Impaired suppressive activities of human MUTYH variant proteins against oxidative mutagenesis

AIM:To investigate the suppressive activity of MUTYH variant proteins against mutations caused by oxidative lesion,8-hydroxyguanine(8OHG),in human cells.METHODS:p.R154H,p.M255V,p.L360P,and p.P377L MUTYH variants,which were previously found in patients with colorectal polyposis and cancer,were selected for use in this study.Human H1299 cancer cell lines inducibly expressing wild-type(WT) MUTYH(type 2) or one of the 4 above-mentioned MUTYH variants were established using the piggyBac transposon vector system,enabling the genomic integration of the transposon sequence for MUTYH expression.MUTYH expression was examined after cumate induction using Western blotting analysis and immunofluorescence analysis.The intracellular localization of MUTYH variants tagged with FLAG was also immunofluorescently examined.Next,the mutation frequency in the supF of the shuttle plasmid pMY189 containing a single 8OHG residue at position 159 of the supF was compared between empty vector cells and cells expressing WT MUTYH or one of the 4 MUTYH variants using a supF forward mutation assay.RESULTS:The successful establishment of human cell lines inducibly expressing WT MUTYH or one of the 4 MUTYH variants was concluded based on the detection of MUTYH expression in these cell lines after treatment with cumate.All of the MUTYH variants and WT MUTYH were localized in the nucleus,and nuclear localization was also observed for FLAG-tagged MUTYH.The mutation frequency of supF was 2.2 × 10-2 in the 8OHG-containing pMY189 plasmid and 2.5 × 10-4 in WT pMY189 in empty vector cells,which was an 86-fold increase with the introduction of 8OHG.The mutation frequency(4.7 × 10-3) of supF in the 8OHG-containing pMY189 plasmid in cells overexpressing WT MUTYH was significantly lower than in the empty vector cells(P < 0.01).However,the mutation frequencies of the supF in the 8OHG-containing pMY189 plasmid in cells overexpressing the p.R154H,p.M255V,p.L360P,or p.P377L MUTYH variant were 1.84 × 10-2,1.55 × 10-2,1.91 × 10-2,and 1.96 × 10-2,respectively,meaning that no significant difference was observed in the mutation frequency between the empty vector cells and cells overexpressing MUTYH mutants.CONCLUSION:The suppressive activities of p.R154H,p.M255V,p.L360P,and p.P377L MUTYH variants against mutations caused by 8OHG are thought to be severely impaired in human cells.

Evaluation of pharmaceutical activities of G-protein coupled receptor targeted pharmaceuticals in Chinese wastewater effluent

The occurre nce of biologically active pharmaceuticals in aquatic enviro nments raised the potential risks to aquatic species.Among these marketed biological active pharmaceuticals,it has been estimated that40%of them target G-protein-coupled receptors(GPCRs).We have illustrated pharmaceutical activities of GPCR targeted pharmaceuticals in English and Japanese wastewater by the in vitro transforming growth factor-α(TGFα)shedding assay.However,as the most important producer and consumer of pharmaceuticals,the occurrence of GPCR targeted pharmaceuticals in China had remained unclear.In this study,we investigated the pharmaceutical activities of GPCR targeted pharmaceuticals in secondary effluents of Chinese wastewater treatment plants.We discovered antagonistic activities against angiotensin(AT1)receptor at up to 7.2×102 ng-valsartan-equivalent quantity/L in Chinese wastewater for the first time as well as agonistic activities against dopamine(D2)receptor.Furthermore,in parallel with the assay,we determined concentrations of GPCR targeted pharmaceuticals in target wastewater by liquid chromatography coupled with tandem mass spectrometry(LC-MS/MS).Through the comparison of predicted antagonistic activities calculated by concentrations and potency values from the assay,we found that the measured antagonistic activities against AT1 receptor from the assay were higher than the predicted AT1 activities from valsartan,irbesartan,and losartan,indicating the potential existence of other unknown AT1 antagonists in wastewater.