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Assis Ecker, Pauline Christ Ledur, Rafael S. da Silva, Daniela Bitencourt Rosa Leal, Oscar E. D. Rodrigues, Daniel Ardisson-Araújo, Emily Pansera Waczuk, João Batista Teixeira da Rocha, Nilda Vargas Barbosa, Chalcogenozidovudine Derivatives With Antitumor Activity: Comparative Toxicities in Cultured Human Mononuclear Cells, Toxicological Sciences, Volume 160, Issue 1, November 2017, Pages 30–46, https://doi.org/10.1093/toxsci/kfx152
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Abstract
Considering a novel series of zidovudine (AZT) derivatives encompassing selenoaryl moieties promising candidates as therapeutics, we examined the toxicities elicited by AZT and derivatives 5′-(4-Chlorophenylseleno)zidovudine (SZ1); 5′-(Phenylseleno)zidovudine (SZ2); and 5′-(4-Methylphenylseleno)zidovudine (SZ3) in healthy cells and in mice. Resting and stimulated cultured human peripheral blood mononuclear cells (PBMCs) were treated with the compounds at concentrations ranging from 10 to 200 µM for 24 and/or 72 h. Adult mice received a single injection of compounds (100 µmol/kg, s.c.) and 72 h after administration, hepatic/renal biomarkers were analyzed. Resting and stimulated PBMCs exposed to SZ1 displayed loss of viability, increased reactive species production, disruption in cell cycle, apoptosis and increased transcript levels and production of pro-inflammatory cytokines. In a mild way, most of these effects were also induced by SZ2. AZT and SZ3 did not cause significant toxicity towards resting PBMCs. Differently, both compounds elicited apoptosis and S phase arrest in stimulated cells. AZT and derivatives administration did not change the body weight and plasma biochemical markers in mice. However, the absolute weight and organ-to-body weight ratio of liver, kidneys and spleen were altered in AZT, SZ1-, and SZ2-treated mice. Our results highlighted the involvement of derivatives SZ1 and SZ2 in redox and immunological dyshomeostasis leading to activation of apoptotic signaling pathways in healthy cells under different division phases. On the other hand, the derivative SZ3 emerged as a promising candidate for further viral infection/antitumor studies as a new effective therapy with low toxicity for immune cells and after acute in vivo treatment.
Therapeutic nucleosides play a crucial role in the current treatment of human immune deficiency virus (HIV) infection by blocking viral complement DNA synthesis carried out by viral reverse transcriptase (RT) (Arts and Hazuda, 2012; Kukhanova, 2012). Nucleoside reverse transcriptase inhibitors (NRTIs) are critical for HIV treatment and consist in an important part of the highly active antiretroviral therapy (HAART) (Kukhanova, 2012). Commonly, the HAART is composed by 3′-Azido-3′-deoxythymidine (AZT or zidovudine), the first NTRI clinically and successfully approved for the treatment of HIV (Khandazhinskaya et al., 2010) Besides its potent antiretroviral effect, AZT has an important role as first-line therapy in several virus-associated human cancers, including Epstein-Barr virus-associated B-cell lymphoma (Abdulkarim and Bourhis, 2001; Gomez et al., 2012). In the therapeutic Guidelines from Brazil, AZT was recently recommended as first-line treatment for cases of T-cell lymphotropic virus type I (HTLV)-I–associated adult T-cell leukemia/lymphoma (CONITEC, Brazilian Ministry of Health, 2015).
The long-term usage of AZT for HIV treatment has been correlated with severe toxic side effects and acquirement of drug resistance. Bone marrow suppression, myopathy, lactic acidosis and hepatic abnormalities are frequently diagnosed in subjects under AZT-based therapy (Richman et al., 1987; Gelmon et al., 1989; Chariot et al., 1999). Additionally, some sub optimal pharmacokinetic properties such as low therapeutic index, short half-life in blood, and low blood-brain barrier permeability contribute considerably for AZT toxicity, since higher doses are required to maintain the drug therapeutic levels (Turk et al, 2002).
A variety of AZT derivatives with distinct structural changes and biological properties have been synthesized in an attempt to circumvent the undesirable side effects of the parent compound and increase the therapeutic efficacy toward HIV and different types of cancer (Khandazhinskaya et al., 2010; Migianu et al., 2005; Tan et al., 1999; Vasilyeva et al., 2015). Unfortunately, only few of these compounds have evolved into approved drugs, for instance, AZT 5′-H-phosphonate (phosphazide, Nikavir) for prevention and treatment of HIV infection (Yurin et al., 2001) and the nucleosides derivatives decitabine (Gore et al., 2006), clofarabine (Bonate et al., 2006), and 5-azacytidine (Issa et al., 2005), approved for the treatment of leukemic and myelodysplasic syndromes.
Recently, Souza and collaborators developed and evaluated the bioactivities of a novel class of compounds derived from zidovudine containing selenium (Se), tellurium (Te), and sulfur (S), called chalcogenozidovudines. Among them, the authors highlighted the prominent efficacy of 3 Se-derivatives as antitumoral agents against human bladder carcinoma cells, namely 5′-(4-Chlorophenylseleno)zidovudine (SZ1); 5′-(Phenylseleno)zidovudine (SZ2) and 5′-(4-Methylphenylseleno)zidovudine (SZ3) (Souza et al., 2015). The addition of Se moieties also conferred to some AZT derivatives thiol peroxidase-like activity. Subsequently, the toxic effects of these molecules were evaluated in an in vitro model of acute exposure with freshly isolated human total leukocytes (Mariano et al., 2016). Cell viability was determined by trypan blue exclusion and genotoxicity was evaluated by comet assay. At low (5–25 µM) and moderate concentrations (50–75 µM), only SZ3 caused cell toxicity. At high concentrations (100–200 µM), SZ1, SZ2, and SZ3 derivatives decreased leukocytes viability.
Although the short-term exposure methodology can give some insight into the gross toxicity of prototypal chemical compounds, it does not allow refined study of the molecular and biochemical processes which can underlie the toxicity of pharmacological agents. Considering the chalcogenozidovudines promising candidates for future studies on viral/cancer diseases, here more accurate battery of methodologies were performed to analyze and compare the potential toxicities elicited by AZT and Se-derivatives. By flow cytometry and gene expression profile we evaluated the effects of AZT and derivatives on cell viability, oxidative stress/inflammatory events and apoptosis in cultured peripheral blood mononuclear cells (PBMCs) from healthy donors. Besides, some gross morphological (body and organs weight, and organ-to-body weight ratio) and biochemical parameters were evaluated as end-points of toxicity in mice acutely treated with the chalcogenozivudine compounds.
MATERIALS AND METHODS
Chemicals
Ficoll-Paque (1.077), RPMI-1640 medium, fetal bovine serum (FBS), penicillin/streptomycin, dihydrorhodamine-123 (DHR-123), triton X-100, methylthiazolyldiphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), propidium iodide (PI), agarose, ethidium bromide solution and the primers used for RT-qPCR analysis were purchased from Sigma-Aldrich (St Louis, Missouri). DNase I, Alexa Fluor 488-conjugated annexin V kit, TRIZOL reagent and DNase-free RNase were purchased from Invitrogen (Carlsbad, California). iScrip cDNA Synthesis Kit was purchased from Bio-Rad (Hercules, California). Lactate dehydrogenase (LDH) assay kit was acquired from Labtest (Minas Gerais, Brazil). The CBA Human Th1/Th2/Th17 Cytokine Kit was acquired from BD (Franklin Lakes, Nova Jersey) (catalog no. 560484CBA).
Synthesis of Chalcogenozidovudine Derivatives
The synthesis of compounds was performed from commercially available nucleoside 3′-azido-3′-deoxythymidine (AZT or zidovudine), as previously reported by Souza et al. (2015). The synthesized derivatives received additional phenyl-seleno- substitutions at 5′ position of AZT molecule, resulting in the chalcogenozidovudine derivatives 5′-(4-Chlorophenylseleno)zidovudine (SZ1), 5′-(Phenylseleno)zidovudine (SZ2), and 5′-(4-Methylphenylseleno)zidovudine (SZ3). Details of synthetic schemes and chemistry characteristics of compounds have already been previously described (Souza et al., 2015). Chemical structures of AZT and Se-derivatives are shown in Figure 1.
Blood Samples
Blood samples were obtained from healthy adult volunteers. Eligibility criteria included age between 20 and 40 years; no alcohol abuse or drug dependence, no exposure to nicotine, no diagnosis of acute or chronic comorbidities and no drug treatment within 30 days before the experiments. The protocol used was conducted following the principles expressed in Declaration of Helsinki, after supervision and specific approval by the Research Ethics Committee of Federal University of Santa Maria (CEP UFSM—Conselho de Ética em Pesquisa da Universidade Federal de Santa Maria—Santa Maria, RS, Brazil, number 0089.0.243.000-07). All subjects were properly instructed and provided a personal written informed consent to the experimental protocol before participating in the study.
