Lack of genotoxicity of formocresol, paramonochlorophenol, and calcium hydroxide on mammalian cells by comet assay

Formocresol, paramonochlorophenol, and calcium hydroxide are widely used in dentistry because of their antibacterial activities in root canal disinfection. However, the results of genotoxicity studies using these materials are inconsistent in literature. The goal of this study was to examine the genotoxic potential of formocresol, paramonochlorophenol, and calcium hydroxide using mouse lymphoma cells and human fibroblasts cells in vitro by the comet assay. Data were assessed by Kruskal-Wallis nonparametric test. The results showed that all compounds tested did not cause DNA damage for the tail moment or tail intensity parameters. These findings suggest that formocresol, paramonochlorophenol, and calcium hydroxide do not promote DNA damage in mammalian cells and that the comet assay is a suitable tool to investigate genotoxicity.

Application of a serum/protein-free medium for the growth of mammalian cell lines and high level production of classical, variant and very virulent strain of infectious bursal disease virus (IBDV)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genotoxicity of the medicinal plant Maytenus robusta in mammalian cells in vivo.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

Erythrina mulungu Alkaloids Are Potent Inhibitors of Neuronal Nicotinic Receptor Currents in Mammalian Cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Telomere and Telomerase Biology

Telomeres are the physical ends of eukaryotic linear chromosomes. Telomeres form special structures that cap chromosome ends to prevent degradation by nucleolytic attack and to distinguish chromosome termini from DNA double-strand breaks. With few exceptions, telomeres are composed primarily of repetitive DNA associated with proteins that interact specifically with double- or single-stranded telomeric DNA or with each other, forming highly ordered and dynamic complexes involved in telomere maintenance and length regulation. In proliferative cells and unicellular organisms, telomeric DNA is replicated by the actions of telomerase, a specialized reverse transcriptase. In the absence of telomerase, some cells employ a recombination-based DNA replication pathway known as alternative lengthening of telomeres. However, mammalian somatic cells that naturally lack telomerase activity show telomere shortening with increasing age leading to cell cycle arrest and senescence. In another way, mutations or deletions of telomerase components can lead to inherited genetic disorders, and the depletion of telomeric proteins can elicit the action of distinct kinases-dependent DNA damage response, culminating in chromosomal abnormalities that are incompatible with life. In addition to the intricate network formed by the interrelationships among telomeric proteins, long noncoding RNAs that arise from subtelomeric regions, named telomeric repeat-containing RNA, are also implicated in telomerase regulation and telomere maintenance. The goal for the next years is to increase our knowledge about the mechanisms that regulate telomere homeostasis and the means by which their absence or defect can elicit telomere dysfunction, which generally results in gross genomic instability and genetic diseases.

Morphological changes of mammalian nucleoli during spermatogenesis and their possible role in the chromatoid body assembling

Chromatoid body (CB) is a typical cytoplasmic organelle of germ cells, and it seems to be involved in RNA/protein accumulation for later germ-cell differentiation. Despite most of the events in mammals spermatogenesis had been widely described in the past decades and the increase in the studies related to the CB molecular composition and physiology, the origins and functions of this important structure of male germ cells are still unclear. The aims of this study were to describe the nucleolar cycle and also to find some relationship between the nucleolar organization and the CB assembling during the spermatogenesis in mammals. Cytochemical and cytogenetics analysis showed nucleolar fragmentation in post-pachytene spermatocytes and nucleolar reorganization in post-meiotic spermatids. Significant difference in the number and in the size of nucleoli between spermatogonia and round spermatids, as well as differences in the nucleolar position within the nucleus were also observed. Ultrastructural analysis showed the CB assembling in the cytoplasm of primary spermatocytes and the nucleolar fragmentation occurring at the same time. In conclusion our results suggest that the CB may play important roles during the spermatogenesis process in mammals and that its origin may be related to the nucleolar cycle during the meiotic cell cycle.

