Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency

The human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test, is based on the augmentation of CD86 and CD54 expression in THP-1 cells following exposure to chemicals. The h-CLAT was found to be capable of determining the hazard of skin sensitization. In contrast, the local lymph node assay (LLNA), widely used as a stand-alone method in Europe and US, identifies the same hazard, but also classifies the potency by using the estimated concentration of SI = 3 (EC3). In this study, several values calculated from the h-CLAT data were evaluated for its correlation to the LLNA EC3 determination. A statistically significant correlation was observed between h-CLAT concentration providing a cell viability of 75% (CV75), h-CLAT estimated concentration of RFI = 150 for CD86 (EC150), and for CD54 (EC200) with LLNA’s EC3. From EC150 and EC200, a minimum induction threshold (MIT) was determined as the smaller of either EC150 or EC200. MIT showed a correlation with EC3 (R = 0.638). Also, MIT had an approximate 80% accuracy for sub-categories of the globally harmonized system (GHS) when a tentative threshold of 13 μg/mL was used. From these data, the h-CLAT values may be one of the useful tools to predict the allergic potency of chemicals.     

Binary test battery with KeratinoSens™ and h-CLAT as part of a bottom-up approach for skin sensitization hazard prediction

Skin sensitization is one of the key safety endpoints for chemicals applied directly to the skin. Several integrated testing strategies (ITS) using multiple non-animal test methods have been developed to accurately evaluate the sensitizing potential of chemicals, but there is no regulatory-accepted ITS to classify a chemical as a non-sensitizer. In this study, the predictive performance of a binary test battery with KeratinoSens™ and h-CLAT compared to the local lymph node assay (LLNA) and human data was examined using comprehensive dataset of 203 chemicals. When two negative results indicate a non-sensitizer, the binary test battery provided sensitivity of 93.4% or 94.4% compared with the LLNA or human data. Taking into account the predictive limitations (i.e. high log Kow, pre-/pro-haptens and acyl transfer agents (or amine-reactive)), the binary test battery had extremely high sensitivity comparable to that of the 3 out of 3 ITS where three negative results of the DPRA, KeratinoSens™ and h-CLAT indicate a non-sensitizer. Therefore, the data from KeratinoSens™ or h-CLAT may provide partly redundant information on the molecular initiating event derived from DPRA. Taken together, the binary test battery of KeratinoSens™ and h-CLAT could be used as part of a bottom-up approach for skin sensitization hazard prediction.     

Non-animal photosafety screening for complex cosmetic ingredients with photochemical and photobiochemical assessment tools

Previously, a non-animal screening approach was proposed for evaluating photosafety of cosmetic ingredients by means of in vitro photochemical and photobiochemical assays; however, complex cosmetic ingredients, such as plant extracts and polymers, could not be evaluated because their molecular weight is often poorly defined and so their molar concentration cannot be calculated. The aim of the present investigation was to establish a photosafety screen for complex cosmetic ingredients by using appropriately modified in vitro photosafety assays. Twenty plant extracts were selected as model materials on the basis of photosafety information, and their phototoxic potentials were assessed by means of ultraviolet (UV)/visible light (VIS) spectral analysis, reactive oxygen species (ROS)/micellar ROS (mROS) assays, and 3T3 neutral red uptake phototoxicity testing (3T3 NRU PT). The maximum UV/VIS absorption value was employed as a judgment factor for evaluating photoexcitability of samples, and the value of 1.0 was adopted as a tentative criterion for photosafety identification. The ROS/mROS assays were conducted at 50 μg/mL, and no false negative prediction was obtained. Furthermore, the ROS/mROS assays at 50 μg/mL had a similar predictive capacity to the ROS/mROS assays in the previous study. A systematic tiered approach for simple and rapid non-animal photosafety evaluation of complex cosmetic ingredients can be constructed using these modified in vitro photochemical assays.     

