Carcinogenicity of insulin analogues

There is epidemiological evidence that the use of some insulin analogues by diabetic patients is correlated with an increased cancer risk. In vitro exposure experiments revealed that insulin glargine (LANTUS) was the only commercial insulin analogue with an increased mitogenic potential. In the human body the injected insulin glargine is rapidly degraded into two main metabolites with a metabolic activity. These two compounds have a mitogenic potential that was not increased compared to regular insulin. Gene expression analysis on a stimulated MCF7-based cell line panel showed that the insulin-like growth factor 1 (IGF1) receptor was the main receptor involved in the insulin analogue induced mitogenic signaling. A chronic exposure experiment with the humanized p53R270H+/-WAPCre mouse model revealed that none of the commercially available insulin analogues induced mammary gland tumor multiplicity or decreased the tumor latency time. However, a follow-up whole transcriptome analysis indicated that some tumors in the insulin glargine treatment group had a higher Warburg potential. Altogether, these results suggest that insulin glargine exposure was not involved in tumor initiation but it might have affected tumor progression.UBL - phd migration 201

Exposure to bacterial signals does not alter pea aphids' survival upon a second challenge or investment in production of winged offspring

Pea aphids have an obligate nutritional symbiosis with the bacteria Buchneraaphidicola and frequently also harbor one or more facultative symbionts. Aphids are also susceptible to bacterial pathogen infections, and it has been suggested that aphids have a limited immune response towards such pathogen infections compared to other, more well-studied insects. However, aphids do possess at least some of the genes known to be involved in bacterial immune responses in other insects, and immune-competent hemocytes. One possibility is that immune priming with microbial elicitors could stimulate immune protection against subsequent bacterial infections, as has been observed in several other insect systems. To address this hypothesis we challenged aphids with bacterial immune elicitors twenty-four hours prior to live bacterial pathogen infections and then compared their survival rates to aphids that were not pre-exposed to bacterial signals. Using two aphid genotypes, we found no evidence for immune protection conferred by immune priming during infections with either Serratia marcescens or with Escherichia coli. Immune priming was not altered by the presence of facultative, beneficial symbionts in the aphids. In the absence of inducible immune protection, aphids may allocate energy towards other defense traits, including production of offspring with wings that could escape deteriorating conditions. To test this, we monitored the ratio of winged to unwinged offspring produced by adult mothers of a single clone that had been exposed to bacterial immune elicitors, to live E. coli infections or to no challenge. We found no correlation between immune challenge and winged offspring production, suggesting that this mechanism of defense, which functions upon exposure to fungal pathogens, is not central to aphid responses to bacterial infections.Toxicolog

Mammary gland tumor promotion by chronic administration of IGF1 and the insulin analogue AspB10 in the p53R270H/⁺WAPCre mouse model

Insulin analogues are structurally modified molecules with altered pharmaco-kinetic and -dynamic properties compared to regular human insulin used by diabetic patients. While these compounds are tested for undesired mitogenic effects, an epidemiological discussion is ongoing regarding an association between insulin analogue therapy and increased cancer incidence, including breast cancer. Standard in vivo rodent carcinogenesis assays do not pick up this possible increased carcinogenic potential. Here we studied the role of insulin analogues in breast cancer development. For this we used the human relevant mammary gland specific p53R270H/⁺WAPCre mouse model. Animals received life long repeated treatment with four different insulin (-like) molecules: normal insulin, insulin glargine, insulin X10 (AspB10) or insulin-like growth factor 1 (IGF1). Insulin-like molecules with strong mitogenic signaling, insulin X10 and IGF1, significantly decreased the time for tumor development. Yet, insulin glargine and normal insulin, did not significantly decrease the latency time for (mammary gland) tumor development. The majority of tumors had an epithelial to mesenchymal transition phenotype (EMT), irrespective of treatment condition. Enhanced extracellular signaling related kinase (Erk) or serine/threonine kinase (Akt) mitogenic signaling was in particular present in tumors from the insulin X10 and IGF1 treatment groups. These data indicate that insulin-like molecules with enhanced mitogenic signaling increase the risk of breast cancer development. Moreover, the use of a tissue specific cancer model, like the p53R270H/⁺WAPCre mouse model, is relevant to assess the intrinsic pro-carcinogenic potential of mitogenic and non-mitogenic biologicals such as insulin analogues. INTRODUCTION METHODS RESULTS CONCLUSION

