Developing In Vitro Models to Define the Role of Direct Mitochondrial Toxicity in Frequently Reported Drug-Induced Rhabdomyolysis

Abstract

The United States Food and Drug Administration Adverse Event Reporting System (FAERS) logged 27,140 rhabdomyolysis cases from 2004 to 31 March 2020. We used FAERS to identify 14 drugs frequently reported in 6583 rhabdomyolysis cases and to investigate whether mitochondrial toxicity is a common pathway of drug-induced rhabdomyolysis by these drugs. Preliminary screening for mitochondrial toxicity was performed using the acute metabolic switch assay, which is adapted here for use in murine L6 cells. Fenofibrate, risperidone, pregabalin, propofol, and simvastatin lactone drugs were identified as mitotoxic and underwent further investigation, using real-time respirometry (Seahorse Technology) to provide more detail on the mechanism of mitochondrial-induced toxicity. To confirm the human relevance of the findings, fenofibrate and risperidone were evaluated in primary human skeletal muscle-derived cells (HSKMDC), using the acute metabolic switch assay and real-time respirometry, which confirmed this designation, although the toxic effects on the mitochondria were more pronounced in HSKMDC. Overall, these studies demonstrate that the L6 model of acute modification may find utility as an initial, cost-effective screen for identifying potential myotoxicants with relevance to humans and, importantly, that drug-induced mitochondrial dysfunction may be a common mechanism shared by some drugs that induce myotoxicity.

Keywords: FAERS; HSKMDC; L6; mitochondrial dysfunction; skeletal muscle toxicity; suspect drug-induced rhabdomyolysis.

Figure 1

The effect of rotenone (0–5 µM, positive control) exposure on ATP levels of L6 cells (2 h), compared to the vehicle control. L6 cells were exposed to rotenone in glucose or galactose serum-free media. Statistical significance compared to the vehicle control was determined by one-way ANOVA with Dunnett’s correction for multiple comparisons. ATP glucose ⁺⁺ p-value < 0.01, ⁺⁺⁺ p-value < 0.001, ⁺⁺⁺⁺ p-value < 0.0001. ATP galactose ^^^^ p-value < 0.0001. ATP levels are reported as the percentage of the vehicle control ± S.D. (n = 3). Key: blue circles represent ATP glucose, and orange squares represent ATP galactose.

Figure 2

The effect of the test compounds on the cellular ATP content of L6 cells (2 h) compared to the vehicle control. Serial concentrations of the compounds were used up to 300 µM (or 100µM for SVL and DAP) in glucose or galactose serum-free media. (A) Compounds were labelled as having a mitochondrial liability. (B) Compounds displayed a multi-mechanistic reduction in ATP. (C) Compounds were negative for mitochondrial toxicity. Values are displayed as mean ± S.D. (n = 3). Statistical significance compared to the vehicle control was determined by one-way ANOVA with Dunnett’s correction for multiple comparisons. ATP glucose ⁺ p-value < 0.05, ⁺⁺ p-value < 0.01, ⁺⁺⁺ p-value < 0.001, ⁺⁺⁺⁺ p-value < 0.001. ATP galactose ^^ p-value < 0.01, ^^^ p-value < 0.001, ^^^^ p-value < 0.0001. ATP levels are reported as the percentage of the vehicle control ± S.D. (n = 3). Key: blue circles represent ATP glucose, and orange squares represent ATP galactose.

Figure 3

Examining the effect of the suspect drugs (6 h) on respiratory parameters in L6 cells. (A) fenofibrate, (B) pregabalin, (C) risperidone, (D) propofol, (E) simvastatin lactone. Values are expressed as a percentage of vehicle control and expressed as mean ± S.D. (n = 3). Statistical significance compared to the vehicle control was determined by two-way ANOVA with Dunnett’s correction for multiple comparisons. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.

Figure 4

The effect of rotenone (0–5 µM, positive control) exposure on ATP levels and LDH retention in HSKMDC (2 h), compared to the vehicle control. HSKMDC cells were exposed to rotenone in glucose or galactose serum-free media. Statistical significance compared to the vehicle control was determined by one-way ANOVA with Dunnett’s correction for multiple comparisons. ATP galactose ^^ p-value < 0.01, ^^^ p-value < 0.001. ATP levels are reported as the percentage of the vehicle control and expressed as mean ± S.D. (n = 3). Key: blue circles represent ATP glucose, orange squares represent ATP galactose, the black triangle represents cytotoxicity-HSKMDC–glucose, and the grey triangle down represents cytotoxicity–HSKMDC–galactose.

Figure 5

The effect of (A) fenofibrate and (B) risperidone exposure (2 h) on ATP content and LDH retention in HSKMDC. Serial concentrations of the compounds were used up to 300 µM in glucose or galactose serum-free media. ATP and cytotoxicity levels are expressed as a percentage of the corresponding media vehicle control and values are displayed as mean ± S.D. (n = 3). Statistical significance compared to the vehicle control was determined by one-way ANOVA with Dunnett’s correction for multiple comparisons. ATP glucose ⁺⁺ p-value < 0.01. ATP galactose ^^ p-value < 0.01, ^^^ p-value < 0.001, ^^^^ p-value < 0.001. Key: blue circles represent ATP glucose, orange squares represent ATP galactose, the black triangle represents cytotoxicity-HSKMDC–glucose, and the grey triangle down represents cytotoxicity–HSKMDC–galactose.

Figure 6

Examining the effect of the acute injection of (A) fenofibrate and (B) risperidone on respiratory parameters in HSKMDC. Values are expressed as a percentage of vehicle control ± S.D. (n = 3). The values were normalised to μg of protein per well. Statistical significance was determined by two-way analysis of variance (ANOVA) with Dunnett’s correction for multiple comparisons. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.001.