A quantitative medium-throughput assay to measure Caenorhabditis elegans development and reproduction.

Growth and offspring count are two commonly determined toxicological endpoints for chemical- or gene-induced developmental and reproductive effects in Caenorhabditis elegans. Here, we present a protocol for a 96 h, medium-throughput assay, assessing both endpoints quantitatively within an automated framework using open-source software. The assay utilizes whole 96-well fluorescence images taken with a high-content screening system. Alternatively, conventional fluorescence images can also be utilized with only a few adjustments. For complete details on the use and execution of this protocol, please refer to Wittkowski et al. (2019).

Repeatability analysis improves the reliability of behavioral data.

Reliability of data has become a major concern in the course of the reproducibility crisis. Especially when studying animal behavior, confounding factors such as novelty of the test apparatus can lead to a wide variability of data which may mask treatment effects and consequently lead to misinterpretation. Habituation to the test situation is a common practice to circumvent novelty induced increases in variance and to improve the reliability of the respective measurements. However, there is a lack of published empirical knowledge regarding reasonable habituation procedures and a method validation seems to be overdue. This study aimed at setting up a simple strategy to increase reliability of behavioral data measured in a familiar test apparatus. Therefore, exemplary data from mice tested in an Open Field (OF) arena were used to elucidate the potential of habituation and how reliability of measures can be confirmed by means of a repeatability analysis using the software R. On seven consecutive days, male C57BL/6J, BALB/cJ and 129S1/SvImJ mice were tested in an OF arena once daily and individual mouse behavior was recorded. A repeatability analysis was conducted with regard to repeated trials of habituation. Our data analysis revealed that monitoring animal behavior during habituation is important to determine when individual differences of the measurements are stable. Repeatability values from distance travelled and average activity increased over the habituation period, revealing that around 60% of the variance of the data can be explained by individual differences between mice. The first day of habituation was significantly different from the following 6 days. A three-day habituation period appeared to be sufficient in this study. Overall, these results emphasize the importance of habituation and in depth analysis of habituation data to define the correct starting point of the experiment for improving the reliability and reproducibility of experimental data.

Analgesic treatment with buprenorphine should be adapted to the mouse strain.

Buprenorphine is a commonly used opioid to treat moderate to severe pain in mice. Although strain differences regarding basal pain sensitivity and the analgesic effect of other opioids have been described for mice, the data for buprenorphine is incomplete. Hence, we investigated basal pain sensitivity and the analgesic effect of buprenorphine (0.42, 4.0 mg·kg-1) in male C57BL/6J, Balb/cJ and 129S1/SvImJ mice using the incremental hot plate. Additionally, we verified single nucleotide polymorphisms in Cytochrome P450 3a (Cyp3a) genes, which encode for enzymes that are relevant for buprenorphine metabolism, and analyzed serum and brain concentrations of buprenorphine and its metabolites. Finally, in a pilot survey we determined µ-opioid receptor (MOR) protein expression in whole brain lysates. Basal pain sensitivity differed significantly between the mouse strains (Balb/cJ > C57BL/6J > 129S1/SvImJ). Additionally, buprenorphine showed a dose- and strain-dependent effect: at a higher dose it led to increased antinociception in C57BL/6J and Balb/cJ mice, whereas in 129S1/SvImJ mice this effect was diminished. Serum and brain concentrations of buprenorphine and its metabolites dose-dependently increased and differed slightly between the strains at the high dose. However, these slight strain differences did not correlate with pain behavior. Furthermore, serum buprenorphine metabolic ratio and distribution of buprenorphine and its metabolites between brain and blood showed no dose- and only some strain-dependent differences independent from nociceptive behavior. Western blot analysis revealed no strain difference in the basal MOR protein expression in brain lysates. Our results indicate that buprenorphine dosing should be determined in a pilot study for the respective mouse strain to optimize pain treatment and to avoid unwanted side effects. The present pharmacokinetic data and the coarse determination of MOR expression do not explain the strain differences in the analgesic effect of buprenorphine. However, follow-up studies focusing on more specific pharmacodynamic factors could further elucidate the reasons.

Caenorhabditis elegans As a Promising Alternative Model for Environmental Chemical Mixture Effect Assessment-A Comparative Study.

