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Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome.

Eakins R, Walsh J, Randle L, Jenkins RE, Schuppe-Koistinen I, Rowe C, Starkey Lewis P, Vasieva O, Prats N, Brillant N, Auli M, Bayliss M, Webb S, Rees JA, Kitteringham NR, Goldring CE, Park BK - Sci Rep (2015)

Bottom Line: Acetaminophen overdose is the leading cause of acute liver failure.Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation.These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Drug Safety Science, University of Liverpool, Liverpool L69 3GE, UK.

ABSTRACT
Acetaminophen overdose is the leading cause of acute liver failure. One dose of 10-15 g causes severe liver damage in humans, whereas repeated exposure to acetaminophen in humans and animal models results in autoprotection. Insight of this process is limited to select proteins implicated in acetaminophen toxicity and cellular defence. Here we investigate hepatic adaptation to acetaminophen toxicity from a whole proteome perspective, using quantitative mass spectrometry. In a rat model, we show the response to acetaminophen involves the expression of 30% of all proteins detected in the liver. Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation. We show that adaptation to acetaminophen has a spatial component, involving a shift in regionalisation of CYP2E1, which may prevent toxicity thresholds being reached. These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury.

No MeSH data available.


Related in: MedlinePlus

Rat liver CYP2E1 activity and localisation changes in response to repeat acetaminophen exposure.Microsomal formation of (a) APAP-GSH and (b) 6′-OH chlorzoxazone in animals which were either vehicle control treated, or repeat dosed with or without toxicity. Microsomal CYP2E1 activity is reduced in toxic (48 h treated, red squares) group and increased in non-toxic (96h treated, blue triangles) group compared to control (black circles). APAP metabolism is significantly higher in non-toxic (96h treated) group (**p < 0.01, ***p < 0.001, ****p < 0.0001). Best fit curves for each group (solid lines) were modelled using literature values for Km and Vmax. (c) Top panel shows representative IHC staining for CYP2E1 across the timecourse; bottom panel shows H&E stain. CV indicates central vein, while PT indicates the portal triad. CYP2E1 redistributes markedly as the timecourse progresses. (d) Densitometric analysis of total liver GLUL, CYP2E1 and actin detected by western blot, showing preferential restitution of CYP2E1 abundance over the similarly centrilobular glutamine synthetase (*p < 0.05, **p < 0.01, ***p < 0.001). (e) Graphical summary of the model.
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f5: Rat liver CYP2E1 activity and localisation changes in response to repeat acetaminophen exposure.Microsomal formation of (a) APAP-GSH and (b) 6′-OH chlorzoxazone in animals which were either vehicle control treated, or repeat dosed with or without toxicity. Microsomal CYP2E1 activity is reduced in toxic (48 h treated, red squares) group and increased in non-toxic (96h treated, blue triangles) group compared to control (black circles). APAP metabolism is significantly higher in non-toxic (96h treated) group (**p < 0.01, ***p < 0.001, ****p < 0.0001). Best fit curves for each group (solid lines) were modelled using literature values for Km and Vmax. (c) Top panel shows representative IHC staining for CYP2E1 across the timecourse; bottom panel shows H&E stain. CV indicates central vein, while PT indicates the portal triad. CYP2E1 redistributes markedly as the timecourse progresses. (d) Densitometric analysis of total liver GLUL, CYP2E1 and actin detected by western blot, showing preferential restitution of CYP2E1 abundance over the similarly centrilobular glutamine synthetase (*p < 0.05, **p < 0.01, ***p < 0.001). (e) Graphical summary of the model.

Mentions: In fact, when we looked in greater detail at another of the key changes visualized in our proteomic analysis, we determined that the process of autoprotection is yet more complicated than straightforward changes in the expression of a specific protein. Our proteomic data for CYP2E1 (Fig. 4a,b) looked to be particularly pertinent in the process of adaptation to APAP, as it shows a loss of the pre-eminent metabolising enzyme responsible for formation of the toxic metabolite of APAP (NAPQI). We therefore looked at the metabolism of both APAP (Fig. 5a) and the CYP2E1 probe substrate chlorzoxazone (Fig. 5b) in our rat model. Whilst overall APAP metabolism was significantly increased at 96 h (Fig. 5a), specific CYP2E1 activity was reduced at 48 h and increased at 96 h (Fig. 5b). Relative CYP2E1 activity was 0.78 at 48 h, and 1.43 at 96 h, compared to control (arbitrary value of 1). These findings, which mirrored our proteomic data for CYP2E1, indicated the potential for an increase in the generation of NAPQI in vivo, which was difficult to resolve with the observed process of adaptation to repeated exposure.


Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome.

Eakins R, Walsh J, Randle L, Jenkins RE, Schuppe-Koistinen I, Rowe C, Starkey Lewis P, Vasieva O, Prats N, Brillant N, Auli M, Bayliss M, Webb S, Rees JA, Kitteringham NR, Goldring CE, Park BK - Sci Rep (2015)

Rat liver CYP2E1 activity and localisation changes in response to repeat acetaminophen exposure.Microsomal formation of (a) APAP-GSH and (b) 6′-OH chlorzoxazone in animals which were either vehicle control treated, or repeat dosed with or without toxicity. Microsomal CYP2E1 activity is reduced in toxic (48 h treated, red squares) group and increased in non-toxic (96h treated, blue triangles) group compared to control (black circles). APAP metabolism is significantly higher in non-toxic (96h treated) group (**p < 0.01, ***p < 0.001, ****p < 0.0001). Best fit curves for each group (solid lines) were modelled using literature values for Km and Vmax. (c) Top panel shows representative IHC staining for CYP2E1 across the timecourse; bottom panel shows H&E stain. CV indicates central vein, while PT indicates the portal triad. CYP2E1 redistributes markedly as the timecourse progresses. (d) Densitometric analysis of total liver GLUL, CYP2E1 and actin detected by western blot, showing preferential restitution of CYP2E1 abundance over the similarly centrilobular glutamine synthetase (*p < 0.05, **p < 0.01, ***p < 0.001). (e) Graphical summary of the model.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4660393&req=5

f5: Rat liver CYP2E1 activity and localisation changes in response to repeat acetaminophen exposure.Microsomal formation of (a) APAP-GSH and (b) 6′-OH chlorzoxazone in animals which were either vehicle control treated, or repeat dosed with or without toxicity. Microsomal CYP2E1 activity is reduced in toxic (48 h treated, red squares) group and increased in non-toxic (96h treated, blue triangles) group compared to control (black circles). APAP metabolism is significantly higher in non-toxic (96h treated) group (**p < 0.01, ***p < 0.001, ****p < 0.0001). Best fit curves for each group (solid lines) were modelled using literature values for Km and Vmax. (c) Top panel shows representative IHC staining for CYP2E1 across the timecourse; bottom panel shows H&E stain. CV indicates central vein, while PT indicates the portal triad. CYP2E1 redistributes markedly as the timecourse progresses. (d) Densitometric analysis of total liver GLUL, CYP2E1 and actin detected by western blot, showing preferential restitution of CYP2E1 abundance over the similarly centrilobular glutamine synthetase (*p < 0.05, **p < 0.01, ***p < 0.001). (e) Graphical summary of the model.
Mentions: In fact, when we looked in greater detail at another of the key changes visualized in our proteomic analysis, we determined that the process of autoprotection is yet more complicated than straightforward changes in the expression of a specific protein. Our proteomic data for CYP2E1 (Fig. 4a,b) looked to be particularly pertinent in the process of adaptation to APAP, as it shows a loss of the pre-eminent metabolising enzyme responsible for formation of the toxic metabolite of APAP (NAPQI). We therefore looked at the metabolism of both APAP (Fig. 5a) and the CYP2E1 probe substrate chlorzoxazone (Fig. 5b) in our rat model. Whilst overall APAP metabolism was significantly increased at 96 h (Fig. 5a), specific CYP2E1 activity was reduced at 48 h and increased at 96 h (Fig. 5b). Relative CYP2E1 activity was 0.78 at 48 h, and 1.43 at 96 h, compared to control (arbitrary value of 1). These findings, which mirrored our proteomic data for CYP2E1, indicated the potential for an increase in the generation of NAPQI in vivo, which was difficult to resolve with the observed process of adaptation to repeated exposure.

Bottom Line: Acetaminophen overdose is the leading cause of acute liver failure.Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation.These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Drug Safety Science, University of Liverpool, Liverpool L69 3GE, UK.

ABSTRACT
Acetaminophen overdose is the leading cause of acute liver failure. One dose of 10-15 g causes severe liver damage in humans, whereas repeated exposure to acetaminophen in humans and animal models results in autoprotection. Insight of this process is limited to select proteins implicated in acetaminophen toxicity and cellular defence. Here we investigate hepatic adaptation to acetaminophen toxicity from a whole proteome perspective, using quantitative mass spectrometry. In a rat model, we show the response to acetaminophen involves the expression of 30% of all proteins detected in the liver. Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation. We show that adaptation to acetaminophen has a spatial component, involving a shift in regionalisation of CYP2E1, which may prevent toxicity thresholds being reached. These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury.

No MeSH data available.


Related in: MedlinePlus