Increased markers of heavy metal exposure and DNA methylation in cluster headache

Background/HypothesisRates of tobacco usage are higher in individuals with cluster headache. Tobacco usage exposes the user to a large variety of toxic compounds, detrimental to health, including heavy metals. We hypothesise that exposure to heavy metals through tobacco usage will drive epigenetic alterations in individuals with cluster headache.To elucidate the extent of heavy metal exposure and global epigenetic alterations arising from tobacco products in relation to cluster headache we performed a case-control study, using DNA methylation, gene and protein expression analysis to evaluate markers for smoking, epigenetic modifications and heavy metal exposure.MethodsRNA was purified from collected blood samples from 16 cluster headache participants and 16 control participants, converted to cDNA, and RNA expression quantified via RT-qPCR. This was done by investigating genes relevant for nicotine exposure; nicotinic acetylcholine receptors (nAChRs), heavy metal exposure; metallothionein (MT) family, markers of liver and kidney damage (ALT, Alb and NGAL), epigenetic enzymes for DNA methylation (DNMTs) and demethylation (TETs), normalized to the housekeeping gene TBP. MT3 protein expression was quantified using an ELISA kit in cerebrospinal fluid from 13 cluster headache participants and 13 control participants. The methylation status of CpG-rich regions of long-interspersed nucleotide element-1 (LINE-1), in bisulfite converted DNA of 20 smoking individuals with cluster headache, 33 non-smoking individuals with cluster headache and 26 control participants, was quantified by pyrosequencing.ResultsAn increased RNA expression of nicotinic acetylcholine receptor subunits CHRNA6 and CHRNA7 was found in the cluster headache group compared to controls. Additionally, there were an increased RNA expression of MT1H and increased protein levels of MT3 along with a decreased RNA expression of DNMT1. A positive correlation was found between the RNA expression of CHRNA7 and MT1H and a negative correlation was found between CHRNA6 and DNMT1.ConclusionIndirect evidence indicates heavy metal exposure in relation to cluster headache as well as a tendency for alterations in global DNA methylation. This supports the hypothesis that heavy metal exposure from smoking contributes towards the pathophysiology of cluster headache.