Lipidomic profiling reveals protective function of fatty acid oxidation in cocaine-induced hepatotoxicity

Abstract

During cocaine-induced hepatotoxicity, lipid accumulation occurs prior to necrotic cell death in the liver. However, the exact influences of cocaine on the homeostasis of lipid metabolism remain largely unknown. In this study, the progression of subacute hepatotoxicity, including centrilobular necrosis in the liver and elevation of transaminase activity in serum, was observed in a three-day cocaine treatment, accompanying the disruption of triacylglycerol (TAG) turnover. Serum TAG level increased on day 1 of cocaine treatment but remained unchanged afterwards. In contrast, hepatic TAG level was elevated continuously during three days of cocaine treatment and was better correlated with the development of hepatotoxicity. Lipidomic analyses of serum and liver samples revealed time-dependent separation of the control and cocaine-treated mice in multivariate models, which was due to the accumulation of long-chain acylcarnitines together with the disturbances of many bioactive phospholipid species in the cocaine-treated mice. An in vitro function assay confirmed the progressive inhibition of mitochondrial fatty acid oxidation after the cocaine treatment. Cotreatment of fenofibrate significantly increased the expression of peroxisome proliferator-activated receptor α (PPARα)-targeted genes and the mitochondrial fatty acid oxidation activity in the cocaine-treated mice, resulting in the inhibition of cocaine-induced acylcarnitine accumulation and other hepatotoxic effects. Overall, the results from this lipidomics-guided study revealed that the inhibition of fatty acid oxidation plays an important role in cocaine-induced liver injury.

Fig. 1.

Histological and biochemical analyses of the influence of cocaine treatment on the C57BL/6 mice. (A) Histology of the liver (magnification: 100×). The location of hepatic central vein (CV) was marked. Dash arrow points to ballooning hepatocytes; solid arrow points to necrotic hepatocytes. (B) Serum ALT activity. (C) TAG level in the serum. (D) TAG level in the liver. (E) Hepatosomatic index. (F) TBARS value in the liver. Values were presented as mean ± SD (n = 4–8). *P < 0.05 and **P < 0.01 indicate statistical significance between control and cocaine-treated samples or between specified pair of samples.

Fig. 2.

Identification of serum metabolites altered by cocaine treatment through LC-MS-based metabolomics. Conditions of LC-MS measurement and procedures of data processing and analysis are described in Experimental Procedures. (A) The scores plot of a PLS-DA model on serum samples from the control mice [labeled as CTL (filled square)] and the mice treated with 30 mg/kg cocaine [labeled as day 1 (open square), day 2 (filled square), and day 3 (open diamond)] (n = 4–8). The t[1] and t[2] values represent the scores of each sample in the principal component 1 and 2, respectively. Fitness (R²) and prediction power (Q²) of this PLS-DA model are 0.82 and 0.41, respectively. (B) The S-loadings plot of serum ions contributing to the classification of serum samples. Major serum ions (I–XXII) interfered by cocaine treatment were labeled and their chemical identities were enlisted in Table 1. (C) The comparison between the chromatogram of serum PalC (XVI) and the chromatogram of PalC standard. (D) The representative MS/MS spectra of serum PalC (XVI) and PalC standard. The fragmentation pattern of PalC is interpreted in the inlaid diagram.

Fig. 3.

Time-dependent changes in serum lipid species after three-day 30 mg/kg cocaine treatment. (A–D) Relative abundance of acylcarnitines, LPCs, PCs, and LPEs in the serum. Values were presented as mean ± SD (n = 4–8). *P < 0.05 and **P < 0.01 indicate statistical significance between control and cocaine-treated samples.

Fig. 4.

Evaluation of mitochondrial β-oxidation function in the liver after three-day 30 mg/kg cocaine treatment. (A) PalC level in the serum. (B) PalC level in the liver. (C) Utilization of PalC in the liver. The in vitro β-oxidation functional assay is described in the Experimental Procedures. Values were presented as mean ± SD (n = 4–8). *P < 0.05 and **P < 0.01 indicate statistical significance between control and cocaine-treated samples.

Fig. 5.

Protective effects of fenofibrate against cocaine-induced hepatotoxicity in the C57BL/6 mice. The protocol of cotreatment of 50 mg/kg fenofibrate with 30 mg/kg cocaine for three days is described in Experimental Procedures. (A) Histology of the liver (magnification: 100×). The location of hepatic central vein (CV) was marked. Solid arrow points to necrotic hepatocytes. (B) Serum ALT activity. (C) TAG level in the serum. (D) TAG level in the liver. (E) Hepatosomatic index. (F) TBARS value in the liver. Values were presented as mean ± SD (n = 3–4). *P < 0.05 and **P < 0.01 indicate statistical significance between control and marked samples.

Fig. 6.

Influence of repeated cocaine exposure and fenofibrate on cocaine metabolism. The procedure of determining the profile of urinary cocaine metabolites is described in Experimental Procedures. Relative abundances of cocaine and eight major urinary metabolites on days 1 and 3 of cocaine-alone treatment (30 mg/kg) and fenofibrate (50 mg/kg)-cocaine cotreatment were compared. Values were presented as mean ± SD (n = 3–4). *P < 0.05 indicates statistical significance between days 1 and 3.

Fig. 7.

Influences of fenofibrate on cocaine-induced changes in the lipidome. (A) The three-dimensional scores plot of a PLS-DA model on serum samples from the control mice treated with CMC [labeled as CTL (filled triangle)] and the mice treated with fenofibrate [labeled as FF (open square)], cocaine [labeled as Coc (filled square)] and fenofibrate-cocaine [labeled as FF+Coc (open diamond)]. The t[1], t[2] and t[3] values represent the scores of each sample in the principal component 1, 2, and 3, respectively. Fitness (R²) and prediction power (Q²) of this PLS-DA model are 0.91 and 0.72, respectively. (B) The S-loadings plot of serum ions contributing to the distinction of the cocaine-alone treatment from the other three treatment groups. Serum lipid markers identified in Fig. 2B were labeled, and their chemical identities are listed in Table 1. (C) Relative abundance of acylcarnitines in the serum. (D) Relative abundance of LPC, PC, and LPE in the serum. Values were presented as mean ± SD (n = 3–4). *P < 0.05 and **P < 0.01 indicate statistical significance between control and marked samples.

Fig. 8.

Protective effects of fenofibrate against cocaine-induced inhibition of fatty acid oxidation function in the liver. (A) PalC level in the serum. (B) PalC level in the liver. (C) Utilization of PalC in the liver. The procedure of in vitro β-oxidation functional assay is described in Experimental Procedures. The utilization of PalC was interpreted as micromoles of PalC that was translocated from the cytosol to the mitochondria of hepatocytes per gram of liver protein per minute. Values were presented as mean ± SD (n = 3–4). *P < 0.05 indicates statistical significance between control and marked samples.

Fig. 9.

Expression of PPARα-targeted genes in the liver after cocaine and fenofibrate treatments. (A) Cpt1a expression level. (B) Cpt2 expression level. (C) Acot1 expression level. The gene expression level of control samples were arbitrarily set as 1. Values were presented as mean ± SD (n = 3–4). *P < 0.05 and **P < 0.01 indicate statistical significance between control and marked samples.