Neutral Red Incorporation Research Articles

VIABILITY OF VERO E6 CELLS EXPOSED TO THE Bothrops leucurus SNAKE CRUDE VENOM

Primary and continuous cell culture systems are a good in vitro alternative for the quantitative assessment of snake venom toxicity. There are still few studies evaluating the cellular response against Bothrops leucurus venom (BlV). This study aimed to evaluate the cytotoxic effect of crude BlV on culture of VERO E6 cells. Neutral Red incorporation (NR) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) tests were performed for viable cells, and Trypan Blue (TB) dye exclusion test was performed for non-viable cells. The differences between the groups were evaluated by ANOVA-One-Way and Dunnet through the program Graph Pad 5.0 with significance of 5%. The 50% cytotoxic concentration (CC50) of BlV was 7.86 μg/mL. There was a trend of dose-dependent reduction on cell viability in all assays evaluated. However, MTT and NR tests were more sensitive for the evaluation of BlV cytotoxicity on the VERO E6 cell line.

The Main Metabolites of Fluorouracil + Adriamycin + Cyclophosphamide (FAC) Are Not Major Contributors to FAC Toxicity in H9c2 Cardiac Differentiated Cells.

In the clinical practice, the combination of 5-fluorouracil (5-FU) + Adriamycin (also known as doxorubicin, DOX) + cyclophosphamide (CYA) (known as FAC) is used to treat breast cancer. The FAC therapy, however, carries some serious risks, namely potential cardiotoxic effects, although the mechanisms are still unclear. In the present study, the role of the main metabolites regarding FAC-induced cardiotoxicity was assessed at clinical relevant concentrations. Seven-day differentiated H9c2 cells were exposed for 48 h to the main metabolites of FAC, namely the metabolite of 5-FU, α-fluoro-β-alanine (FBAL, 50 or 100 μM), of DOX, doxorubicinol (DOXOL, 0.2 or 1 μM), and of CYA, acrolein (ACRO, 1 or 10 μM), as well as to their combination. The parent drugs (5-FU 50 μM, DOX 1 μM, and CYA 50 μM) were also tested isolated or in combination with the metabolites. Putative cytotoxicity was evaluated through phase contrast microscopy, Hoechst staining, membrane mitochondrial potential, and by two cytotoxicity assays: the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and the neutral red (NR) lysosomal incorporation. The metabolite DOXOL was more toxic than FBAL and ACRO in the MTT and NR assays. When in combination, neither FBAL nor ACRO increased DOXOL-induced cytotoxicity. No nuclear condensation was observed for any of the tested combinations; however, a significant mitochondrial potential depolarization after FBAL 100 μM + DOXOL 1 μM + ACRO 10 μM or FBAL 100 μM + DOXOL 1 μM exposure was seen at 48 h. When tested alone DOX 1 μM was more cytotoxic than all the parent drugs and metabolites in both the cytotoxicity assays performed. These results demonstrated that DOXOL was the most toxic of all the metabolites tested; nonetheless, the metabolites do not seem to be the major contributors to FAC-induced cardiotoxicity in this cardiac model.

Petrochemical residue-derived silica-supported titania-magnesium catalysts for the photocatalytic degradation of imidazolium ionic liquids in water