Cell Culture and Treatments
Fresh PBMCs were isolated from blood of individual donors and subsequently used in the experiments. In order to obtain cells for all assays, 6 different subjects were selected for the study, and more than 1 collection was carried out in the same donor at different days of sampling. Afterwards, the fresh isolated cells were prepared using the blood obtained from individual donors (the samples of different donors were not pooled). Briefly, PBMCs were isolated from EDTA-treated peripheral blood by a density gradient centrifugation according to the standard technique of Ficoll-Paque (1.077), following previously published procedures (Posel et al., 2012). Resting cells were cultured for 24 or 72 h at concentration of 1 × 106/ml in RPMI-1640 medium supplemented with 10% FBS, penicillin (100 IU) and streptomycin (100 µg/ml). During the treatments, cells were maintained at 37 °C in humidified atmosphere in the presence of 5% CO2. Before, cells were counted with a Neubauer chamber and plated into a culture medium containing 10, 25, 50, 100, and 200 μM of AZT and Se-derivatives. Control cells were incubated only with medium. A DMSO control group (0.5%) was included as negative control (solvent of the compounds). The lowest concentration of AZT used in the assays reflects the steady-state plasma peak concentration observed in patients under antiretroviral regimen (Matteucci, 2009). Cells were monitored periodically for mycoplasma contamination. The quality of resting PBMCs isolation was assessed to determine the percentages of contaminating granulocytes and red blood cells (RBCs). On average, PBMCs isolation by Ficoll gradient contained insignificant numbers of granulocytes and RBCs. The percentages of CD3+ T cells, CD19+ B cells, and CD14+ monocytes within the PBMCs populations were not determined.
In order to compare the effects of AZT and Se-derivatives on proliferating and non-proliferating cells, we also performed some key experiments with PBMCs stimulated with the mitogen phytohemaglutinin (PHA). Fresh PBMCs were placed in a medium supplemented with PHA (5 µg/ml) and in the log-phase of growth, they were exposed to AZT and Se-derivatives. PHA-stimulated PBMCs were treated with the different compounds for 72 h under the same conditions cited above for resting cells. Cell viability, apoptosis rate, reactive species levels, cell cycle, and BAX/Bcl-2mRNA expression were the specific parameters evaluated in stimulated PBMCs.
Cell Viability Measurement
Cell viability was evaluated by 3 different protocols: tetrazolium dye colorimetric assay (MTT), PI staining and LDH activity determination. For each test, 200 μl of the cell suspension (2 × 105 cells/well) was incubated in 96-well plates containing AZT and Se-derivatives at final concentrations of 10, 25, 50, 100, and 200 µM for 24 and 72 h.
MTT assay
MTT assay was based on the enzymatic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to form formazan crystal by cellular dehydrogenase enzymes in metabolically active cells (Babot et al., 2005). Briefly, 20 μl of MTT (dissolved in PBS 1%) was added in each sample and incubated for 2 h at 37 °C in 5% CO2 atmosphere. Cells were incubated in the dark to avoid unexpected phototoxicity. Subsequently, plates were centrifuged at 500× g for 10 min and the supernatant was removed by aspiration. Formazan crystals were dissolved with 200 μl DMSO and the spectrophotometric absorbance of solution was recorded at 570 nm using a microplate reader. Cell viability was assessed as the percentage of inhibition in relation to the control. Absorbance of control cells was assumed as 100%.
PI staining
PI staining was measured by flow cytometry analysis. This dye is able to enter into PBMCs with compromised cell membranes, binding to the double-stranded DNA/RNA. Cultured cells were collected from microplates and washed twice in PBS buffer before staining with 3 µM of PI for 30 min. Afterwards, cell samples were immediately analyzed by flow cytometry (BD Accuri). Fluorescence intensity was collected in the FL-3 channel (630/30 nm). One hundred thousand events were acquired for each sample and the results were expressed as % of stained cells.
LDH activity
The release of cytosolic enzyme LDH in the culture medium from cells was used as parameter of plasma membrane damage. The nicotinamide adenine dinucleotide (NADH) produced by enzyme after oxidation of lactate to pyruvate was colorimetrically quantified by standard spectroscopy following the protocol recommended by the manufacturer. The results were expressed as Units of LDH per liter of sample (U/L).
Determination of Reactive Species (RS)
RS levels in PBMCs were determined by established assay of dihydrorhodamine-123 (DHR-123) staining, following Royall and Ischiropoulos (1993). DHR-123 readily diffuses across cell membranes and is efficiently oxidized by RS to form the fluorescent cationic and lipophilic probe rhodamine 123 (Rho-123) (Yazdani, 2015). Briefly, isolated PBMCs were seeded at 2 × 105 cells per well into 96-well plates with AZT and Se-derivatives at final concentrations of 10, 25, 50, 100, and 200 µM for 24 and 72 h. Afterwards, cells were incubated with DHR-123 in the dark for 30 min. Then, cells were washed with PBS and immediately subjected to flow cytometry analysis (BD accuri). Green fluorescence intensity was collected in the FL-1 channel (530/30 nm, standard FITC filter set), and a fluorescence histogram was plotted for each sample. Rho-123 fluorescence intensity was proportional to the concentration of RS generated in PBMCs. A life-gate based on forward scatter (FSC) and side scatter (SSC) parameters was made to analyze only PBMCs and exclude cell debris and remaining granulocytes. One hundred thousand events were collected for each sample.
Cell Size and Granularity
Digital signals of FSC and SSC were also recorded for further analysis of cell size and granularity, respectively, and to estimate the percentage of cells undergoing apoptosis. One hundred thousand events were acquired for each sample.
Apoptosis/Necrosis Analysis
Cell apoptosis and necrosis indexes were detected with Alexa Fluor 488-conjugated annexin V kit, using annexin V to measure one of the earliest features of apoptosis, the plasma membrane phosphatidylserine (PS) translocation from the inner to the outer leaflet. Since this assay is usually combined with PI to concurrently assess membrane integrity, this method is able to distinguish early apoptotic cells from late apoptotic or necrotic ones. The harvested cells were adjusted to the concentration of 1 × 106/ml and then treated with 10, 25, 50, 100, and 200 µM of AZT and Se-derivatives for 72 h. Afterwards, 200 µl of suspension (2 × 105 cells) were stained with PI solution (1.5 µM) and FITC-conjugated annexin V, according to the manufacturer's directions. Signals generated by annexin V and PI were analyzed immediately on flow cytometer using FL1 and FL3 channels. PBMCs stained positively with Annexin V-FITC but remaining impermeable to PI (AV+/PI−) were regarded as early apoptotic cells. PBMCs stained double positively with annexin V and PI (AV+/PI+) were regarded as late apoptotic cells. PBMCs stained positively only with PI were considered as necrotic cells (AV-/PI+). Appropriate fluorescence compensation was manually set for FL1 (annexin) and FL3 channel (PI) to avoid signal overlap. Fifty thousand events were acquired for each sample.
Analysis of Cell Cycle Distribution by PI Staining
Cell cycle analyses were carried out according to William-Faltaos et al. (2006), with minor modifications. PBMCs were seeded at 2 × 105 cells per well into 96-well plates containing AZT and Se-derivatives at final concentrations of 10, 25, 50, 100, and 200 µM for 72 h. Afterwards, cells were harvested, washed with PBS and fixed with 1 ml of cold 70% ethanol for 24 h at −20 °C. Cells were then centrifuged at 500 × g for 10 min and washed once with PBS. Then, cells were resuspended in 200 µl of a labeling solution (1% PBS containing 30 µM PI, 200 µg/ml DNase-free RNase and 0.1% Triton-X) for 30 min in the dark. Subsequently, cell cycle profile was analyzed by flow cytometry using FL2 channel to estimate the percentage of cells in G0/G1, S, and G2/M phases. The percentage of hypodiploid cells to the left of G0/G1peak (sub-G0/G1) was taken as percentage of apoptotic cells. One hundred thousand events were acquired for each sample.
