an exposure to P. lima and may be more susceptible to O. cf. ovata. Furthermore, the latter clearly impaired oyster physiology and defense mechanisms, thus highlighting that harmful algal blooms of O. cf. ovata could potentially lead to increased susceptibility of C. gasar oysters to parasite infections.

Single and combined toxicity of amino-functionalized polystyrene nanoparticles with potassium dichromate and copper sulfate on brine shrimp Artemia franciscana larvae.

The increasing use and disposal of plastics has become a persistent problem in the marine environment, calling for studies that refer to realistic scenarios to understand their effects on biota. Particularly, the understanding about the effects of co-exposure with nanoplastic particles and metals on aquatic organisms is still limited. The present work aimed to investigate the acute toxicity of amino-functionalized polystyrene nanoparticles (PS-NH2; 50 nm) as proxy for nanoplastics on brine shrimp Artemia franciscana larvae under different culture conditions and at different stages of development, as well as the combined effect with two reference toxicants - potassium dichromate (K2Cr2O7) and copper sulfate (CuSO4). Nauplii (instar II or III larval stages) were exposed to different concentrations of PS-NH2 (0.005 to 5 µg mL-1) for up to 48 h, with or without agitation in order to mimic a more realistic environmental scenario. Larval mobility and PS-NH2 accumulation were monitored under microscopy. PS-NH2 alone showed toxicity only at the highest concentration tested (5 µg mL-1) regardless the incubation method used (61.2 + 3.1% and 65.0 + 4.5% with and without agitation, respectively). Moreover, instar III stage was the most sensitive to PS-NH2 exposure (38.2% immobility in 24 h of exposure; 5 µg mL-1). Evidence of PS-NH2 retention in the gastrointestinal tract in a concentration- and time-dependent manner was also obtained. Mixtures of PS-NH2 (0.005 and 5 µg mL-1) with different concentrations of K2Cr2O7 increased the immobilization rate of the larvae after 48 h of exposure, when compared to the K2Cr2O7 alone. Similar results were observed for CuSO4 in the co-exposure conditions at different concentrations. However, exposing nauplii to a mixture of PS-NH2 (0.005 µg mL-1) and CuSO4 decreased immobilization rate, in comparison to the group exposed to CuSO4 alone. The present work highlights the potential risk posed by nanoplastics to zooplanktonic species through their interaction with other toxicants.

Mitochondrial permeability transition pore in sea urchin female gametes.

Mitochondrial permeability transition pore (MPTP) has been associated to calcium homeostasis and reactive oxygen species (ROS) generation in several cell types. While extensively investigated in somatic cells, there are few data regarding MPTP phenomenon in gametes. The aim of the present work was to investigate MPTP occurrence in sea urchin female gametes. The protonophores CCCP and FCCP, and the Ca2+ ionophore ionomycin, were used as pore inductors. Pore opening was monitored by mitochondrial potential sensitive probes and cobalt-quenched calcein assay. The pore desensitizer cyclosporin A (CsA) prevented the loss of mitochondrial inner membrane potential (ΔΨm) and pore opening induced by MPTP activators. The disruption of ΔΨm led to an increase in ROS generation, which was completely prevented by CsA. Our data also demonstrated that the increase in ROS production induced by MPTP opening requires extracellular Ca2+. In summary, the current study provides evidence about the occurrence of MPTP in sea urchin eggs in a similar manner as described in vertebrate somatic cells - CsA-sensitive, voltage- and Ca2+-triggered - and shows MPTP as a highly conserved physiological event through the evolution.

ABCB1 and ABCC1-like transporters in immune system cells from sea urchins Echinometra lucunter and Echinus esculentus and oysters Crassostrea gasar and Crassostrea gigas.