Safety evaluation of Whole Algalin Protein (WAP) from Chlorella protothecoides

Microalgae such as Chlorella spp., were once consumed as traditional human foods; now they are being developed as ingredients for modern diets. Whole Algalin Protein (WAP) from dried milled Chlorella protothecoides was evaluated for dietary safety in a 13-week feeding trial in rodents with genotoxic potential evaluated using in vitro and in vivo assays and the likelihood of food allergy potential evaluated via human repeat-insult patch test (HRIPT). In the subchronic study, rats consumed feed containing 0, 25,000, 50,000 or 100,000 ppm WAP for 92–93 days. No treatment-related mortalities or effects in general condition, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, gross pathology, organ weights, and histopathology occurred. Several endpoints exhibited statistically significant effects, but none was dose-related. The no-observed-adverse-effect level (NOAEL) was based on the highest WAP concentration consumed by the rats and was equivalent to 4805 mg/kg/day in males and 5518 mg/kg/day in females. No mutagenicity occurred in Salmonella typhimurium or Escherichia coli tester strains (⩽5000 μg/plate WAP) with or without mutagenic activation. No clastogenic response occurred in bone marrow from mice administered a single oral dose (2000 mg/kg WAP). Skin sensitization was not induced by WAP via HRIPT, indicating little potential for food allergy.     

Simultaneous determination of ranitidine and metronidazole in pharmaceutical formulations at poly(chromotrope 2B) modified activated glassy carbon electrodes

A simple and sensitive electrochemical method for the simultaneous and quantitative detection of ranitidine (RT) and metronidazole (MT) was developed, based on a poly(chromotrope 2B) modified activated glassy carbon electrode (PCHAGCE). The PCHAGCE showed excellent electrocatalytic activity toward the reduction of both RT and MT in 0.1 mol/L phosphate buffer solution (pH 6.0). The peak-to-peak separations for the simultaneous detection of RT and MT between the two reduction waves in cyclic voltammetry were increased significantly from ∼0.1 V at activated GCE, to ∼0.55 V at PCHAGCE. By differential pulse voltammetry techniques, the reduction peak currents of RT and MT were both linear over the range of 1.0 × 10⁻⁵–4.0 × 10⁻⁴ mol/L. The detection limits (S/N = 3) were 5.4 × 10⁻⁷ mol/L and 3.3 × 10⁻⁷ mol/L for RT and MT, respectively. The modified electrode was successfully applied to the determination of RT and MT in pharmaceutical preparations and human serum as real samples with stable and reliable recovery data.     

Development and validation of an OECD reproductive toxicity test guideline with the pond snail Lymnaea stagnalis (Mollusca,Gastropoda)

The OECD test guideline development program has been extended in 2011 to establish a partial life-cycle protocol for assessing the reproductive toxicity of chemicals to several mollusk species, including the great pond snail Lymnaea stagnalis. In this paper, we summarize the standard draft protocol for a reproduction test with this species, and present inter-comparison results obtained in a 56-day prevalidation ring-test using this protocol.Seven European laboratories performed semi-static tests with cultured snails of the strain Renilys® exposed to nominal concentrations of cadmium chloride (from 53 to 608 μg Cd L⁻¹). Cd concentrations in test solutions were analytically determined to confirm accuracy in the metal exposure concentrations in all laboratories. Physico-chemical and biological validity criteria (namely dissolved oxygen content >60% ASV, water temperature 20 ± 1 °C, control snail survival >80% and control snail fecundity >8 egg-masses per snail over the test period) were met in all laboratories which consistently demonstrated the reproductive toxicity of Cd in snails using the proposed draft protocol. Effect concentrations for fecundity after 56 days were reproducible between laboratories (68 < EC_50–56d < 124 μg L⁻¹) and were consistent with literature data. EC_50–56d and EC_10–56d values were comprised within a factor of 1.8 and 3.6, respectively, which is in the range of acceptable variation defined for reference chemicals in OECD test guidelines for invertebrates. The inter-laboratory reproducibility coefficient of variation (CV) for the Cd LC_50–56d values was 8.19%. The inter-laboratory comparison of fecundity within the controls gave a CV of 29.12%, while exposure to Cd gave a CV of 25.49% based on the EC_50–56d values. The OECD has acknowledged the success of this prevalidation exercise and a validation ring-test involving 14 laboratories in Europe, North- and South-America is currently being implemented using four chemicals (Cd, prochloraz, trenbolone and tributyltin).     

