Primary hepatocytes from mice lacking cysteine dioxygenase show increased cysteine concentrations and higher rates of metabolism of cysteine to hydrogen sulfide and thiosulfate

Abstract

The oxidation of cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by cysteine dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H2S) and thiosulfate (an intermediate in the oxidation of H2S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine β-synthase (CBS) in cysteine desulfhydration by liver, we investigated the metabolism of cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. The greater flux of cysteine through the cysteine desulfhydration reactions catalyzed by CTH and CBS in hepatocytes from Cdo1-null mice appeared to be the consequence of their higher cysteine levels, which were due to the lack of CDO and hence lack of catabolism of cysteine by the cysteinesulfinate-dependent pathways. Both CBS and CTH appeared to contribute substantially to cysteine desulfhydration, with estimates of 56 % by CBS and 44 % by CTH in hepatocytes from wild-type mice, and 63 % by CBS and 37 % by CTH in hepatocytes from Cdo1-null mice.

Fig. 1

Metabolism of methionine sulfur and cysteine by direct oxidation and desulfhydration-oxidation pathways. The direct oxidation pathway is usually the major route for disposal of excess cysteine, as indicated by the heavy lines and arrows in the pathway shown on the right. Little is known about the reaction by which hypotaurine is converted to taurine. The dotted lines indicate that sulfite produced in the direct oxidation pathway may be incorporated into thiosulfate, and the sulfite produced by mitochondrial sulfide oxidation may be directly oxidized to sulfate without incorporation into thiosulfate. Details of the major desulfhydration reactions catalyzed by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH) are shown in the inset on the left. CBS-ts, CBS-catalyzed transsulfuration; CBS-ds, CBS-catalyzed cysteine desulfhydration; CTH-ts, CTH-catalyzed transsulfuration; CTH-ds, CTH-catalyzed cysteine desulfhydration; CDO, cysteine dioxygenase; CSAD, cysteinesulfinic acid decarboxylase; ETHE1, mitochondrial persulfide dioxygenase; GOT, aspartate (cysteinesulfinate) aminotransferase; SQRDL, sulfide quinone reductase-like protein; SUOX, sulfite oxidase; TR, thiosulfate reductase; TST, thiosulfate sulfur transferase

Fig. 2

HSip-1 method for assay of H₂S production over time. a An example of a standard curve for HSip-1 fluorescence as a function of amount of Na₂S added to the medium at time zero. DMEM to which 0.3 mM Cys, 0.05 mM BCS, 0.03 mM HSip-1, and varying levels of added Na₂S monohydrate, but no cells, had been added was maintained under standard culture conditions. Aliquots of medium were removed after 24 and 48 h of culture to measure HSip-1 fluorescence. Fluorescence developed over time and was stable between 24 and 48 h. Fluorescence readings were corrected for blank cultures that did not contain any Na₂S. b Demonstration of specificity of HSip-1 assay for quantitation of H₂S. HEK293T cells were transfected with CTH cDNA or empty vector and then incubated in standard DMEM with 0.05 mM BCS and 0.03 mM HSip-1, and with/without 5 mM DL-propargylglycine (PPG). Fluorescence was read after removing 100 μl of medium from each well at 6 h and again at 12 h. Fluorescence readings were corrected for blank cultures that did not contain cells. Values are mean ± SEM for 3 replicates. Values not denoted by the same letter are significantly different (p ≤ 0.05) by Tukey’s mean comparison procedure

Fig. 3

a H₂S production in hepatocytes from wild-type and Cdo1-null mice as assessed by trapping and measurement of H₂S released into the medium using the HSip-1 probe. Hepatocytes were cultured in standard DMEM or in DMEM supplemented with 0.3 mM Cys for 48 h. Medium also contained 0.05 mM BCS and 25 μM HSip-1. Fluorescence was read after removing 100 μl of medium from each well at 48 h. Fluorescence readings were corrected for blank cultures that did not contain cells. b Thiosulfate production in hepatocytes from wild-type and Cdo1-null mice as assessed by accumulation of thiosulfate in the medium. Hepatocytes were cultured in standard DMEM or in DMEM supplemented with 0.3 mM Cys for 48 h. Medium also contained 0.05 mM BCS. Results are the mean ± SEM from 6 to 7 independent experiments. Values for a metabolite that are not denoted by the same letter are significantly different (p ≤ 0.05) by Tukey’s mean comparison procedure

Fig. 4

Relative amounts of enzymes involved in cysteine desulfhydration and sulfide oxidation in cultured hepatocytes of wild-type and Cdo1-null mice. a Western blots of enzymes involved in cysteine desulfhydration: (cystathionine γ-lyase (CTH), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MPST). b Western blots of mitochondrial enzymes involved in sulfide oxidation/sulfide quinone reductase (SQRDL), persulfide dioxygenase (ETHE1) and thiosulfate sulfurtransferase (TST, rhodanese). Representative blots are shown. c Densities of bands in Western blots were normalized using β-actin. Values in the bar graphs represent mean ± SEM for 3 separate experiments. d Activities of CTH, MPST and TST measured in hepatocytes isolated from wild-type and Cdo1-null mice after the cells had been cultured in standard DMEM for 24 h. Activities were measured using V_max assays

Fig. 5

Total cellular cysteine levels in hepatocytes from wild-type and Cdo1-null mice. Hepatocytes were cultured in standard DMEM or in DMEM supplemented with 0.3 mM Cys for 24 h or 48 h. Results are the mean ± SEM from 3 to 5 independent experiments. Values for a metabolite that are not denoted by the same letter are significantly different (p ≤ 0.05) by Tukey’s mean comparison procedure

Fig. 6

a H₂S production in hepatocytes from wild-type and Cdo1-null mice as assessed by trapping and measurement of H₂S released into the medium with the HSip-1 probe. b Thiosulfate production in hepatocytes from wild-type and Cdo1-null mice as assessed by accumulation of thiosulfate in the medium. Hepatocytes were cultured in DMEM supplemented with 0.3 mM Cys and with 1 mM PPG, 2 mM PPG or no PPG for 24 h. Results are the mean ± SEM from 3 independent experiments. Values for thiosulfate that are not denoted by the same letter are significantly different (p ≤ 0.05) by Tukey’s mean comparison procedure; values for H₂S did not reach statistical significance for individual comparisons

Fig. 7

Effect of PPG on total cellular cysteine levels in hepatocytes from wild-type and Cdo1-null mice. Hepatocytes were cultured in DMEM supplemented with 0.3 mM Cys with or without 2 mM PPG for 24 h or 48 h. Results are the mean ± SEM from 3 independent experiments. Values for a metabolite that are not denoted by the same letter are significantly different (p ≤ 0.05) by Tukey’s mean comparison procedure

Fig. 8

Cellular (a) and medium (b) cystathionine levels and medium lanthionine levels (c) for cultures of hepatocytes from wild-type and Cdo1-null mice that were cultured in DMEM supplemented with 0.3 mM Cys. Cystathionine was measured at both 24 h and 48 h; lanthionine was measured only at 24 h. Results are the mean ± SEM from 3 to 5 independent experiments. Values for cystathionine that are not denoted by the same letter are significantly different (p ≤ 0.05) by Tukey’s mean comparison procedure; values for lanthionine did not reach statistical significance