Analysis of mRNA Expression by RT-qPCR (Quantitative Reverse Transcription PCR)
PBMCs were grown for 72 h at a density of 1 × 106 cells/well in a 12-well plate containing AZT and Se-derivatives at final concentrations of 10, 25, 50, 100, and 200 µM for 72 h. A total of 3 × 106 cells were used for RNA extraction in each control and treated group. Following treatments, cells were centrifuged at 500 × g for 10 min and the supernatant was aspirated. The resulting pellet was washed once with PBS and the RNA was extracted using TRIZOL reagent according to the manufacturer’s instructions. The purity and yield of total RNA were determined spectrophotometrically using the Nanodrop-1000 spectrophotometer. The integrity of RNA bands (18 and 28 S) was checked on 1% agarose gel electrophoresis (w/v) stained with ethidium bromide (0.5 μg/ml) and identified by visualizing the bands of expected size under UV transilluminator. Later, cells were treated with DNase I to exclude genomic DNA contamination. According to the manufacturer’s instructions, cDNA was synthesized from 1 µg DNase I-treated RNA using the Bio-Rad Reverse Transcriptase. The cDNA aliquots were then utilized in qPCR reactions. The thermal cycle was carried out using a StepOne Plus real-time PCR system (Applied Biosystems) as follows: DNA polymerase was activated at 95 °C for 3 min, followed by 40 cycles of amplification at 95 °C for 15 s (denaturing step), 60 °C for 15 s (annealing step), and 72 °C for 25 s (extension), followed by a melting curve analysis to control the absence of nonspecific products. Threshold and baselines were manually determined using the StepOneSoftware v2.3 (Applied Biosystems) and the CT (cycle threshold) value for each sample was calculated and recorded using 2−ΔΔCT. The gene-specific primer sequences were based on published sequences in GenBank Overview (http://www.ncbi.nlm.nih.gov/genbank/). The primers were designed using Primer3 program version 0.4.0 (http://frodo.wi.mit.edu/primer3/). The primers used in the present study are listed in Table 1. GPDH was applied as reference gene. The levels of gene expression were expressed as ratio of the standard control group value.
Gene . | Gene bank . | Direction . | Primer Sequence . | Annealing Temperature . | MgCl2 . |
---|---|---|---|---|---|
GAPDH | NM_001256799.2 | Foward | 5′-TTCGTCATGGGTGTGAACC-3′ | 60 °C | 3.0 mM |
Reverse | 5′-AGTTGTCATGGATGACCTTGG-3′ | ||||
BAD | NM_004322.3 | Foward | 5′-TGAACCAGAGTTGTGCTTGTCT-3′ | 59 °C | 3.0 mM |
Reverse | 5′-CCACATCTACCATTCCTTCCTT-3′ | ||||
BAX | NM_001291428.1 | Foward | 5′-TAACATGGAGCTGCAGAGGA-3′ | 58 °C | 3.0 mM |
Reverse | 5′-CAGTTTGCTGGCAAAGTAGAAA-3′ | ||||
Bcl-2 | NM_000633.2 | Foward | 5′-AAAGATCCGAAAGGAATTGGA-3′ | 58 °C | 3.0 mM |
Reverse | 5′-TGTGCTTTGCATTCTTGGAC-3′ | ||||
CASP3 | NM_004346.3 | Foward | 5′-GAACTGGACTGTGGCATTGA-3′ | 58 °C | 3.0 mM |
Reverse | 5′-CCTTTGAATTTCGCCAAGAA-3′ | ||||
CASP9 | NM_001229.4 | Foward | 5′-ACCAGAGATTCGCAAACCAG-3′ | 58 °C | 1.5 mM |
Reverse | 5′-TCACCAAATCCTCCAGAACC-3′ | ||||
NF-κB | NM_001077494.3 | Foward | 5′-CAGCTGGCTGAAGATGTGAA-3′ | 59 °C | 3.0 mM |
Reverse | 5′-GTGTTTTGGAAGGAGCAGGA-3′ | ||||
p53 | NM_000701.7 | Foward | 5′-AAAGTCTAGAGCCACCGTCCA -3′ | 58 °C | 1.5 mM |
Reverse | 5′-CAGTCTGGCTGCCAATCCA -3′ | ||||
IL-2 | NM_000637.3 | Foward | 5′-CTGGAGCATTTACTGCTGGATTT-3′ | 58 °C | 3.0 mM |
Reverse | 5′-TGTTTCAGTTCTGTGGCCTTCT-3′ | ||||
IL-6 | NM_000250.1 | Foward | 5′-ACCCCCAATAAATATAGGACTGGA-3′ | 60 °C | 3.0 mM |
Reverse | 5′-CGAAGGCGCTTGTGGAGA-3′ | ||||
IL-10 | NM_000454.4 | Foward | 5′-CGAGATGCCTTCAGCAGAGT-3′ | 60 °C | 3.0 mM |
Reverse | 5′-GGCAACCCAGGTAACCCTTA-3′ | ||||
TNF-α | NM_000636.3 | Foward | 5′-GTTGTAGCAAACCCTCAAGCTG-3′ | 60 °C | 3.0 mM |
Reverse | 5′-TATCTCTCAGCTCCACGCCA-3′ | ||||
INF-γ | NM_001752.3 | Foward | 5′-CAGGTCATTCAGATGTAGCGGA-3′ | 60 °C | 3.0 mM |
Reverse | 5′-TGTCACTCTCCTCTTTCCAATTCT-3′ |
Gene . | Gene bank . | Direction . | Primer Sequence . | Annealing Temperature . | MgCl2 . |
---|---|---|---|---|---|
GAPDH | NM_001256799.2 | Foward | 5′-TTCGTCATGGGTGTGAACC-3′ | 60 °C | 3.0 mM |
Reverse | 5′-AGTTGTCATGGATGACCTTGG-3′ | ||||
BAD | NM_004322.3 | Foward | 5′-TGAACCAGAGTTGTGCTTGTCT-3′ | 59 °C | 3.0 mM |
Reverse | 5′-CCACATCTACCATTCCTTCCTT-3′ | ||||
BAX | NM_001291428.1 | Foward | 5′-TAACATGGAGCTGCAGAGGA-3′ | 58 °C | 3.0 mM |
Reverse | 5′-CAGTTTGCTGGCAAAGTAGAAA-3′ | ||||
Bcl-2 | NM_000633.2 | Foward | 5′-AAAGATCCGAAAGGAATTGGA-3′ | 58 °C | 3.0 mM |
Reverse | 5′-TGTGCTTTGCATTCTTGGAC-3′ | ||||
CASP3 | NM_004346.3 | Foward | 5′-GAACTGGACTGTGGCATTGA-3′ | 58 °C | 3.0 mM |
Reverse | 5′-CCTTTGAATTTCGCCAAGAA-3′ | ||||
CASP9 | NM_001229.4 | Foward | 5′-ACCAGAGATTCGCAAACCAG-3′ | 58 °C | 1.5 mM |
Reverse | 5′-TCACCAAATCCTCCAGAACC-3′ | ||||
NF-κB | NM_001077494.3 | Foward | 5′-CAGCTGGCTGAAGATGTGAA-3′ | 59 °C | 3.0 mM |
Reverse | 5′-GTGTTTTGGAAGGAGCAGGA-3′ | ||||
p53 | NM_000701.7 | Foward | 5′-AAAGTCTAGAGCCACCGTCCA -3′ | 58 °C | 1.5 mM |
Reverse | 5′-CAGTCTGGCTGCCAATCCA -3′ | ||||
IL-2 | NM_000637.3 | Foward | 5′-CTGGAGCATTTACTGCTGGATTT-3′ | 58 °C | 3.0 mM |
Reverse | 5′-TGTTTCAGTTCTGTGGCCTTCT-3′ | ||||
IL-6 | NM_000250.1 | Foward | 5′-ACCCCCAATAAATATAGGACTGGA-3′ | 60 °C | 3.0 mM |
Reverse | 5′-CGAAGGCGCTTGTGGAGA-3′ | ||||
IL-10 | NM_000454.4 | Foward | 5′-CGAGATGCCTTCAGCAGAGT-3′ | 60 °C | 3.0 mM |
Reverse | 5′-GGCAACCCAGGTAACCCTTA-3′ | ||||
TNF-α | NM_000636.3 | Foward | 5′-GTTGTAGCAAACCCTCAAGCTG-3′ | 60 °C | 3.0 mM |
Reverse | 5′-TATCTCTCAGCTCCACGCCA-3′ | ||||
INF-γ | NM_001752.3 | Foward | 5′-CAGGTCATTCAGATGTAGCGGA-3′ | 60 °C | 3.0 mM |
Reverse | 5′-TGTCACTCTCCTCTTTCCAATTCT-3′ |
The gene-specific primer sequences were based on published sequences in GenBank Overview. Primers were designed using Primer3 program version 0.4.0. The specific annealing temperature and MgCl2 concentration used for amplification were optimized for each primer.