A key challenge of mixture toxicity testing is that a multitude of substances with even more combinations need to be tested in a broad dose range. Consequently testing in rodent bioassays, the current gold standard of toxicity testing, is hardly feasible. High-throughput compatible cell culture systems, however, suffer from limitations with respect to toxicokinetics, tissue interactions, and compensatory mechanisms. Therefore, simple organisms like the nematode Caenorhabditis elegans, combining relevant advantages of complex in vivo and fast in vitro assays might prove highly valuable within a testing strategy for mixtures. To investigate the comparability between results obtained with C. elegans and traditional rodent assays, we used five azole fungicides as well investigated model substances. Our findings suggest that azoles act additively in C. elegans which is in line with previous results in rats. Additionally, we show that toxicokinetics are one important factor for the differences in the relative toxicity of the azoles in both species. Importantly, we also demonstrate that in contrast to most rodent in vivo studies, C. elegans assays provide well-defined concentration-response relationships which are a very good basis for the prediction of mixture effects. We conclude that C. elegans may be an appropriate model for mixture toxicity testing at least within a first step to identify and prioritize relevant mixtures for further testing.

Update of the DevTox data database for harmonized risk assessment and alternative methodologies in developmental toxicology: Report of the 9th Berlin Workshop on Developmental Toxicity.

Representatives of applied science (e.g. governmental organizations, academia, and industry) met to discuss the progress towards a harmonized human health risk assessment in developmental toxicology of plant protection products, biocidal products, and other environmental chemicals at the 9th Berlin Workshop on Developmental Toxicity held in September 2018. Within the focus of the scientific discussion were the future of in-vitro methods for developmental and reproductive toxicology, the potential relevance of alternative species in testing of developmental effects, and risk and hazard assessment of developmental and endocrine effects. Furthermore, the need for a harmonized terminology for classification of anomalies in laboratory animals in developmental toxicity studies aiming for human health risk assessment was determined. Here, the DevTox database was identified as an extremely valuable tool. Overall, the participants agreed that still one of the biggest challenges for testing developmental toxicity in the 21st century is the development of animal-free test strategies and alternatives to animal testing that could provide human-relevant information in a rapid, efficient, and mechanistically informative manner.

Simple changes of individual studies can improve the reproducibility of the biomedical scientific process as a whole.

We developed a new probabilistic model to assess the impact of recommendations rectifying the reproducibility crisis (by publishing both positive and 'negative' results and increasing statistical power) on competing objectives, such as discovering causal relationships, avoiding publishing false positive results, and reducing resource consumption. In contrast to recent publications our model quantifies the impact of each single suggestion not only for an individual study but especially their relation and consequences for the overall scientific process. We can prove that higher-powered experiments can save resources in the overall research process without generating excess false positives. The better the quality of the pre-study information and its exploitation, the more likely this beneficial effect is to occur. Additionally, we quantify the adverse effects of both neglecting good practices in the design and conduct of hypotheses-based research, and the omission of the publication of 'negative' findings. Our contribution is a plea for adherence to or reinforcement of the good scientific practice and publication of 'negative' findings.

Liver lobe and strain differences in the activity of murine cytochrome P450 enzymes.

The cytochrome P450 (CYP) enzyme superfamily is the most important enzyme system for phase I biotransformation. For toxico- and pharmacokinetic studies, use of liver-based microsomes, including those of mice, is state-of-the-art to study CYP-dependent metabolism. However, reproducibility and interpretation of these data is still very variable, partly because current testing guidelines do not cover details on organ sampling and potential liver lobe differences. Hence, we analyzed CYP activity, CYP protein content, mRNA expression of CYP1A, CYP2C, CYP2D and CYP3A isozymes, and cytochrome P450 reductase (CPR) activity of the four different liver lobes and processus papillaris of male C57BL/6J mice in comparison to whole liver. Additionally, we used whole liver of Balb/cJ and 129S1/SvImJ for strain comparison. Our data show significant differences in CYP activity, being most prominent in lobus sinister lateralis and lobus medialis, and lowest in processus papillaris. These differences were not caused by varying Cyp gene expression or CYP protein level, but partly correspond with lobe specific CPR activities. We also observed significant strain differences in CYP mRNA expression and activities with overall high activities in 129S1/SvImJ mice and low activities in Balb/cJ mice compared to C57BL/6J mice. In addition, strain specific differences in CYP2C and CYP2D activity seem to be reflected in strain dependent differences in CPR activity. In summary, our results indicate that in mice CYP activity and gene expression are strain dependent and may vary highly between liver lobes. To ensure reproducibility and comparability of different probes and studies, this should be taken into account when liver samples are collected for the analysis of CYP-dependent metabolism.

