Substance abuse as compensation for stress factors involved in the performance of helping professions

THE ABSTRACT It has been shown recently that workload, stress, and burnout syndrome among the staff of the medical rescue service may be major risk factors in terms of triggering the use of psychoactive substances. Representing what is understandably a delicate issue, substance use among emergency medical staff has not been thoroughly studied in our country. Emergency medical workers' difficult working conditions and the chronic stress they are exposed to, in combination with a lack of support and care on the part of their employers, result in exhaustion and general distress, accompanied by the development of symptoms associated with both physical and mental disorders. This condition may lead to the use of psychoactive substances as a negative coping strategy. Consisting of both theoretical background and case studies, the paper points out the relationship between the chronic effect of stressors pertaining to the job of emergency medical workers and the use of psychoactive substances as a way of coping with and compensating for the implications of work-related stress and fatigue. Thorough case studies are presented to demonstrate the onset and development of addictive behaviour within a wider context, with special emphasis being placed on its association with coping with both acute and chronic occupational..

Methotrexate induces CerS6-dependent formation of ER membrane aggregates in A549 non-small cell lung carcinoma cells.

<p>(A) Treatment with MTX (10 nM) resulted in the increase of autophagosome number, but CerS6 was not associated with these organelles. (B) MTX (10 nM) induced aggregation of ER membranes and the accumulation of CerS6 in the aggregates. Arrows indicate ER membrane aggregates. Note the lack of such aggregates after lometrexol (1.0 μM) treatment. (C) MTX (10 nM) induced ER aggregation (detected by monitoring CALR-RFP) in the absence of exogenous CerS6. (D) A549 cells lacking CerS6 (siRNA silencing) did not form ER aggregates in response to MTX. Cells were co-transfected with GFP-CerS6 and LC3-RFP (panel A) or CALR-RFP (panel B), MTX was added 6 h after transfection and live cell images were captured 48 h later. In experiments with endogenous CerS6, cells were co-transfected with GFP and CALR-RFP. Six hours before co-transfection, cells were transfected with scrambled siRNA (<i>panel C</i>) or CerS6 siRNA (<i>panel D</i>). MTX was added 6 h after the second transfection and images were captured 48 h later. Pearson’s coefficient for co-localization (calculated using Fiji software) is indicated for each panel.</p

CerS6 is a target of MTX in mediation of antiproliferative effect.

<p>(A) Silencing CerS6 by siRNA rescues A549 cells from cytotoxic effect of MTX while silencing CerS4 does not change the drug toxicity. Cells were transfected with scrambled siRNA, CerS6 siRNA or CerS4 siRNA (25 pmol), MTX was added 6 h later and live cells were assessed by MTT assay 48 h after the beginning of MTX treatment. Data represent an average of two independent experiments, each performed in quadruplicates with error bars representing SD. Student’s <i>t</i> test was performed for statistical analysis (difference in cell number between control and CerS6-silenced cells were statistically significant with <i>p<</i>0.005; there were no significant differences in cell number between control and CerS4-silenced cells). Panels on the right show the efficiency of CerS6 and CerS4 silencing (evaluated at 48 h of MTX treatment). (B) Levels of ceramide in control A549 cells (<i>Cntr</i>, transfected with scrambled siRNA), MTX-treated cells transfected with scrambled siRNA (<i>MTX</i>) and MTX-treated cells after CerS6 silencing (<i>CerS6 siRNA/MTX</i>, transfected with CerS6 siRNA). Differences between the groups were analyzed by one-way ANOVA and asterisks indicate statistically significant changes (p<0.05). (C) Silencing of CerS6 by siRNA protects cancer cells from MTX cytotoxic effect. Student’s <i>t</i>-test was performed and the differences between MTX effect in control and CerS6-silenced cells were statistically significant (p<0.01). (D) MTX (10 nM) leads to elevation of CerS6 but not of CerS4 in A549 cells.</p

Targeting of CerS6 by MTX is p53-dependent.

<p>(A) MTX (10 nM) treatment strongly elevates CerS6 in p53^+/+ but not p53^-/- cells. (B) Antifolate lometrexol (LTX) bypasses p53 and does not affect CerS6: <i>left panel</i>, LTX inhibits both p53^+/+ and p53^-/- A549 cells (Experiments were performed three times with six wells per concentration point in each experiment; error bars, SD) no statistically significant differences between p53^+/+ and p53^-/- cells were observed (p>0.1); <i>right panel</i>, levels of p53 and CerS6 (Western blot) in the cells after LTX treatment. Cells were exposed to LTX for 48 h.</p

Effects of MTX on CerS6, ceramide levels and cellular proliferation.