Gene . | Gene bank . | Direction . | Primer Sequence . | Annealing Temperature . | MgCl2 . |
---|---|---|---|---|---|
GAPDH | NM_001256799.2 | Foward | 5′-TTCGTCATGGGTGTGAACC-3′ | 60 °C | 3.0 mM |
Reverse | 5′-AGTTGTCATGGATGACCTTGG-3′ | ||||
BAD | NM_004322.3 | Foward | 5′-TGAACCAGAGTTGTGCTTGTCT-3′ | 59 °C | 3.0 mM |
Reverse | 5′-CCACATCTACCATTCCTTCCTT-3′ | ||||
BAX | NM_001291428.1 | Foward | 5′-TAACATGGAGCTGCAGAGGA-3′ | 58 °C | 3.0 mM |
Reverse | 5′-CAGTTTGCTGGCAAAGTAGAAA-3′ | ||||
Bcl-2 | NM_000633.2 | Foward | 5′-AAAGATCCGAAAGGAATTGGA-3′ | 58 °C | 3.0 mM |
Reverse | 5′-TGTGCTTTGCATTCTTGGAC-3′ | ||||
CASP3 | NM_004346.3 | Foward | 5′-GAACTGGACTGTGGCATTGA-3′ | 58 °C | 3.0 mM |
Reverse | 5′-CCTTTGAATTTCGCCAAGAA-3′ | ||||
CASP9 | NM_001229.4 | Foward | 5′-ACCAGAGATTCGCAAACCAG-3′ | 58 °C | 1.5 mM |
Reverse | 5′-TCACCAAATCCTCCAGAACC-3′ | ||||
NF-κB | NM_001077494.3 | Foward | 5′-CAGCTGGCTGAAGATGTGAA-3′ | 59 °C | 3.0 mM |
Reverse | 5′-GTGTTTTGGAAGGAGCAGGA-3′ | ||||
p53 | NM_000701.7 | Foward | 5′-AAAGTCTAGAGCCACCGTCCA -3′ | 58 °C | 1.5 mM |
Reverse | 5′-CAGTCTGGCTGCCAATCCA -3′ | ||||
IL-2 | NM_000637.3 | Foward | 5′-CTGGAGCATTTACTGCTGGATTT-3′ | 58 °C | 3.0 mM |
Reverse | 5′-TGTTTCAGTTCTGTGGCCTTCT-3′ | ||||
IL-6 | NM_000250.1 | Foward | 5′-ACCCCCAATAAATATAGGACTGGA-3′ | 60 °C | 3.0 mM |
Reverse | 5′-CGAAGGCGCTTGTGGAGA-3′ | ||||
IL-10 | NM_000454.4 | Foward | 5′-CGAGATGCCTTCAGCAGAGT-3′ | 60 °C | 3.0 mM |
Reverse | 5′-GGCAACCCAGGTAACCCTTA-3′ | ||||
TNF-α | NM_000636.3 | Foward | 5′-GTTGTAGCAAACCCTCAAGCTG-3′ | 60 °C | 3.0 mM |
Reverse | 5′-TATCTCTCAGCTCCACGCCA-3′ | ||||
INF-γ | NM_001752.3 | Foward | 5′-CAGGTCATTCAGATGTAGCGGA-3′ | 60 °C | 3.0 mM |
Reverse | 5′-TGTCACTCTCCTCTTTCCAATTCT-3′ |
Gene . | Gene bank . | Direction . | Primer Sequence . | Annealing Temperature . | MgCl2 . |
---|---|---|---|---|---|
GAPDH | NM_001256799.2 | Foward | 5′-TTCGTCATGGGTGTGAACC-3′ | 60 °C | 3.0 mM |
Reverse | 5′-AGTTGTCATGGATGACCTTGG-3′ | ||||
BAD | NM_004322.3 | Foward | 5′-TGAACCAGAGTTGTGCTTGTCT-3′ | 59 °C | 3.0 mM |
Reverse | 5′-CCACATCTACCATTCCTTCCTT-3′ | ||||
BAX | NM_001291428.1 | Foward | 5′-TAACATGGAGCTGCAGAGGA-3′ | 58 °C | 3.0 mM |
Reverse | 5′-CAGTTTGCTGGCAAAGTAGAAA-3′ | ||||
Bcl-2 | NM_000633.2 | Foward | 5′-AAAGATCCGAAAGGAATTGGA-3′ | 58 °C | 3.0 mM |
Reverse | 5′-TGTGCTTTGCATTCTTGGAC-3′ | ||||
CASP3 | NM_004346.3 | Foward | 5′-GAACTGGACTGTGGCATTGA-3′ | 58 °C | 3.0 mM |
Reverse | 5′-CCTTTGAATTTCGCCAAGAA-3′ | ||||
CASP9 | NM_001229.4 | Foward | 5′-ACCAGAGATTCGCAAACCAG-3′ | 58 °C | 1.5 mM |
Reverse | 5′-TCACCAAATCCTCCAGAACC-3′ | ||||
NF-κB | NM_001077494.3 | Foward | 5′-CAGCTGGCTGAAGATGTGAA-3′ | 59 °C | 3.0 mM |
Reverse | 5′-GTGTTTTGGAAGGAGCAGGA-3′ | ||||
p53 | NM_000701.7 | Foward | 5′-AAAGTCTAGAGCCACCGTCCA -3′ | 58 °C | 1.5 mM |
Reverse | 5′-CAGTCTGGCTGCCAATCCA -3′ | ||||
IL-2 | NM_000637.3 | Foward | 5′-CTGGAGCATTTACTGCTGGATTT-3′ | 58 °C | 3.0 mM |
Reverse | 5′-TGTTTCAGTTCTGTGGCCTTCT-3′ | ||||
IL-6 | NM_000250.1 | Foward | 5′-ACCCCCAATAAATATAGGACTGGA-3′ | 60 °C | 3.0 mM |
Reverse | 5′-CGAAGGCGCTTGTGGAGA-3′ | ||||
IL-10 | NM_000454.4 | Foward | 5′-CGAGATGCCTTCAGCAGAGT-3′ | 60 °C | 3.0 mM |
Reverse | 5′-GGCAACCCAGGTAACCCTTA-3′ | ||||
TNF-α | NM_000636.3 | Foward | 5′-GTTGTAGCAAACCCTCAAGCTG-3′ | 60 °C | 3.0 mM |
Reverse | 5′-TATCTCTCAGCTCCACGCCA-3′ | ||||
INF-γ | NM_001752.3 | Foward | 5′-CAGGTCATTCAGATGTAGCGGA-3′ | 60 °C | 3.0 mM |
Reverse | 5′-TGTCACTCTCCTCTTTCCAATTCT-3′ |
The gene-specific primer sequences were based on published sequences in GenBank Overview. Primers were designed using Primer3 program version 0.4.0. The specific annealing temperature and MgCl2 concentration used for amplification were optimized for each primer.
Cytokines Measurement by Cytometric Bead Array
The CBA assay (BD CBA Human Th1/Th2/Th17 Cytokine Kit) provides a method for capturing soluble analytes with known dimensions and fluorescence using flow cytometry. Secreted cytokines levels were quantified according to the protocol recommended by the manufacturer. The harvested cells were adjusted to a concentration of 6 × 105/well and treated with AZT and Se-derivatives at final concentrations of 10, 25, 50, 100, and 200 µM for 72 h. Afterwards, cells were centrifuged at 500 × g for 10 min. The supernatant was aspirated and stored in −80 °C freezer to measure cytokines levels. In brief, 50 μl of PBMCs supernatant or standard sample was used to capture antibody bead reagent. After incubation at room temperature for 3 h in the dark, cells were washed with 1 ml of wash buffer and centrifuged at 200 × g for 5 min. The fluorescence produced by CBA beads was measured on a flow cytometer (BD accuri) and analyzed with its software.
In Vivo Treatment
Animals
Since chalcogenodivudines had relatively low toxicity in vitro, we determined their acute toxicity in mice. Twenty 2-month-old male Swiss albino mice (30–40 g) were used for in vivo experiments. The animals were purchased from our own breeding colony and, then allowed to acclimatize for 2 weeks before the beginning of the experiments. They were housed in groups of 4 per cages (30 × 20 × 13 cm) on a 12-h light/dark cycle with free access to food (Supra, Brazil) and water. The temperature and relative humidity were controlled at 22 °C and 50%, respectively. All procedures used were performed in accordance with guidelines of the Committee on Care and Use of Experimental Animal Resources and accepted by the local Ethical Committee of the UFSM under the number 4622031115.
Treatment, biochemical, and behavior parameters
Mice were randomly separated into 5 groups (n = 4 animals per group), and subjected to a single subcutaneous injection of 100 µmol/Kg of AZT or Se-derivatives. The dose used was based in acute treatments conducted with other organoselenium compounds such as diphenyl diselenide and ebselen (Meotti et al., 2003; Nogueira et al., 2003). The control group received the vehicle DMSO (1 ml/kg). After administration, mice were observed individually for 72 h for clinical signs of toxicity such as mortality rate, gross changes in behavior, piloerection (hair bruising), diarrhea and increased diuresis. The gross behavioral parameters of animals were recorded using closed circuit cameras. Seventy-two hours after the administration of compounds, the animals were euthanized by decapitation and blood collected by exsanguination. Blood samples were transferred into heparin-anticoagulant vials and immediately centrifuged at 300 × g for 10 min. Plasma was used for measuring aspartate aminotransferase (AST), alanine aminotransferase (ALT), LDH, creatinine, urea, alkaline phosphatase (ALK), and uric acid levels. All samples were analyzed colorimetrically at 340 nm using commercial kits according to the manufacturer's instructions in triplicate (Labtest Diagnóstica S/A; Minas Gerais, Brazil). After blood collection, liver, kidneys, spleen and brain were dissected and weighed.
