Effect of Mn²⁺ on sirtuins and the DNA damage response in C. elegans

Abstract

Manganese (Mn) is an essential trace element found ubiquitously in almost all soils and ground water. In food, Mn is mainly found in plant foods such as legumes, nuts and whole grain products. The estimated daily requirement for adults is about 2.3 mg and due to its wide occurrence in food products Mn deficiency is a very rare concern. However, Mn is in humans capable to accumulate in the brain, which in excess is associated with dementia and Parkinson's-like movement disorders, referred to as manganism. Excessive exposure to Mn can cause among others impaired energy metabolism with subsequent mitochondrial dysfunction and radical production. Radicals and reactive oxygen species (ROS) may modify macromolecules like lipids and proteins, as well as the DNA. ROS are tolerable in moderate amounts, but in disbalance with antioxidants it leads to oxidative stress, which in turn can induce among others oxidative DNA damage and strand breaks. To prevent DNA damage, organisms like the nematode Caenorhabditis elegans (C. elegans) are equipped with sophisticated DNA repair and DNA damage response systems, for example the base excision repair or single strand break repair. Parts of these repair systems that we were interested in are sirtuins and the poly-ADP-ribosylation (PARylation). PARylation is catalyzed by poly-(ADP-ribosyl) polymerases in case of DNA damages and degraded by poly-(ADP-ribosyl) glycohydrolases after the damage is repaired. To investigate the DNA damage response in relation to Mn overexposure, the multicellular organism C. elegans was used. We examined the effects of Mn overexposure on the DNA damage response in C. elegans and inquired, whether the absence of the proteins sirtuin 1 (sirt 1) or poly-(ADP-ribosyl) glycohydrolases 1 and 2 (parg 1 and 2) leads to hypersensitivity to Mn and an altered DNA damage response. Therefore, we used deletion mutants, which did not express the desired protein. For all nematode strains, a survival assay was performed following incubation with Mn for 1 and 4 h (L4 larvae) and bioavailability was determined by optical emission spectrometry with inductively coupled plasma (ICP-OES). In addition, the impact of Mn, tert-butyl-hydroperoxide (t-BOOH) and Olaparib on PARylation was investigated by usage of liquid chromatography with mass spectrometry coupling (LC-MS/MS). We were able to show that t-BOOH induces PARylation and in contrast Olaparib has an inhibitory impact. However, Mn showed no significant effect. In addition, parg 1 was shown to be the prevailing poly-(ADP-ribosyl) glycohydrolase in C. elegans. Altogether, we have shown that PARylation in C. elegans can accurately be measured by LC-MS/MS and also that oxidative stress inducers such as t-BOOH and PAR inhibitors like Olaparib have an impact on PARylation in nematodes. Nevertheless, we had to conclude that the incubation with Mn has no effect on the level of PARylation in any of the strains. Therefore, it is necessary to conduct further studies to investigate the relationship between Mn overexposure and the DNA dam...