Individual and combined in vitro (anti)androgenic effects of certain food additives and cosmetic preservatives

The individual and combined (binary mixtures) (anti)androgenic effect of butylparaben (BuPB), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG) was evaluated using the MDA-kb2 cell line. Exposing these cells to AR agonists results in the expression of the reporter gene (encoding for luciferase) and luminescence can be measured in order to monitor the activity of the reporter protein. In case of the evaluation of the anti-androgenic effect, the individual test compounds or binary mixtures were tested in the presence of a fixed concentration of a strong AR agonist (1000 pM 5-alpha-dihydrotestosterone; DHT). Cell viability was assessed using a resazurin based assay. For PG, this is the first report in the literature concerning its (anti)androgenic activity. In case of both individual and mixture testing none of the compounds or binary combinations showed androgenic activity. When tested in the presence of DHT, BuPB, BHA and BHT proved to be weak anti-androgens and this was confirmed during the evaluation of binary mixtures (BuPB + BHA, BuPB + BHT and BHA + BHT). Besides performing the in vitro testing of the binary combinations, two mathematical models (dose addition and response addition) were evaluated in terms of accuracy of prediction of the anti-androgenic effect of the selected binary mixtures. The dose addition model guaranteed a good correlation between the experimental and predicted data. However, no estimation was possible in case of mixtures containing PG, due to the lack of effect of the compound in case of the individual testing.     

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.     

Hair mercury concentrations and in vitro fertilization (IVF) outcomes among women from a fertility clinic

Total hair mercury (Hg) was measured among 205 women undergoing in vitro fertilization (IVF) treatment and the association with prospectively collected IVF outcomes (229 IVF cycles) was evaluated. Hair Hg levels (median = 0.62 ppm, range: 0.03–5.66 ppm) correlated with fish intake (r = 0.59), and exceeded the recommended EPA reference of 1 ppm in 33% of women. Generalized linear mixed models with random intercepts accounting for within-woman correlations across treatment cycles were used to evaluate the association of hair Hg with IVF outcomes adjusted for age, body mass index, race, smoking status, infertility diagnosis, and protocol type. Hair Hg levels were not related to ovarian stimulation outcomes (peak estradiol levels, total and mature oocyte yields) or to fertilization rate, embryo quality, clinical pregnancy rate or live birth rate.     

Maturin acetate from Psacalium peltatum (Kunth) Cass. (Asteraceae) induces immunostimulatory effects in vitro and in vivo

Maturin acetate (MA) is one of main constituents in Psacalium peltatum. The cytotoxic effects of MA on tumorigenic cells were evaluated using the MTT assay. The in vitro immunostimulatory effects of maturin acetate (MA) were evaluated on the viability of murine splenocytes and macrophages, and human peripheral blood mononuclear cells (PBMC). The effects of MA on the production of nitrous oxide, pinocytosis and lysosomal enzyme activity were assayed in murine macrophages RAW 264.7. The effects of MA on the NK cell activity were also assayed. The in vivo immunostimulatory activities of MA were evaluated on BALB/c mice immunosuppressed with cyclophosphamide (CY). MA lacks cytotoxic activity against human cancer cells (IC₅₀ > 200 μM). In the absence of LPS, MA 10 μM or higher stimulated significantly (P ? 0.05), compared to untreated cells (-LPS), the viability of murine macrophages and splenocytes. In the absence of LPS, MA 10 μM or higher stimulated significantly (P ? 0.05), compared to untreated cells (-LPS), the lysosomal enzyme activity and pinocytosis. In immunosuppressed mice, MA increases significantly (P ? 0.05), compared to CY-treated mice, the production of IL-2 and IL-15 and IFN-γ. In conclusion, MA exerts immunostimulatory activities in vitro and in vivo.     