Statistical Analysis
All parameters were analyzed by One-Way ANOVA followed by Bonferroni test when appropriate, in order to detect significant differences in measured variables among the groups. p < .05 was considered to indicate a statistically significant difference. The graphics were made using the GraphPad Prism (GraphPad Software, San Diego, California, USA).
RESULTS
Cell Viability and Membrane Integrity of AZT and Se-Derivatives Treated-PBMCs
Results from MTT assay show that resting PBMCs treated with 100 and 200 µM of SZ1 displayed loss of viability after 24 h exposure (Figure 2A). The toxicity of SZ1 increased after 72 h exposure and cell viability loss was observed in PBMCs exposed from 10 to 200 µM. In contrast, AZT, SZ2, and SZ3 did not decrease resting PBMCs viability after 24 and 72 h exposure (Figure 2A). The effects induced by AZT and Se-derivatives in PHA-stimulated cells after 72 h exposure tended to be similar to those observed in resting cells (Supplementary Figure 1A). Accordingly, SZ1 derivative decreased the reduction of MTT in all the concentrations tested (10–200 µM), whereas SZ2 decreased MTT reduction only at 200 µM (Supplementary Figure 1A).
All tested concentrations of SZ1 induced a significant increase in PI incorporation into resting PBMCs after both treatment periods, indicating cell membrane integrity impairments. Exposure of resting PBMCs to 10 µM SZ1 increased the PI labeling from 1% (control cells) to more than 7% after 24 h. The exposure to 100 and 200 µM SZ1 increased the PI labeling to >80% (Figure 2B). The toxicity of SZ2 was much lower than that of SZ1 after 72 h exposure. The labeling of vehicle-treated cells (about 5%) increased to 12% (100 µM) and 21% (200 µM) in the presence of SZ2. In contrast, membrane integrity of resting PBMCs treated with AZT and SZ3 was not altered when compared with the control.
In PHA-stimulated cells, the basal PI staining was higher than in resting cells and the treatment of cells with SZ1 (20–200 µM) and SZ2 (200 µM) for 72 h also increased the PI staining.
LDH leaking was increased significantly only in resting PBMCs exposed to 100 and 200 µM SZ1 for 72 h (Figure 2C). LDH leaking from cells exposed to AZT, SZ2 and SZ3 did not differ significantly from control. LDL leaking was also more elevated in PHA-stimulated PBMCs than in resting cells and the treatment with SZ1 for 72 h increased LDH leaking (Supplementary Figure 1C).
RS Levels in AZT and Se-Derivatives Treated- PBMCs
The exposure of resting PMBCs to SZ1 increased the oxidation of DHR-123 and the labeling of cells was enhanced about 2 times at all the tested concentrations and sampling times (Figs. 3A and B). RS levels in resting PBMCs exposed to AZT, SZ2 and SZ3 did not differ from control group after 24 and 72 h exposure. In PHA-stimulated PBMCs, SZ1 also increased the levels of RS production after 72 h of exposure (Supplementary Figure 1D).
Morphological Changes in PBMCs After Treatment With AZT and Se-Derivatives
Changes in the scatter parameters (decreased size and increased granularity) precede DNA fragmentation and might therefore be an early indicator of apoptosis (Wesselborg and Kabelitz, 1993). We analyzed the scatter pattern of resting PBMCs exposed to AZT, SZ1, SZ2 and SZ3 by flow cytometry (Figure 4). Resting PBMCs cultured for 72 h in the presence of SZ1concentrations >25 µM exhibited significant increase in granularity, corresponding to 1.50- to 1.75-fold the levels found in control group (Figs. 4A and C). Concomitant with the increased granularity, the size of resting PBMCs decreased approximately 15% in the cells treated with 50, 100, and 200 µM of SZ1 for 72 h (Figs. 4A and B). A decrease in cell size profile was also observed after 24 h of exposure to SZ1. However, AZT, SZ2, and SZ3 did not modify PBMCs size/granularity profile (Figure 4A). Similar pattern of morphological changes tended to occur in PHA-stimulated cells exposed to the SZ1 (a significant decrease in size at the highest concentration and an increase in granularity at 10 and 25 µM). The highest concentration of SZ2 also caused a significant increase in PHA-stimulated cells granularity (Supplementary Figs. 1E–G).
Apoptosis in AZT and Se-Derivatives Treated-PBMCs
To check RS-mediated apoptosis, we used Annexin V-FITC labeling analysis to stain cells after treatment with AZT and Se-derivatives. The treatment of resting PBMCs with SZ1 for 72 h caused late apoptosis/necrosis (Figs. 5A and B). There was approximately 11%, 14%, 40%, 75%, 88%, and 87% of annexin V-FITC/PI-positive cells in the groups treated with vehicle, 10, 25, 50, 100, and 200 µM of SZ1, respectively (Figure 5). Late apoptosis/necrosis rate was also increased in resting cells exposed to 100 and 200 µM SZ2 (24.5% and 34.6% of annexin V-FITC/PI-positive cells, respectively). No significant apoptotic/necrotic cell death was observed in resting PBMCs treated with both AZT and SZ3, when compared with the control (Figs. 5A and B).
In contrast to obtained with resting PBMCs, AZT and all Se-derivatives induced apoptotic events in PHA-stimulated cells treated for 72 h. There was a significant increase of annexin V-FITC positive cells in the groups treated with 100 and 200 µM of AZT and SZ2 when compared with control group (Figure 6). The index of early apoptosis also increased in stimulated PBMCs exposed to SZ1 and SZ3 from 10 to 50 µM, respectively (Figure 6).
Cell Cycle Profile of AZT and Se-Derivatives Treated-PBMCs
Exposure of resting PBMCs to 200 µM SZ1 for 72 h arrested cells in the S phase and decreased the G0/G1 ratio. Data from cell cycle revealed that 63.1%, 12.8%, and 8.8% of these cells were in G1, S, and G2/M phase, respectively (Figure 7). In control group these values were respectively 73%, 8.1%, and 9.6%. Exposure of resting PBMCs to 100 and 200 µM SZ1 resulted in the appearance of a new cell population in the sub-G1, the typical region where apoptotic cells are found (Ormerod, 1998) (Figs. 7A and 7B). The occurrence of a sub-G1 peak is associated with inter-nucleosomal DNA cleavage, which occurs during the late apoptosis stage (Kajstura et al, 2007). The presence of the sub-G1 peak (apoptotic state) was not significantly modified by AZT, SZ2 and SZ3 treated-resting PBMCs when compared with vehicle-treated cells (Figure 7).
In PHA-stimulated cells, exposure to AZT and all Se-derivatives arrested cells in the S-phase of cycle and increased the cell population in the sub-G1 region, effects that in resting cells had been elicited only by SZ1 (Figs. 8A and B). The treatment of PHA-stimulated PBMCs with AZT, SZ1, and SZ2 also caused a mild decrease in G0/G1 ratio. The highest concentration of AZT decreased the number of cells in G2 phase, phenomenon that was not elicited by Se-derivatives (Figs. 8A and B).
mRNA Expression of Apoptotic and Inflammatory Genes in AZT and Se-Derivatives Treated-PBMCs
The influence of AZT and Se-derivatives on the expression of mRNA genes related to apoptosis and inflammation was firstly determined in cells exposed to 100 μM SZ1, which was as effective as 200 μM after 72 h exposure. As this treatment caused a pronounced degradation of RNA bands and loss in total RNA (data not shown), PBMCs cultured in medium containing 100 μM SZ1 were not used to evaluate mRNA levels. Then, RT-qPCR mRNA analyzes were carried out in PBMCs exposed to 10 and 25 µM of SZ1; 10, 25, and 100 µM of SZ2 and 25, 100 µM of AZT and SZ3.
mRNA Expression of Apoptotic/Anti-Apoptotic Responsive Genes
All pro-apoptotic mRNA genes analyzed here (Caspase-3, Caspase-9, BAX, BAD, and p53) were over expressed after exposure of resting cells to 25 µM SZ1 for 72h. The mRNA levels of caspase 3, caspase 9, BAX, and BAD increased more than 2 fold in comparison to the control groups (Figure 9). In contrast, the anti-apoptotic Bcl-2 mRNA gene expression was significantly down regulated in resting PBMCs exposed to 10 µM SZ1 (1.8-fold lower than in control groups). The BAX/Bcl-2 ratio was significantly increased in PBMCs exposed to 10 and 25 µM SZ1 and 100 µM SZ2 (Figure 9). AZT and SZ3 did not induce changes in the relative transcript levels of any apoptosis responsive genes tested (Figure 9).
