No abstract

Abstract. Methylmercury (MeHg) is a well-known environmental toxicant. With its lipophilic nature and high reactivity to sulfhydryl groups, it is widely distributed and accumulated in the body to damage cells. Oxidative stress is proposed as a major mechanism underlying the cytotoxic action of MeHg. In the present study, we found that L-glutamate (L-Glu) concentrationdependently increased MeHg cytotoxicity in HeLa S3 cells. The enhancement of the toxicity was accompanied by enhanced apoptosis, increased production of reactive oxygen species, and decreased glutathione level. An anti-oxidant N-acetylcysteine largely alleviated the cytotoxicity, suggesting enhanced oxidative stress behind L-Glu-elicited increase of MeHg toxicity. The effect was specific to L-Glu and L-α-aminoadipate, whereas D-Glu, L-aspartate, and D-aspartate were not effective. In addition, the cystine uptake by the cells was mostly mediated by a L-Glu / L-α-aminoadipate-sensitive amino acid transport system x − C . All these results suggest that the inhibition of system x − C by L-Glu underlies the enhancement of MeHg cytotoxicity. The enhancement was highly synergistic because MeHg and L-Glu alone had little toxic effect in the conditions used. This synergism was confirmed in neural cells (neuroblastoma cell lines). It is proposed that similar mechanisms may underlie the neural toxicity of MeHg, particularly in the locality of lesions characteristic of MeHg toxicity.

Urate is the final metabolite of purine in humans. Renal urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.

Background Neutrophil extracellular traps (NETs) were first described as web-like structures that trap and neutralize microbes at sites of infection. NETs are comprised of chromatin components and neutrophil cytoplasmic proteins. Because these components of NETs might provide an immunogenic substrate for autoimmune responses during regular encounters with commensal and pathogenic microbes, it has been reported that NETs are involved in autoimmunity such as SLE or ANCA associated vasculitis (AAV). It has also been reported that autoantibodies themselves for important cytoplasmic auto-antigens of neutrophils such as MPO or PR3 induce NETs production. However, the role of other neutrophil cytoplasmic antigens for the production of NETs is unclear. Objectives Thus, we investigated how auto-antibodies for these antigens other than PR3 and MPO are involved in NETs productions by neutrophils. Methods Human peripheral blood neutrophils were obtained from healthy donors. Neutrophils were seed onto Polystyrene chamber slide system and incubated with PMA (control) or PMA plus several kinds of anti-neutrophil cytoplasmic antigens for 3 hours. Cells were fixed and stained with Hoechst 33342 and Sytox Green for DNA and anti-MPO antibody. The percentage NETs producing cells and the quantitation of nuclear decondensation were analyzed using Image J software. We distinguished cells that released NETs fiber from cells that just died of NETosis. Results Anti-MPO antibody strongly promoted both NETosis induction and release of NETs fiber. Although anti-PR3 antibody promoted NETosis induction, it did not increase the release of NETs fiber. Antibody for cathepsin G, found in the azunophil granule, also promoted NETosis induction only. There were some other antibodies, such as anti-lactoferrin and anti-neutrophil elastase, that promoted both NETosis and release of NETs fiber. Conclusions It has been thought that ANCAs directed against granule proteins of neutrophils are implicated in the pathogenesis of AAV, partly because ANCAs promote NETs production by neutrophils. We showed that not only anti-MPO and anti-PR3 but also antibodies for other neutrophil cytoplasmic antigens promote NETs production. The mechanism these antibodies for cytoplasmic protein induce NETosis and how important they are for the pathogenesis of AAV are to be elucidated. Disclosure of Interest None Declared