The ionic liquids 1-n-butyl-3-methylimidazolium chloride (C4MImCl), 1-n-butyl-3-methylimidazolium hexafluorophosphate (C4MImPF6), 1-n-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (C4MImNTf2), 1-n-butyl-2,3-dimethylimidazolium chloride (C4MMImCl) and 1-n-butyl-2,3-dimethylimidazolium bis(trifluoromethylsulfonyl)imide (C4MMImNTf2) were degraded under UV and visible radiation in the presence of a silica-supported photocatalyst (SiTi), which was derived from a petrochemical residual catalyst slurry rich in titanium and magnesium. These ionic liquid advanced oxidation processes were studied by UV spectroscopy and electrospray ionization mass spectrometry. After 120 min of irradiation, 50.2, 48.7, 45.3, 40.7 and 35.3% of C4MImCl, C4MImPF6, C4MImNTf2, C4MMImCl and C4MMImNTf2 were degraded under UV and 45.4, 43.6, 40.2, 36.8 and 32.7% of C4MImCl, C4MImPF6, C4MImNTf2, C4MMImCl and C4MMImNTf2 were degraded under visible light, respectively. The commercial titania P25 catalyst degraded 66.3 and 50.2% of C4MImCl under UV and visible radiation, respectively. Regarding the stability of photocatalyst SiTi, it was reused five times with only minor loss of catalytic activity. Doping of SiTi with 25 wt% Mg2+/SiO2 resulted in improved degradation values of 64.6% (UV irradiation) and 65.3% (visible irradiation) of C4MImCl. Cytotoxicity assays (MTT reduction and Neutral Red incorporation) on NIH/3T3 cells after 24 h of irradiation of C4MImCl indicated the absence of in vitro cytotoxicity. Altogether, this study opens new opportunities for the management of petrochemical waste, providing access to effective and stable heterogeneous photocatalysts for the degradation of imidazolium ionic liquids.

CYTOTOXICITY OF STRAWBERRY EXTRACT ON ORAL CANCER CELL LINE

Objective: Berries including strawberry may have beneficial effects against oxidative stress-mediated diseases such as cancer. The purpose of the present study was to investigate the cytotoxic effects of strawberry extract on oral cancer cell line.Methods: Strawberry methanolic extract (SBE) was prepared, and cytotoxic activity of different concentration of SBE on KB cell lines was determined by (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and neutral red dye incorporation test.Results: Strawberry extract exhibits cytotoxic activity over the oral cancer cell lines. On administration of about 100 μg/ml of strawberry extract, about 50% of cell viability could be observed and assessed from the cell lines.Conclusion: Strawberries have a cytotoxic effect on oral cancer cell line due to the presence of anticancer constituents in the berries. These berries can be used as a natural medicine for cancer sufferers.

Effect of neutral red incorporation on Al-doped ZnO thin films and its bio-electrochemical interaction with NAD+/NADP+ dependent enzymes

A new approach to deposition of electroactive ZnO thin films have been carried out, by one-pot chemical bath deposition with Al dopant and incorporation of neutral red as organic mediator. The morphological, structural and functional characterization of the neutral red incorporated, Al-doped ZnO (NR-AZO) film was carried out using electron microscopy, FTIR, XRD and EIS respectively. The incorporated neutral red was found to induce strain in the crystal of AZO proportional to the concentration used in depositing solution which further affected the charge transfer resistance of the films in solution. One mM neutral red was found to be the optimum concentration for both conductivity and response to NADH/NADPH. The response of the films was further validated by immobilizing NAD+ dependent alcohol dehydrogenase (ADH) and NADP+ dependent glucose dehydrogenase (GDH) independently. The ADH/NR-AZO showed a sensitivity of 3.2 μA cm−2 mM−1 with a LoD of 1.7 μM of ethanol in the range 5.6 μM–7 mM, whereas GDH/NR-AZO showed a sensitivity of 4.33 μA cm−2 mM−1 with a LoD of 27 μM of glucose in the range 90 μM–4 mM. This method serves as a simple alternative to immobilize the organic redox dyes into the inorganic thin films in a single step making it electroactive towards specific biomolecules.

Mitoxantrone is More Toxic than Doxorubicin in SH-SY5Y Human Cells: A 'Chemobrain' In Vitro Study.

