Maneb alters central carbon metabolism and thiol redox status in a toxicant model of Parkinson's disease

Abstract

The dithiocarbamate fungicide maneb (MB) has attracted interest due to increasing concern of the negative health effects of pesticides, as well as its association with Parkinson's disease (PD). Our laboratory has previously reported distinct phenotypic changes of neuroblastoma cells exposed to acute, sub-toxic levels of MB, including decreased mitochondrial respiration, altered lactate dynamics, and metabolic stress. In this study, we aimed to further define the specific molecular mechanisms of MB toxicity through the comparison of several thiol-containing compounds and their effects on cellular energy metabolism and thiol redox nodes. Extracellular flux analyses and stable isotope labeled tracer metabolomics were employed to evaluate alterations in energy metabolism of SK-N-AS human neuroblastoma cells after acute exposure of an array of compounds, including dithiocarbamates (maneb, nabam, zineb) and other thiol-containing small molecules (glutathione, N-acetylcysteine). These studies revealed MB and its methylated form (MeDTC) as unique toxicants with significant alterations to mitochondrial respiration, proliferation, and glycolysis. We observed MB to significantly impact cellular thiol redox status by oxidizing cellular glutathione and altering the thiol redox status of peroxiredoxin 3 (Prx3, mitochondrial) after acute exposure. Redox Western blotting revealed a MB-specific modification of cellular Prx3, strengthening the argument that MB can preferentially target mitochondrial enzymes containing reactive cysteine thiols. Further, stable isotope tracer metabolomics confirmed our energetics assessments, and demonstrated that MB exposure results in acute derangement of central carbon metabolism. Specifically, we observed shunting of cellular glucose into the pentose-phosphate pathway and reduction of TCA intermediates derived from glucose and glutamine. Also, we report novel lactate utilization for TCA enrichment and glutathione synthesis after MB exposure. In summary, our results further confirm that MB exerts its toxic effects via thiol modification, and significantly transforms central carbon metabolism.

Keywords: Dithiocarbamate; Fungicide; Parkinson's disease; Protein thiol; Redox.

FIGURE 1:. Chemical methylation of DTC fungicides to yield the MeDTC compound which displays variable toxicity in neuroblastoma.

A) Chemical Reaction scheme in which NB(1), MB(2), and ZB(3) are converted to MeDTC(4). i) MeI + EtOH @ RT for 1 HR; ii) MeI + DMSO @ RT for 2 HR. B) WST-1 activity assay after a 2 hr exposure to DTC compounds (N = 10, mean ± SEM). C) Flow Cytometry using the Annexin V / Dead Cell Assay after a 24 hr exposure to DTC compounds (N = 9–10, mean ± SEM) with representative plots for D) DMSO, E) H202 Positive Control, F) 50μM MB, and G) 50μM MeDTC.

FIGURE 2:. Maneb shows unique toxicity compared to similar DTC compounds in neuroblastoma.

A) Confluence as measured by the Incucyte Live Cell Analyzer over a 48 Hour exposure of DTC compounds using phase microscopy at 10X (N = 12, mean ± SEM). Percent change from basal is represented at B) 2 hours and C) 24 hours. D) Cytotoxicity as measured by green fluorescence over a 48 hour exposure to DTC compounds (N = 12, mean ± SEM) with percent change at E) 2 hours and F) 24 hours. G) Apoptosis as measured by red fluorescence over a 48 hour exposure to DTC compounds (N = 12, mean ± SEM) with percent change at H) 2 hours and I) 24 hours.

FIGURE 3:. MB directly alters the mitochondrial Peroxiredoxin (Prx3) in neuroblastoma.

A) Glutathione redox state as measured by HPLC after a 2 hr exposure to DTC compounds (N = 6, mean ± SEM). Western blot of protein abundance for both B) Prx1 (cytosolic) and C) Prx3 (mitochondrial) (N = 6, mean ± SEM). D) Representative redox Western blot of E) Prx1 and F) Prx3 (N = 6, mean ± SEM). G) Representative redox Western blot without NEM for H) Prx1 and I) Prx3 (N = 6, mean ± SEM). J) Relative quantification of the unidentified band (arrows to G). K) Prx3 Oxidation after addition of 100 μM H₂O₂ to cell lysates (N = 6, mean ± SEM). L) Prx3 Oxidation with varied concentrations of MB (N = 6, mean ± SEM). Blots not shown here can be found in SUPP FIG 5.

FIGURE 4:. MeDTC does not act as other DTC compounds, with maneb showing most drastic effects on both mitochondrial function and glycolysis in neuroblastoma.

A) Seahorse XF tracer for the cell mitochondrial stress test with an acute injection of 50 μM of each compound or DMSO (0.5%) (N = 15–18, mean ± SEM). Parameters for B) Non-mitochondrial Oxygen Consumption, C) Basal Respiration, D) Maximal Respiration, E) ATP Synthesis, F) Spare Respiratory Capacity, and G) Acute Response were calculated per Agilent. H) Seahorse XF tracer for the glycolysis stress test with an acute injection yielding parameters for I) Glycolysis, J) Glycolytic Capacity, and K) Glycolytic Reserve (N = 15 – 18, mean ± SEM).

FIGURE 5:. MB and NB acute exposure cause an increase in glutamine dependency in neuroblastoma.

An acute Mitochondrial Fuel Flex Test was utilized on the Seahorse XF analyzer, yielding dependency and capacity measures for A) Glutamine, B) Fatty Acids, and C) Glucose (N = 5 – 6, mean ± SEM).

Figure 6:. [1,2,3-13C]- and [U-13C]-Glucose SIL metabolomics.

(A) demonstrates redox regulation of glycolysis (B-E) resulting in increased usage of the pentose phosphate pathway (F) and decreased ATP production (G). (H) A schematic of altered glycolytic pathways.

FIGURE 7:. Metabolism of [¹³C₅,¹⁵N₂] Glutamine.

(A) shows decreased incorporation of labeled glutamine (B) into GSH (D) and increased TCA cycle usage (C,E,F) and possible transamination (G,H).

FIGURE 8:. Assessment of [¹³C₃] Lactate Metabolism.

(A) demonstrates that MB-treatment results in uptake and usage of exogenous lactate in the TCA cycle (B-D), as well as incorporation into GSH (E).

FIGURE 9:. MB is not methylated in neuroblastoma.

A) ELISA on cell lysates for S-Adenosyl Methionine (SAM) after a 2 hr exposure to DTC compounds (N = 3 – 6, mean ± SEM). B) Peak area and line-of-best-fit for linear regression of spiked MeDTC standards in SK-N-AS cells for 2 hours by LC/MS/MS (N = 3, mean ± SEM). C) Peak area of 50 μM equivalents of each DTC compounds after a 2 hour exposure in SK-N-AS (N = 3, mean ± SEM).

FIGURE 10:

Acute MB exposure causes a diversion of energy substrates to gluconeogenesis in neuroblastoma cells.