ABC transporters activity and expression have been associated with the multixenobiotic resistance phenotype (MXR). The activity of these proteins leads to a reduction in the intracellular concentration of several xenobiotics, thus reducing their toxicity. However, little attention has been given to the expression of ABC transporters in marine invertebrates and few studies have investigated their role in immune system cells of sea urchins and shellfish bivalves. The aim of the present study was to investigate the activity of the ABC transporters ABCB1 and ABCC1 in immune system cells of sea urchins (coelomocytes) and oysters (hemocytes) from different climatic regions (Brazil and France). Sea urchins and oysters were collected at Paraíba coast; Brazil (Echinometra lucunter and Crassostrea gasar) and Rade of Brest; France (Echinus esculentus and Crassostrea gigas). Coelomocytes and hemocytes were stained with the ABC transporter substrate calcein-AM and dye accumulation analyzed under flow cytometry. Reversin 205 (ABCB1 transporter blocker) and MK571 (ABCC1 transporter blocker) were used as pharmacological tools to investigate ABC transporter activity. A different pattern of calcein accumulation was observed in coelomocytes: phagocytes > colorless spherulocytes > vibrate cells > red spherulocytes. The treatment with MK571 increased calcein fluorescence levels in coelomocytes from both species. However, reversin 205 treatment was not able to increase calcein fluorescence in E. esculentus coelomocytes. These data suggest that ABCC1-like transporter activity is present in both sea urchin species, but ABCB1-like transporter activity might only be present in E. lucunter coelomocytes. The activity of ABCC1-like transporter was observed in all cell types from both bivalve species. However, reversin 205 only increased calcein accumulation in hyalinocytes of the oyster C. gasar, suggesting the absence of ABCB1-like transporter activity in all other cell types, including hyalinocytes from the oyster C. gigas. Additionally, our results showed that C. gigas exhibited higher activity of ABCC1-like transporter in all hemocyte types than C. gasar. The present work is the first to characterize ABCB1 and ABCC1-like transporter activity in the immune system cells of sea urchins E. lucunter and E. esculentus and oysters. Our findings encourage the performing studies regarding ABC transporters activity/expression in immune system cells form marine invertebrates under stress conditions and the possible use of ABC transporters as biomarkers.

Effects of cyanobacteria Synechocystis spp. in the host-parasite model Crassostrea gasar-Perkinsus marinus.

Perkinsosis is a disease caused by protozoan parasites from the Perkinsus genus. In Brazil, two species, P. beihaiensis and P. marinus, are frequently found infecting native oysters (Crassostrea gasar and C. rhizophorae) from cultured and wild populations in several states of the Northeast region. The impacts of this disease in bivalves from Brazil, as well as the interactions with environmental factors, are poorly studied. In the present work, we evaluated the in vitro effects of the cyanobacteria Synechocystis spp. on trophozoites of P. marinus and haemocytes of C. gasar. Four cyanobacteria strains isolated from the Northeast Brazilian coast were used as whole cultures (WCs) and extracellular products (ECPs). Trophozoites of P. marinus were exposed for short (4h) and long (48h and 7days, the latter only for ECPs) periods, while haemocytes were exposed for a short period (4h). Cellular and immune parameters, i.e. cell viability, cell count, reactive oxygen species production (ROS) and phagocytosis of inert (latex beads) and biological particles (zymosan and trophozoites of P. marinus) were measured by flow cytometry. The viability of P. marinus trophozoites was improved in response to WCs of Synechocystis spp., which could be a beneficial effect of the cyanobacteria providing nutrients and reducing reactive oxygen species. Long-term exposure of trophozoites to ECPs of cyanobacteria did not modify in vitro cell proliferation nor viability. In contrast, C. gasar haemocytes showed a reduction in cell viability when exposed to WCs, but not to ECPs. However, ROS production was not altered. Haemocyte ability to engulf latex particles was reduced when exposed mainly to ECPs of cyanobacteria; while neither the WCs nor the ECPs modified phagocytosis of the biological particles, zymosan and P. marinus. Our results suggest a negative effect of cyanobacteria from the Synechocystis genus on host immune cells, in contrast to a more beneficial effect on the parasite cell, which could together disrupt the balance of the host-parasite interaction and make oysters more susceptible to P. marinus as well as opportunistic infections.

