24-h Post-incubation Research Articles

Evaluation of potential toxicity of Steriplant©N aerosolization toward human alveolar cells A459 in vitro.

Protection of patients against hospital-acquired infections is of major importance. Disinfection of magnetic resonance imaging suites is, due to their unique properties and environment particularly, difficult to implement. We developed an OPTI-JET CS MD 2ZE aerosolizator for disinfection of a magnetic resonance imaging suite using the electrolyzed oxidizing water biocide Steriplant©N. The disinfection of the magnetic resonance imaging suite with this system reduced from the number of colony formed unit/m3 air by 87% and 96% in 6 and 15min of disinfection, respectively. It is well known that exposure of personnel or patients to aerosols may represent risk to the respiratory system; therefore, the aim of this study was to assess potential cytotoxicity and genotoxicity of Steriplant©N aerosolization toward human alveolar cells A459 in vitro. The A459 cells were exposed to aerosol containing different concentrations (50% and 100% v/v) of Steripalnt©N for 6min in a chamber that had been constructed to simulate the conditions in the magnetic resonance imaging suite. The cytotoxicity was evaluated by measuring iodide uptake, and the genotoxicity was determined by measuring formation of phosphorylated H2AX histones, a marker for deoxyribonucleic acid double-strand breaks, immediately after the aerosolization and after 1, 4, and 24h postincubation. The results demonstrated that aerosolization with Steriplant©N at conditions reflecting aerosolization in a magnetic resonance imaging suite is not cytotoxic and does not exhibit genotoxic potential in vitro.

Green design “bioinspired disassembly-reassembly strategy” applied for improved tumor-targeted anticancer drug delivery

In this study, a simple and green approach ‘bioinspired disassembly-reassembly strategy’ was employed to reconstitute lipoprotein nanoparticles (RLNs) using whole-components of endogenous ones (contained dehydrated human lipids and native apolipoproteins). These RLNs were engineered to mimic the configuration and properties of natural lipoproteins for efficient drug delivery. In testing therapeutic targeting to microtubules, paclitaxel (PTX) was reassembled into RLNs to achieve improved targeted anti-carcinoma treatment and minimize adverse effects, demonstrating ultimately more applicable than HDL-like particles which are based on exogenous lipid sources. We have characterized that apolipoprotein-decoration of PTX-loaded RLNs (RLNs-PTX) led to favoring uniformly dispersed distribution, increasing PTX-encapsulation with a sustained-release pattern, while enhancing biostability during blood circulation. The innate biological RLNs induced efficient intracellular trafficking of cargos in situ via multi-targeting mechanisms, including scavenger receptor class B type I (SR-BI)-mediated direct transmembrane delivery, as well as other lipoprotein-receptors associated endocytic pathways. The resulting anticancer treatment from RLNs-PTX was demonstrated a half-maximal inhibitory concentration of 0.20μg/mL, cell apoptosis of 18.04% 24h post-incubation mainly arresting G2/M cell cycle in vitro, and tumor weight inhibition of 70.51% in vivo. Collectively, green-step assembly-based RLNs provided an efficient strategy for mediating tumor-targeted accumulation of PTX and enhanced anticancer efficacy.

