Assessment of the predictive capacity of the 3T3 Neutral Red Uptake cytotoxicity test method to identify substances not classified for acute oral toxicity (LD50 > 2000 mg/kg): Results of an ECVAM validation study

Assessing chemicals for acute oral toxicity is a standard information requirement of regulatory testing. However, animal testing is now prohibited in the cosmetics sector in Europe, and strongly discouraged for industrial chemicals. Building on the results of a previous international validation study, a follow up study was organised to assess if the 3T3 Neutral Red Uptake cytotoxicity assay could identify substances not requiring classification as acute oral toxicants under the EU regulations. Fifty-six coded industrial chemicals were tested in three laboratories, each using one of the following protocols: the previously validated protocol, an abbreviated version of the protocol and the protocol adapted for an automation platform. Predictions were very similar among the three laboratories. The assay exhibited high sensitivity (92–96%) but relatively low specificity (40–44%). Three chemicals were under predicted. Assuming that most industrial chemicals are not likely to be acutely toxic, this test method could prove a valuable component of an integrated testing strategy, a read-across argument, or weight-of-evidence approach to identify non toxic chemicals (LD₅₀ > 2000 mg/kg). However, it is likely to under predict chemicals acting via specific mechanisms of action not captured by the 3T3 test system, or which first require biotransformation in vivo.     

Anti-thyroid hormone activity of bisphenol A,tetrabromobisphenol A and tetrachlorobisphenol A in an improved reporter gene assay

Previously, we transiently transfected Gal4-fused thyroid hormone receptor (TR) expressing vector and the Gal4 response reporter structure pUAS-tk-luc into HepG2 cell, developed a TR beta-1 mediated reporter gene assay to screen for compounds that acted on the TR signaling pathway. In this study, we improved the test efficiency by changing the transfected cell line into CV-1 cell. Triiodothyronine (T3) and thyroxine (T4) induced higher luciferase expression, with the median effective concentration (EC₅₀) of 1.16 × 10^?8 and 1.36 × 10^?7 M, respectively. Bisphenol A (BPA) was selected as a positive antagonist, exhibiting weak anti-thyroid hormone activity with the median inhibitory concentration (IC₅₀) of 1.64 × 10^?5 M. The assay showed acceptable repeatability to T3 with inter coefficient of variability (CV) of 27.5% and intra CV of 18.6%. Two flame retardants, tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA), were tested for their agonist and antagonist activities. As a result, we found that both of them possessed TR antagonist activity and neither of them showed agonist activity. These results suggested that TBBPA and TCBPA could act as TH antagonists. This assay provided a useful tool for the assessment of environmental chemicals as thyroid system disruptors.     

Potential method to determine irritant potency in vitro – Comparison of two reconstructed epidermal culture models with different barrier competency

Testing chemicals for their ability to cause skin irritation is required for all ingredients of products that come into contact with the skin. Here, we describe a potential method for determining the irritant potency of a chemical in vitro and apply the method to two different reconstructed epidermis models which exhibit different barrier properties. Two surfactants: sodium dodecyl sulphate, Triton X100 and two non-surfactants: 2-4-di-nitro-chloro-benzene, cinnamaldehyde were applied topically in a dose response for 24 h. Biomarkers IL-1alpha, IL-1RA, IL-8 and MTT were assessed and EC₅₀ values determined. Variation in barrier properties between the epidermal models led to variation in the extent of penetration of surfactants, but not of non-surfactants which in turn influenced the EC₅₀ value obtained from surfactants. Furthermore, EC₅₀ values showed that no single biomarker could be classed as the most sensitive biomarker since biomarker sensitivity differed between the different chemicals studied. However, the ranking of the chemicals in order of strong to weak irritant was the same irrespective of the model used and also independent of the biomarker used (Triton X100 > DNCB > SDS > CA). This study describes a method which not only distinguishes an irritant from a non-irritant but which may possibly also be used to determine irritant potency.     

