Chronotoxicity of Acrylamide in Mice Fed a High-Fat Diet: The Involvement of Liver CYP2E1 Upregulation and Gut Leakage

Abstract

Acrylamide (ACR) is produced under high-temperature cooking of carbohydrate-rich foods via the Maillard reaction. It has been reported that ACR has hepatic toxicity and can induce liver circadian disorder. A high fat diet (HFD) could dysregulate liver detoxification. The current study showed that administration of ACR (100 mg/kg) reduced the survival rate in HFD-fed mice, which was more pronounced when treated during the night phase than during the day phase. Furthermore, ACR (25 mg/kg) treatment could cause chronotoxicity in mice fed a high-fat diet, manifested as more severe mitochondrial damage of liver during the night phase than during the day phase. Interestingly, HFD induced a higher CYP2E1 expressions for those treated during the night phase, leading to more severe DNA damage. Meanwhile, the expression of gut tight junction proteins also significantly decreases at night phase, leading to the leakage of LPSs and exacerbating the inflammatory response at night phase. These results indicated that a HFD could induce the chronotoxicity of ACR in mice liver, which may be associated with increases in CYP2E1 expression in the liver and gut leak during the night phase.

Keywords: CYP2E1; acrylamide; chronotoxicity; high-fat diet.

Figure 1

The effects of a HFD on the survival rate in ACR-treated mice at ZT0 and ZT12. A total of 40 mice were randomly divided into 4 groups. The control mice and the HFD-fed mice were treated with 100 mg/kg ACR by means of intragastric administration at ZT0 or ZT12. The survival rate is shown for the four groups of mice after treatment with 100 mg/kg ACR at ZT0 and ZT12 for 48 h (n = 10/group).

Figure 2

The differential effects of ACR administration on liver damage at ZT0 and ZT12 in HFD-fed mice. A total of 40 mice received a HFD diet to induce an obese model and were then randomly divided into 4 groups (n = 10/group). Mice were treated with or without ACR for 7 days at ZT0 or at ZT12. (A) Representative H&E staining of the liver and the black arrows indicated the liver fibrosis in HFD + ACR ZT12 group. (B,C) The serum ALT and AST activity, respectively. (D) The serum GSH content. The data are presented as the mean ± SEM, n ≥ 6 mice/group. * p < 0.05, versus the HFD group at ZT0. ^# p < 0.05, ^## p < 0.01, versus the HFD + ACR group at ZT0. ^∆ p < 0.05, ^∆∆ p < 0.01, versus the HFD group at ZT12.

Figure 3

The differential effects of ACR administration on liver mitochondrial function at ZT0 and ZT12 in HFD-fed mice. (A) Representative EM images of liver sections from HFD-fed mice with or without ACR treatment at ZT0 and ZT12 (25,000×) and red arrow indicated the swollen phenotype and irregular shape mitochondrial. (B,C) Mitochondrial surface and coverage, respectively, were calculated from EM images. The mRNA levels of mitochondrial fission, fusion, and mitophagy genes (D) Fis1, (E) Drp1, (F) Mfn1, (G) Pink1, (H) Opa1, and (I) Binp3 were measured by RT-qPCR with or without ACR treatment at ZT0 and ZT12; GAPDH was used as the loading control. (J) Representative Western blots of complexes I, II, III, and IV; α-tubulin was used as a loading control. (K) The densitometric analyses. The data are presented as the mean ± SEM, n ≥ 6 mice/group. * p < 0.05, ** p < 0.01, versus the HFD group at ZT0. ^# p < 0.05, ^## p < 0.01, versus the HFD + ACR group at ZT0. ^∆ p < 0.05, ^∆∆ p < 0.01, versus the HFD group at ZT12.

Figure 4

The differential effects of ACR administration on CYP2E1 expression at ZT0 and ZT12 in HFD-fed mice. (A) Representative IHC image of CYP2E1 in the mouse liver and the positive cells were stained tan. (B) Representative Western blots of CYP2E1 and Cry1 in the mouse liver; α-tubulin was used as a loading control. (C,D) The densitometric analyses. (E) The mRNA level of liver CYP2E1 (F) The liver CYP2E1 activity. (G) The mRNA level of POR in the mouse liver; GAPDH was used as the loading control. The data are presented as the mean ± SEM, n ≥ 6 mice/group. ** p < 0.01, versus the HFD group at ZT0. ^## p < 0.01, versus the HFD + ACR group at ZT0. ^∆ p < 0.05, versus the HFD group at ZT12.

Figure 5

The differential effects of ACR administration on DNA damage in the liver at ZT0 and ZT12 in HFD-fed mice. (A) Images of DNA damage as revealed by the comet assay (800×). (B) Percent with comet. The data are presented as the mean ± SEM, n ≥ 6 mice/group. ^## p < 0.01, versus the HFD + ACR group at ZT0.

Figure 6

The differential effects of ACR administration on the gut barrier integrity at ZT0 and ZT12 in HFD-fed mice. (A) Immunofluorescence staining images of Claudin-1 and Occludin isolated from the mouse gut. The blue fluorescence was DAPI staining and the green fluorescence was fluorescent secondary antibody of Claudin-1 and Occludin. (B,C) The mRNA levels of Claudin-1 and Occludin, respectively; GAPDH was used as the loading control. (D) The LPS content in the serum. The data are presented as the mean ± SEM, n ≥ 6 mice/group. ** p < 0.01, versus the HFD group at ZT0. ^## p < 0.01, versus the HFD + ACR group at ZT0.

Figure 7

The differential effects of ACR administration on inflammatory responses at ZT0 and ZT12 in HFD-fed mice. (A) Western blots with loading controls of p-NF-κB and NF-κB, p-p38 and p38, and p-JNK and JNK. (B) The densitometric analyses of p-NF-κB/NF-κB, p-ERK/ERK, p-P38/P38, and p-JNK/JNK. (C) The mRNA levels of TNF-α and IL-1β in the livers of ACR-treated, HFD-fed mice at ZT0 and ZT12; GADPH was used as the loading control. (D) The mRNA levels of TNF-α and IL-1β in the gut of ACR-treated, HFD-fed mice at ZT0 and ZT12; GADPH was used as the loading control. The data are presented as the mean ± SEM, n ≥ 6 mice/group. ^## p < 0.01, versus the HFD + ACR group at ZT0.