Mechanical loading and the synthesis of 1,25(OH)2D in primary human osteoblasts

The metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D) is synthesized from its precursor 25-hydroxyvitamin D (25(OH)D) by human osteoblasts leading to stimulation of osteoblast differentiation in an autocrine or paracrine way. Osteoblast differentiation is also stimulated by mechanical loading through activation of various responses in bone cells such as nitric oxide signaling. Whether mechanical loading affects osteoblast differentiation through an enhanced synthesis of 1,25(OH)2D by human osteoblasts is still unknown. We hypothesized that mechanical loading stimulates the synthesis of 1,25(OH)2D from 25(OH)D in primary human osteoblasts. Since the responsiveness of bone to mechanical stimuli can be altered by various endocrine factors, we also investigated whether 1,25(OH)2D or 25(OH)D affect the response of primary human osteoblasts to mechanical loading.Primary human osteoblasts were pre-incubated in medium with/without 25(OH)D3 (400nM) or 1,25(OH)2D3 (100nM) for 24h and subjected to mechanical loading by pulsatile fluid flow (PFF). The response of osteoblasts to PFF was quantified by measuring nitric oxide, and by PCR analysis. The effect of PFF on the synthesis of 1,25(OH)2D3 was determined by subjecting osteoblasts to PFF followed by 24h post-incubation in medium with/without 25(OH)D3 (400nM).We showed that 1,25(OH)2D3 reduced the PFF-induced NO response in primary human osteoblasts. 25(OH)D3 did not significantly alter the NO response of primary human osteoblasts to PFF, but 25(OH)D3 increased osteocalcin and RANKL mRNA levels, similar to 1,25(OH)2D3. PFF did not increase 1,25(OH)2D3 amounts in our model, even though PFF did increase CYP27B1 mRNA levels and reduced VDR mRNA levels. CYP24 mRNA levels were not affected by PFF, but were strongly increased by both 25(OH)D3 and 1,25(OH)2D3.In conclusion, 1,25(OH)2D3 may affect the response of primary human osteoblasts to mechanical stimuli, at least with respect to NO production. Mechanical stimuli may affect local vitamin D metabolism in primary human osteoblasts. Our results suggest that 1,25(OH)2D3 and mechanical loading, both stimuli of the differentiation of osteoblasts, interact at the cellular level.

Effects of essential oils supplementation on in vitro and in situ feed digestion in beef cattle

The objective of this study was to investigate the effect of lemongrass oil (LMO) and a mixture of garlic and ginger oil (CEO) on gas production (GP) and feed digestibility using the batch culture and in situ ruminal technique. Four feeds: wheat distillers dried grains with solubles (DDGS), barley grain, grass hay, and a total mixed ration (TMR) were tested with varying essential oil (EO) dosages. The TMR consisted of 350g/kg grass hay, 150g/kg alfalfa hay, 200g/kg barley grain, 100g/kg corn DDGS, 100g/kg wheat DDGS, 50g/kg canola meal, and 50g/kg vitamin and mineral supplement. The in vitro study was a complete randomized design with 2×4 factorial arrangement of two EO (LMO and CEO) combined with four dosages of EO (i.e., 0, 100, 200, and 300mg/kg substrate DM). Digestibilities of DM (DMD) and neutral detergent fibre (NDFD) were measured at 24h and 48h post incubation, while GP was read at 3, 6, 12, 24, 36, and 48h post incubation. In situ ruminal degradability was measured using three ruminally fistulated beef heifers with incubation time of 4, 12, 24 or 48h. There was no interaction on in vitro DMD and NDFD between EO source and its dose. The DMD and NDFD were greater with CEO compared to LMO for wheat DDGS (P<0.01; 48h) and barley grain (P<0.01; 24h), but lower for TMR (P<0.05; 24 or 48h). Increasing the dosage of EO linearly (P<0.01) increased the DMD of wheat DDGS and barley grain at 24h post incubation, and linearly (P<0.01) and quadratically (P<0.05) improved in vitro DMD and NDFD of grass hay and TMR with addition of LMO and CEO at 24 or 48h post incubation. The cumulative GP was overall affected (P<0.01) by both LMO and CEO in quadratic manner after 24, 36 or 48h of incubation. In situ ruminal DMD of wheat DDGS and barley grain were higher (P<0.05) at 4 or 24h of incubation with CEO than with control or LMO which had no differences in DMD. However, the in situ DMD of grass hay and TMR were improved by both LMO and CEO supplementation after 24h (P<0.01) or 48h (P<0.05) post incubation. The dose of 200mg/kg DM was likely cost-effective to improve DMD for both LMO and CEO. The LMO and CEO appeared to be more effective to improve the DMD of fibrous feeds since the in vitro DMD and NDFD of grass hay and TMR were consistently improved at 24h or 48h post incubation. These results suggested that the LMO and CEO used in the present study could be potentially developed as rumen modifier to improve feed digestibility in the rumen.