Toxicological assessment of combined lead and cadmium: Acute and sub-chronic toxicity study in rats

The exposure to chemical mixtures is a common and important determinant of toxicity and receives concern for their introduction by inhalation and ingestion. However, few in vivo mixture studies have been conducted to understand the health effects of chemical mixtures compared with single chemicals. In this study, the acute and 90 day sub-chronic toxicity tests of combined Pb and Cd were conducted. In the acute toxicity test, the LD₅₀ value of Pb(NO₃)₂ and CdCl₂ mixture by the oral route was 2696.54 mg/kg by Bliss method. The sub-chronic treatment revealed that the low-dose combination of Pb and Cd exposures can significantly change the physiological and biochemical parameters of the blood of Sprague–Dawley (SD) rats with dose–response relationship and causes microcytic hypochromic anemia and the damages of liver and kidney of the SD rats to various degrees. Histopathological exams showed that the target organs of Pb and Cd were testicle, liver, and kidneys. These observations suggest that Pb and Cd are practically additive-toxic for the SD rats in oral acute toxicity studies. The lowest observed adverse-effect level in rats may be lower than a dose of 29.96 mg/(kg bw day) when administered orally for 90 consecutive days.     

What are the differences between aerobic and anaerobic toxic effects of sulfonamides on Escherichia coli?

Bacteria in the environment face the threat of antibiotics. However, most studies investigating the toxicity and toxicity mechanisms of antibiotics have been conducted on microorganisms in aerobic conditions, while studies examining the anaerobic toxicity and toxicity mechanisms of antibiotics are still limited. In this study, we determined the aerobic and anaerobic toxicities of sulfonamides (SAs) on Escherichia coli. Next, a comparison of the aerobic and anaerobic toxicities indicated that the SAs could be divided into three groups: Group I: log(1/EC_50-anaerobic) > log(1/EC_50-aerobic) (EC_50-anaerobic/EC_50-aerobic, the median effective concentration under anaerobic/aerobic conditions), Group II: log(1/EC_50-anaerobic) ≈ log(1/EC_50-aerobic), and Group III: log(1/EC_50-anaerobic) < log(1/EC_50-aerobic). Furthermore, this division was not based on the reactive oxygen species (ROS) level or the interaction energy (E_binding) value, which represents the affinity between SAs and dihydropteroate synthase (dhps) but rather on the total binding energy. Furthermore, SAs with greatly similar structures were categorized into different groups. This deep insight into the difference between aerobic and anaerobic toxicities will benefit environmental science, and the results of this study will serve as a reference for the risk assessment of chemicals in the environment.     

Adaptation of the validated SkinEthic™ Reconstructed Human Epidermis (RHE) skin corrosion test method to 0.5 cm2 tissue sample

At its 25th meeting the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed that the SkinEthic™ Reconstructed Human Epidermis (RHE) model could be used for distinguishing between corrosive and non-corrosive chemicals within the context of the Organisation Economic for Co-operation and Development (OECD) test guideline, TG 431 (ESAC 16–17 November 2006). Both test method development and multi-center study were performed using 0.63 cm² RHE tissue samples.The purpose of the present study was to demonstrate that similar results could be obtained using the validated test method adapted to 0.5 cm² RHE tissue samples. Test method adaptation only consisted in applying a reduced volume of test substance (40 μL instead of 50 μL for liquids and 20 μL water + 20 mg test substance instead of 25 μL water + 25 mg test substance for solids) and a reduced propan-2-ol extraction volume (1.5 mL instead of 2 mL) during the MTT reduction assay.The test method was assessed with 25 representative test substances of different chemical classes. Among the latter, the 12 OECD reference test substances (6 corrosives and 6 non-corrosives) were evaluated and showed to be similarly classified as in vivo. More generally, the SkinEthic™ skin corrosion test adapted to 0.5 cm² RHE tissue samples fully complies with the OECD performance and reproducibility requirements with the 25 test substances.     