The potential neurotoxic effects of anticancer drugs, like doxorubicin (DOX) and mitoxantrone (MTX; also used in multiple sclerosis), are presently important reasons for concern, following epidemiological data indicating that cancer survivors submitted to chemotherapy may suffer cognitive deficits. We evaluated the in vitro neurotoxicity of two commonly used chemotherapeutic drugs, DOX and MTX, and study their underlying mechanisms in the SH-SY5Y human neuronal cell model. Undifferentiated human SH-SY5Y cells were exposed to DOX or MTX (0.13, 0.2 and 0.5 μM) for 48 h and two cytotoxicity assays were performed, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction and the neutral red (NR) incorporation assays. Phase contrast microphotographs, Hoechst, and acridine orange/ethidium bromide stains were performed. Mitochondrial membrane potential was also assessed. Moreover, putative protective drugs, namely the antioxidants N-acetyl-l-cysteine (NAC; 1 mM) and 100 μM tiron, the inhibitor of caspase-3/7, Ac-DEVD-CHO (100 μM), and a protein synthesis inhibitor, cycloheximide (CHX; 10 nM), were tested to prevent DOX- or MTX-induced toxicity. The MTT reduction assay was also done in differentiated SH-SY5Y cells following exposure to 0.2 μM DOX or MTX. MTX was more toxic than DOX in both cytotoxicity assays and according to the morphological analyses. MTX also evoked a higher number of apoptotic nuclei than DOX. Both drugs, at the 0.13 μM concentration, caused mitochondrial membrane potential depolarization after a 48-h exposure. Regarding the putative neuroprotectors, 1 mM NAC was not able to prevent the cytotoxicity caused by either drug. Notwithstanding, 100 μM tiron was capable of partially reverting MTX-induced cytotoxicity in the NR uptake assay. One hundred μM Ac-DEVD-CHO and 10 nM cycloheximide (CHX) also partially prevented the toxicity induced by DOX in the NR uptake assay. MTX was more toxic than DOX in differentiated SH-SY5Y cells, while MTX had similar toxicity in differentiated and undifferentiated SH-SY5Y cells. In fact, MTX was the most neurotoxic drug tested and the mechanisms involved seem dissimilar among drugs. Thus, its toxicity mechanisms need to be further investigated as to determine the putative neurotoxicity for multiple sclerosis and cancer patients.

Cytotoxicity potential of flaxseed oil on oral cancer cell lines

Flax seed, one of the oldest crops is a potential dietary supplement for healthy lifestyle. It is a very nutritious food that is rich in omega 3 fatty acids than any other plant source. It contains lignans which is a potential component in reducing tumorous growth effectively. To prove the cytotoxicity of flax seed oil on oral cancer cell lines, this study was conducted on KB cells, oral cancer cell lines. Flax seed oil is the main material for study and certain salts and chemicals are used for the cell viability assays. The cell viability assays used was the MTT assay and neutral red incorporation assay. The cell viability assays are done and the results showed the cytotoxicity of flax seed oil on the oral cancer cell lines. Hence, it can be used as a biomedicine against cancer which can reduce the adverse effects of cancer treatment and prove to be a life saving drug for oral cancer suffering people which can help many in the future.

Evaluation of the antimicrobial activity and cytotoxic effect of hydroxyapatite containing Brazilian propolis

The aim of this work was to produce hydroxyapatite powder (HA) containing the dry extract of green and red propolis, and to evaluate the possible bactericidal activity of these materials over a short period of time through a fast release system. The ethanolic extracts of green and red propolis (EEP) were incorporated into the material by spray drying. After release tests, powders containing dry EEP were characterized regarding the content of total phenolics and flavonoids. Material characterization was undertaken by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The antimicrobial activity was evaluated by plate colony counting, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against Staphylococcus aureus (S. aureus). The cytotoxicity of the materials was determined by the neutral red incorporation method. The materials showed apparently spherical morphology, indicating a decrease in the degree of agglomeration with the addition of propolis. Characteristic HA and propolis functional groups were observed in the FTIR. The materials showed a higher release of phenolics and lower amounts of flavonoids when compared to the EEP, with the higher amounts of flavonoids observed for HA with red propolis. A bactericidal effect was observed for all materials within the interval of 0.5 and 1 h, showing lower inhibitory activity (MIC) and higher bactericidal activity (MBC) when compared to the EEP, with the best results attributed to HA with red propolis. The IC50 values (which is the concentration needed to inhibit cell growth by 50%) obtained from the cytotoxicity assay for HA with the green and red propolis lay between MIC and MCB. Considering these results, it is suggested that HA and propolis may be used as a possible antimicrobial agent, inhibiting the growth of S. aureus, although further in vivo biocompatibility should be investigated before using this material as a medical device with bactericidal potential.