Transcription of selected immune-related genes in spleen cells of cod, Gadus morhua following incubation with alginic acid and β-glucan

The stimulatory effects of alginic acid and β-glucan on the transcription of selected immune-related genes in the spleen of Atlantic cod, Gadus morhua were evaluated in vitro. Spleen cells were incubated with 100μgml−1 of the test substances and the transcription levels of the selected genes related to antibacterial defense, inflammation, antioxidant defense, and glucose metabolism were determined at 3- and 24-h post-incubation. G-type lysozyme, interleukin-8 (IL-8), Cu, Zn-superoxide dismutase (Cu, Zn-SOD) and glucose transporter-1 and -4 (GLUT-1 and -4) genes were the early responders to the test substances as shown by their increased expression levels at 3-h post-incubation. Interleukin-1β (IL-1β) and GLUTs 2 and 3 showed significantly high levels of expression at 24-h post-incubation with the test substances and these genes could be the late responders. The expression levels of the bactericidal/permeability-increasing protein/lipopolysaccharide-binding protein (BPI-LBP) and catalase were significantly upregulated in cells incubated with β-glucan but not with alginic acid at 24-h after exposure. Most of the genes studied, except Cu, Zn-SOD and GLUT-1 had enhanced transcription levels at 24-h post-incubation with β-glucan. In contrast, the expression of most of the genes in cells incubated with alginic acid, returned to the pre-incubation transcription levels by 24h. Thus both alginic acid and β-glucan are able to differentially enhance the transcription of the selected immune-related genes in the spleen cells. This information could be valuable for further practical studies that aim to test these immunostimulants in fish.

COMPARISON OF EFFECTS INDUCED BY FORMALDEHYDE EXPOSURE ON ALVEOLAR AND BRONCHIAL EPITHELIAL CELLS IN VITRO

Background and Aims Exposure to formaldehyde (FA) at environmental concentrations is suspected to be associated to asthma and respiratory disorders. Nevertheless, the physiopathology of this relation is still unclear and experimental data are needed. In vitro experimentation is complementary to epidemiological studies. However experimental procedure parameters need to be adapted, such as the choice of target cells of respiratory tract. The aim of this study was to compare the inflammatory response of FA exposure at environmental level on alveolar and bronchial epithelial cells. Methods Human alveolar (A549) and bronchial (BEAS-2B) epithelial cells were exposed at the air-liquid interface in a Vitrocell exposure module to FA (50 μg/m3) (or air for control) during 30 min. After 24h post-incubation, cellular viability was assessed using LDH release. Two chemokines (IL-8, MCP-1) were assayed in the apical supernatant by ELISA. In addition, to modulate inflammatory response, pre-sensitization was performed using LPS (5 μg/mL) (to mimic a bacterian activation), TNFα (1 ng/mL) or macrophages conditioned media (to mimic the macrophages/epithelial cells cooperation) during 16h before exposure. Results Whatever the cell type, cell viability was unaffected by air or FA exposure compared to cells not exposed at the air-liquid interface. Without presensitization or after LPS presensitization, FA had not effect on A549 or BEAS-2B, compared to air-exposed cells. FA exposure, after TNFα presensitization, resulted in an inflammatory response, an increase of IL-8 production by A549 cells; after macrophage conditioned media presensitization, an increase of IL-8 production by A549 cells, and decrease of MCP-1 production by BEAS-2B cells was observed. Conclusions After presensitization FA exposure at environmental levels provokes an inflammatory response in epithelial cells of the respiratory tract. Moreover, this response is differently modulated depending on the level of the cells in the respiratory tract.