CYP2C9 genotype vs. metabolic phenotype for individual drug dosing--a correlation analysis using flurbiprofen as probe drug.

Currently, genotyping of patients for polymorphic enzymes responsible for metabolic elimination is considered a possibility to adjust drug dose levels. For a patient to profit from this procedure, the interindividual differences in drug metabolism within one genotype should be smaller than those between different genotypes. We studied a large cohort of healthy young adults (283 subjects), correlating their CYP2C9 genotype to a simple phenotyping metric, using flurbiprofen as probe drug. Genotyping was conducted for CYP2C9*1, *2, *3. The urinary metabolic ratio MR (concentration of CYP2C9-dependent metabolite divided by concentration of flurbiprofen) determined two hours after flurbiprofen (8.75 mg) administration served as phenotyping metric. Linear statistical models correlating genotype and phenotype provided highly significant allele-specific MR estimates of 0.596 for the wild type allele CYP2C9*1, 0.405 for CYP2C9*2 (68 % of wild type), and 0.113 for CYP2C9*3 (19 % of wild type). If these estimates were used for flurbiprofen dose adjustment, taking 100 % for genotype *1/*1, an average reduction to 84 %, 60 %, 68 %, 43 %, and 19 % would result for genotype *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3, respectively. Due to the large individual variation within genotypes with coefficients of variation ≥ 20 % and supposing the normal distribution, one in three individuals would be out of the average optimum dose by more than 20 %, one in 20 would be 40 % off. Whether this problem also applies to other CYPs and other drugs has to be investigated case by case. Our data for the given example, however, puts the benefit of individual drug dosing to question, if it is exclusively based on genotype.

Influence of CYP2D6-genotype on tamoxifen efficacy in advanced breast cancer.

The influence of CYP2D6 genotype on the efficacy of tamoxifen (Tam) has been extensively analyzed in early breast cancer with conflicting results. However, there is only scarce data regarding this potential influence in advanced breast cancer (ABC). We hypothesize that Tam is more effective in patients with a functional CYP2D6 allele than in patients with impaired CYP2D6 activity. ABC patients with prior or ongoing palliative Tam treatment (20 mg/d) were eligible. Genomic DNA was extracted from blood (n = 51) and formalin-fixed, paraffin-embedded tissue (n = 43). CYP2D6*2, *3, *4, *5, *6, *10, *17, *29, *41, CYP2D6 duplication and multiplication were determined in blood and CYP2D6*4 in tissue samples. Primary endpoint was progression free survival (PFS); secondary endpoints included clinical benefit (CB), and overall survival (OS). The clinical charts were retrospectively analyzed regarding survival and treatment effects. Genotyping was performed blinded and clinical data were analyzed separately. 94 patients were identified with a median age of 59 years (29-90 years). In 6 patients genotyping did not show conclusive results, therefore these patients were excluded from further analysis. Genotyping results were as follows: 1.1 % ultrarapid, 84.1 % extensive, 3.4 % intermediate, and 11.4 % poor metabolizers. Patients without any fully functional allele (IM/IM, IM/PM, PM/PM) had a significant shorter PFS and OS compared to patients with at least one functional allele (EM/EM, EM/IM, EM/PM) (PFS: p = 0.017; HR = 2.19; 95 % CI 1.15-4.18; OS: p = 0.028; HR = 2.79; 95 % CI 1.12-6.99). The CB rate was 73 % for EM-group and 38.5 % for IM + PM-group (p = 0.019). Our results show a significant influence of the CYP2D6 genotype on the efficacy of Tam in the treatment of ABC. In contrast to the adjuvant setting, the evidence in the palliative setting is congruent. CYP2D6 testing in ABC should be considered.