CYTOTOXICITY OF IMIDACLOPRID AND MYCLOBUTANIL PESTICIDES ON THREE CANCER CELL LINES

Three cancer cell lines, i.e. HEpG-2 (human liver carcinoma), MCF-7 (human breast adeno-carcinoma), and PC3 (Prostatic Small Cell Carcinoma) were used to determine the cytotoxic effects of the neonicotinoid insecticide (imidacloprid) and conazole fungicide (myclobutanil). Cytotoxicity was measured by neutral red incorporation (NRI) assay. The lowest concentration of the tested pesticides (0.5 μg/ml) was toxic. With the increase of the concentration up to 80 μg/ml, the Department of plant protection, Faculty of Agric., Ain Shams University, shoubra Elkheima, Cairo, Egypt Department of Genetics, Faculty of Agric., Ain Shams University, Cairo, Egypt damage degree of the cellular form and size was more serious. The midpoint cytotoxicity value, (NRI50) for imidacloprid and myclobutanil for HEpG-2, MCF-7, and PC3 cancer cell lines were 110.5, 67.7 and 67.6 μg/ml and 38.12, 41 and 27.5 μg/ml, respectively. In general, myclobutanil was very toxic in the three cancer cell lines compared with imidacloprid.

Cytotoxic and genotoxic effects of abamectin, chlorfenapyr, and imidacloprid on CHOK1 cells.

The cytotoxicity and genotoxicity of abamectin, chlorfenapyr, and imidacloprid have been evaluated on the Chinese hamster ovary (CHOK1) cells. Neutral red incorporation (NRI), total cellular protein content (TCP), and methyl tetrazolium (MTT) assays were followed to estimate the mid-point cytotoxicity values, NRI50, TCP50, and MTT50, respectively. The effects of the sublethal concentration (NRI25) on glutathione S-transferase (GST), glutathione reductase (GRD), glutathione peroxidase (GPX), and total glutathione content have been evaluated in the presence and absence of reduced glutathione (GSH), vitamin C, and vitamin E. The genotoxicity was evaluated using chromosomal aberrations (CA), micronucleus (MN) formation, and DNA fragmentation techniques in the presence and absence of the metabolic activation system, S9 mix. Abamectin was the most cytotoxic pesticide followed by chlorfenapyr, while imidacloprid was the least cytotoxic one. The glutathione redox cycle components were altered by the tested pesticides in the absence and presence of the tested antioxidants. The results of genotoxicity indicate that abamectin, chlorfenapyr, and imidacloprid have potential genotoxic effects on CHOK1 cells under the experimental conditions.

Nutritional ingredients modulate adipokine secretion and inflammation in human primary adipocytes.

Nutritional factors such as casein hydrolysates and long chain polyunsaturated fatty acids have been proposed to exert beneficial metabolic effects. We aimed to investigate how a casein hydrolysate (eCH) and long chain polyunsaturated fatty acids could affect human primary adipocyte function in vitro. Incubation conditions with the different nutritional factors were validated by assessing cell vitality with lactate dehydrogenase (LDH) release and neutral red incorporation. Intracellular triglyceride content was assessed with Oil Red O staining. The effect of eCH, a non-peptidic amino acid mixture (AA), and long-chain polyunsaturated fatty acids (LC-PUFAs) on adiponectin and leptin secretion was determined by enzyme-linked immunosorbent assay (ELISA). Intracellular adiponectin expression and nuclear factor-κB (NF-κB) activation were analyzed by Western blot, while monocyte chemoattractant protein-1 (MCP-1) release was explored by ELISA. The eCH concentration dependently increased adiponectin secretion in human primary adipocytes through its intrinsic peptide bioactivity, since the non-peptidic mixture, AA, could not mimic eCH’s effects on adiponectin secretion. Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and DHA combined with arachidonic acid (ARA) upregulated adiponectin secretion. However, only DHA and DHA/ARA exerted a potentanti-inflammatory effect reflected by prevention of tumor necrosis factor-α (TNF-α) induced NF-κB activation and MCP-1 secretion in human adipocytes. eCH and DHA alone or in combination with ARA, may hold the key for nutritional programming through their anti-inflammatory action to prevent diseases with low-grade chronic inflammation such as obesity or diabetes.

