ESAC Opinion on the BASF-coordinated Performance Standards-based validation of the LuSens test method for skin sensitisation testing

ESAC, the EURL ECVAM Scientific Advisory Committee, advises EURL ECVAM on scientific issues. Its main role is to conduct independent peer review of validation studies of alternative test methods and to assess their scientific validity for a given purpose. The committee reviews the appropriateness of study design and management, the quality of results obtained and the plausibility of the conclusions drawn. ESAC peer reviews are formally initiated with a EURL ECVAM Request for ESAC Advice, which provides the necessary background for the peer-review and establishes its objectives, timelines and the questions to be addressed. The peer review is normally prepared by specialised ESAC Working Groups. These are typically composed of ESAC members and other external experts relevant to the test method under review. These experts may be nominated by ESAC, EURL ECVAM and partner organisations within the International Cooperation on Alternative Test Methods (ICATM). ESAC ultimately decides on the composition of these Working Groups. ESAC's advice to EURL ECVAM is formally provided as 'ESAC Opinions' and 'Working Group Reports' at the end of the peer review. ESAC may also issue Opinions on other scientific issues of relevance to the work and mission of EURL ECVAM but not directly related to a specific alternative test method. The ESAC Opinion expressed in this report relates to the peer-review of the BASF-coordinated Performance Standards-based validation of the LuSens test method for skin sensitisation testing.JRC.F.3-Chemicals Safety and Alternative Method

Genome-Wide Association Studies of the PR Interval in African Americans

The PR interval on the electrocardiogram reflects atrial and atrioventricular nodal conduction time. The PR interval is heritable, provides important information about arrhythmia risk, and has been suggested to differ among human races. Genome-wide association (GWA) studies have identified common genetic determinants of the PR interval in individuals of European and Asian ancestry, but there is a general paucity of GWA studies in individuals of African ancestry. We performed GWA studies in African American individuals from four cohorts (n = 6,247) to identify genetic variants associated with PR interval duration. Genotyping was performed using the Affymetrix 6.0 microarray. Imputation was performed for 2.8 million single nucleotide polymorphisms (SNPs) using combined YRI and CEU HapMap phase II panels. We observed a strong signal (rs3922844) within the gene encoding the cardiac sodium channel (SCN5A) with genome-wide significant association (p<2.5×10−8) in two of the four cohorts and in the meta-analysis. The signal explained 2% of PR interval variability in African Americans (beta  = 5.1 msec per minor allele, 95% CI  = 4.1–6.1, p = 3×10−23). This SNP was also associated with PR interval (beta = 2.4 msec per minor allele, 95% CI = 1.8–3.0, p = 3×10−16) in individuals of European ancestry (n = 14,042), but with a smaller effect size (p for heterogeneity <0.001) and variability explained (0.5%). Further meta-analysis of the four cohorts identified genome-wide significant associations with SNPs in SCN10A (rs6798015), MEIS1 (rs10865355), and TBX5 (rs7312625) that were highly correlated with SNPs identified in European and Asian GWA studies. African ancestry was associated with increased PR duration (13.3 msec, p = 0.009) in one but not the other three cohorts. Our findings demonstrate the relevance of common variants to African Americans at four loci previously associated with PR interval in European and Asian samples and identify an association signal at one of these loci that is more strongly associated with PR interval in African Americans than in Europeans

Alterations in locomotor activity of feeding zebrafish larvae as a consequence of exposure to different environmental factors

Behavioral studies are important tools for understanding the development and pathology of neurological diseases. Zebrafish are an emerging alternative model in behavioral and neurological studies as the behavioral repertoire of zebrafish (Danio rerio) is similar to humans, and nervous system structures and functions are highly conserved. In this study, we investigated alterations in day/night locomotor activity of free swimming, feeding wild-type zebrafish larvae (8–15dpf) due to changes in the rhythm of light/dark cycles or caloric content of food. We furthermore exposed zebrafish larvae to continuous stress by applying alternated minor vibrations. Under altered rhythms of light/dark cycle’s zebrafish larvae still expressed a distinct light/dark activity pattern but the total activity was reduced compared to control animals. When the larvae were exposed to continuous light, they still had coordinated resting cycles but maximal activity and excitation rates after feeding were increased, indicating that food became the new zeitgeber. Feeding food of high caloric content induced continuously high activity levels during light cycles and significantly elevated activity levels during the dark. Exposure to continuous vibrations lowered total activity levels. We showed previously that changes in environmental factors like light/dark cycles or changes in caloric content of food can affect adipogenesis, lipid composition, and circadian rhythm of free swimming, feeding larvae but this is the first time showing how theses factor alter behavior

Prolonged hypoxia increases survival even in Zebrafish (Danio rerio) showing cardiac arrhythmia.