Influence of alginic acid and fucoidan on the immune responses of head kidney leukocytes in cod

The present study investigated the immunomodulatory activities of alginic acid and fucoidan, both derived from brown seaweeds, on selected cellular immune responses and antibacterial activity of head kidney (HK) leukocytes of cod, Gadus morhua. Primary cultures of HK leukocytes were incubated with either 10 or 100 μg ml⁻¹ of the substances and the effects on respiratory burst, cellular proliferation, acid and alkaline phosphatase activity and cellular myeloperoxidase were measured at 3- and 24-h post-incubation. The antibacterial activity of the supernatants collected from the cell cultures incubated with 100 μg ml⁻¹ of the substances were tested against Vibrio anguillarum and Aeromonas salmonicida. Respiratory burst was significantly elevated in cells incubated with either alginic acid or fucoidan in a dose-dependent manner. Incubation with a higher dose of alginic acid and fucoidan resulted in lower cellular proliferation at 3- and 24-h, respectively. Both acid and alkaline phosphatase activities of HK leukocytes were not significantly modulated, except for a slight elevation of acid phosphatase in cells incubated with 100 μg ml⁻¹ of alginic acid for 24-h. Fucoidan, but not alginic acid significantly increased cellular myeloperoxidase activity at a concentration of 100 μg ml⁻¹. The growth of the bacteria in both the treated and control supernatants was significantly lower than what was observed in the bacterial culture medium. However, the supernatants from the treated cells had significantly higher bacterial growth compared with supernatants of the control cells. Taken together, these results showed that at the tested concentrations, both alginic acid and fucoidan are able to differentially stimulate some cellular immune responses of cod HK leukocytes in vitro and the respiratory burst activity was significantly stimulated by these brown algal derivatives. These substances could be tested as potential immunostimulants in future in vivo studies.

Antimony trichloride induces DNA damage and apoptosis in mammalian cells

Antimony compounds are widely used in various manufacturing and semiconducting industries. Previously, it has been shown that antimony trichloride (SbCl 3) elevates sister chromatid exchange (SCE) rates in V79 cells after a 28-h incubation. However, only limited data on its genotoxic effects are available so far. The present results demonstrate that a 4-h exposure to >50 μM SbCl 3 could induce micronuclei (MN) formation in cultured Chinese hamster ovary (CHO-K1) cells, human bronchial epithelial (BES-6) cells and human fibroblasts (HF). The order of sensitivity to SbCl 3 determined by Sulforhodamine B (SRB)-staining survival assay is HF>BES-6 cells>CHO-K1 cells, with LD 50 values in these cells being ≈40, 80 and 180 μM, respectively. Apoptosis and DNA fragmentation was not found in cells immediately following 4-h SbCl 3 treatment. However, DNA fragmentation was detected in CHO-K1 cells after 4-h SbCl 3 treatment and a 16 h or more post incubation in fresh medium by 1.5% agarose gel electrophoresis. The delayed apoptosis was also observed under microscopic examination in HF, BES-6 and CHO-K1 cells after similar treatment protocol. In addition, an increase in calcium accumulation appeared in CHO-K1 cells and HF immediately after a 4-h SbCl 3 treatment, or after a 24-h post incubation in fresh medium. The present results provide important genotoxic and cytotoxic information characterizing the cellular changes induced by short-term SbCl 3 exposure in rodent and human cells.

The formation and repair of cisplatin-DNA adducts in wild-type and cisplatin-resistant L1210 cells: comparison of immunocytochemical determination with detection in isolated DNA