Cytotoxic effects of acephate, ethoprophos, and monocrotophos in CHO-K1 cells

This work evaluates the cytotoxicity of acephate, ethoprophos, and monocrotophos in the Chinese hamster ovary cell line. The neutral red incorporation (NRI), total cellular protein (TCP) content, and methyl tetrazolium (MTT) assays were followed to estimate the midpoint cytotoxicity values, NRI50, TCP50, and MTT50, respectively. The targeted cells were exposed to the tested pesticides in the presence and absence of fetal calf serum. The effects of the sublethal concentration (NRI25) on glutathione S-transferase, glutathione reductase, glutathione peroxidase, and total glutathione content have been evaluated. In addition, the ameliorative effects of extracellular reduced glutathione (GSH, 1 mM), vitamin C (70 µM), and vitamin E (30 µM) were investigated. The exposure period and protein content in the culture medium affected the cytotoxicity of the tested pesticides. Unexpectedly, acephate showed the least MTT50 value, 56.6 µM. The tested antioxidants caused different and significant effects on the components of the glutathione redox cycle.

Cytotoxicity of benalaxyl, metalaxyl, and triadimefon on Chinese hamster ovary cells

The cytotoxicity of the fungicides benalaxyl, metalaxyl, and triadimefon was evaluated in vitro using the Chinese hamster ovary (CHO-K1) cell line. The midpoint cytotoxicity values of neutral red (NR) incorporation (NRI50), total cellular protein content (TCP50), and the methyl tetrazolium assay (MTT50) were estimated. Benalaxyl was the most cytotoxic fungicide, followed by metalaxyl and triadimefon. Fetal calf serum (10%) caused a reduction in benalaxyl, metalaxyl, and triadimefon cytotoxicity by factors of 1.8, 1.3, and 1.3. The effects of sublethal concentrations (NRI25) of the three fungicides on the glutathione redox cycle components glutathione S-transferase, glutathione reductase, glutathione peroxidase, and total glutathione content were studied. The ameliorative effects of extracellular glutathione (1 mmol L−1), vitamin C (70 µmol L−1), and vitamin E (30 µmol L−1) were also investigated. The three antioxidants led to significant effects on the glutathione redox cycle components.

Biocompatibility of magnesium particles evaluated by in vitro cytotoxicity and genotoxicity assays

Mg is a biodegradable biomaterial which may release particles (MP) to the environment. The possible cyto- and genotoxic effects of MP derived from magnesium powder (mesh 325) were analyzed on rat osteosarcoma UMR106 cells in order simulate the effect of Mg debris. Neutral red (NR) incorporation and acridine orange/ethidium bromide (AO/EB) staining techniques were used as endpoints to analyze the cytotoxic effects at 25-1000 μg/mL concentration range. Genotoxicity was estimated according to micronucleus (MN) formation and the Comet assay (CA). Results showed that MP size changes with time due to corrosion. Changes in lysosomal activity were observed after 24 h only at 1000 μg/mL. Accordingly, AO/EB staining showed a significant decrease in the number of living cells at 500 μg/mL. Transmission electronic microscopy showed MP internalization (60 and 200 nm diameter) in cells after 2-h treatment, whereas no MP was detected after 24 h. A significant dose-dependent increase in MN frequencies was observed at 25-100 μg/mL range (nontoxic range). DNA damage induction was assessed by CA only at 500 μg/mL. Results showed dose-dependent cytotoxic and genotoxic effects of MP on UMR106 cells with different threshold values of MP concentration.

