Integrated redox proteomics and metabolomics of mitochondria to identify mechanisms of cd toxicity

Abstract

Cadmium (Cd) exposure contributes to human diseases affecting liver, kidney, lung, and other organ systems, but mechanisms underlying the pleotropic nature of these toxicities are poorly understood. Cd accumulates in humans from dietary, environmental (including cigarette smoke), and occupational sources, and has a twenty-year biologic half-life. Our previous mouse and cell studies showed that environmental low-dose Cd exposure altered protein redox states resulting in stimulation of inflammatory signaling and disruption of the actin cytoskeleton system, suggesting that Cd could impact multiple mechanisms of disease. In the current study, we investigated the effects of acute Cd exposure on the redox proteome and metabolome of mouse liver mitochondria to gain insight into associated toxicological mechanisms and functions. We analyzed redox states of liver mitochondrial proteins by redox proteomics using isotope coded affinity tag (ICAT) combined mass spectrometry. Redox ICAT identified 2687 cysteine-containing peptides (peptidyl Cys) of which 1667 peptidyl Cys (657 proteins) were detected in both control and Cd-exposed samples. Of these, 46% (1247 peptidyl Cys, 547 proteins) were oxidized by Cd more than 1.5-fold relative to controls. Bioinformatics analysis using MetaCore software showed that Cd affected 86 pathways, including 24 Cys in proteins functioning in branched chain amino acid (BCAA) and 14 Cys in proteins functioning in fatty acid (acylcarnitine/carnitine) metabolism. Consistently, high-resolution metabolomics data showed that Cd treatment altered levels of BCAA and carnitine metabolites. Together, these results show that mitochondrial protein redox and metabolites are targets in Cd-induced hepatotoxicity. The results further indicate that redox proteomics and metabolomics can be used in an integrated systems approach to investigate complex disease mechanisms.

Keywords: cysteine proteome; environmental toxicant; metabolome; pathway maps; thiol/disulfide redox state.

FIG. 1.

GSH, GSSG, and GSH/GSSG redox potential in plasma and liver by acute Cd exposure. Plasma (A, B) and organ tissues (C–E) collected from mice (9 mice per group) exposed to saline control (0 Cd) or Cd (10 mg/kg body weight, 10 Cd) for 6 h were analyzed for redox states of thiol/disulfide couples, GSH, GSSG (A, top) and Cys and CySS amounts (B–E, top), and respective GSH/GSSG (A, C–E; bottom) and Cys/CySS (B, bottom) redox potentials (E_hGSSG and E_hCySS). *p < 0.05 versus CR group.

FIG. 2.

Cd decreased total protein-bound thiols and increased mRNA expression of metallothionein (MT). Tissues including liver, lung, and kidney isolated from mice treated with Cd as described above were measured for total protein-bound thiol (Pr-SH) by Ellman's reagent (A). MT-1 (B) and MT-2 (C) expression levels (mRNA level) were measured by quantitative real-time PCR in different tissues as a measure of cellular response to the metal stress, Cd. *p < 0.05 versus CR group.

FIG. 3.

Liver mitochondrial GSH, protein thiol, and functional impairment by Cd exposure. Isolated mitochondria from mice exposed to saline CR and Cd (10 mg/kg body weight) for 6 h were used to measure mitochondrial permeability transition (MPT; A, B) and determine GSH/GSSG related oxidative stress (C–F). Mitochondria were suspended in media containing 3mM inorganic phosphate and 5mM sodium succinate with or without 60μM Ca²⁺ at 20°C, and activation of MPT was monitored at 540 nm. The MPT from Cd-exposed mice (B) were more activated than CR (A) in both presence and absence of Ca²⁺. Redox states of isolated mitochondria from CR and Cd were examined by measuring amounts of GSH/GSSG (C), GSH/GSSG redox potential (E_hGSSG, D), and concentration of protein-bound thiol (Pr-SH, E) and glutathionylated protein (Pr-SSG, F). *p < 0.05 versus CR group. Data are representative of experiments from 9 mice for each CR and Cd treatment.

FIG. 4.

Cd-induced oxidation in liver mitochondrial redox proteome. Freshly isolated liver mitochondria (120 μg protein) obtained from saline CR (3 mice) and Cd (3 mice) were analyzed for redox proteomics using redox ICAT-based mass spectrometry (MS). (A) Distribution of the oxidized states (% oxidized) of the Cys-including peptide (peptidyl Cys) relevant to 655 proteins from CR (blue; n = 1667) and Cd (red; n = 1667). The mean value of % oxidation from 1667 peptidyl Cys from CR and Cd were 8.9% and 15.7%, respectively. (B) Pie chart showing the distribution of peptidyl Cys according to the measured fold oxidation. Of 1667 peptidyl Cys, 77% were oxidized more than 50% (1.5-fold) by Cd treatment. (C) MetaCore Bioinformatics software identified significant pathway maps that are regulated by Cd-oxidized peptidyl Cys (fold oxidation ≥ 1.5, n = 1247). Of the 86 statistically significant pathways, the top 10 pathways are shown (C).

FIG. 5.

Cd-affected significant mitochondrial pathways for branched chain amino acid metabolism. Among significant pathways associated with Cd-oxidized proteins/peptidyl Cys and Cd-increased metabolites, the pathways for branched chain amino acids (leucine, isoleucine, and valine) metabolism are shown. Redox ICAT/MS-identified 24 peptidyl Cys (rectangular box; p = 1.5 × 10⁻¹⁷) oxidized by Cd and high-resolution metabolomics-identified 3 metabolites (oval) increased by Cd are shown in these pathways including total 80 proteins and metabolites.

FIG. 6.

Cd-altered mitochondrial metabolome. Isolated mitochondria prepared for redox proteomics were used for metabolomics by high-resolution mass spectrometry. Top 10 pathways associated with 542 small molecules (m/z features) that are significantly affected by Cd were ranked by MetaCore Bioinformatics software according to p-value.

FIG. 7.

Cd-affected significant mitochondrial pathways for long-chain fatty acid oxidation metabolism. Redox ICAT/MS-identified 13 peptidyl Cys (rectangular box; p = 1.9 × 10⁻⁶) oxidized by Cd and high-resolution metabolomics-identified 12 metabolites (oval) are shown in these pathways including total 83 proteins and metabolites.

FIG. 8.

Cd-affected carnitine/acylcarnitine metabolism. A schematic diagram for Cd-affected carnitine/acylcarnitine transport from cytoplasm to mitochondria is shown (A). Several critical proteins (CAT, CPTI, CPTII, and CAC carrier) regulating this transport system are indicated with oxidation in specific Cys residues by Cd exposure. Metabolites identified by metabolomics, including carnitine, acetylcarnitine, palmitoylcarnitine, and stearylcarnitine, are shown with m/z and altered abundance in the mitochondria by Cd treatment (A). (B) Mitochondrial palmitoylcarnitine level (²H₃-palmitoylcarnitine) was measured by mass spectrometer (m/z, 403.3617) after treating a mouse with Cd or saline for 6 h. *p = 0.008, n = 3.