In PHA-stimulated cells, the mRNA levels of apoptotic factor BAX was overexpressed after treatment with 50 µM of SZ1 (1.5-fold), 50 µM of AZT (1.7-fold) and 10 µM of SZ2 (1.9-fold) (Figure 10A). The relative transcript levels of anti-apoptotic Bcl-2 gene was not affected by AZT and Se-derivatives, but the BAX/Bcl-2 ratio was significantly increased in stimulated PBMCs exposed to AZT, SZ1, and SZ2 (Figs. 10B and C).
mRNA Levels of Inflammation Responsive Genes
Cytokine mRNA expression levels (INF-γ and TNF-α mRNA) were significantly upregulated in resting PBMCs exposed to 25 µM of SZ1 (3.28- and 2.17-fold, respectively). IL-2 and IL-6 mRNA expression levels were increased after exposure of resting PBMCs to 10 µM SZ1 (1.94- and 3.01-fold, respectively). Exposure of cells to SZ2 caused a similar change in the pattern of INF-γ, TNF-α, and IL-6 mRNA expression to those obtained with SZ1. In contrast, 10 µM SZ1 caused a down-regulation on the levels of mRNA that encode IL-10 and NF-κB (2.56- and 2.0-fold, respectively), while the derivative SZ2 increased approximately 1.6-fold NF-κB mRNA levels in relation to the control (Figure 11). AZT and SZ3 treatment increased the expression of INF-γ mRNA and SZ3 decreased the expression of IL-10 and IL-2 (Figure 11).
Cytokines Levels in AZT and Se-Derivatives Treated-PBMCs
To determine the levels of cytokines in the supernatant from cultured resting PBMCs, we utilized the Th1/Th2 Cytometric Bead Array (CBA), which enabled the measurement of secreted cytokines IL-2, IL-10, TNF-α, and INF-γ. The treatment of resting PBMCs with lower concentrations of SZ1 and SZ2 induced T-helper type 1 (Th1) activation that was characterized by hyper-production of pro-inflammatory cytokines TNF-α and INF-γ (Table 2). Likewise, the levels of IL-10 were increased in the supernatant from resting PBMCs exposed to SZ1 and SZ2. AZT and SZ3 treatments did not alter the levels of any cytokines secreted by cells when compared with the control cells (Table 2).
pmol/ml . | Control . | AZT . | SZ1 . | SZ2 . | SZ3 . | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
25 µM . | 100 µM . | 10 µM . | 25 µM . | 100 µM . | 10µM . | 25µM . | 100µM . | 25µM . | 100µM . | ||
IL-2 | 1.63 ± 0.84 | 1.12 ± 1.12 | 1.18 ± 0.44 | 5.02 ± 1.82 | 0.08 ± 0.06 | 0.71 ± 0.54 | 4.66 ± 4.13 | 0.66 ± 0.66 | 0.71 ± 0.48 | 2.52 ± 2.52 | 3.68 ± 1.85 |
IL-10 | 38.94 ± 11.94 | 55.02 ± 2.16 | 53.01 ± 34.17 | 173.8 ± 85.87* | 6.42 ± 4.94 | 0 | 102.8 ± 40.23* | 83.17 ± 20.68 | 88.77 ± 15.68 | 75.57 ± 28.27 | 104.0 ± 22.6 |
TNF-α | 0.61 ± 0.34 | 1.77 ± 1 | 0.44 ± 0.28 | 5.54 ± 2.33* | 3.61 ± 1.89* | 0.64 ± 0.33 | 3.34 ± 2.49* | 0.44 ± 0.25 | 0.1 ± 0.05 | 0.83 ± 0.41 | 0.41 ± 0.28 |
INF-γ | 0.97 ± 0.97 | 1.57 ± 1.34 | 1.63 ± 1.63 | 14.44 ± 3.27* | 0 | 0 | 12.47 ± 12.34* | 0 | 0 | 1.56 ± 1.56 | 1.56 ± 1.56 |
pmol/ml . | Control . | AZT . | SZ1 . | SZ2 . | SZ3 . | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
25 µM . | 100 µM . | 10 µM . | 25 µM . | 100 µM . | 10µM . | 25µM . | 100µM . | 25µM . | 100µM . | ||
IL-2 | 1.63 ± 0.84 | 1.12 ± 1.12 | 1.18 ± 0.44 | 5.02 ± 1.82 | 0.08 ± 0.06 | 0.71 ± 0.54 | 4.66 ± 4.13 | 0.66 ± 0.66 | 0.71 ± 0.48 | 2.52 ± 2.52 | 3.68 ± 1.85 |
IL-10 | 38.94 ± 11.94 | 55.02 ± 2.16 | 53.01 ± 34.17 | 173.8 ± 85.87* | 6.42 ± 4.94 | 0 | 102.8 ± 40.23* | 83.17 ± 20.68 | 88.77 ± 15.68 | 75.57 ± 28.27 | 104.0 ± 22.6 |
TNF-α | 0.61 ± 0.34 | 1.77 ± 1 | 0.44 ± 0.28 | 5.54 ± 2.33* | 3.61 ± 1.89* | 0.64 ± 0.33 | 3.34 ± 2.49* | 0.44 ± 0.25 | 0.1 ± 0.05 | 0.83 ± 0.41 | 0.41 ± 0.28 |
INF-γ | 0.97 ± 0.97 | 1.57 ± 1.34 | 1.63 ± 1.63 | 14.44 ± 3.27* | 0 | 0 | 12.47 ± 12.34* | 0 | 0 | 1.56 ± 1.56 | 1.56 ± 1.56 |
The supernatant of resting PBMCs was collected after 72 h of exposure and used in CBA assay as described in “Materials and Methods” Section. Levels of IL-2, IL-10, TNF-α, and IFN-γ are expressed as ρmol/ml and represent the mean ± SEM of 3 independent experiments.
indicates significant difference from control (p < .05, One-Way ANOVA). Value zero indicates cytokine production below the lower detection.
pmol/ml . | Control . | AZT . | SZ1 . | SZ2 . | SZ3 . | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
25 µM . | 100 µM . | 10 µM . | 25 µM . | 100 µM . | 10µM . | 25µM . | 100µM . | 25µM . | 100µM . | ||
IL-2 | 1.63 ± 0.84 | 1.12 ± 1.12 | 1.18 ± 0.44 | 5.02 ± 1.82 | 0.08 ± 0.06 | 0.71 ± 0.54 | 4.66 ± 4.13 | 0.66 ± 0.66 | 0.71 ± 0.48 | 2.52 ± 2.52 | 3.68 ± 1.85 |
IL-10 | 38.94 ± 11.94 | 55.02 ± 2.16 | 53.01 ± 34.17 | 173.8 ± 85.87* | 6.42 ± 4.94 | 0 | 102.8 ± 40.23* | 83.17 ± 20.68 | 88.77 ± 15.68 | 75.57 ± 28.27 | 104.0 ± 22.6 |
TNF-α | 0.61 ± 0.34 | 1.77 ± 1 | 0.44 ± 0.28 | 5.54 ± 2.33* | 3.61 ± 1.89* | 0.64 ± 0.33 | 3.34 ± 2.49* | 0.44 ± 0.25 | 0.1 ± 0.05 | 0.83 ± 0.41 | 0.41 ± 0.28 |
INF-γ | 0.97 ± 0.97 | 1.57 ± 1.34 | 1.63 ± 1.63 | 14.44 ± 3.27* | 0 | 0 | 12.47 ± 12.34* | 0 | 0 | 1.56 ± 1.56 | 1.56 ± 1.56 |
pmol/ml . | Control . | AZT . | SZ1 . | SZ2 . | SZ3 . | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
25 µM . | 100 µM . | 10 µM . | 25 µM . | 100 µM . | 10µM . | 25µM . | 100µM . | 25µM . | 100µM . | ||
IL-2 | 1.63 ± 0.84 | 1.12 ± 1.12 | 1.18 ± 0.44 | 5.02 ± 1.82 | 0.08 ± 0.06 | 0.71 ± 0.54 | 4.66 ± 4.13 | 0.66 ± 0.66 | 0.71 ± 0.48 | 2.52 ± 2.52 | 3.68 ± 1.85 |
IL-10 | 38.94 ± 11.94 | 55.02 ± 2.16 | 53.01 ± 34.17 | 173.8 ± 85.87* | 6.42 ± 4.94 | 0 | 102.8 ± 40.23* | 83.17 ± 20.68 | 88.77 ± 15.68 | 75.57 ± 28.27 | 104.0 ± 22.6 |
TNF-α | 0.61 ± 0.34 | 1.77 ± 1 | 0.44 ± 0.28 | 5.54 ± 2.33* | 3.61 ± 1.89* | 0.64 ± 0.33 | 3.34 ± 2.49* | 0.44 ± 0.25 | 0.1 ± 0.05 | 0.83 ± 0.41 | 0.41 ± 0.28 |
INF-γ | 0.97 ± 0.97 | 1.57 ± 1.34 | 1.63 ± 1.63 | 14.44 ± 3.27* | 0 | 0 | 12.47 ± 12.34* | 0 | 0 | 1.56 ± 1.56 | 1.56 ± 1.56 |
The supernatant of resting PBMCs was collected after 72 h of exposure and used in CBA assay as described in “Materials and Methods” Section. Levels of IL-2, IL-10, TNF-α, and IFN-γ are expressed as ρmol/ml and represent the mean ± SEM of 3 independent experiments.
indicates significant difference from control (p < .05, One-Way ANOVA). Value zero indicates cytokine production below the lower detection.
