The TET enzymes are members of the 2-oxoglutarate-dependent dioxygenase family and comprise three isoenzymes in humans: TETs 1-3. These TETs convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA, and high 5-hmC levels are associated with active transcription. The importance of the balance in these modified cytosines is emphasized by the fact that TET2 is mutated in several human cancers, including myeloid malignancies such as acute myeloid leukemia (AML). We characterize here the kinetic and inhibitory properties of Tets and show that the K m value of Tets 1 and 2 for O 2 is 30 M, indicating that they retain high activity even under hypoxic conditions. The AML-associated mutations in the Fe 2؉ and 2-oxoglutaratebinding residues increased the K m values for these factors 30 -80-fold and reduced the V max values. Fumarate and succinate, which can accumulate to millimolar levels in succinate dehydrogenase and fumarate hydratase-mutant tumors, were identified as potent Tet inhibitors in vitro, with IC 50 values ϳ400 -500 M. Fumarate and succinate also down-regulated global 5-hmC levels in neuroblastoma cells and the expression levels of some hypoxia-inducible factor (HIF) target genes via TET inhibition, despite simultaneous HIF␣ stabilization. The combination of fumarate or succinate treatment with TET1 or TET3 silencing caused differential effects on the expression of specific HIF target genes. Altogether these data show that hypoxia-inducible genes are regulated in a multilayered manner that includes epigenetic regulation via TETs and 5-hmC levels in addition to HIF stabilization.The 2-oxoglutarate-dependent dioxygenases (2-OGDDs) 2 comprise an enzyme family of about 70 members in humans (1, 2). These enzymes all share the same basic reaction mechanism, in which the substrate is hydroxylated by molecular oxygen in the presence of a divalent metal cofactor (most commonly Fe 2ϩ ) and the 2-oxoglutarate cosubstrate is decarboxylated to succinate and CO 2 (1). The substrates for 2-OGDDs vary from proteins to DNA, RNA, and fatty acids (1). Interestingly, a large number of 2-OGDDs act on the chromatin structure, most notably the ten-eleven-translocation 5-methylcytosine dioxygenases (TETs) and the Jumonji domain-containing histone demethylases (1-3). The stability of the ␣ subunit of the key regulator of the hypoxia response, the hypoxia-inducible factor (HIF), is also regulated by 2-OGDDs, namely the HIF-prolyl 4-hydroxylases (HIF-P4Hs), also known as PHDs and EglNs (1, 2, 4).The TET enzymes convert the 5-methylcytosine (5-mC) in DNA sequentially to 5-hydroxymethylcytosine (5-hmC), 5-formylcytocine, and 5-carboxylcytocine, leading to DNA demethylation (3, 5-7). 5-hmC is also likely to have its own epigenetic function beyond simply being a demethylating base (3). The highest levels of 5-hmC are found in stem cells of various origins and in neural tissues (6, 7). There are three human TET isoenzymes. TET1 is highly expressed in embryonic stem cells, whereas TETs 2 and 3 are required for normal hematopoiesis...

Background: Wood combustion emissions have been studied previously either by in vitro or in vivo models using collected particles, yet most studies have neglected gaseous compounds. Furthermore, a more accurate and holistic view of the toxicity of aerosols can be gained with parallel in vitro and in vivo studies using direct exposure methods. Moreover, modern exposure techniques such as air-liquid interface (ALI) exposures enable better assessment of the toxicity of the applied aerosols than, for example, the previous state-of-the-art submerged cell exposure techniques. Methods: We used three different ALI exposure systems in parallel to study the toxicological effects of spruce and pine combustion emissions in human alveolar epithelial (A549) and murine macrophage (RAW264.7) cell lines. A wholebody mouse inhalation system was also used to expose C57BL/6 J mice to aerosol emissions. Moreover, gaseous and particulate fractions were studied separately in one of the cell exposure systems. After exposure, the cells and animals were measured for various parameters of cytotoxicity, inflammation, genotoxicity, transcriptome and proteome.

Background: Emissions from road traffic are under constant discussion since they pose a major threat to human health despite the increasingly strict emission targets and regulations. Although the new passenger car regulations have been very effective in reducing the particulate matter (PM) emissions, the aged car fleet in some EU countries remains a substantial source of PM emissions. Moreover, toxicity of PM emissions from multiple new types of biobased fuels remain uncertain and different driving conditions such as the sub-zero running temperature has been shown to affect the emissions. Overall, the current literature and experimental knowledge on the toxicology of these PM emissions and conditions is scarce. Methods: In the present study, we show that exhaust gas PM from newly regulated passenger cars fueled by different fuels at sub-zero temperatures, induce toxicological responses in vitro. We used exhaust gas volume-based PM doses to give us better insight on the real-life exposure and included one older diesel car to estimate the effect of the new emissions regulations. Results: In cars compliant with the new regulations, gasoline (E10) displayed the highest PM concentrations and toxicological responses, while the higher ethanol blend (E85) resulted in slightly lower exhaust gas PM concentrations and notably lower toxicological responses in comparison. Engines powered by modern diesel and compressed natural gas (CNG) yielded the lowest PM concentrations and toxicological responses. Conclusions: The present study shows that toxicity of the exhaust gas PM varies depending on the fuels used. Additionally, concentration and toxicity of PM from an older diesel car were vastly higher, compared to contemporary vehicles, indicating the beneficial effects of the new emissions regulations.

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