Antiviral evaluation of octadecyloxyethyl esters of (S)-3-hydroxy-2-(phosphonomethoxy)propyl nucleosides against herpesviruses and orthopoxviruses

Our previous studies showed that esterification of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) or 1-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine (HPMPC) with alkoxyalkyl groups such as hexadecyloxypropyl (HDP) or octadecyloxyethyl (ODE) resulted in large increases in antiviral activity and oral bioavailability. The HDP and ODE esters of HPMPA were shown to be active in cells infected with human immunodeficiency virus, type 1 (HIV-1), while HPMPA itself was virtually inactive. To explore this approach in greater detail, we synthesized four new compounds in this series, the ODE esters of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]guanine (HPMPG), 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]thymine (HPMPT), 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2-amino-6-cyclopropylaminopurine (HPMP-cPrDAP) and evaluated their antiviral activity against herpes simplex virus, type 1 (HSV-1), human cytomegalovirus (HCMV), and vaccinia, cowpox and ectromelia. Against HSV-1, subnanomolar EC₅₀ values were observed with ODE–HPMPA and ODE–HPMPC while ODE–HPMPG had intermediate antiviral activity with an EC₅₀ of 40 nM. In HFF cells infected with HCMV, the lowest EC₅₀ values were observed with ODE–HPMPC, 0.9 nM. ODE–HPMPA was highly active with an EC₅₀ of 3 nM, while ODE–HPMPG and ODE–HPMPDAP were also highly active with EC₅₀s of 22 and 77 nM, respectively. Against vaccinia and cowpox viruses, ODE–HPMPG and ODE–HPMPDAP were the most active and selective compounds with EC₅₀ values of 20–60 nM and selectivity index values of 600–3500. ODE–HPMPG was also active against ectromelia virus with an EC₅₀ value of 410 nM and a selectivity index value of 166. ODE–HPMPG and ODE–HPMPDAP are proposed for further preclinical evaluation as possible candidates for treatment of HSV, HCMV or orthopoxvirus diseases.     

Comparative analysis of goitrogenic effects of phenylthiourea and methimazole in zebrafish embryos

Craniofacial malformations, reduced locomotion and induction of genes encoding for enzymes involved in thyroid hormone synthesis were assessed using methimazole and N-phenylthiourea in zebrafish embryos. Gene expression, the most sensitive endpoint (EC_{50_MMI} = 372–765 μM, EC_{50_PTU} = 7.6–8.6 μM), was analysed in wild-type and in a transgenic strain, tg(tg:mCherry), expressing mCherry fluorescence protein under the control of the thyroglobulin gene. Reduction of locomotion and craniofacial malformations were observed at one or two orders of magnitude above concentrations affecting gene expression, respectively. Both effects could be linked to the malformations caused by reduced thyroxin levels. Our results show that due to the presence of the autoregulatory loop of the hypothalamus–pituitary–thyroid axis, various molecular initiating events of thyroid disruption are amenable for the zebrafish embryo. We propose the tg(tg:mCherry) bioassay as a sensitive tool in medium scale screening of goitrogens, given the minimal effort for sample preparation and analysis of gene expression.     

90-Day subchronic toxicity study of sodium molybdate dihydrate in rats

This study investigated the subchronic toxicity of molybdenum (Mo) in Sprague–Dawley rats given sodium molybdate dihydrate in the diet for 90 days at dose levels of 0, 5, 17 or 60 mg Mo/kg_bw/day. The study complied with OECD Test Guideline (TG) 408, with additional examination of estrus cycles and sperm count, motility, and morphology from OECD TG 416. The overall no-observed-adverse-effect level was 17 mg Mo/kg_bw/day, based on effects on body weight, body weight gain, food conversion efficiency and renal histopathology (females only) at 60 mg Mo/kg_bw/day. No treatment-related adverse effects on reproductive organ weights or histopathology, estrus cycles or sperm parameters were observed at any dose level. No adverse effects were observed in the high dose animals after the 60-day recovery period, with the exception that male rats did not fully recover from reduced body weight. Serum blood, liver and kidney samples were analyzed for molybdenum, copper, zinc, manganese, iron, cobalt and selenium; high levels of molybdenum and copper were found in the serum, blood, liver and kidneys of rats treated with 60 mg Mo/kg_bw/day. In conclusion, the LOAEL and NOAEL for molybdenum were determined to be 60 and 17 mg Mo/kg_bw/day, respectively.     