The protective effects of guaraná extract (Paullinia cupana) on fibroblast NIH-3T3 cells exposed to sodium nitroprusside

The antioxidant effects of the hydro-alcoholic guaraná extract (Paullinia cupana var. sorbilis Mart.) on nitric oxide (NO) and other compounds generated from the degradation of sodium nitroprusside (SNP) in an embryonic fibroblast culture (NIH-3T3 cells) were evaluated. The guaraná bioactive compounds were initially determined by high-performance liquid chromatography: caffeine = 12.240 mg/g, theobromine = 6.733 mg/g and total catechins = 4.336 mg/g. Cells were exposed to 10 μM SNP during a 6 h period because the cells exhibited >90% mortality at this concentration. Guaraná was added to the cultures in five concentrations (0.5, 1, 5, 10 and 20 mg/mL). The guaraná antioxidant effect was evaluated by viability assays, biochemical oxidation [lipid peroxidation, catalase and superoxide dismutase (SOD) activity] and genotoxicity (DNA Comet assay) analysis. Additionally, oxidative stress was evaluated by a 2,7-dihydrodichlorofluorescein diacetate fluorescence assay. Guaraná reverted the SNP toxicity mainly at lower concentrations (<5 mg), which decreased cell mortality, lipid peroxidation, DNA damage and cell oxidative stress as well as increased the SOD levels. These results demonstrate that guaraná has an antioxidant effect on NO metabolism in situations with higher cellular NO levels.     

Anti-oxidant and natural killer cell activity of Korean red ginseng (Panax ginseng) and urushiol (Rhus vernicifera Stokes) on non-alcoholic fatty liver disease of rat

Anti-oxidative and immunologic effects of the Korea red ginseng (KRG; Panax ginseng) and urushiol (Rhus vernicifera Stokes) on non-alcoholic fatty liver disease (NAFLD) were evaluated. Forty-five rats (five Long-Evans Tokushima Otsuka and 40 Otsuka Long-Evans Tokushima Fatty [OLETF] rats) received chew diets for 10 months; after this period. The OLETF rats were divided into the following four groups according to diet for 2 months: NAFLD (chew), KRG (chew + KRG [200 mg/kg/day]), urushiol (chew + urushiol [0.5 mg/kg/day]), and ursodeoxycholic acid (UDCA) (chew + UDCA [15 mg/kg/day]) groups. Liver function, lipid profiles and anti-oxidant activity of liver and serum, natural killer (NK) cell activity, and pathology were compared. In KRG and urushiol groups, the level of serum triglyceride ([302.0 ± 70.4 and 275.2 ± 63.8] vs. 527.7 ± 153.3 mg/dL) were lower compared with that of NAFLD group (p < 0.05). The levels of HDL-cholesterol (liver tissue: [4.8 ± 0.2 and 4.8 ± 0.5] vs. 4.2 ± 0.2 mg/g) and NK cell activity ([3485 ± 910 and 3559 ± 910] vs. 2486 ± 619 counts) were significantly higher than those of the NAFLD group (p < 0.001). Inflammation with neutrophil infiltration was observed in only two rats in the NAFLD group. These results suggest that 2 months of oral KRG or urushiol administration improves lipid profiles and stimulates NK cell activity, while inhibiting steatohepatitis in OLEFT rats.     

Evaluation of cytochrome P450 1 (CYP1) and N-acetyltransferase 1 (NAT1) activities in HaCaT cells: Implications for the development of in vitro techniques for predictive testing of contact sensitizers