Involvement of TSSA (trypomastigote small surface antigen) in Trypanosoma cruzi invasion of mammalian cells

TSSA (trypomastigote small surface antigen) is a polymorphic mucin-like molecule displayed on the surface of Trypanosoma cruzi trypomastigote forms. To evaluate its functional properties, we undertook comparative biochemical and genetic approaches on isoforms present in parasite stocks from extant evolutionary lineages (CL Brener and Sylvio X-10). We show that CL Brener TSSA, but not the Sylvio X-10 counterpart, exhibits dose-dependent and saturable binding towards non-macrophagic cell lines. This binding triggers Ca2+-based signalling responses in the target cell while providing an anchor for the invading parasite. Accordingly, exogenous addition of either TSSA-derived peptides or specific antibodies significantly inhibits invasion of CL Brener, but not Sylvio X-10, trypomastigotes. Non-infective epimastigote forms, which do not express detectable levels of TSSA, were stably transfected with TSSA cDNA from either parasite stock. Although both transfectants produced a surface-associated mucin-like TSSA product, epimastigotes expressing CL Brener TSSA showed a similar to 2-fold increase in their attachment to mammalian cells. Overall, these findings indicate that CL Brener TSSA functions as a parasite adhesin, engaging surface receptor(s) and inducing signalling pathways on the host cell as a prerequisite for parasite internalization. More importantly, the contrasting functional features of TSSA isoforms provide one appealing mechanism underlying the differential infectivity of T. cruzi stocks.

Mutations in CTC1, encoding conserved telomere maintenance component 1, cause Coats plus

Coats plus is a highly pleiotropic disorder particularly affecting the eye, brain, bone and gastrointestinal tract. Here, we show that Coats plus results from mutations in CTC1, encoding conserved telomere maintenance component 1, a member of the mammalian homolog of the yeast heterotrimeric CST telomeric capping complex. Consistent with the observation of shortened telomeres in an Arabidopsis CTC1 mutant and the phenotypic overlap of Coats plus with the telomeric maintenance disorders comprising dyskeratosis congenita, we observed shortened telomeres in three individuals with Coats plus and an increase in spontaneous gamma H2AX-positive cells in cell lines derived from two affected individuals. CTC1 is also a subunit of the alpha-accessory factor (AAF) complex, stimulating the activity of DNA polymerase-alpha primase, the only enzyme known to initiate DNA replication in eukaryotic cells. Thus, CTC1 may have a function in DNA metabolism that is necessary for but not specific to telomeric integrity.

Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na, K-ATPase activity by mammalian FXYD2 peptide

This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (gamma C-33) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K+, ATP and N-4(+)center dot K-0.5 for Na+ was unaffected. Exogenous pig FXYD2 increased the V-max for stimulation of gill Na,K-ATPase activity by Na+, K+ and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na, K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity. (C) 2012 Elsevier B.V. All rights reserved.

1,4-Diamino-2-butanone, a putrescine analogue, promotes redox imbalance in Trypanosoma cruzi and mammalian cells

The putrescine analogue 1,4-diamino-2-butanone (DAB) is highly toxic to various microorganisms, including Trypanosoma cruzi. Similar to other a-aminocarbonyl metabolites. DAB exhibits pro-oxidant properties. DAB undergoes metal-catalyzed oxidation yielding H2O2, NH4+ ion, and a highly toxic alpha-oxoaldehyde. In vitro. DAB decreases mammalian cell viability associated with changes in redox balance. Here, we aim to clarify the DAB pro-oxidant effects on trypomastigotes and on intracellular T. cruzi amastigotes. DAB (0.05-5 mM) exposure in trypomastigotes, the infective stage of T. cruzi, leads to a decline in parasite viability (IC50 c.a. 0.2 mM DAB; 4 h incubation), changes in morphology, thiol redox imbalance, and increased TcSOD activity. Medium supplementation with catalase (2.5 mu M) protects trypomastigotes against DAB toxicity, while host cell invasion by trypomastigotes is hampered by DAB. Additionally, intracellular amastigotes are susceptible to DAB toxicity. Furthermore, pre-treatment with 100-500 mu M buthionine sulfoximine (BSO) of LLC-MK2 potentiates DAB cytotoxicity, whereas 5 mM N-acetyl-cysteine (NAC) protects cells from oxidative stress. Together, these data support the hypothesis that redox imbalance contributes to DAB cytotoxicity in both T. cruzi and mammalian host cells. (C) 2012 Elsevier Inc. All rights reserved.

