Role for human arylamine N-acetyltransferase 1 in the methionine salvage pathway.

The Phase II drug metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) has been implicated in the growth and survival of cancer cells, although the mechanisms that underlies these effects are unknown. Here, a focused metabolomics approach was used to identify changes in folate catabolism as well as the S-adenosylmethionine (SAM) cycle following NAT1 knockdown with shRNA. Although acetylation of the folate catabolite p-aminobenzoylglutamate (pABG) was significantly decreased, there were no changes in intracellular pABG or the various components of the SAM cycle. By contrast, the flux of homocysteine in the medium was different following NAT1 knockdown after the methionine content was exhausted suggesting a need for this metabolite in methionine synthesis. Analysis of the growth of various cancer cells in methylthioadenosine-supplemented medium showed that NAT1 knockdown inhibited the methionine salvage pathway in HT-29 cells but not in HeLa or MDA-MB-436 cells. The cause of this was a low level of expression of the isomerase MRI-1 in the HT-29 cells. Knocking down both NAT1 and MRI-1 in HeLa cells with siRNA further demonstrated a redundancy between these 2 enzymes, although direct isomerase activity by NAT1 could not be demonstrated. The present study has identified a novel endogenous role for human NAT1 that might explain some of its effects in cancer cell growth and survival.

Autoantibodies against homocysteinylated protein in a mouse model of folate deficiency-induced neural tube defects.

BACKGROUND: Periconceptional supplementation with folic acid results in a significant reduction in the incidence of neural tube defects (NTDs). Nonetheless, NTDs remain a leading cause of perinatal morbidity and mortality worldwide, and the mechanism(s) by which folate exerts its protective effects are unknown. Homocysteine is an amino acid that accumulates under conditions of folate-deficiency, and is suggested as a risk factor for NTDs. One proposed mechanism of homocysteine toxicity is its accumulation into proteins in a process termed homocysteinylation. METHODS & RESULTS: Herein, we used a folate-deficient diet in pregnant mice to demonstrate that there is: (i) a significant inverse correlation between maternal serum folate levels and serum homocysteine; (ii) a significant positive correlation between serum homocysteine levels and titers of autoantibodies against homocysteinylated protein; and (iii) a significant increase in congenital malformations and NTDs in mice deficient in serum folate. Furthermore, in mice administered the folate-deplete diet before conception, supplementation with folic acid during the gestational period completely rescued the embryos from congenital defects, and resulted in homocysteinylated protein titers at term that are comparable to that of mice administered a folate-replete diet throughout both the pre- and postconception period. These results demonstrate that a low-folate diet that induces NTDs also increases protein homocysteinylation and the subsequent generation of autoantibodies against homocysteinylated proteins. CONCLUSION: These data support the hypotheses that homocysteinylation results in neo-self antigen formation under conditions of maternal folate deficiency, and that this process is reversible with folic acid supplementation.

5-methyl-tetrahydrofolate and the S-adenosylmethionine cycle in C57BL/6J mouse tissues: gender differences and effects of arylamine N-acetyltransferase-1 deletion.

Folate catabolism involves cleavage of the C(9)-N(10) bond to form p-aminobenzoylgluamate (PABG) and pterin. PABG is then acetylated by human arylamine N-acetyltransferase 1 (NAT1) before excretion in the urine. Mice null for the murine NAT1 homolog (Nat2) show several phenotypes consistent with altered folate homeostasis. However, the exact role of Nat2 in the folate pathway in vivo has not been reported. Here, we examined the effects of Nat2 deletion in male and female mice on the tissue levels of 5-methyl-tetrahydrofolate and the methionine-S-adenosylmethionine cycle. We found significant gender differences in hepatic and renal homocysteine, S-adenosylmethionine and methionine levels consistent with a more active methionine-S-adenosylmethionine cycle in female tissues. In addition, methionine levels were significantly higher in female liver and kidney. PABG was higher in female liver tissue but lower in kidney compared to male tissues. In addition, qPCR of mRNA extracted from liver tissue suggested a significantly lower level of Nat2 expression in female animals. Deletion of Nat2 affected liver 5- methyl-tetrahydrofolate in female mice but had little effect on other components of the methionine-S-adenosylmethionine cycle. No N-acetyl-PABG was observed in any tissues in Nat2 null mice, consistent with the role of Nat2 in PABG acetylation. Surprisingly, tissue PABG levels were similar between wild type and Nat2 null mice. These results show that Nat2 is not required to maintain tissue PABG homeostasis in vivo under normal conditions.