Biochemical and Behavioral Parameters in Mice Acutely Treated With AZT and Se-Derivatives
The plasma levels of AST/TGO, ALT/TGP, LDH, ALK, creatinine, urea, and uric acid as well as the body weight of mice that received a single injection of AZT and Se-derivatives did not differ from control after 72 h (Supplementary Table 1). However, there was a significant decrease in the absolute weight of spleen from mice treated with AZT, SZ1, and SZ2. AZT, SZ1, and SZ2 also caused a decrease in the body weight-to-organ weight ratio of spleen (Table 3). Additionally, the body weight-to-organ weight ratios of liver and kidney were increased in mice treated with SZ1 and AZT, respectively (Table 3). These effects were not observed in SZ3-treated mice (Table 3). No gross behavioral alterations and clinical signals such as piloerection, diarrhea, increased diuresis and mortality were observed after the treatment with AZT and Se-derivatives SZ1, SZ2, and SZ3 (data not shown).
. | CTL . | AZT . | SZ1 . | SZ2 . | SZ3 . |
---|---|---|---|---|---|
Absolute weight (g) | |||||
Brain | 0.43 ± 0.04 | 0.43 ± 0.04 | 0.39 ± 0.01 | 0.39 ± 0.009 | 0.41 ± 0.01 |
Liver | 1.50 ± 0.40 | 1.83 ± 0.21 | 1.96 ± 0.62 | 1.78 ± 0.17 | 2.06 ± 0.13 |
Spleen | 0.12 ± 0.03 | 0.08 ± 0.01* | 0.06 ± 0.003* | 0.08 ± 0.01* | 0.12 ± 0.009 |
Kidneys | 0.42 ± 0.08 | 0.39 ± 0.03 | 0.38 ± 0.05 | 0.41 ± 0.05 | 0.46 ± 0.005 |
Body-to-organ weight (g) | |||||
Brain | 1.38 ± 0.20 | 1.28 ± 0.18 | 1.37 ± 0.10 | 1.34 ± 0.14 | 1.21 ± 0.06 |
Liver | 5.10 ± 0.35 | 5.36 ± 0.53 | 7.02 ± 1.10* | 6.08 ± 0.21 | 5.98 ± 0.49 |
Spleen | 0.41 ± 0.03 | 0.26 ± 0.04* | 0.26 ± 0.04* | 0.29 ± 0.04* | 0.35 ± 0.006 |
Kidneys | 1.40 ± 0.03 | 1.20 ± 0.09* | 1.45 ± 0.20 | 1.39 ± 0.06 | 1.34 ± 0.09 |
. | CTL . | AZT . | SZ1 . | SZ2 . | SZ3 . |
---|---|---|---|---|---|
Absolute weight (g) | |||||
Brain | 0.43 ± 0.04 | 0.43 ± 0.04 | 0.39 ± 0.01 | 0.39 ± 0.009 | 0.41 ± 0.01 |
Liver | 1.50 ± 0.40 | 1.83 ± 0.21 | 1.96 ± 0.62 | 1.78 ± 0.17 | 2.06 ± 0.13 |
Spleen | 0.12 ± 0.03 | 0.08 ± 0.01* | 0.06 ± 0.003* | 0.08 ± 0.01* | 0.12 ± 0.009 |
Kidneys | 0.42 ± 0.08 | 0.39 ± 0.03 | 0.38 ± 0.05 | 0.41 ± 0.05 | 0.46 ± 0.005 |
Body-to-organ weight (g) | |||||
Brain | 1.38 ± 0.20 | 1.28 ± 0.18 | 1.37 ± 0.10 | 1.34 ± 0.14 | 1.21 ± 0.06 |
Liver | 5.10 ± 0.35 | 5.36 ± 0.53 | 7.02 ± 1.10* | 6.08 ± 0.21 | 5.98 ± 0.49 |
Spleen | 0.41 ± 0.03 | 0.26 ± 0.04* | 0.26 ± 0.04* | 0.29 ± 0.04* | 0.35 ± 0.006 |
Kidneys | 1.40 ± 0.03 | 1.20 ± 0.09* | 1.45 ± 0.20 | 1.39 ± 0.06 | 1.34 ± 0.09 |
Values are expressed as mean ± SEM of 4 mice/group.
p < .05 indicates statistical difference from control group by One-Way ANOVA followed by Bonferroni test.
. | CTL . | AZT . | SZ1 . | SZ2 . | SZ3 . |
---|---|---|---|---|---|
Absolute weight (g) | |||||
Brain | 0.43 ± 0.04 | 0.43 ± 0.04 | 0.39 ± 0.01 | 0.39 ± 0.009 | 0.41 ± 0.01 |
Liver | 1.50 ± 0.40 | 1.83 ± 0.21 | 1.96 ± 0.62 | 1.78 ± 0.17 | 2.06 ± 0.13 |
Spleen | 0.12 ± 0.03 | 0.08 ± 0.01* | 0.06 ± 0.003* | 0.08 ± 0.01* | 0.12 ± 0.009 |
Kidneys | 0.42 ± 0.08 | 0.39 ± 0.03 | 0.38 ± 0.05 | 0.41 ± 0.05 | 0.46 ± 0.005 |
Body-to-organ weight (g) | |||||
Brain | 1.38 ± 0.20 | 1.28 ± 0.18 | 1.37 ± 0.10 | 1.34 ± 0.14 | 1.21 ± 0.06 |
Liver | 5.10 ± 0.35 | 5.36 ± 0.53 | 7.02 ± 1.10* | 6.08 ± 0.21 | 5.98 ± 0.49 |
Spleen | 0.41 ± 0.03 | 0.26 ± 0.04* | 0.26 ± 0.04* | 0.29 ± 0.04* | 0.35 ± 0.006 |
Kidneys | 1.40 ± 0.03 | 1.20 ± 0.09* | 1.45 ± 0.20 | 1.39 ± 0.06 | 1.34 ± 0.09 |
. | CTL . | AZT . | SZ1 . | SZ2 . | SZ3 . |
---|---|---|---|---|---|
Absolute weight (g) | |||||
Brain | 0.43 ± 0.04 | 0.43 ± 0.04 | 0.39 ± 0.01 | 0.39 ± 0.009 | 0.41 ± 0.01 |
Liver | 1.50 ± 0.40 | 1.83 ± 0.21 | 1.96 ± 0.62 | 1.78 ± 0.17 | 2.06 ± 0.13 |
Spleen | 0.12 ± 0.03 | 0.08 ± 0.01* | 0.06 ± 0.003* | 0.08 ± 0.01* | 0.12 ± 0.009 |
Kidneys | 0.42 ± 0.08 | 0.39 ± 0.03 | 0.38 ± 0.05 | 0.41 ± 0.05 | 0.46 ± 0.005 |
Body-to-organ weight (g) | |||||
Brain | 1.38 ± 0.20 | 1.28 ± 0.18 | 1.37 ± 0.10 | 1.34 ± 0.14 | 1.21 ± 0.06 |
Liver | 5.10 ± 0.35 | 5.36 ± 0.53 | 7.02 ± 1.10* | 6.08 ± 0.21 | 5.98 ± 0.49 |
Spleen | 0.41 ± 0.03 | 0.26 ± 0.04* | 0.26 ± 0.04* | 0.29 ± 0.04* | 0.35 ± 0.006 |
Kidneys | 1.40 ± 0.03 | 1.20 ± 0.09* | 1.45 ± 0.20 | 1.39 ± 0.06 | 1.34 ± 0.09 |
Values are expressed as mean ± SEM of 4 mice/group.
p < .05 indicates statistical difference from control group by One-Way ANOVA followed by Bonferroni test.