Relative CO(2)/NH(3) selectivities of mammalian aquaporins 0-9

Previous work showed that aquaporin 1 (AQP1), AQP4-M23, and AQP5 each has a characteristic CO(2)/NH(3) and CO(2)/H(2)O permeability ratio. The goal of the present study is to characterize AQPs 0-9, which traffic to the plasma membrane when heterologously expressed in Xenopus oocytes. We use video microscopy to compute osmotic water permeability (P(f)) and microelectrodes to record transient changes in surface pH (ΔpH(S)) caused by CO(2) or NH(3) influx. Subtracting respective values for day-matched, H(2)O-injected control oocytes yields the channel-specific values P(f)* and ΔpH(S)*. We find that P(f)* is significantly >0 for all AQPs tested except AQP6. (ΔpH(S)*)(CO(2)) is significantly >0 for AQP0, AQP1, AQP4-M23, AQP5, AQP6, and AQP9. (ΔpH(S)*)(NH(3)) is >0 for AQP1, AQP3, AQP6, AQP7, AQP8, and AQP9. The ratio (ΔpH(S)*)(CO(2))/P(f)* falls in the sequence AQP6 (∞) > AQP5 > AQP4-M23 > AQP0 ≅ AQP1 ≅ AQP9 > others (0). The ratio (ΔpH(S)*)(NH(3))/P(f)* falls in the sequence AQP6 (∞) > AQP3 ≅ AQP7 ≅ AQP8 ≅ AQP9 > AQP1 > others (0). Finally, the ratio (ΔpH(S)*)(CO(2))/(-ΔpH(S)*)(NH(3)) falls in the sequence AQP0 (∞) ≅ AQP4-M23 ≅ AQP5 > AQP6 > AQP1 > AQP9 > AQP3 (0) ≅ AQP7 ≅ AQP8. The ratio (ΔpH(S)*)(CO(2))/(-ΔpH(S)*)(NH(3)) is indeterminate for both AQP2 and AQP4-M1. In summary, we find that mammalian AQPs exhibit a diverse range of selectivities for CO(2) vs. NH(3) vs. H(2)O. As a consequence, by expressing specific combinations of AQPs, cells could exert considerable control over the movements of each of these three substances

Rapid screening of potential autophagic inductor agents using mammalian cell lines

Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye-staining of autophagic vesicles, and LC3-II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used assays to determine cell viability - Crystal Violet staining (CVS), 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) - to predict autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16-F10, HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a unique single microplate.

Processing of O 6 - methylguanine in mammalian cells

DNA methylating compounds are widely used as anti-cancer chemotherapeutics. The pharmaceutical critical DNA lesion induced by these drugs is O6-methylguanine (O6MeG). O6MeG is highly mutagenic and genotoxic, by triggering apoptosis. Despite the potency of O6MeG to induce cell death, the mechanism of O6MeG induced toxicity is still poorly understood. Comparing the response of mouse fibroblasts wild-type (wt) and deficient for ataxia telangiectasia mutant protein (ATM), a kinase responsible for both the recognition and the signalling of DNA double-strand breaks (DSBs), it was shown that ATM deficient cells are more sensitive to the methylating agents N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), methyl methansulfonate (MMS) and the anti-cancer drug temozolomide, in both colony formation and apoptosis assays. This clearly shows that DSBs are involved in O6MeG toxicity. By inactivating the O6MeG repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) with the specific inhibitor O6-benzylguanine (O6BG), ATM wt and deficient cells became more sensitive to MNNG and MMS. The opposite effect was observed when over-expressing MGMT in ATM -/- cells. The results show that O6MeG is the critical DNA lesion causing death in ATM cells following MNNG treatment, and is partially responsible for the toxicity observed following MMS treatment. Furthermore, by inhibiting the ATM kinase activity with caffeine, it was shown that the resistance of wt cells to MNNG was due to the kinase activity of ATM, as wt cells underwent more apoptosis following methylating agent treatment in the presence of caffeine. Apoptosis and caspase-3 activation were late events, starting 48h after treatment. This lends support to the model where O6MeG lesions are converted into DSBs during replication. As ATM wt and deficient cells showed similar G2/M blockage and Chk1 activation following MNNG and MMS treatment, it was concluded that the protective effect of ATM is not due to cell cycle progression control. The hypersensitivity of ATM deficient cells was accompanied by their inability to activate the anti-apoptotic NFkB pathway. In a second part of this study, it was shown that the inflammatory cytokine IL-1 up-regulates the DNA repair gene apurinic endonuclease 2 (APEX2). Up-regulation of APEX2 occurred by transcriptional regulation as it was abrogated by actinomycin D. APEX2 mRNA accumulation was accompanied by increase in APEX2 protein level. IL-1 induced APEX2 expression as well as transfection of cells with APEX2 cDNA positively correlated with a decrease in apoptosis after treatment with genotoxic agents, particularly affecting cell death after H2O2. This indicates an involvement of APEX2 in the BER pathway in cells responding to IL-1.