We have studied the formation and repair of cisplatin-DNA adducts in wild-type mouse leukemia L1210/0 cells and in the sublines L1210/2 and L1210/5, which differ in cisplatin sensitivity. In a colony-formation assay these sublines were 9- and 22-fold more resistant compared to L1210/0, respectively. Cisplatin-induced DNA modification was studied at the cellular level by immunocytochemistry with antiserum NKI-A59 raised against cisplatin-treated DNA. Levels of nuclear staining immediately after a 1-h treatment were similar to those seen after a 24-h post-incubation in drug-free medium. Clear differences in DNA platination were found between the cell lines: immediately after exposure, L1210/2 and L1210/5 showed only 32 and 14%, respectively, of the nuclear staining observed in L1210/0, and 48 and 13% after 24 h. In these experiments, adduct-specific nuclear staining was quantified as the area under the adduct versus concentration curves (AUC). The formation and repair in these cell lines of the bifunctional adducts cis-Pt(NH 3) 2d(pGpG) (Pt-GG), cis-Pt(NH 3) 2d(pApG) (Pt-AG) and cis-Pt(NH 3) 2(dGMP) 2 (G-Pt-G) were studied with an enzyme-linked immunosorbent assay (ELISA). No relation between repair and resistance was observed. The results suggest that differences in induced DNA platination levels, rather than in repair, are responsible––at least in part––for the differences in cisplatin resistance. A mechanism such as an increased tolerance of the resistant cells to platinum-DNA damage may also be involved.

Schedule-dependent modulation of idarubicin cytotoxicity by lonidamine in human lymphoma cell lines

The ability of lonidamine (LND), an energolytic derivative of indazol-carboxylic acid, to modulate the cytotoxic activity of idarubicin (IDA) and doxorubicin (DX) was investigated in two human lymphoma cell lines (H9 and U937). A different pattern of interaction between the drugs was observed as a function of treatment sequence. Specifically, a 24-h postincubation with a non-cytotoxic concentration of LND (75 mu M) increased the activity of a 1-h anthracycline treatment in both cell lines. However, the extent of potentiation for IDA was more than twofold that of DX. No enhancement of anthracycline activity was observed when LND preceded IDA. For comparative purposes, the modulating effect of all-trans-retinoic acid (ATRA) on the cytotoxicity of IDA was evaluated according to different treatment schemes in both lymphoma cell lines. In U937 cells, which undergo monocytic differentiation after exposure to retinoids, a marked increase in LDA activity was obtained following a 48-h postincubation with 1.5 mu M ATRA. No potentiation of anthracycline activity was obtained using the opposite drug sequence. In H9 cells, no significant interference between ATRA and IDA was observed independent of the modality of drug administration. The ability of LND to potentiate IDA activity, and the consideration that LND causes side effects different from those caused by anthracyclines, make this compound an attractive candidate for multidrug combination therapy in hematological neoplasms.

Studies on the relationship between treatment condition and micronucleus induction in V79 cells exposed to silica and glass fibers

Studies have been carried out to determine the relationship between treatment condition and frequencies of micronucleated cells (MNC) and multinucleated cells (MTC) in Chinese hamster lung fibroblasts (V79 cells) exposed to dusts and fibers. Cells were treated with Min-U-Sil 5 silica or Owens Corning AAA-10 glass fibers under three different conditions: 24-h exposure (24E), 24-h exposure followed by 24-h post-incubation in fresh medium (24E–24P), and 48-h exposure (48E). Results showed that the frequency of MNC increased in a concentration-related manner in silica-treated V79 cells only under the condition of 24E–24P. The increase in MNC frequency after 24-h exposure was not concentration-related. No significant increase in MNC was detected in cells sampled after 48-h treatment. The frequencies of MTC in the treatment groups were higher than that in the control group. However, the increase was not statistically significant. Compared with silica, glass fibers were more active for MTC and MNC induction on a mass basis. The highest response was also observed under the condition of 24E–24P. These results indicate that 24-h exposure followed by 24-h post-incubation is a suitable treatment condition for the micronucleus assay on mineral dusts and fibers.