Antiviral activity of some Tunisian medicinal plants against Herpes simplex virus type 1

Fifteen species of Tunisian traditional medicinal plants, belonging to 10 families, were selected for this study. They were Inula viscosa (L.) Ait and Reichardia tingitana (L.) Roth ssp. discolor (Pom.) Batt. (Asteraceae), Mesembryanthemum cristallinum L. and M. nodiflorum L. (Aizoaceae), Arthrocnemum indicum (Willd.) Moq., Atriplex inflata Muell., A. parvifolia Lowe var. ifiniensis (Caball) Maire, and Salicornia fruticosa L. (Chenopodiaceae), Cistus monspeliensis L. (Cistaceae), Juniperus phoenicea L. (Cupressaceae), Erica multiflora L. (Ericaceae), Frankenia pulverulenta L. (Frankeniaceae), Hypericum crispum L. (Hypericaceae), Plantago coronopus L. ssp. eu-coronopus Pilger var. vulgaris G.G. (Plantaginaceae) and Zygophyllum album L. (Zygophyllaceae). Fifty extracts prepared from those plants were screened in order to assay their antiviral activity against Herpes simplex virus type 1 (HSV-1), using neutral red incorporation. Extracts from eight plants among these 15 showed some degree of antiviral activity, while the methanolic extract of E. multiflora was highly active with EC50 of 132.6 µg mL−1. These results corroborate that medicinal plants from Tunisia can be a rich source of potential antiviral compounds.

Chemical composition and antimicrobial activities of the essential oil of (Tunisian) Chrysanthemum trifurcatum (Desf.) Batt. and Trab. flowerheads

Abstract The chemical composition of essential oil isolated from the flowerheads of Chrysanthemum trifurcatum (Desf.) Batt. and Trab. var. macrocephalum (viv.) Beg. (Asteraceae) by hydrodistillation was analysed by GC and GC/MS. A total of 56 compounds representing 97.48% of the oil were identified: limonene (20.89%), γ-terpinene (19.13%), 1,8-cineole (10.64%), β-pinene (8.77%), α-pinene (5.32%), 2-hexenal (4.85%), 4-terpenyl acetate (3.42%), β-myrcene (2.31%), germacrene-B (2.01%), β-spathulenol (1.62%), longifolene (1.39%), α-cadinol (1.39%), α-thujene (1.23%) and β-bourbobene (1.06%) were found to be the major components. Essential oil of flowerheads of C. trifurcatum was tested for antibacterial activity against eight strains, using a microdilution method, and for cytotoxicity and antiviral activity against Herpes simplex virus type 1 using a neutral red incorporation method. The oil showed a great potential of antibacterial effect against Staphylococcus epidermidis and Bacillus subtilis, in the inhibition range of 64–66% and IC50 ranging from 62.5 to 125 μg/ml. On Vero cells, the CC50 of the oil was 735.9 μg/ml and it did not exhibit a significant antiviral activity.

Alterations in the glutathione-redox balance induced by the bio-insecticide Spinosad in CHO-K1 and Vero cells

The biopesticide Spinosad causes a drop in cell viability in two mammalian cellular models CHO-K1 and Vero, using the neutral red incorporation assay as endpoint. Dose–response curves were assessed after 24, 48, and 72 h under different conditions i.e. presence of 10% fetal calf serum or 1% bovine serum albumin or antioxidants. The presence of antioxidant agents, viz. reduced glutathione (1 mM), vitamin C (100 μM), and vitamin E (20 μM) reduced significantly the cytotoxic effect of Spinosad, thus pointing to an oxidative damage mediated by this compound. An increase in malondialdehyde production was observed after 24-h treatment with Spinosad in both Vero and CHO-K1 cells, using fractions of NRU 50 as final concentrations. At concentrations equivalent to its NRU 20, NRU 10 and NRU 5 Spinosad caused significant alterations in the glutathione-redox cycle in the form of significant decrease in total and reduced glutathione, large increase in glutathione peroxidase activity, little induction of glutathione reductase, and significant decline of glutathione S-transferase activities.