Xenobiotic metabolizing enzymes like cytochrome P450s and N-acetyltransferase are expressed in keratinocytes and professional antigen-presenting cells. Thus, biotransformation of chemicals applied to the skin can be relevant for their potential to cause skin toxicity and immune responses like allergic contact dermatitis. Considering the keratinocyte cell line HaCaT as a relevant in vitro tool for epidermal biotransformation, we specifically investigated CYP1 (EROD) and N-acetyltransferase 1 (NAT1) activities of three different HaCaT shipments and human primary keratinocytes (NHEK). Solvent treated HaCaT showed EROD levels near the detection limit (0.047 pmol/mg/min), primary keratinocytes (n = 4) were in a range between 0 and 0.76 pmol/mg/min. B[a]P (1 μM) induced EROD activities of 19.0 ± 0.9 pmol/mg/min (n = 11) in HaCaT and 5.8 ± 0.5 pmol/mg/min (n = 4) in NHEK. N-acetylation activities for para-aminobenzoic acid (PABA) were in average 3.4-fold higher in HaCaT compared to NHEK (8 ± 0.5 nmol/mg/min) and varied between the HaCaT shipments (range 12.0–44.5 nmol/mg/min). This was in good agreement with NAT1 promoter P1 dependent mRNA level and N-acetylation of the contact allergen para-phenylenediamine (PPD) under typical cell-based assay conditions. We conclude that HaCaT represent a suitable in vitro model for studying the qualitative contribution of epidermal phase1/phase2 metabolism to toxicological endpoints such as skin sensitization.     

Impact of assay selection and study design on the outcome of cytotoxicity testing of medical devices: The case of multi-purpose vision care solutions

Medical device biocompatibility testing usually includes a cytotoxicity component. Assay selection and protocol design often depend on a specific testing standard rather than on the characteristics of the medical device. To better understand the impact of assay selection on study outcome of unstructured medical devices, we evaluated contact lens multi-purpose solutions (MPS) in the agar diffusion, direct contact and two elution cytotoxicity assays. To simulate the conditions of use, MPS were evaluated alone and in combination with contact lenses. All MPS passed the agar diffusion assay (n = 3) and extracts prepared from contact lenses soaked in MPS passed the USP elution assay (n = 3). Both the duration of contact and MPS concentration impacted the outcome of a modified elution assay. When tested at 25% strength for 48 h, all MPS evaluated were non-cytotoxic (n > 3). Test article movement and mechanical damage were significant issues with the direct contact assay. Movement was effectively controlled by manipulating contact lens orientation while using 0.8 mL culture medium. All MPS passed the USP direct contact cytotoxicity test when evaluated using this optimized methodology (n = 3). These data are consistent with MPS results in ocular irritation studies in rabbits (n = 3).     

Towards the bioequivalence of pressurised metered dose inhalers 2. Aerodynamically equivalent particles (with and without glycerol) exhibit different biopharmaceutical profiles in vitro

Two solution-based pressurised metered dose inhaler (pMDI) formulations were prepared such that they delivered aerosols with identical mass median aerodynamic diameters, but contained either beclomethasone dipropionate (BDP) alone (glycerol-free formulation) or BDP and glycerol in a 1:1 mass ratio (glycerol-containing formulation). The two formulations were deposited onto Calu-3 respiratory epithelial cell layers cultured at an air interface. Equivalent drug mass (∼1000 ng or ∼2000 ng of the formulation) or equivalent particle number (1000 ng of BDP in the glycerol-containing versus 2000 ng of BDP in the glycerol-free formulation) were deposited as aerosolised particles on the air interfaced surface of the cell layers. The transfer rate of BDP across the cell layer after deposition of the glycerol-free particles was proportional to the mass deposited. In comparison, the transfer of BDP from the glycerol-containing formulation was independent of the mass deposited, suggesting that the release of BDP is modified in the presence of glycerol. The rate of BDP transfer (and the extent of metabolism) over 2 h was faster when delivered in glycerol-free particles, 465.01 ng ± 95.12 ng of the total drug (20.99 ± 4.29%; BDP plus active metabolite) transported across the cell layer, compared to 116.17 ng ± 3.07 ng (6.07 ± 0.16%) when the equivalent mass of BDP was deposited in glycerol-containing particles. These observations suggest that the presence of glycerol in the maturated aerosol particles may influence the disposition of BDP in the lungs.     

