Grouping approaches based on structure alone are insufficient to conclude about toxicological properties-the example of monoamine-based chelates.

Aminocarboxylic acid (monoamine-based) chelating agents such as GLDA, MGDA, NTA, and EDG are widely used in a variety of products and processes. In the European Union, based on the Green Deal and the Chemicals Strategy for Sustainability (CSS), there is an increasing tendency to speed up chemical hazard evaluation and to regulate chemicals by grouping substances based on molecular structure similarity. Recently, it was proposed to group polycarboxylic acid monoamines, hydroxy derivatives and their salts with monovalent cations, and to consider all group members as potential carcinogens based on the official CLP classification of one group member, viz. NTA, which is classified as suspected carcinogen Cat. 2. In this review, we show that a grouping approach for harmonized classification and labeling based on molecular structure alone, disregarding existing animal test data as well as current scientific and regulatory knowledge, would result in incorrect classification. Using such a simplistic, although considered pragmatic approach, classification of all group members upfront would not improve protection of human health. Instead, it could not only lead to unnecessary additional vertebrate animal testing but also to onerous and disproportionate restrictions being placed on the use of these valuable substances; some of these even being considered as green chemicals.

Regulation of Claspin by the p38 stress-activated protein kinase protects cells from DNA damage.

Stress-activated protein kinases (SAPKs) enhance survival in response to environmental changes. In yeast, the Hog1 SAPK and Mrc1, a protein required for DNA replication, define a safeguard mechanism that allows eukaryotic cells to prevent genomic instability upon stress during S-phase. Here we show that, in mammals, the p38 SAPK and Claspin-the functional homolog of Mrc1-protect cells from DNA damage upon osmostress during S-phase. We demonstrate that p38 phosphorylates Claspin and either the mutation of the p38-phosphorylation sites in Claspin or p38 inhibition suppresses the protective role of Claspin on DNA damage. In addition, wild-type Claspin but not the p38-unphosphorylatable mutant has a protective effect on cell survival in response to cisplatin treatment. These findings reveal a role of Claspin in response to chemotherapeutic drugs. Thus, this pathway protects S-phase integrity from different insults and it is conserved from yeast to mammals.

Loss of pro-apoptotic Bax and Bak increases resistance to dihydroartemisinin-mediated cytotoxicity in normoxia but not in hypoxia in HCT116 colorectal cancer cells.

Tumor hypoxia is a major biological factor that drives resistance to chemotherapy and radiotherapy. We previously demonstrated that the pro-oxidative drug dihydroartemisinin (DHA) efficiently targeted normoxic and hypoxic cancer cells. Although well studied in normoxia, the mechanism behind DHA-mediated cytotoxicity in hypoxia is insufficiently explored. Here, we analyzed the effect of DHA in HCT116 wild type (wt) cells and in HCT116 Bax-/-Baksh cells with a defective intrinsic apoptosis pathway. Normoxic HCT116 wt cells underwent apoptosis shortly after treatment with DHA. Autophagy-associated cell death contributes to short-term cytotoxicity of DHA in normoxia. These cells switched to an apoptosis- and autophagy-independent cell death after treatment with DHA in hypoxia and displayed similar long-term survival in response to DHA in normoxia and hypoxia. In HCT116 Bax-/-Baksh cells, DHA induced cell cycle arrest shortly after treatment irrespective of oxygen levels. Later, HCT116 Bax-/-Baksh cells induced a delayed cell death after treatment with DHA in hypoxia followed by return to normoxia, while treatment with DHA in normoxia was hardly toxic. We identified lower glutathione levels in hypoxic HCT116 cells which correlated with higher lipid peroxidation after treatment with DHA. Moreover, insufficient expression of Bax/Bak counteracted hypoxia-mediated downregulation of mitochondrial function, thereby adding to DHA-induced ROS production and lipid peroxidation in hypoxia. In summary, DHA-mediated cytotoxicity in normoxia depended on Bax/Bak expression, while cytotoxicity after treatment with DHA in hypoxia was regulated independently of Bax/Bak in HCT116 colorectal cancer cells.

Activation of anti-oxidant Keap1/Nrf2 pathway modulates efficacy of dihydroartemisinin-based monotherapy and combinatory therapy with ionizing radiation.

The efficacy of radiotherapy depends not only on DNA damage but also on ROS production, both induced by ionizing radiation. Massive ROS production can induce cell death or activate protective pathways such as Keap1/Nrf2 pathway, which regulates intracellular cysteine availability through upregulation of SLC7A11, a subunit of xCT transporter, and subsequently glutathione synthesis, thus improving antioxidative defense. The anti-malaria drug dihydroartemisinin (DHA) shows anti-neoplastic potential. Previous publications suggested that DHA increased ROS production. We intended to enhance oxidative stress with DHA to improve the efficacy of radiotherapy. Therefore, we first analyzed the oxidative response to DHA in HCT116 colorectal and NCI-H460 lung adenocarcinoma cells. In response to DHA, we detected lipid peroxidation and protein oxidation, which resulted in mitochondrial damage and eventually in iron-dependent cell death. Concurrently, DHA activated Keap1/Nrf2 pathway in HCT116 cells, leading to increased SLC7A11 expression and glutathione level. In Keap1-mutant NCI-H460 cells, Nrf2 was constantly activated and responsible for high SLC7A11 and glutathione levels. Pancancer analysis revealed that lung cancer is the tumor entity with the most frequent Keap1 alterations. Although NCI-H460 cells reacted more refractory to DHA-induced cell death than HCT116 cells, eradication of clonogenic cells by DHA was more efficient in both cell lines when Keap1/Nrf2 pathway was inhibited. When applied simultaneously, radiotherapy and DHA more efficiently eradicated clonogenic cells than either therapy alone, but treatment schedule can mitigate the combinatory effect in HCT116 cells. In summary, DHA improved efficacy of radiotherapy, but treatment schedule must be considered with care especially in Keap1-wildtype cells.