Pesticides and their binary combinations as P-glycoprotein inhibitors in NIH 3T3/MDR1 cells

The purpose of the present study was to assess do selected pesticides as well as their binary combinations act as inhibitors of P-glycoprotein (P-gp) activity of NIH 3T3 mouse fibroblasts stably transfected with human MDR1 gene (NIH 3T3/MDR1). As a result of P-gp inhibition, the increase of intracellular accumulation of a model P-gp substrate fluorescent calcein acetoxymethyl ester was measured. Pesticide and verapamil individual dose–response data were scaled and expressed as percent of maximum effect. Results showed that out of 14 pure pesticides tested, endosulfan, phosalone and propiconazole were nearly as potent as model inhibitor verapamil (EC₅₀ = 1.5 μM), while diazinon showed a lower potency of inhibiting P-gp transport activity (EC₅₀ = 58.4 μM). Concentrations of pesticides that produced the same inhibiting effect (isoboles) were combined binary. Results calculated using the isobole method revealed that diazinon caused synergistic effect in inhibiting P-gp transport activity in all combinations.     

Boar spermatozoa as a biosensor for detecting toxic substances in indoor dust and aerosols

The presence, quantity and origins of potentially toxic airborne substances were searched in moisture damaged indoor environments, where building related ill health symptoms were suspected and reference sites with no health complaints. Boar spermatozoa were used as the toxicity sensor. Indoor aerosols and dusts were collected from kindergartens, schools, offices and residences (n = 25) by electrostatic filtering, vacuuming, wiping from elevated surfaces and from the interior of personal computers. Toxicity was measured from the ethanol or methanol extracts of the dusts and aerosols. EC₅₀ was expressed as the lowest concentration of the airborne substance that inhibited motility of >50% of the exposed sperm cells compared to vehicle control, within 30 min, 1 day or 3–4 days of exposure. Remarkably toxic aerosols (EC₅₀ ⩽6 μg ml⁻¹) were found from 11 sites, all of these were sites with known or suspected for building related ill health. Toxic microbial cultures were obtained from subsamples of the toxic aerosols/dusts. From these cereulide, amylosin, valinomycin and a novel indoor toxin, stephacidin B were identified and toxicities measured. Airborn dispersal of valinomycin from Streptomyces griseus cultures was evaluated using a flow-through chamber. Significant amounts of valinomycin (LC–MS assay) and toxicity (boar sperm motility assay) were carried by air and were after 14 days mainly recovered from the interior surfaces of the flow chamber.     

Contractile effects of endothelins on isolated ampullar segment of human oviduct in luteal phase of menstrual cycle

Endothelin-1 induces contractions of human oviduct ampullar segment in follicular phase of menstrual cycle, acting on ET_A receptors. The aim of our study was to investigate effects of endothelin-1, endothelin-2 and endothelin-3 on isolated ampullar segment of human oviducts, taken from the patients in luteal phase of menstrual cycle. Fallopian tubes were taken from 20 female patients (one tube from each patient) during abdominal hysterectomy with adnexectomy, due to extensive uterine fibroids. The oviduct ampulla was mounted in an organ bath longitudinally, and the tension of the isolated preparation was recorded with the isometric transducer. Endothelin-1 produced concentration-dependent tonic contraction of the isolated ampullar segment (EC₅₀ = 6.80 ± 1.2 × 10^?10 M), and concentration-dependent inhibition of its rhythmic contractions (EC₅₀ = 7.86 ± 2.3 × 10^?10 M). Endothelin-2 produced concentration-dependent tonic contraction of the isolated ampullar segment (EC₅₀ = 4.56 ± 0.3 × 10^?10 M), without affecting its rhythmic contractions. Endothelin-3 did not affect either tone or rhythmic contractions of the isolated preparations. Selective antagonist for ET_A receptor subtype, BQ 123, produced inhibition of endothelin-1 effects on both tone (pA₂ = 9.50) and spontaneous rhythmic contractions (pA₂ = 10.73), while selective antagonist for ET_B receptor subtype, BQ 788, produced only inhibition of endothelin-1 effects on tone (pA₂ = 9.61), while the effect of endothelin-1 on spontaneous rhythmic contractions remained unaffected. The results of our study suggest that in the luteal phase both ET_A and ET_B receptors regulate tone, and only ET_A receptors regulate rhythmic activity of human oviduct's ampullar segment.     

Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya

Isothiocyanates are electrophiles that are able to induce phase II biotransformation enzyme gene expression via an electrophile-responsive element (EpRE) in the gene regulatory region. To study the potency of different isothiocyanates to induce the expression of EpRE-regulated genes, a Hepa-1c1c7 luciferase reporter cell line was exposed to structurally different isothiocyanates. The reporter cell line, EpRE(mGST-Ya)–LUX, contains the EpRE from the regulatory region of the mouse glutathione S-transferase Ya gene. Isothiocyanates containing a methyl-sulfur side chain, e.g. sulforaphane, showed a lower EC₅₀ (0.8–3.2 μM) and a comparable induction factor (17–22.4) compared to the structurally different isothiocyanates containing an alkyl or aromatic side chain, e.g. allyl and phenylethyl isothiocyanate (EC₅₀ 3.9–6.5 μM, induction factor 17.5–23). After 24 h of exposure, on average (±SD) 23 ± 5% of the isothiocyanate was found in the cells and 77% in the cell medium. Isothiocyanates prove to be strong inducers of electrophile-responsive element-mediated gene expression at physiological concentrations. The here described luciferase reporter cell line is a suitable assay to measure the potency of compounds to induce EpRE-regulated gene expression.     

Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells

IntroductionPrestoBlue (PB) is a new, simple and extremely fast live assay to monitor cell viability and cytotoxicity.Herein, we compared two in vitro cytotoxicity assays, new (PB) and classic (MTT), in the assessment of viability of human umbilical vein endothelial cells (HUVECs) in the presence of selected plant extracts.MethodsThe anti-proliferative effects of two extracts from medicinal plants, i.e., walnut husk extract and spent hop extract, used at the concentration range of 1–200 μg/ml of gallic acid equivalent, were compared with the effects recorded for resveratrol — a natural polyphenolic compound. Reduction of dyes by endothelial cells was determined colorimetrically (MTT and PB) and fluorometrically (PB).ResultsAt higher concentrations, all tested compounds caused significant loss of cell viability. Regardless of plant compound, the PB assay, when measured colorimetrically, produced higher EC₅₀ values compared to other modes of measurement, however, the statistically significant differences in EC₅₀ values among the assays were revealed only for spent hop extract. Conversely, the EC₅₀ values for each plant compound obtained in MTT (colorimetric assay) and PB (fluorometric assay) were similar. According to EC₅₀ values, the cytotoxicity of plant compounds ranked as follows: spent hop extract > resveratrol > walnut husk extract. Furthermore, the MTT assay showed overall lower inter-assay variability and higher signal-to-noise ratio compared to PB assay.DiscussionIn conclusion, we recommend fluorometric PrestoBlue assay for cytotoxicity assessment in human endothelial cells. Due to substantial differences in EC₅₀ values and S/N ratios between spectrophotometric PB and MTT or fluorometric PB assays, colorimetric quantification of HUVECs' viability with the use of PB reagent should be avoided.     

Evaluated the adverse effects of cadmium and aluminum via drinking water to kidney disease patients: Application of a novel solid phase microextraction method

