Substantiate a read-across hypothesis by using transcriptome data—A case study on volatile diketones

This case study explores the applicability of transcriptome data to characterize a common mechanism of action within groups of short-chain aliphatic α-, β-, and γ-diketones. Human reference in vivo data indicate that the α-diketone diacetyl induces bronchiolitis obliterans in workers involved in the preparation of microwave popcorn. The other three α-diketones induced inflammatory responses in preclinical in vivo animal studies, whereas beta and gamma diketones in addition caused neuronal effects. We investigated early transcriptional responses in primary human bronchiolar (PBEC) cell cultures after 24 h and 72 h of air-liquid exposure. Differentially expressed genes (DEGs) were assessed based on transcriptome data generated with the EUToxRisk gene panel of Temp-O-Seq®. For each individual substance, genes were identified displaying a consistent differential expression across dose and exposure duration. The log fold change values of the DEG profiles indicate that α- and β-diketones are more active compared to γ-diketones. α-diketones in particular showed a highly concordant expression pattern, which may serve as a first indication of the shared mode of action. In order to gain a better mechanistic understanding, the resultant DEGs were submitted to a pathway analysis using ConsensusPathDB. The four α-diketones showed very similar results with regard to the number of activated and shared pathways. Overall, the number of signaling pathways decreased from α-to β-to γ-diketones. Additionally, we reconstructed networks of genes that interact with one another and are associated with different adverse outcomes such as fibrosis, inflammation or apoptosis using the TRANSPATH-database. Transcription factor enrichment and upstream analyses with the geneXplain platform revealed highly interacting gene products (called master regulators, MRs) per case study compound. The mapping of the resultant MRs on the reconstructed networks, visualized similar gene regulation with regard to fibrosis, inflammation and apoptosis. This analysis showed that transcriptome data can strengthen the similarity assessment of compounds, which is of particular importance, e.g., in read-across approaches. It is one important step towards grouping of compounds based on biological profiles

Effects of spatial variation in dose delivery: what can we learn from radon-related lung cancer studies?

Exposure to radon progeny results in heterogeneous dose distributions in many different spatial scales. The aim of this review is to provide an overview on the state of the art in epidemiology, clinical observations, cell biology, dosimetry, and modelling related to radon exposure and its association with lung cancer, along with priorities for future research. Particular attention is paid on the effects of spatial variation in dose delivery within the organs, a factor not considered in radiation protection. It is concluded that a multidisciplinary approach is required to improve risk assessment and mechanistic understanding of carcinogenesis related to radon exposure. To achieve these goals, important steps would be to clarify whether radon can cause other diseases than lung cancer, and to investigate radon-related health risks in children or persons at young ages. Also, a better understanding of the combined effects of radon and smoking is needed, which can be achieved by integrating epidemiological, clinical, pathological, and molecular oncology data to obtain a radon-associated signature. While in vitro models derived from primary human bronchial epithelial cells can help to identify new and corroborate existing biomarkers, they also allow to study the effects of heterogeneous dose distributions including the effects of locally high doses. These novel approaches can provide valuable input and validation data for mathematical models for risk assessment. These models can be applied to quantitatively translate the knowledge obtained from radon exposure to other exposures resulting in heterogeneous dose distributions within an organ to support radiation protection in general

A read-across study on diketones

The alpha-diketone diacetyl (2,3-butanedione) is used as butter flavorant in the microwave popcorn industry and suspected to cause a rare obstructive pulmonary disease (popcorn lung) in workers. E-cigarette smoking is also a potential route of exposure to diacetyl and the potentially less toxic alternative 2,3-pentanedione. In this case study we are developing new-approach methodologies (NAMs) to reduce the uncertainty of read-across predictions. Primary bronchial epithelial cells were exposed to the alpha-diketones diacetyl, 2,3-pentanedione and 2,3-hexanedione or the beta-diketone 2,4-pentanedione under air-liquid interface (ALI) conditions using the P.R.I.T.® ExpoCube® device. This unique exposure device provides a highly efficient exposure situation by preventing contact between the test compound and the culture medium. Primary human bronchial epithelial cells (PBECs) from tumor-free lung tissues from four donors were differentiated into airway epithelium at ALI conditions. Test atmospheres were generated by evaporation of the volatile test compounds and diluted in clean air. FT-IR spectroscopy enabled online analysis of the exposure concentration. PBECs were exposed for 1h once or repeatedly on three consecutive days. Cellular viability was measured by LDH-leakage and monolayer integrity by measuring the transepithelial electrical resistance (TEER) 24h after the final exposure. Exposure concentrations ranged from 100 to 1840 ppm (diacetyl) and from 50 to 5000 ppm (other diketone analogues). Lowest observed adverse effect levels (LOAELs) were lower after repeated exposure compared to the single exposure protocol. NAM read outs correlated with alpha- and beta-diketone specific toxicity since 2,4-pentanedione displayed significantly lower cytotoxic effects in comparison to its analogues. In conclusion, in vitro testing of volatile gases enabled ranking of test compounds with regard to inhalation toxicity. Moreover, further comprehensive evaluations may adequately predict specific lung toxicity of structurally related compounds. Acknowledgement: This project received funding from the European Union’s Horizon 2020 research and innovation programme (grant agreement No 681002)

Xenobiotic metabolism in differentiated human bronchial epithelial cells

Pathogenesis and treatment of chronic pulmonary disease

Detection of genotoxic and non-genotoxic renal carcinogens in vitro in NRK-52E cells using a transcriptomics approach

There is a need to develop quick, cheap, sensitive and specific methods to detect the carcinogenic potential of chemicals. Currently there is no in vitro model system for reliable detection of non-genotoxic carcinogens (NGTX) and current tests for detection of genotoxic carcinogens (GTX) can have low specificity. A transcriptomics approach holds promise and a few studies have utilised this technique. However, the majority of these studies have examined liver carcinogens with little work on renal carcinogens which may act via renal-specific NGTX mechanisms. In this study the normal rat renal cell line (NRK-52E) was exposed to sub-toxic concentrations of selected rat renal carcinogens and non-carcinogens (NC) for 6 h, 24 h and 72 h. Renal carcinogens were classified based on their presumed mode of action into GTX and NGTX classes. A whole-genome transcriptomics approach was used to determined genes and pathways as potential signatures for GTX, NGTX and those common to both carcinogenic events in vitro. For some of the GTX compounds an S9 drug metabolising system was included to aid pro-carcinogen activation. Only three genes were commonly deregulated after carcinogen (GTX + NGTX) exposure, one Mdm2 with a detection rate of 67%, and p21 and Cd55 with a detection rate of 56%. However, examination of enriched pathways showed that 3 out of 4 NGTX carcinogens and 4 out of 5 GTX carcinogens were related to known pathways involved in carcinogenesis giving a detection rate of 78%. In contrast, none of the NC chemicals induced any of the above genes or well-established carcinogenic pathways. Additionally, five genes (Lingo1, Hmox1, Ssu72, Lyrm and Usp9x) were commonly altered with 3 out of 4 NGTX carcinogens but not with NC or GTX carcinogens. However, there was no clear separation of GTX and NGTX carcinogens using pathway analysis with several pathways being common to both classes. The findings presented here indicate that the NRK-52E cell line has the potential to detect carcinogenic chemicals, although a much larger number of chemicals need to be used to confirm these findings