1. Biotransformation of L-cysteine S-conjugates and N-acetyl-L-cysteine S-conjugates of the sevoflurane degradation product fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (compound A) in human kidney in vitro: interindividual variability in N-acetylation, N-deacetylation, and beta-lyase-catalyzed metabolism
T Gul Altuntas, Evan D Kharasch Drug Metab Dispos. 2002 Feb;30(2):148-54. doi: 10.1124/dmd.30.2.148.
Fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE; 1) is a fluoroalkene formed by the base-catalyzed degradation of the anesthetic sevoflurane. FDVE is nephrotoxic in rats. In both rats and humans, FDVE undergoes glutathione-dependent conjugation, cleavage to cysteine S-conjugates, and renal beta-lyase-catalyzed metabolism to reactive intermediates, which may cause nephrotoxicity. Interindividual variability in renal metabolism of FDVE is unknown. Therefore, this investigation quantified beta-lyase-catalyzed bioactivation and N-acetyltransferase-catalyzed inactivation of FDVE cysteine S-conjugates and reactivation of mercapturates by N-deacetylase in cytosol and microsomes from 20 human kidneys. In cytosol, N-acetylation ranged from 0.008 to 0.045 (0.024 +/- 0.01) nmol of mercapturate/mg/min and 0.001 to 0.07 (0.024 +/- 0.02) nmol of mercapturate/mg/min for alkane and alkene cysteine S-conjugates, respectively. Similar results for microsomal N-acetylation were obtained; N-acetylation ranged from 0.005 to 0.055 (0.025 +/- 0.02) nmol of mercapturate/mg/min and 0.001 to 0.06 (0.030 +/- 0.02) nmol of mercapturate/mg/min for alkane and alkene cysteine S-conjugates, respectively. Beta-lyase-catalyzed metabolism to pyruvate varied from 0.004 to 0.14 (0.051 +/- 0.04) nmol/mg/min and from 0.10 to 0.40 (0.26 +/- 0.08) nmol/mg/min for alkane and alkene cysteine-S-conjugates, respectively. N-deacetylation of mercapturates ranged from 0.8 to 2.5 (1.25 +/- 0.57) nmol of cysteine S-conjugate formed/mg/min and 0.05 to 0.37 (0.17 +/- 0.10) nmol of cysteine S-conjugate formed/mg/min for alkane and alkene FDVE mercapturates. Cytosolic cysteine S-conjugates metabolism by renal beta-lyase predominated over N-acetylation (ratio of activities was 0.2-6 and 3-146 for the alkane and alkene cysteine S-conjugates). N-deacetylation predominated over N-acetylation (ratio of activities was 20-205 and 2-54 for alkane and alkene S-conjugates). There was considerable (up to 50-fold) interindividual variability in rates of FDVE toxication (beta-lyase metabolism and N-deacetylation) and detoxication. This interindividual variability may effect individual susceptibility to the nephrotoxicity of FDVE and other haloalkenes.
2. Reactive intermediate formation from the 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A)-derived cysteine S-conjugate S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine in pyridoxal model systems
Zeen Tong, M W Anders Chem Res Toxicol. 2002 May;15(5):623-8. doi: 10.1021/tx010148b.
2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A) is a degradation product of the anesthetic sevoflurane and undergoes cysteine conjugate beta-lyase-dependent bioactivation to nephrotoxic metabolites in rats. The present experiments were designed to identify reactive intermediates formed from S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine, a compound A-derived cysteine S-conjugate, in two pyridoxal model systems, namely Cu2+/pyridoxal and N-dodecylpyridoxal in cetyltrimethylammonium micelles. S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine was incubated in the model systems with benzyl bromide, pentafluorobenzyl bromide, aniline, and o-phenylenediamine as trapping agents. The products were purified by TLC and identified by 19F and 1H NMR spectroscopy and by GC/MS. In the absence of trapping agents, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid and 3,3,3-trifluorolactic acid, which have been identified previously in biotransformation studies, were formed. With the chemical models, 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropanethiolate, the expected first intermediate, was not trapped with benzyl bromide. Rather, the dehydrofluorination product 2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenylthiolate was trapped with benzyl bromide to give benzyl 2-(fluoromethoxy)-3,3,3-trifluoropropanethioate, which was formed in both chemical models. When pentafluorobenzyl bromide was used as a trapping agent, GC/MS analysis showed that the expected thiolate was trapped to give pentafluorobenzyl 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl sulfide in the N-dodecylpyridoxal model. In both chemical models, 2-(fluoromethoxy)-3,3,3-trifluorothioacyl fluoride was trapped with aniline to give N-phenyl 2-(fluoromethoxyl)-3,3,3-trifluoropropanethioamide, which cyclized to give 3-phenyl-4-thiono-5-(trifluoromethyl)-1,3-oxazolane. The results demonstrate that most of the reactive intermediates and products formed by the beta-lyase-catalyzed biotransformation of compound A-derived cysteine S-conjugates are also formed in the two chemical systems studied. Some products were, however, formed in chemical systems that have not been observed in previous in vivo and in vitro studies; it is not known whether these products are formed in biological systems and whether they contribute to the observed nephrotoxicity of cysteine S-conjugates.
3. TNFAIP3 promotes survival of CD4 T cells by restricting MTOR and promoting autophagy
Yu Matsuzawa, et al. Autophagy. 2015;11(7):1052-62. doi: 10.1080/15548627.2015.1055439.
Autophagy plays important roles in metabolism, differentiation, and survival in T cells. TNFAIP3/A20 is a ubiquitin-editing enzyme that is thought to be a negative regulator of autophagy in cell lines. However, the role of TNFAIP3 in autophagy remains unclear. To determine whether TNFAIP3 regulates autophagy in CD4 T cells, we first analyzed Tnfaip3-deficient naïve CD4 T cells in vitro. We demonstrated that Tnfaip3-deficient CD4 T cells exhibited reduced MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) puncta formation, increased mitochondrial content, and exaggerated reactive oxygen species (ROS) production. These results indicate that TNFAIP3 promotes autophagy after T cell receptor (TCR) stimulation in CD4 T cells. We then investigated the mechanism by which TNFAIP3 promotes autophagy signaling. We found that TNFAIP3 bound to the MTOR (mechanistic target of rapamycin) complex and that Tnfaip3-deficient cells displayed enhanced ubiquitination of the MTOR complex and MTOR activity. To confirm the effects of enhanced MTOR activity in Tnfaip3-deficient cells, we analyzed cell survival following treatment with Torin1, an MTOR inhibitor. Tnfaip3-deficient CD4 T cells exhibited fewer cell numbers than the control cells in vitro and in vivo. In addition, the impaired survival of Tnfaip3-deficient cells was ameliorated with Torin1 treatment in vitro and in vivo. The effect of Torin1 was abolished by Atg5 deficiency. Thus, enhanced MTOR activity regulates the survival of Tnfaip3-deficient CD4 T cells. Taken together, our findings illustrate that TNFAIP3 restricts MTOR signaling and promotes autophagy, providing new insight into the manner in which MTOR and autophagy regulate survival in CD4 T cells.