In present study aluminum (Al) and cadmium (Cd) were determined in ground water samples and assesses human health risks associated with elevated concentrations of toxic metals in dissolved form, using a novel solid phase microextraction (SPμE). Ground water sample (n = 200) and biological sample (blood) of patients having chronic kidney disorders (CKD) along with healthy control subjects of same area (southern part of Pakistan) were collected. A simple system, including the micropipette tip packed with modified ionic liquid-activated carbon cloth (IL-ACC) coated with 8-hydroxyqunilone (8-HQ) attached to syringe. The analytes in water and acid digested blood samples were manually drawn for 2–10 cycles (drawing/discharging) at different pH range. The analytes sorbed on coated ACC were then desorbed with 2.0 mol L⁻¹ HNO₃ in ethanol by drawing/discharging cycles for 1–5 times. The concentration of extracted analytes was determined by electrothermal atomic absorption spectrometer. The influence of different variables on the extraction efficiency of Cd and Al, were optimized. The Al and Cd concentrations in groundwater were found to be elevated than recommended limits by the World Health Organization. The urinary N-acetyl-h-glucosaminidase values were significantly higher in CKD patients as compared to refrent subjects (p < 0.001). The significant variation in levels of Cd and Al were observed in blood samples of CKD patients than referents subjects (p < 0.01). The strong positive correlation among Al and Cd levels in groundwater versus blood samples of CKD patients (r = 0.82–0.85) p < 0.01) was observed than those values calculated for referent subjects (r = 0.425–0.536).     

Sulforaphane mitigates cadmium-induced toxicity pattern in human peripheral blood lymphocytes and monocytes

Cadmium (Cd) is a highly toxic and widely distributed heavy metal that induces various diseases in humans through environmental exposure. Therefore, alleviation of Cd-induced toxicity in living organisms is necessary. In this study, we investigated the protective role of sulforaphane on Cd-induced toxicity in human peripheral blood lymphocytes and monocytes. Sulforaphane did not show any major reduction in the viability of lymphocytes and monocytes. However, Cd treatment at a concentration of 50 μM induced around 69% cell death. Treatment of IC₁₀-Cd and 100 μM sulforaphane combination for 24 and 48 h increased viability by 2 and 9% in cells subjected to Cd toxicity, respectively. In addition, IC₂₅ of Cd and 100 μM sulforaphane combination recovered 17–20% of cell viability. Cd induced apoptotic and necrotic cell death. Sulforaphane treatment reduced Cd-induced cell death in lymphocytes and monocytes. Our results clearly indicate that when the cells were treated with Cd + sulforaphane combination, sulforaphane decreased the Cd-induced cytotoxic effect in lymphocytes and monocytes. In addition, sulforaphane concentration plays a major role in the alleviation of Cd-induced toxicity.     

KeraSkin™-VM: A novel reconstructed human epidermis model for skin irritation tests

Several alternative in vitro methods to evaluate skin irritants have been developed recently. In July 2010, OECD officially endorsed the validated reference method (VRM) that uses reconstituted human epidermis (RhE) models as replacements for the in vivo skin irritation test. This study evaluated the KeraSkin™-VM model, a novel human epidermis model that was reconstructed with Asian skin tissue using 20 reference chemicals according to the OECD TG 439 performance standard. The test chemicals were applied to the epidermal surface side for 45 min and then rinsed, and then incubated for 42 h post-treatment. An overall accuracy of 80%, sensitivity of 90% and specificity of 70% were obtained when the results from KeraSkin™-VM were compared with UN GHS categories, which was comparable to the EpiDerm™ Skin irritation test (SIT) rates. Furthermore, KeraSkin™-VM demonstrated good performance in terms of within-laboratory reproducibility and predictive capacity to screen skin irritants.     

Cosmetics Europe multi-laboratory pre-validation of the EpiOcular™ reconstituted human tissue test method for the prediction of eye irritation

Cosmetics Europe, The Personal Care Association (known as Colipa before 2012), conducted a program of technology transfer and within/between laboratory reproducibility of MatTek Corporation’s EpiOcular? Eye Irritation Test (EIT) as one of the two human reconstructed tissue test methods. This EIT EpiOcular? used a single exposure period for each chemical and a prediction model based on a cut-off in relative survival [?60% = irritant (I) (GHS categories 2 and 1); >60% = no classification (NC)]. Test substance single exposure time was 30 min with a 2-h post-exposure incubation for liquids and 90 min with an 18-h post-exposure incubation for solids. Tissue viability was determined by tetrazolium dye (MTT) reduction. Combinations of 20 coded chemicals were tested in 7 laboratories. Standardized laboratory documentation was used by all laboratories. Twenty liquids (11 NC/9 I) plus 5 solids (3 NC/2 I) were selected so that both exposure regimens could be assessed. Concurrent positive (methyl acetate) and negative (water) controls were tested in each trial. In all, 298 independent trials were performed and demonstrated 99.7% agreement in prediction (NC/I) across the laboratories. Coefficients of variation for the% survival for tissues from each treatment group across laboratories were generally low. This protocol has entered in 2010 the experimental phase of a formal ECVAM validation program.     