Towards an advanced testing strategy for genotoxicity using image-based 2D and 3D HepG2 DNA damage response fluorescent protein reporters

In vitro assessment of mutagenicity is an essential component in the chemical risk assessment. Given the diverse modes of action by which chemicals can induce DNA damage, it is essential that these in vitro assays are carefully evaluated for their possibilities and limitations. In this study, we used a fluorescent protein HepG2 reporter test system in combination with high content imaging. To measure induction of the DNA damage response (DDR), we used three different green fluorescent protein (GFP) reporters for p53 pathway activation. These allowed for accurate quantification of p53, p21 and BTG2 (BTG anti-proliferation factor 2) protein expression and cell viability parameters at a single cell or spheroid resolution. The reporter lines were cultured as 2D monolayers and as 3D spheroids. Furthermore, liver maturity and cytochrome P450 enzyme expression were increased by culturing in an amino acid rich (AAGLY) medium. We found that culture conditions that support a sustained proliferative state (2D culturing with DMEM medium) give superior sensitivity when genotoxic compounds are tested that do not require metabolization and of which the mutagenic mode of action is dependent on replication. For compounds, which are metabolically converted to mutagenic metabolites, more differentiated HepG2 DDR reporters (e.g., 3D cultures) showed a higher sensitivity. This study stratifies how different culture methods of HepG2 DDR reporter cells can influence the sensitivity towards diverse genotoxicants and how this provides opportunities for a tiered genotoxicity testing strategy.Toxicolog

Mammary gland tumor promotion by chronic administration of IGF1 and the insulin analogue AspB10 in the p53(R270H/+)WAPCre mouse model

Insulin analogues are structurally modified molecules with altered pharmaco-kinetic and -dynamic properties compared to regular human insulin used by diabetic patients. While these compounds are tested for undesired mitogenic effects, an epidemiological discussion is ongoing regarding an association between insulin analogue therapy and increased cancer incidence, including breast cancer. Standard in vivo rodent carcinogenesis assays do not pick up this possible increased carcinogenic potential. Here we studied the role of insulin analogues in breast cancer development. For this we used the human relevant mammary gland specific p53R270H/⁺WAPCre mouse model. Animals received life long repeated treatment with four different insulin (-like) molecules: normal insulin, insulin glargine, insulin X10 (AspB10) or insulin-like growth factor 1 (IGF1). Insulin-like molecules with strong mitogenic signaling, insulin X10 and IGF1, significantly decreased the time for tumor development. Yet, insulin glargine and normal insulin, did not significantly decrease the latency time for (mammary gland) tumor development. The majority of tumors had an epithelial to mesenchymal transition phenotype (EMT), irrespective of treatment condition. Enhanced extracellular signaling related kinase (Erk) or serine/threonine kinase (Akt) mitogenic signaling was in particular present in tumors from the insulin X10 and IGF1 treatment groups. These data indicate that insulin-like molecules with enhanced mitogenic signaling increase the risk of breast cancer development. Moreover, the use of a tissue specific cancer model, like the p53R270H/⁺WAPCre mouse model, is relevant to assess the intrinsic pro-carcinogenic potential of mitogenic and non-mitogenic biologicals such as insulin analogues. INTRODUCTION METHODS RESULTS CONCLUSIONSToxicolog

Mapping the dynamics of Nrf2 antioxidant and NFκB inflammatory responses by soft electrophilic chemicals in human liver cells defines the transition from adaptive to adverse responses