<p>(A) Levels of CerS6 (Western blot) in cancer cells with and without MTX treatment. Cells were collected 48 h after MTX was added. (B) Changes in CerS6 levels after treatment with 10 nM MTX for 48 h (calculated from A). The intensity of bands was quantified using ImageJ software. In all cases, normalization for levels of actin was performed. (C) Changes in levels of selected ceramides in A549 cells after treatment with 10 nM MTX; P_i, total phospholipid inorganic phosphate present in cell extracts. Student’s <i>t</i>-test was performed for statistical analysis of the changes in ceramide levels and asterisks indicate statistically significant differences (p<0.05). (D) Antiproliferative effect of MTX (10 and 100 nM) in a panel of cancer cell lines. Live cells were assessed by MTT assay. Error bars show SD of four measurements. Differences between control and drug treated groups were statistically significant (calculated by one-way ANOVA) with <i>p</i> value below 0.0007 in all cases with the exception of A549 p53^-/- cells (p = 0.0357). (E) Scatter plot of CerS6 protein levels (from panel B) <i>versus</i> cell survival after MTX treatment (from panel D) for ten cell lines indicated in the above panels. Pearson’s coefficient (<i>r</i> = −0.83; n = 10; <i>p</i><0.01) indicates strong negative correlation between changes in CerS6 levels and cell survival upon MTX treatment.</p

Proposed two-step mechanism for CerS6-dependent MTX cytotoxicity.

<p>Step 1: MTX induces p53, which functions as the transcriptional activator of CerS6 and increases levels of the protein in ER. Step 2: MTX induces ER stress leading to aggregation of CerS6-containing ER membranes. Enhanced generation of C₁₆-ceramide by CerS6 further promotes cytotoxic effect.</p

Disposition of GNMT in cellular metabolism.

<p>GNMT converts SAM to SAH, methylating glycine to sarcosine. This reaction regulates SAM/SAH ratio and shuttles methyl groups, from activated methyl cycle back to the folate pool. Inhibitory effect of 5-CH3-THF (5-MTHF) on GNMT catalysis is indicated. Hcy, homocysteine; Sarc, sarcosine; THF, tetrahydrofolate.</p

Catalytically inactive or folate-binding deficient GNMT mutants are capable of the antiproliferative effect.

<p><b>A</b>. Crystal structure of GNMT tetramer (RCSB Protein Data Bank 3ths; subunits are shown in different colors) with bound 5-MTHF monoglutamate (two molecules shown in spacefill mode are bound per tetramer). <b>B</b>. Positions of amino acid residues in the GNMT catalytic center (from RCSB Protein Data Bank 1XVA). Acetate (Ac) is the competitive inhibitor of Gly and presumably occupies the same position in the active center. Glu 15 (E15*) is from a different subunit. Dotted lines indicate hydrogen bonds. <b>C</b> and <b>D</b>. The enzyme activities and CD spectra of GNMT mutants, analyzed in this study. <b>E</b>. The MTT proliferation assay of cells transfected with empty vector (control), wild type GNMT (WT), or corresponding mutants. <i>Error bars</i> represent ± S.D., <i>n =3</i>. <b>F</b>. Folate binding site at the GNMT subunit interface (as shown in panel <b>A</b>); Selected for mutagenesis are residues within close distance to 5-MTHF molecule (these residues are from all four subunits, which are denoted in parentheses). <b>G</b>. Binding of 5-MTHF by GNMT mutants. <i>Error bars</i> represent ± S.D., <i>n =2</i>. <b>H</b>. The MTT proliferation assay of cells transfected with empty vector (control), wild type GNMT (WT), or folate-binding deficient mutants mutants. <i>Error bars</i> represent ± S.D., <i>n =3</i>. <b>I</b>. The supplementation with excessively high media folate or SAM does not rescue cells from the GNMT antiproliferative effect. <i>Error bars</i> represent ± S.D., <i>n =3</i>.</p

Cellular responses to GNMT expression.

<p>A. Distribution of GNMT-expressing cells (<i>right panel</i>) between cell cycle phases (propidium iodide staining) compared to control (<i>left panel</i>) GNMT-deficient cells. <b>B</b>. Assessment of DNA damage in GNMT expressing cells by the Comet assay. <b>C</b>. Apoptotic cells assessed by Annexin V/propidium iodide staining after GNMT expression (<i>bottom right quadrant</i>, early apoptotic cells; <i>upper right quadrant</i>, late apoptotic cells); only green cells (expressing GFP-GNMT) were evaluated. <b>D</b>. Calculation of apoptotic cells from C. <b>E</b>. Activation of ERK phosphorylation in response to GNMT expression. <b>F</b>. zVAD-fmk, but not ERK inhibitor PD98059, partially rescues cells from the antiproliferative effect of GNMT (data for A549 cells are shown).</p

Effect of GNMT transient transfection on cellular proliferation.

<p>Cell viability was assessed by MTT assay (absorbance at 570 nm reflects the number of live cells). <i>Error bars</i> represent ± S.D., <i>n =3</i>. Insets show levels of GNMT (Western blot) at different time points after transfection.</p