The value of selected in vitro and in silico methods to predict acute oral toxicity in a regulatory context: Results from the European Project ACuteTox

ACuteTox is a project within the 6th European Framework Programme which had as one of its goals to develop, optimise and prevalidate a non-animal testing strategy for predicting human acute oral toxicity. In its last 6 months, a challenging exercise was conducted to assess the predictive capacity of the developed testing strategies and final identification of the most promising ones. Thirty-two chemicals were tested blind in the battery of in vitro and in silico methods selected during the first phase of the project. This paper describes the classification approaches studied: single step procedures and two step tiered testing strategies. In summary, four in vitro testing strategies were proposed as best performing in terms of predictive capacity with respect to the European acute oral toxicity classification. In addition, a heuristic testing strategy is suggested that combines the prediction results gained from the neutral red uptake assay performed in 3T3 cells, with information on neurotoxicity alerts identified by the primary rat brain aggregates test method. Octanol–water partition coefficients and in silico prediction of intestinal absorption and blood–brain barrier passage are also considered. This approach allows to reduce the number of chemicals wrongly predicted as not classified (LD₅₀ > 2000 mg/kg b.w.).     

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.     

Sensitivity of continuous performance test (CPT) at age 14 years to developmental methylmercury exposure

Hit Reaction Time latencies (HRT) in the Continuous Performance Test (CPT) measure the speed of visual information processing. The latencies may involve different neuropsychological functions depending on the time from test initiation, i.e., first orientation, learning and habituation, then cognitive processing and focused attention, and finally sustained attention as the dominant demand. Prenatal methylmercury exposure is associated with increased reaction time (RT) latencies. We therefore examined the association of methylmercury exposure with the average HRT at age 14 years at three different time intervals after test initiation. A total of 878 adolescents (87% of birth cohort members) completed the CPT. The RT latencies were recorded for 10 min, with visual targets presented at 1000 ms intervals. After confounder adjustment, regression coefficients showed that CPT-RT outcomes differed in their associations with exposure biomarkers of prenatal methylmercury exposure: During the first 2 min, the average HRT was weakly associated with methylmercury (beta (SE) for a ten-fold increase in exposure, (3.41 (2.06)), was strongly for the 3-to-6 min interval (6.10 (2.18)), and the strongest during 7–10 min after test initiation (7.64 (2.39)). This pattern was unchanged when simple reaction time and finger tapping speed were included in the models as covariates. Postnatal methylmercury exposures did not affect the outcomes. Thus, these findings suggest that sustained attention as a neuropsychological domain is particularly vulnerable to developmental methylmercury exposure, indicating probable underlying dysfunction of the frontal lobes. When using CPT data as a possible measure of neurotoxicity, test results should therefore be analyzed in regard to time from test initiation and not as overall average reaction times.     

Effects of GABAB receptor agonists on cocaine hyperlocomotor and sensitizing effects in rats

The present study was designed to find out whether pharmacological activation of GABA_B receptors played a role in cocaine sensitization. To this end, male Wistar rats were injected with baclofen or 3-aminopropyl(methyl)phosphinic acid (SKF 97541), the potent and selective GABA_B receptor agonists. The rats, which were repeatedly (for 5 days) administered with cocaine (10 mg/kg) and then challenged with cocaine (10 mg/kg) after 5-day withdrawal period, showed significantly higher locomotor hyperactivity in comparison with the effect observed in saline-pretreated and cocaine challenged rats. Baclofen (1.25, 2.5 and 5 mg/kg), administered for 5 days prior to cocaine, dose-dependently attenuated cocaine sensitization. When injected in the same treatment regimen, SKF 97541 (0.03 mg/kg) reduced the development of cocaine sensitization. To examine the effects of baclofen and SKF 97541 on the expression of cocaine sensitization, the drugs were given acutely before a challenge dose of cocaine (10 mg/kg) on day 10. Either baclofen (2.5 and 5 mg/kg) or SKF 97541 (0.1 mg/kg) decreased sensitization to cocaine. Our findings implicate a role of GABA_B receptors in locomotor responses to cocaine. More specifically, they show that stimulation of GABA_B receptors exerted inhibitory actions on acute locomotor responses to cocaine and on the expression of cocaine sensitization, what may offer a therapeutic potential of GABA_B receptor agonists in the treatment of cocaine dependence.