Lasting effect of preceding culture conditions on the susceptibility of C6 cells to peroxide-induced oxidative stress

The aim of the present study was to investigate the influence of the maintenance culture conditions on the competence of C6 rat glioma cells to cope with peroxide-induced oxidative stress. C6 cells were maintained either in Ham’s nutrient mixture F-10 supplemented with 15% horse serum and 2.5% foetal bovine serum (FBS) or in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% FBS. The differently cultured cells were exposed under identical conditions to hydrogen peroxide (H₂O₂) and cumene hydroperoxide (CHP) in serum-free DMEM. The cells maintained in high serum Ham’s F-10 medium (1) were less sensitive towards the cytotoxic action of both peroxides (EC₅₀-values: H₂O₂: 193 ± 23 μM; CHP: 94 ± 16 μM) than the cells maintained in low serum DMEM (EC₅₀-values: H₂O₂: 51 ± 10 μM; CHP: 27 ± 11 μM), (2) eliminated the peroxides (initial concentration: 100 μM) with higher rates (H₂O₂: 56 ± 5.5 vs. 32 ± 2.7, CHP: 32 ± 6 vs. 3.4 ± 0.6 nmol/min mg protein), (3) contained more glutathione (30 ± 2.5 vs. 14 ± 1.1 nmol/mg protein) and (4) owned a higher glutathione peroxidase activity (28 ± 3.4 vs. 9.5 ± 0.8 mU/mg protein). Glutathione reductase and catalase activities were not affected. These results demonstrate that the preceding culture conditions have a lasting effect on the susceptibility of cultured cells to oxidative stressors like peroxides. As cause for these differences a dissimilar supply of the cells with serum born antioxidants like selenium and α-tocopherol is discussed.     

Synthesis and antihistaminic activity of 3H-benzo [4,5] thieno [2,3-d][1,2,3] triazin-4-ones

In the present study the antihistaminic activity of tricyclic benzothieno 1,2,3-triazine derivatives namely CP-3 (3-(phenyl)-5,6,7,8-tetrahydro,3H-benzo[4,5] thieno [2,3-d][1,2,3] triazin-4-one), CP-5 (3-(3-methyl phenyl)-5,6,7,8-tetrahydro,3H-benzo[4,5] thieno [2,3-d][1,2,3] triazin-4-one) and CP-8 (3-(4-chloro phenyl)-5,6,7,8-tetrahydro,3H-benzo[4,5] thieno [2,3-d][1,2,3] triazin-4-one) were evaluated using in vitro (isolated guinea pig ileum) and in vivo (bronchodilator activity in guinea pigs) models and the sedative potential of the test compounds were evaluated using actophotometer in mice. In in vitro antihistaminic study, the CP-3, CP-5, CP-8 and chlorpheniramine maleate (CPM) have shown a rightward shift in concentration response curve (CRC) of histamine with a change in EC₅₀ values of histamine in all the four tissue preparations. The slope obtained in the schild plot indicated that CP-5, CP-8 and CPM were competitive in nature for H₁-receptors. However, CP-3 has shown non-competitive antagonism. In in vivo antihistaminic study, the CP-3, CP-5, CP-8 and CPM have shown mean increase in exposition time against histamine challenge compared to control group (p < 0.001). All the test drugs (10 mg/kg) and CPM (2 mg/kg) have offered a significant (p < 0.001) protection against preconvulsive dyspnoea (PCD) compared to control. In conclusion, all the test drugs have shown very good antihistaminic activity and the test drugs have very little sedative action compared to CPM.