Tolerance towards hypoxia is highly pronounced in zebrafish. In this study even beneficial effects of hypoxia, specifically enhanced survival of zebrafish larvae, could be demonstrated. This effect was actually more pronounced in breakdance mutants, which phenotypically show cardiac arrhythmia. Breakdance mutants (bre) are characterized by chronically reduced cardiac output. Despite an about 50% heart rate reduction, they become adults, but survival rate significantly drops to 40%. Normoxic bre animals demonstrate increased hypoxia inducible factor 1 a (Hif-1α) expression, which indicates an activated hypoxic signaling pathway. Consequently, cardiovascular acclimation, like cardiac hypertrophy and increased erythrocyte concentration, occurs. Thus, it was hypothesized, that under hypoxic conditions survival might be even more reduced. When bre mutants were exposed to hypoxic conditions, they surprisingly showed higher survival rates than under normoxic conditions and even reached wildtype values. In hypoxic wildtype zebrafish, survival yet exceeded normoxic control values. To specify physiological acclimation, cardiovascular and metabolic parameters were measured before hypoxia started (3 dpf), when the first differences in survival rate occurred (7 dpf) and when survival rate plateaued (15 dpf). Hypoxic animals expectedly demonstrated Hif-1α accumulation and consequently enhanced convective oxygen carrying capacity. Moreover, bre animals showed a significantly enhanced heart rate under hypoxic conditions, which reached normoxic wildtype values. This improvement in convective oxygen transport ensured a sufficient oxygen and nutrient supply and was also reflected in the significantly higher mitochondrial activity. The highly optimized energy metabolism observed in hypoxic zebrafish larvae might be decisive for periods of higher energy demand due to organ development, growth and increased activity. However, hypoxia increased survival only during a short period of development and starting hypoxia before or after this phase reduced survival, particularly in bre animals. Thus, the physiological plasticity, which enables zebrafish larvae to benefit from a hypoxia, occurs only within a narrow developmental window

Physiological, Diurnal and Stress-Related Variability of Cadmium-Metallothionein Gene Expression in Land Snails.

The terrestrial Roman snail Helix pomatia has successfully adapted to strongly fluctuating conditions in its natural soil habitat. Part of the snail's stress defense strategy is its ability to express Metallothioneins (MTs). These are multifunctional, cysteine-rich proteins that bind and inactivate transition metal ions (Cd(2+), Zn(2+), Cu(+)) with high affinity. In Helix pomatia a Cadmium (Cd)-selective, inducible Metallothionein Isoform (CdMT) is mainly involved in detoxification of this harmful metal. In addition, the snail CdMT has been shown to also respond to certain physiological stressors. The aim of the present study was to investigate the physiological and diurnal variability of CdMT gene expression in snails exposed to Cd and non-metallic stressors such as desiccation and oxygen depletion. CdMT gene expression was upregulated by Cd exposure and desiccation, whereas no significant impact on the expression of CdMT was measured due to oxygen depletion. Overall, Cd was clearly more effective as an inducer of the CdMT gene expression compared to the applied non-metallic stressors. In unexposed snails, diurnal rhythmicity of CdMT gene expression was observed with higher mRNA concentrations at night compared to daytime. This rhythmicity was severely disrupted in Cd-exposed snails which exhibited highest CdMT gene transcription rates in the morning. Apart from diurnal rhythmicity, feeding activity also had a strong impact on CdMT gene expression. Although underlying mechanisms are not completely understood, it is clear that factors increasing MT expression variability have to be considered when using MT mRNA quantification as a biomarker for environmental stressors

Microarray analysis.

<p>Comparison of mRNA expression of 3 and 7 dpf old normoxic control or <i>bre</i> larvae. Transcripts, which were up or down-regulated in control as well as <i>bre</i> animals, are listed.</p