Mitochondrial permeability transition pore (MPTP) desensitization increases sea urchin spermatozoa fertilization rate.

Mitochondrial permeability transition pore (MPTP) is a protein complex whose opening promotes an abrupt increase in mitochondrial inner membrane permeability. Calcium signaling pathways are described in gametes and are involved in the fertilization process. Although mitochondria may act as Ca(2+) store and have a fast calcium-releasing mechanism through MPTP, its contribution to fertilization remains unclear. The work aimed to investigate the MPTP phenomenon in sea urchin spermatozoa and its role on the fertilization. Several pharmacological tools were used to evaluate the MPTP's physiology. Our results demonstrated that MPTP occurs in male gametes in a Ca(2+) - and voltage-dependent manner and it is sensitive to cyclosporine A. Additionally, our data show that MPTP opening does not alter ROS generation in sperm cells. Inhibition of MPTP in spermatozoa strongly improved the fertilization rate, which may involve mechanisms that increase the spermatozoa lifespan. The present work is the first report of the presence of a voltage- and Ca(2+) -dependent MPTP in gametes of invertebrates and indicates MPTP opening as another evolutionary feature shared by sea urchins and mammals. Studies about MPTP in sea urchin male gametes may contribute to the elucidation of several mechanisms involved in sperm infertility.

Effects of salinity and temperature on in vitro cell cycle and proliferation of Perkinsus marinus from Brazil.

Field and in vitro studies have shown that high salinities and temperatures promote the proliferation and dissemination of Perkinsus marinus in several environments. In Brazil, the parasite infects native oysters Crassostrea gasar and Crassostrea rhizophorae in the Northeast (NE), where the temperature is high throughout the year. Despite the high prevalence of Perkinsus spp. infection in oysters from the NE of Brazil, no mortality events were reported by oyster farmers to date. The present study evaluated the effects of salinity (5, 20 and 35 psu) and temperature (15, 25 and 35 °C) on in vitro proliferation of P. marinus isolated from a host (C. rhizophorae) in Brazil, for a period of up to 15 days and after the return to the control conditions (22 days; recovery). Different cellular parameters (changes of cell phase's composition, cell density, viability and production of reactive oxygen species) were analysed using flow cytometry. The results indicate that the P. marinus isolate was sensitive to the extreme salinities and temperatures analysed. Only the highest temperature caused lasting cell damage under prolonged exposure, impairing P. marinus recovery, which is likely to be associated with oxidative stress. These findings will contribute to the understanding of the dynamics of perkinsiosis in tropical regions.

Antimitotic activity of the pyrimidinone derivative py-09 on sea urchin embryonic development.

Chemotherapy is the main cancer treatment and consists of drug administration that interferes with several metabolic pathways, leading to tumor cell death. Antimitotic drugs have a relevant role in chemotherapy. This study aimed to investigate the effect of a pyrimidinone derivative (6-(p-Anisyl)-2-(p-chlorophenyl)-4-oxo-3,4-dihydropyrimidine-5-carbonitrile, Py-09) on sea urchin embryonic development model. The effects of the compound were analyzed on fertilization, embryonic development, mitochondrial membrane potential (ΔΨm), production of reactive oxygen species (ROS) and ABC transporter activity. Py-09 inhibited the fertilization and the embryonic development in a time and dose-dependent pattern, with the maximum effect at 50 µM (EC50=12.5 µM). Py-09 induced the loss of ΔΨm without altering ROS intracellular levels. Morphological changes were observed in the pattern of embryo cleavage (unequal cleavage) and at larval stages (fissures of spicules and pigment cell leakage). We also demonstrated that Py-09 is not an ABC transporter substrate and the derivative does not circumvent the MXR phenomenon. Our study reports--for the first time--the antimitotic activity of Py-09 and stimulates new research on the potential of Py-09 as a pharmacological tool for in vitro studies, as well as its use as a new anticancer drug.

