RNA expression profiling in sulfamethoxazole-treated patients with a range of in vitro lymphocyte cytotoxicity phenotypes

Abstract

The lymphocyte toxicity assay (LTA) is a proposed surrogate marker of sulfonamide antibiotic hypersensitivity. In the LTA, peripheral blood mononuclear cells (PBMCs) undergo apoptosis more readily in hypersensitive versus tolerant patients when exposed to drug-hydroxylamine metabolites in vitro. The purpose of this study was to identify key gene transcripts associated with increased cytotoxicity from sulfamethoxazole-hydroxylamine in human PBMCs in the LTA. The LTA was performed on PBMCs of 10 patients hypersensitive to trimethoprim-sulfamethoxazole (HS) and 10 drug-tolerant controls (TOL), using two cytotoxicity assays: YO-PRO (n = 20) and MTT (n = 12). mRNA expression profiles of PBMCs, enriched for CD8⁺ T cells, were compared between HS and TOL patients. Transcript expression was interrogated for correlation with % cytotoxicity from YO-PRO and MTT assays. Correlated transcripts of interest were validated by qPCR. LTA results were not significantly different between HS and TOL patients, and no transcripts were found to be differentially expressed between the two groups. 96 transcripts were correlated with cytotoxicity by YO-PRO (r = ±.63-.75, FDR 0.188). Transcripts were selected for validation based on mechanistic plausibility and three were significantly over-expressed by qPCR in high cytotoxicity patients: multi-specific organic anion transporter C (ABCC5), mitoferrin-1 (SLC25A37), and Porimin (TMEM123). These data identify novel transcripts that could contribute to sulfonamide-hydroxylamine induced cytotoxicity. These include SLC25A37, encoding a mitochondrial iron transporter, ABCC5, encoding an arylamine drug transporter, and TMEM123, encoding a transmembrane protein that mediates cell death.

Keywords: cell death; hypersensitivity; idiosyncratic drug reactions; in vitro toxicity; reactive metabolites.

Figure 1

Relationship between clinical status and cytotoxicity from sulfamethoxazole‐hydroxylamine (SMX‐HA; 800 μmol·L⁻¹) in human PBMCs as detected by the YO‐PRO and MTT methods. (A) Scatter plot of % cytotoxicity with YO‐PRO detection for SMX tolerant (TOL; n = 10) vs hypersensitive (HS; n = 10) patients, (P = .67 between groups); the horizontal line represents the mean for each group. (B) Scatter plot of % cytotoxicity with MTT detection for TOL (n = 6) versus HS (n = 6) patients, (P = .52 between groups); the horizontal line represents the mean for each group

Figure 2

Heat map of expression of 96 transcripts correlated with SMX‐HA cytotoxicity in PBMCs from 20 patients using by the YO‐PRO method. The 20 samples are listed along the x‐axis in order of lowest (left) to highest (right) % cytotoxicity. Red indicates highest expression, blue lowest expression

Figure 3

Waterfall plot of pathway analysis for 96 transcripts correlated with % cytotoxicity for PBMCs exposed to SMX‐HA in vitro and assessed by the YO‐PRO method of detection. Gene ontology (GO) terms are listed along the y‐axis, and numbers of transcripts identified for each term are indicated on the x‐axis

Figure 4

Correlation between % PBMC cytotoxicity by YO‐PRO detection and expression of ABCC5 by qPCR in PBMCs from 20 patients (Pearson's r = −.486, P = .030)

Figure 5

Correlation between % cytotoxicity by YO‐PRO detection and expression of SLC25A37 by qPCR (Pearson's r = −.632, P = .037) in PBMCs from 20 patients exposed in vitro to SMX‐HA (800 mmol·L⁻¹), without inclusion of an extreme outlier (triangle)

Figure 6

Correlation between % cytotoxicity by YO‐PRO detection and expression of TMEM123 by qPCR (Pearson's r = .526, P = .017)

Figure 7

Correlation between % cytotoxicity by YO‐PRO detection and expression of RPL37 (Pearson's r = .322, P = .166) by qPCR