Interactive Effects of N6AMT1 and As3MT in Arsenic Biomethylation

Abstract

In humans, arsenic is primarily metabolized by arsenic (+3 oxidation state) methyltransferase (As3MT) to yield both trivalent and pentavalent methylated metabolites. We recently reported that the putative N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) can biotransform monomethylarsonous acid (MMA(III)) to dimethylarsinic acid, conferring resistance of human cells to arsenic exposure. To further decipher the role of N6AMT1 and its interaction with As3MT in arsenic biomethylation, we examined the relative contribution of N6AMT1 and As3MT in metabolizing arsenic using several newly modified UROtsa human urothelial cells, ie, UROtsa cells with either a constant level of N6AMT1 or As3MT in combination with an inducible level of As3MT or N6AMT1, respectively. Our analysis confirmed the involvement of N6AMT1 in MMA(III) biomethylation but not for inorganic arsenic. In a comparable level of N6AMT1 and As3MT, the effect of N6AMT1 mediated MMA(III) biomethylation was obscured by the action of As3MT. Furthermore, we showed that the levels of N6AMT1 and As3MT proteins varied among and within human normal and cancerous tissues. Overall, the data showed that N6AMT1 has a role in MMA(III) biomethylation, but its effect is relatively minor and limited compared with As3MT. In addition, the varied levels and distributions of N6AMT1 and As3MT among human tissues may potentially contribute to the tissue specificity and susceptibility to arsenic toxicity and carcinogenicity.

Keywords: As3MT; N6AMT1; arsenic metabolism.

FIG. 1.

Establish stabilized UROtsa cell lines with a constant level of N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) or arsenic (+3 oxidation state) methyltransferase (As3MT) in combination with an inducible level of As3MT or N6AMT1. A, Representative images of UROtsa cells infected with either retrovirus control or retrovirus containing N6AMT1 or As3MT, showing the cell lines were positive with the expression of ZsGreen fluorescent marker; B, Representative images of established UROtsa cell lines post 24 h Dox treatment. Administration of Dox induced a dose-dependent increase of the m-cherry fluorescence, indicating a workable induction cell model.

FIG. 2.

Quantification of N6AMT1 and As3MT protein levels in established UROtsa cell lines. Western blot analysis shows increased levels of N6AMT1 and As3MT proteins in URO-R/N6 and URO-R/As3 cells, respectively. A gradually increased level of N6AMT1 or As3MT was observed post the administration of Dox in URO-R/As3-L/N6 and URO-R/N6-L/As3 cells.

FIG. 3.

Elevated levels of human N6AMT1 and/or As3MT protein have no overall impact on monomethylarsonous acid (MMA^III)-induced cytotoxicity. A dose-dependent decrease in viability of all cell lines was observed after MMA^III exposure (A, URO-R; B, URO-R/N6; C, URO-R/As3; D, URO-R-L; E, URO-R/N6-L/As3, and F, URO-R/As3-L/N6). Jonckheere’s 2-sided trend test was performed and showed that there were no significant differences of MMA^III-induced cytotoxicity in the comparison between any cell lines (P value > .05). Elevated As3MT protein levels in UROtsa cells led to resistance to MMA^III at 1 and 3µM levels (D and F; *P value < .05). Dox induced increase of As3MT protein level could alter the susceptibility of UROtsa cells to MMA^III exposure when N6AMT1 protein presented (E; ^#P value < .05).