Photoprotective effect of coumarin and 3-hydroxycoumarin in sea urchin gametes and embryonic cells.

Ultraviolet radiation B (UVB) represents 5% of all solar UV radiation and chronic exposure can induce harmful biological responses, including skin cancer. Prospection of new drugs with photoprotective properties and less toxic effects is constant and natural products have been the main options in this field. Coumarins are a group of natural phenolic compounds that shows several pharmacological activities. The aim of present work was to investigate the effect of coumarin and six derivatives in sea urchin gametes and zygotes exposed to UVB. Embryonic development assay was used to monitor UVB embryotoxicity. Firstly, we demonstrated that coumarin inhibited first embryonic cell division from 5 µM (EC50 = 52.9 µM) and its derivatives showed an embryotoxic effect ten times higher. Then, gametes or zygotes were treated with coumarin compounds before or after UVB exposure (UVB doses ranged from 0.056 to 0.9 kJm(-2)). Pretreatment of gametes or zygotes with coumarin or 3-hydroxycoumarin (1 µM, both) decreased UVB embryotoxic effect. Protective effect of the compounds was observed only when cells were treated previous to UVB exposure. Coumarin derivatives 4-hydroxycoumarin, 6-hydroxycoumarin, 7-hydroxycoumarin, 6,7-dihydroxycoumarin and 6-methoxy-7-hydroxycoumarin did not exhibit photoprotective activity. Our data provides evidences that coumarin and 3-hydroxycoumarin can be a promising class of photoprotective drugs.

ATP-binding cassette transporters protect sea urchin gametes and embryonic cells against the harmful effects of ultraviolet light.

Embryos of marine organisms whose development occurs externally are particularly sensitive to ultraviolet (UV) light (bands A and B, respectively, UVA and UVB). ATP-binding cassette (ABC) transporters are the first line of cellular defense against chemical or physical stress. The present work investigated the involvement of ABC transporters on UVA or UVB effects on eggs, spermatozoa, and embryonic cells of the sea urchin Echinometra lucunter. Gametes or embryos were exposed to UVA (3.6-14.4 kJ m(-2)) or UVB (0.112-14.4 kJ m(-2)), and embryonic development was monitored by optical microscopy at different developmental stages in the presence or absence of the ABC-transporter blockers reversin205 (ABCB1 blocker) or MK571 (ABCC1 blocker). E. lucunter eggs, spermatozoa and embryos were resistant to UVA exposure. Resistance to the harmful effects of UVB was strongly associated to ABC transporter activity (embryos > eggs > spermatozoa). ABCB1 or ABCC1 blockage promoted the injurious effects of UVA on spermatozoa. ABCC1 transporter blockage increased UVB-dependent damage in eggs while ABCB1 transporter inhibition increased harmful effects of UVB in embryonic cells. ABC-transporter activity was not, however, affected by UVB exposure. In conclusion, the present study is the first report on the protective role of ABC transporters against harmful effects of UVA and UVB on sea urchin eggs and embryonic cells.

Immunological responses of the mangrove oysters Crassostrea gasar naturally infected by Perkinsus sp. in the Mamanguape Estuary, Paraíba state (Northeastern, Brazil).