Cytotoxic effects of two antimolting insecticides in mammalian CHO-K1 cells

Cytotoxicity of two insect growth regulators, diflubenzuron, a benzoylphenylurea derivative that inhibits the synthesis of new chitin in target organisms, and pyriproxyfen, an insect juvenile hormone analogue, were tested on CHO-K1 cultures, using the neutral red incorporation assay. Both compounds displayed cytotoxic effects that rise with time exposure. The presence of either fetal calf serum or bovine serum albumin diminished significantly the cytotoxicity of both compounds, thus pointing to a strong protein binding. In addition, extensive metabolization with rat liver submitochondrial fraction gave rise to metabolites less toxic than the parent compounds, implying the relative safety of both diflubenzuron and pyriproxyfen in mammals.

Changes in glutathione-redox balance induced by hexachlorocyclohexane and lindane in CHO-K1 cells

1. The basal cytotoxic effect of the organochlorine pesticides hexachlorocyclohexane and lindane on CHO-K1 cultures was assessed at fractions of their lethal doses as determined by the neutral red incorporation (NRI) assay (NRI6.25, NRI12.5 and NRI25). The sulphur-redox cycle enzymes glutathione peroxidase, glutathione reductase and glutathione S-transferase, and total and oxidized glutathione were evaluated at several points during the standard growth curve of the cultures. 2. After incubation with each compound for 24h, both glutathione peroxidase and reductase showed a substantial increase at the lowest exposure doses (NRI6.25) - more significantly for lindane than for 1,2,3,4,5,6-hexachlorocyclohexane (HCH) - and dropped at higher doses of both compounds. The reduced and oxidized glutathione content was greatly diminished at the lower exposures, whereas the total glutathione content was higher at NRI12.5 values. 3. Changes in cell membrane integrity were assessed for a wide range of pesticide concentrations with the lactate dehydrogenase release assay and lipid peroxidation. Membrane leakage and peroxide production were significantly enhanced at concentrations of HCH 50µg ml−1, although this effect was not significant at lindane concentrations <200µg ml−1. 4. Lipid peroxidation increased with exposure to HCH at concentrations as low as NRI6.25, whereas in the case of lindane, this increase was only significant at doses of NRI25 and above.

Evaluation of the cytotoxicity effect of dimethyl sulfoxide (DMSO) on Caco2/TC7 colon tumor cell cultures.

Dimethyl sulfoxide (DMSO) is usually used to solubilize poorly soluble drugs in permeation assays such as that using Caco2 enterocyte-like cells. The objective of this study was to evaluate the toxicity of DMSO on Caco2/TC7 cells and determinate the maximal concentration usable in permeation experiments. Caco2/TC7 cells were cultured for 21 d on 96-well plates for evaluation of toxicity. The determination of lactate dehydrogenase (LDH) release in cell supernatant and the measurement of Neutral Red (NR) uptake are used for cytotoxicity assays. DMSO solutions (0-100%) in Hank's balanced salt solution containing HEPES (25 mM), pH 7.4, were incubated with Caco-2/TC7 cells on 96 well plates. Caco2/TC7 cells were cultured on Transwell-Clear inserts to evaluate the influence of DMSO on the apparent permeability of the paracellular marker mannitol. DMSO 10% did not induce any significant increase in LDH release whereas a significant increase in LDH activity (ANOVA, p<0.05) occurred at a DMSO concentration of 20 to 50%. NR incorporation in viable cells was statistically reduced by 27 to 36% at DMSO concentration of 20% up to 100% (ANOVA, p>0.05). No statistical difference (p<0.05) in apparent mannitol permeability was observed between the control and 10% DMSO groups. In conclusion, at concentrations of up to 10%, DMSO did not produce any significant alteration in apical membrane permeability or on cell-to-cell tight junctional complexes.