A comprehensive understanding of the dynamic activation and crosstalk between different cellular stress response pathways that drive cell adversity is crucial in chemical safety assessment. Various chemicals have electrophilic properties that drive cell injury responses in particular oxidative stress signaling and inflammatory signaling. Here we used bacterial artificial chromosome-based GFP cellular stress reporters with live cell confocal imaging, to systematically monitor the differential modulation of the dynamics of stress pathway activation by six different soft electrophiles: sulforaphane, andrographolide, diethyl maleate, CDDO-Me, ethacrynic acid and tert-butyl hydroquinone. The various soft electrophiles showed differential potency and dynamics of Nrf2 activation and nuclear translocation. These differences in Nrf2 dynamics correlated with distinct activation pattern of Nrf2 downstream targets SRNX1 and HMOX1. All soft electrophiles caused a strong dose dependent suppression of a cytokine-induced NFĸB response represented by suppression of NFĸB nuclear oscillation and inhibition of the downstream target gene activation A20 and ICAM1, which followed the potency of Nrf2 modulation but occurred at higher concentration close to saturation of Nrf2 activation. RNAi-based depletion of RelA resulted in a prolonged presence of Nrf2 in the nucleus after soft electrophile treatment; depletion of Nrf2 caused the induction of NFĸB signaling and activation of its downstream targets A20 and ICAM1. A systematic transcriptome analysis confirmed these effects by soft electrophiles on Nrf2 and NFκB signaling crosstalk in human induced-pluripotent stem cell-derived hepatocyte-like cells. Altogether our data indicate that modulation of Nrf2 by soft electrophiles may have consequences for efficient inflammatory signaling.</p

The distribution and abundance of chironomids in high-latitude Eurasian lakes with respect to temperature and continentality: development and application of new chironomid-based climate-inference models in northern Russia

The large landmass of northern Russia has the potential to influence global climate through amplification of climate change. Reconstructing the climate in this region over millennial timescales is crucial for understanding the processes that affect the climate system. Chironomids, preserved in lake sediments, have the potential to produce high resolution, low error, quantitative summer air temperature reconstructions. Canonical correspondence analysis (CCA) of modern surface sediments from 100 high-latitude lakes, located in northern European Russia to central Siberia, showed chironomid distribution was primarily driven by July air temperatures. The strong relationship enabled the development of chironomid-inference model based on 81 lake and 89 taxa to reconstruct July air temperature. Analysis of a range of chironomid-inferred temperature model suggest the best to be a two component weighted averaging and partial least squares (WA-PLS model) with r2jack = 0.92 and RMSEP = 0.89°C. Comparison of species responses to July temperature with the Norwegian training set showed the temperature optima of individual species was 1-3°C in the Russian data regardless of modelling technique. This suggests that chironomid-based inference models should only be applied to sediment cores collected within the geographic source area of the training set. The differing responses between the Norwegian and Russian faunas led to the development of a 149 lake, 120 taxa chironomid-continentality inference model. The 2-component WA-PLS model was the minimal adequate model with r2jack = 0.73 and RMSEP = 9.9. Recent warming in the Arctic has been spatial and seasonal heterogeneous; in many areas warming is more pronounced in the spring and autumn leading to a lengthening of the summer, while summer temperatures have remained relatively stable. A continentality model has the potential to detect these seasonal changes in climate. The Russian inference model also improves the representation of a number of taxa, such as Corynocera oliveri-type, Constempellina and Paracladius, which frequently occur in subfossil assemblages from arctic Russian lakes, but are poorly represented in European training sets. These are cold-adapted taxa and their absence from the training sets could lead to overestimations of July temperatures in fossil samples where these taxa form a major component (for example see Andreev et al. 2005). Comparison of reconstructed July air temperatures and continentality indices from a tundra lake in north-east European Russia showed close agreement with local instrumental records over the past 70 years and suggests the models may produce reliable estimates of past climate