Perkinsus genus includes protozoan parasites of marine mollusks, especially bivalves. In the last four years, this parasite has been detected in mangrove oysters Crassostrea rhizophorae and Crassostrea gasar from the Northeastern region of Brazil. Hemocytes are the key cells of the oyster immune system, being responsible for a variety of cellular and humoral reactions, such as phagocytosis, encapsulation and the release of several effector molecules that control the invasion and proliferation of microorganisms. In Brazil, there is little information on perkinsosis and none on the immune responses of native oysters' species against Perkinsus spp. The objective of this study was to determine the effects of natural infection by Perkinsus sp. on the immunological parameters of mangrove oysters C. gasar cultured in the Mamanguape River Estuary (Paraíba, Brazil). Adults oysters (N = 40/month) were sampled in December 2011, March, May, August and October 2012. Gills were removed and used to determine the presence and intensity of the Perkinsus sp. infection, according to a scale of four levels (1-4), using the Ray's fluid thioglycollate medium assay. Immunological parameters were measured in hemolymph samples by flow cytometry, including: total hemocyte count (THC), differential hemocyte count (DHC), cell mortality, phagocytic capacity, and production of Reactive Oxygen Species (ROS). The plasma was used to determine the hemagglutination activity. The results showed the occurrence of Perkinsus sp. with the highest mean prevalence (93.3%) seen so far in oyster populations in Brazil. Despite that, no oyster mortality was associated. In contrast, we observed an increase in hemocyte mortality and a suppression of two of the main defense mechanisms, phagocytosis and ROS production in infected oysters. The increase in the percentage of blast-like cells on the hemolymph, and the increase in THC in oysters heavily infected (at the maximum intensity, 4) suggest an induction of hemocytes proliferation. The immunological parameters varied over the studied months, which may be attributed to the dynamics of infection by Perkinsus sp. The results of the present study demonstrate that Perkinsus sp. has a deleterious effect on C. gasar immune system, mainly in high intensities, which likely renders oysters more susceptible to other pathogens and diseases.

Involvement of ABCB1 and ABCC1 transporters in sea urchin Echinometra lucunter fertilization.

Fertilization is an ordered sequence of cellular interactions that promotes gamete fusion to form a new individual. Since the pioneering work of Oskar Hertwig conducted on sea urchins, echinoderms have contributed to the understanding of cellular and molecular aspects of the fertilization processes. Studies on sea urchin spermatozoa reported the involvement of a plasma membrane protein that belongs to the ABC proteins superfamily in the acrosome reaction. ABC transporters are expressed in membranes of eukaryotic and prokaryotic cells, and are associated with the transport of several compounds or ions across biomembranes. We aimed to investigate ABCB1 and ABCC1 transporter activity in sea urchin spermatozoa and their involvement in fertilization. Our results indicate that Echinometra lucunter spermatozoa exhibit a low intracellular calcein accumulation (18.5% stained cells); however, the ABC blockers reversin205, verapamil, and MK571 increased dye accumulation (93.0-96.6% stained cells). We also demonstrated that pharmacologically blocking ABCB1 and ABCC1 decreased spermatozoa fertilizing capacity (70% inhibition), and this phenotype was independent of extracellular calcium. These data suggest that functional spermatozoa ABCB1 and ABCC1 transporters are crucial for a successful fertilization. Additional studies must be performed to investigate the involvement of membrane lipid homeostasis in the fertilization process.

Extracellular Ca2+ influx is crucial for the early embryonic development of the sea urchin Echinometra lucunter.

The involvement of Ca(2+) in the activation of eggs and in the first steps of the embryonic development of several species is a well-known phenomenon. An association between Ca(2+) sources with the fate of the blastopore during embryonic development has been investigated by several authors. Ca(2+) influx mediated by voltage-gated channels and Ca(2+) mobilization from intracellular stores are the major sources of Ca(2+) to egg activation and succeeding cell divisions. Studies on sea urchins embryonic development show that intracellular Ca(2+) stores are responsible for egg activation and early embryogenesis. In the present work we investigated the involvement of extracellular Ca(2+) in the first stages of the embryonic development of the sea urchin Echinometra lucunter. Divalent cation chelators EDTA and EGTA strongly blocked the early embryonic development. Adding to this, we demonstrated the involvement of voltage-gated Ca(2+) channels in E. lucunter embryogenesis since Ca(2+) channel blockers powerfully inhibited the early embryonic development. Our data also revealed that Ca(2+) influx is crucial for embryonic development during only the first 40 min postfertilization. However, intracellular Ca(2+) remains mandatory to embryonic development 40 min postfertilization, seen that both the intracellular Ca(2+) chelator BAPTA-AM and calmodulin antagonists trifluoperazine and chlorpromazine inhibited the first stages of development when added to embryos culture 50 min postfertilization. Our work highlights the crucial role of extracellular Ca(2+) influx through voltage-gated Ca(2+) channels for the early embryonic development of the sea urchin E. lucunter and characterizes an exception in the phylum Echinodermata.