DISCUSSION
The AZT-derivatives containing selenium moieties SZ1, SZ2, and SZ3 exhibited potent antitumoral activity in human bladder carcinoma cells in a previous study; however few analyses have been done on healthy cells. In an attempt to estimate the safety of AZT-derivatives toward healthy cells, we choose cultured PBMCs as target for screening and elucidating the toxicological mechanisms of this new class of compounds. We carried out several experiments in a wide range of concentrations, which vary from 10 µM (the maximum steady-state plasma levels achieved in HIV regimen with AZT) (Matteucci et al., 2009) until the suprapharmacological concentration of 200 µM, using AZT as parental control. As AZT has antiviral activity against HIV-infected quiescent T-cells, we opted to perform cell assays preferably with resting PBMCs. However, given that studies have shown that the cytotoxicity of AZT may differ between proliferating and nonproliferating cells, some key experiments were repeated using PHA-stimulated PBMCs. In view that the AZT-derivatives had relatively low toxicity in vitro, adult mice were treated with a single dose of respective compounds, in order to provide insights about their acute toxicity in vivo.
In general, our findings demonstrated that among AZT-derivatives, only SZ3 did not induce toxic signals in resting PBMCs, exhibiting similar effects to AZT in all parameters evaluated. Contrary, SZ1 followed by SZ2 were toxics, eliciting events that encompassed from redox/immune responses activation until viability loss and cell death in these cells. In contrast, AZT and SZ3 affected the cell cycle in PHA-stimulated PBMCs and these cells were also more sensitive to SZ1 and SZ2. Interestingly, the lower toxicity of SZ3 in relation to the other AZT derivatives was also evidenced after acute in vivo administration in mice.
In addition to the key role in signaling and regulation pathways, it is well known that excessive levels of RS are implicated in cell damage, which can lead to activation of cellular death such as apoptosis (Elmore, 2007; Fulda et al., 2010). In order to test this assumption, we firstly conducted complementary assays using flow cytometry-based protocols to check whether AZT and its Se-derivatives could trigger apoptosis in resting PBMCs. The results from these tests, taken together with MTT, LDH, PI staining and RS production, pointed the remarkable cytotoxic and apoptotic effects mainly of derivative SZ1, followed by SZ2.
Regarding molecular mechanisms for SZ1, we observed a significant overexpression of pro-apoptotic genes, followed by a downregulation of Bcl-2 in resting PBMCs. These cell responses could be related with either NF-κB or p53-regulated pathways since the mRNA expression of these transcript factors was also modulated by SZ1. It is noteworthy that p53 is responsible to suppress cell cycle progression and trigger the initiation of the apoptotic cascade through transcriptional regulation of target genes, such as the members of Bcl-2 family (Kannan et al., 2001; Lane, 1992; Schuler et al., 2000). Otherwise, in addition to activation of different patterns of gene expression involved in the control of the immune and inflammatory responses, NF-κB regulates genes that protect cells from undergoing apoptosis in response to DNA damage or cytokines (Barkett and Gilmore, 1999; Yamamoto and Gaynor, 2001). Importantly, the inhibition of NF-κB activation can reduce cyclin D1 activity, a positive regulator of G1-to-S-phase progression, resulting in delayed/impaired cell cycle (Guttridge et al., 1999; Yamamoto and Gaynor, 2001). Thus, the down-regulation of NF-κB observed in SZ1-exposed PBMCs could render the cells more susceptible to apoptosis through p53-mediated BAX transcription pathway. In conformity with our results, studies have reported that NF-κB inhibition causes apoptosis in B lymphocytes (Arsura et al., 1996; Wu et al., 1996) and that RS like H2O2 may activate p53 (Meyer et al., 1993), which upregulate death proteins as well as downregulate Bcl-2 (Chen and Pervaiz, 2009).
Overall, the effects elicited by SZ1 and SZ2 in PHA-stimulated PBMCs were in the same direction to those observed in resting cells; however, the effects of all derivatives and AZT were stronger than in resting cells. AZT and SZ3 also caused apoptosis and changed the cell cycle in proliferating PBMCs. We thought that the increased apoptotic death in dividing cells treated with AZT and Se-derivatives is, at least in part, associated with their effects in arresting the S phase of cell cycle. In accordance, literature has indicated that AZT can arrest the S and G2/M phases in human cancer cell lines (HepG2, NIH 3T3, and HeLa) (Fang et al., 2009; Fang and Beland, 2009; Olivero et al, 2005).
We emphasize 2 aspects that could be related with the different profile of effects of AZT and derivatives in proliferating cells: (1) Since the majority of the circulating lymphocytes are quiescent, the stimulated cells have higher thymidine turnorver that contribute to their increased sensitivity to the compounds (Gao et al., 1993; Humer et al., 2008; Melana et al., 1998); (2) The higher rate of phosphorylation in proliferating cells increase AZT-triphosphate incorporation into DNA, event that might lead to DNA strand breaks, increasing the cytotoxicity generated by AZT (Enomoto et al., 2011; Fang et al., 2009; Gao et al., 1993).
Immune response is a redox state regulated process whereas the activation of immune cells such as lymphocytes and macrophages can be markedly enhanced by RS (Kesarwani et al., 2013). Of toxicological importance, a number of cytokines are likely to influence the rate of programmed cell death by inducing BAD dephosphorylation, release of cytochrome c, caspases activation, mitochondrial membrane depolarization and release of nitric oxide in a variety of cells (Barbu et al., 2002; Chang et al., 2004; Wall et al., 2003; Wang et al., 2006). In this scenario, our data show that both SZ1 and SZ2 stimulated the release of cytotoxic cytokines and apoptosis in resting PBMCs. It is plausible suppose that the downregulation of Bcl-2 and overregulation of proapoptotic genes, important features of apoptotic signaling found in cells cultured with SZ1, could be modulated by cytokines, as previously reported in Nakagawa and Yamaguchi (2005) and Grunnet et al. (2009). In contrast to SZ1, we did not observe a relationship between RS overproduction and cytokines on SZ2-induced cell death.
As previously mentioned, Souza et al. (2015) evidenced that the AZT-derivatives tested here had significant antitumoral activity against bladder carcinoma 5637 cells by inducing apoptosis via overexpression of BAX, Caspases-3 and 9 and down-regulation of Bcl-2. Interestingly, herein we did not verify apoptotic signals in healthy resting PBMCs treated with AZT and SZ3; but AZT and all Se-derivatives elicited early apoptosis in PHA-stimulated PBMCs. Together, these data show that there is a differential susceptibility between malignant/nonmalignant and proliferating/non-proliferating cells to the compounds.
In general, SZ3 showed considerable safety in a short-term treatment, exhibiting poor toxicity both in vitro and in vivo, encouraging the investigation of its biological effect as anti-tumor agent in chronic in vivo models. Another advantageous aspect of AZT analogs is the Se per se. This element is widely known to be present in selenoproteins and selenoenzymes that play an important role as antioxidant for cells (Cardoso et al., 2015; Steinbrenner and Sies, 2009). Therefore, derivatives containing Se could optimize AZT action by adding an antioxidant aspect, which is not found in the parental nucleoside. Consequently, Se-containing AZT derivatives could afford protection against oxidative damage caused by chronic diseases commonly treated with this nucleoside. In this sense, we emphasize the findings showing that Se levels are decreased in HIV positive subjects and Se supplementation has been shown to promote health benefits in HIV infection (Beck et al., 1990; Constans et al., 1995; Hori et al., 1997; Hurwitz et al., 2007).
In addition to the antioxidant potential, the substitution of hydrophilic functional group OH from AZT molecule by phenylseleno groups conferred increased lipophilicity to the derivatives. In this regard, some studies have been published pointing out that the introduction of an additional ring to the pyrimidine core of nucleosides tends to improve the biological properties for these molecules by increasing their lipophilic characteristics and bioavailability (Abdel-Latif, 2007; Aly et al., 2012; Mohamed et al., 2010).
CONCLUSION
This study provided new findings concerning the injurious aspects of 3 novel structurally modified AZT derivatives against healthy immune cells in different stages of division. We highlighted the SZ3 molecule as the most promising candidate for further biological investigations with a relevant anticancer property and low toxicity against quiescent and stimulated healthy cells and after acute in vivo administration in mice. Unfortunately, we did not test yet the likely antiviral action of SZ3 during in vitro HIV infection. However, the antitumor selective activity observed for SZ3 makes it an interesting candidate to test for HIV infection blocking. Moreover, SZ3 contains a Se moiety, encouraging additional studies on oxidative and inflammatory processes. Last, complementary in vivo experiments are needed to confirm and clarify SZ3 safety.
SUPPLEMENTARY DATA
Supplementary data are available at Toxicological Sciences online.
FUNDING
The financial support by FAPERGS/CNPq/PRONEX n° 16/2551-0000 499-4, Pronem, CAPES and CNPq is gratefully acknowledged. J.B.T.R, D.B.R.L and N.V.B are the recipients of CNPq fellowships.
REFERENCES
CONITEC: National Committee for Health Technology Incorporation. Brazilian Ministry of Health. Report n° 50. September 29, 2015.
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