DNA breaks and repair in the mouse leukemia L1210 cells exposed to three different types of interstrand DNA cross-linkers

DNA breaks and repair in mouse leukemia L1210 cells treated with 3 different types of cross-linkers, mitomycin C (MMC), 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) and SN-07 (a macromolecular antibiotic), were studied. Measured in D37 values, MMC gave the highest number of cross-links per lethal ‘hit’ directly after the 1-h treatment in the alkaline elution assay, followed by ACNU and SN-07. A good dose-response increase in induced interstrand DNA cross-linking frequency was observed in cells treated with 2.5–10 μg/ml MMC and with 10–100 μg/ml ACNU for 1 h with and without 24-h post-incubation. After 6-h post-incubation, the highest frequency of cross-linking was observed in cells treated with 2.5 μg/ml MMC and 30 μg/ml ACNU, while cross-link production continued in the cells treated with SN-07 for 12-h post-incubation. No significant increase in DNA breaks was observed in cells treated with MMC throughout 24-h post-incubation. The highest frequency of single-strand DNA breaks in cells treated with ACNU was observed immediately after the treatment and they disappeared after 6-h post-incubation. After 24-h post-incubation, a marked enhancement of the DNA breaks was observed in cells treated with SN-07 and the cells contained double-strand DNA breaks also. RNA synthesis was not affected in the cells treated with 10 μg/ml MMC and slightly inhibited to 70% of control in those treated with 100 μg/ml ACNU, while DNA synthesis in both cells was significantly inhibited after 24-h post-incubation. By contrast, both RNA and DNA synthesis were completely inhibited in cells treated with 8.0 μg/ml SN-07.

Modes of genotoxicity of a macromolecular antibiotic, SN-07, a novel type of interstrand DNA cross-linker

The modes of genotoxicity of a novel macromolecular antitumor antibiotic (SN-07) were examined using both prokaryotic and eukaryotic cells in vitro. The antibiotic induced a frameshift-type reverse mutation in Ames Salmonella typhimurium TA98 at 1.6–400 ng/plate with and without S9 mix. SN-07 also induced chromosomal aberrations and a forward mutation (6-TG r) in Chinese hamster V79 cells after 1 h treatment at 12.5–100 ng/ml without metabolic activation. The alkaline elution technique revealed that SN-07 induced interstrand DNA cross-linking dose-dependently after treatment with 2.5–10 μg/ml for 1 h followed by elution at pH 12.1, but it did not induce the dose-dependent cross-linking after the same treatment followed by elution at pH 12.6. It was also found that SN-07 induced single-strand DNA breaks (pH 12.1) and alkali-labile (pH 12.6) sites after treatment with 0.1–10 μg/ml for 1 h followed by 24-h post-incubation.

Excision repair of DNA base damage in human cells treated with the chemical carcinogen 4-nitroquinoline 1-oxide

Excision repair of DNA damage produced by 4-nitroquinoline 1-oxide (4NQO), a potent chemical carcinogen, was compared in a normal human amnion FL cell line and a xeroderma pigmentosum (XP) cell line unable to repair ultraviolet-induced pyramidine dimers. The main objective of this study was to investigate, by a direct assay of the loss of damage from DNA, whether DNA damage induced by 4NQO in human cells is repaired by the excision-repair system as in Escherichia coli cells. DNA was extracted from FL and XP cells treated with [ 3H]4NQO, hydrolyzed and subjected to radiochromatographic analysis in order to quantitate the initial formation of 4NQO damage and subsequent disappearance during post-incubation. Two peaks of stable 4NQO-quanine adducts appeared on the chromatogram, together with one peak of stable 4NQO-adenine adduct and a peak due to 4-aminoquinoline 1-oxide (4AQO) released from a labile fraction of 4NQO-guanine adduct during hydrolysis. The three kinds of stable 4NQO-purine adduct disappeared from DNA of the FL cells at almost the same rate of about 60% during 24-h post-incubation in culture medium, and 4AQO disappeared somewhat faster. In the XP cells, however, the stable adducts did not disappear from DNA, whereas about 40% of the 4AQO-releasing adduct disappeared from DNA. These findings at the molecular level quantitatively parallel the previous findings at the cellular level that the XP cells are several times as sensitive as normal cells to killing by 4NQO. These results lead to the conclusion that in human cells 4NQO-induced lethality is mainly due to the four kinds of 4NQO-purine adduct as it is in E. coli, and that the adducts are excisable by the same excision-repair mechanism that works on pyramidine dimers.