Influence of fetal bovine serum on cytotoxic and genotoxic effects of lectins in MCF-7 cells.

Canavalia ensiformis (ConA), Canavalia brasiliensis (Conbr), and Cratylia floribunda (CFL) lectins have exhibited glucose-mannose binding specificity. We investigated the effect of fetal bovine serum (FBS) concentrations (1, 5, 10, and 20%) on the cytotoxic effect of these lectins against breast tumor cell line MCF-7. Cell viability was examined using the MTT reduction assay. When cells were grown in a medium supplemented with a higher serum concentration (10 or 20%), all lectins were much less toxic. When we used 1% FBS, it was possible to achieve a concentration-dependent activity by all examined lectins, with an IC(50) of 3.5, 25, and 60 µg/mL for ConA, Conbr, and CFL, respectively. All lectins incubated with 1% FBS induced apoptosis and DNA damage in MCF-7 cells. We conclude that ConA, Conbr, and CFL lectins' cytotoxic and genotoxic effects were observed only at low concentrations of serum.

Characterization of functional activity of ABCB1 and ABCC1 proteins in eggs and embryonic cells of the sea urchin Echinometra lucunter.

ABC transporter (ATP-binding-cassette transporter) proteins have been strongly associated with the phenomenon of multidrug resistance in cancer cells. Furthermore, their physiological expression has been studied in many organisms, including bacteria, fungi, plants and vertebrate or invertebrate animals. Their widespread expression through the evolution demonstrates their relevance to the survival of living things. In the present study, we characterized the functional activity of ABCB1 and ABCC1 proteins in gametes and embryonic cells of the sea urchin Echinometra lucunter. The ABC transporter proteins' functional activity was up-regulated post-fertilization. Eggs and spermatozoa of E. lucunter accumulated more C-AM (calcein acetoxymethyl ester), a fluorescent substrate of ABCB1 and ABCC1 proteins, than embryonic cells. Verapamil, reversin 205 and indomethacin were able to increase C-AM influx in eggs and embryos. However, verapamil and reversin 205 were more efficient than indomethacin, suggesting a predominance of ABCB1 protein over ABCC1 protein activity. Multidrug resistance modulating agents, at the concentration range that inhibited ABC transporter proteins, did not block the embryonic development until blastula stage. However, inhibition of ABCB1-mediated efflux by reversin 205 circumvented resistance of embryos to the antimitotic vinca alkaloid vinblastine. Embryonic development was more efficiently blocked when colchicine was previously added to eggs than to embryos 5 min after fertilization. This set of results suggests that these proteins act as a fundamental biochemical barrier conferring a protective physiological role against toxic xenobiotics in E. lucunter embryos.

CPT-11-induced cell death in leukemic cells is not affected by the MDR phenotype.

CPT-11 is a topoisomerase I (Topo I) inhibitor which was initially described as active in multi-drug resistance (MDR) tumors. The MDR phenomenon is characterized by the overexpression of efflux pumps which are able to extrude a range of drugs non-related chemical or functionally. In this work, we treated leukemic cells with CPT-11 300 microM at 24h and compared its cytotoxicity with the activity of efflux pumps and with cell cycle phase. Our findings show that CPT-11 has a potent anti-tumor activity in leukemic cells regardless MDR phenotype and the cell cycle phase, suggesting new avenues to be explored in leukemia treatment.