Assessment of the biological pathways targeted by isocyanate using N-succinimidyl N-methylcarbamate in budding yeast Saccharomyces cerevisiae

Abstract

Isocyanates, a group of low molecular weight aromatic and aliphatic compounds possesses the functional isocyanate group. They are highly toxic in nature hence; we used N-succinimidyl N-methylcarbamate (NSNM), a surrogate chemical containing a functional isocyanate group to understand the mode of action of this class of compounds. We employed budding yeast Saccharomyces cerevisiae as a model organism to study the pathways targeted by NSNM. Our screening with yeast mutants revealed that it affects chromatin, DNA damage response, protein-ubiquitylation and chaperones, oxidative stress, TOR pathway and DNA repair processes. We also show that NSNM acts as an epigenetic modifier as its treatment causes reduction in global histone acetylation and formation of histone adducts. Cells treated with NSNM exhibited increase in mitochondrial membrane potential as well as intracellular ROS levels and the effects were rescued by addition of reduced glutathione to the medium. We also report that deletion of SOD1 and SOD2, the superoxide dismutase in Saccharomyces cerevisiae displayed hypersensitivity to NSNM. Furthermore, NSNM treatment causes rapid depletion of total glutathione and reduced glutathione. We also demonstrated that NSNM induces degradation of Sml1, a ribonucleotide reductase inhibitor involved in regulating dNTPs production. In summary, we define the various biological pathways targeted by isocyanates.

Figure 1. NSNM inhibits growth of wild type yeast cells in a dose-dependent manner.

A) Growth curves of wild-type S. cerevisiae in the presence or absence of drug. Exponentially growing yeast cultures of the wild-type strain were treated with the indicated concentrations of NSNM. Growth was monitored by measuring OD₆₀₀ at regular intervals for 8 h. B) Growth Assay; 1588-4C (Wild-type) was grown up to log phase. 3 μl of each undiluted and 10-fold serially diluted culture was spotted onto control SCA plates and SCA plates containing 100, 200 or 300 μM NSNM. All plates were incubated at 30°C for 72 h and photographed. C) Clonogenic assay; equal number of cells from mid-log phase of untreated and NSNM treated cultures (3 h) were spread on standard SCA plates in triplicate. All plates were incubated at 30°C and the colony forming ability was analyzed after 36 h. Number of colonies were counted and shown in the form of bar diagram. D and E) FACS analysis, showing the effect of the NSNM on yeast cell cycle. Wild-type cells were cultured in SC medium to exponential phase and treated with alpha factor to synchronize all cells in G1 phase. After synchronization cells were released in either DMSO (control) or 50 μM NSNM containing media. The culture was sampled at indicated time points and cellular DNA content was analysed by FACS.

Figure 2. Screening of yeast deletion-mutants for NSNM sensitivity.

A–E) Growth Assay; yeast deletion mutants of various pathways were grown up to log phase. 3 μl of each undiluted and 10-fold serially diluted culture was spotted onto control SCA plates and SCA plates containing 100, 200 and 300 μM NSNM. All plates were incubated at 30°C for 72 h and photographed. Mutant yeast strains of A) Histone tails, B) HATs and HDACs, C) Molecular chaperones, D) Protein-ubiquitination pathways, E) TOR pathway.

Figure 3. NSNM induces alterations in histone modifications and makes adduct with histone H3 and H4.

A) Wild-type (1588-4C) cells were cultured up to log phase and treated with increasing concentrations of NSNM (0, 10, 25, 50, 100 μM) for 3 hr. Whole cell extracts were prepared by TCA extraction method and samples were subjected to western blot analysis using indicated antibodies. B) Wild type yeast cells were treated with DMSO and 100 μM NSNM for 3 h at OD₆₀₀ (0.8) and equal numbers of cells were processed for MNase digestion. MNase was used at different concentrations (0, 50, 100, 200, 300, 500 and 1000 U/ml). Samples were run on 1.2% agarose gel, together with DNA ladder, and stained with ethidium-bromide. C) Core histones were incubated with increasing concentrations of NSNM for 1 h at 37 °C. Proteins were resolved by 12% SDS-PAGE. The interaction of NSNM with histones was analyzed by probing with histone H3 and H4 antibodies. Arrow indicates the appearance of high molecular weight band detected by H3 and H4 antibody respectively. * represents non-specific band detected by H3 antibody.

Figure 4. NSNM causes depletion in total glutathione levels and increase intracellular ROS levels.

(A) Wild type cells were grown in DMSO or indicated concentration of NSNM for 3 h. GSH, GSSG, and the GSH: GSSG ratios were determined. Values are means S.D. of three independent cultures. (B & C) Mitochondrial membrane permebility and reactive oxygen species (ROS) production detected by MitoTracker and DCF-DA respectively in control cells and cells treated with 50 or 100 μM of NSNM for 3 h. Cells treated with 5 mM H₂O₂ served as positive control. Upper panel of (C) shows phase contrast microscopic images; the lower panel show florescence microscopic image of the same cells. (D) Wild-type yeast strain grown in SC media and treated with increasing concentration of NSNM (25, 50 or 100 μM) for 1 3 h. Yeast cells were processed for FACS analysis after staining with either DCF-DA or MitoTracker Red. % value depicts the proportion of cells showing fluorescence after staining with indicated dyes.

Figure 5. SODs deletions are hypersensitive to the NSNM while GSH supplementation rescues the effect.

(A) Growth Assay; Wild type, sod1Δ or sod2Δ was grown up to log phase. 3 μl of each undiluted and 10-fold serially diluted culture was spotted onto control SCA plates, SCA plates containing 100, 200, 300 μM NSNM, and SCA plates impregnated with GSH (10 mM) in combination with 300 μM NSNM. All plates were incubated at 30°C for 72 h and photographed. (B &C) Wild-type yeast strain grown in SC media supplemented with or without 10 mM GSH for 1 h followed by exposure to 100 μM NSNM for 3 h. Yeast cells were processed for FACS analysis after staining with either DCF-DA (B) or MitoTracker Red (C). % value depicts the proportion of cells showing florescence after staining with indicated dyes.

Figure 6. NSNM exposure leads to degradation of Sml1 without activating RNR genes or Rad52 foci formation.

(A) Whole cell extracts were prepared by TCA extraction method and samples were subjected to western blot analysis with indicated antibodies. Blotting with antibodies against Tbp and Rap1 or Ponceau S staining of representative blot were used as loading controls. (B) Sml1-YFP tagged strain were treated with NSNM (100 μM) for 3 h. For control same strains were treated with MMS (0.03%), images were taken as described in materials and methods. (C and D) Growth Assay; wild type and different mutant yeast strains were spotted onto control SCA (DMSO) plates or SCA plates containing 100, 200 or 300 μM NSNM. All plates were incubated at 30°C for 72 h and photographed. (E) NSNM exposure does not lead to Rad52 foci formation. Rad52-YFP tagged yeast strain was treated with 100 μM NSNM for 3 h, same strain was treated with 0.03% MMS (control) prior to visualization of foci by confocal microscopy.