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Title: Investigations on the effect of fasting on liver function and the response to acetaminophen overdose in two mouse models
Author: Saulol Hamid, Nur Fazila
ISNI:       0000 0004 6495 3863
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2016
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Laboratory mice are widely used in biomedical research as models for human diseases and also to provide insight into the toxicity of various xenobiotics. Fasting of mice prior to dosing is common practise in toxicological studies to ensure uniform drug absorption. However, many studies ignore the potential effect of fasting on parameters that are affected by the circadian rhythm. Model hepatotoxicants like acetaminophen (APAP) are commonly used to provide better insight into, for example, chemical structure, drug metabolism and the response of cells to toxicants. The APAP mouse model has been extensively used for studies on pathogenesis and intervention of drug induced liver injury (DILI) based on the CYP450 mediated formation of N-acetyl-pbenzo-quinoneimine (NAPQI). More recently, it also served for the identification of new biomarkers for liver injury. In addition, it can also serve to gain further insight into liver regeneration, the mechanisms of which need to be further elucidated. So far, liver regeneration has mainly been studied in partial hepatectomy (PHx) models, which could have a different mechanism than regeneration after DILI. The work presented in this thesis has used two mouse models, out-bred CD-1 and in-bred C57BL/6J mice to investigate the differences in their response to APAP overdose and the effect of fasting prior to dosing. This was done over a time course expected to cover both acute liver injury and regeneration. The results on saline dosed control mice show that fasting alone significantly reduces the body weight (up to 12%) with onset of the fasting period during day time (16:00 to 18:45). Interestingly, C57BL/6J mice have higher basic hepatic GSH levels than CD-1 mice. While the hepatic GSH content followed a circadian rhythm in fed mice, it overshot in animals refed after fasting. In contrast, the restitution of ATP after feeding was slow in CD-1 mice where even after 24 hours of refeeding the ATP levels were still lower than in fed mice. In fasted C57BL/6J mice, however, they returned to control animal levels within 20 hours of refeeding. Surprisingly, after 10 hours of refeeding, the GSH levels showed a significantly higher fold increase than the ATP levels in both strains. Fasting did not induce any histopathological changes, however there was evidence of complete hepatocellular glycogen depletion after fasting and full glycogen restitution within 1 hour of refeeding in C57BL/6J mice but 4 hours in CD-1 mice. Fasting neither had an effect on the transcription of cytokines TNF-a, IL-6, and IL-10 nor on hepatocellular proliferation, assessed on the basis of NF-kB and cyclin-D1 transcription and in situ PCNA expression. The determination of the hepatic GSH and ATP content, serum ALT levels and the histopathological changes with an established scoring system served to assess the extent of liver injury following APAP overdose. A reduction of the GSH content was observed as early as 1 hour post dosing (hpd) in CD-1 mice and at 3 hpd in C57BL/6J mice; after that the levels increased gradually again until the end of the study period. In fasted CD-1 mice, GSH levels returned to control levels with delay; this was variable in C57BL/6J mice until the levels were similar to those of control animals at 36 hpd. ATP levels remained lower in fasted mice of both strains over the entire time course. In both strains, APAP overdose resulted in a progressive increase in serum ALT activity with significantly higher levels in fasted mice at the later time points. This was matched by the histological findings, i.e. centrilobular hepatocyte loss. In fed mice, cell death via apoptosis was initially seen and most abundant at 3 and 5 hpd in CD-1 mice. However, in C57BL/6J, only a few individual apoptotic hepatocytes were seen in the affected centrilobular areas at 5 hpd, whereas they were abundant in the same location at 24 hpd. The first sign of liver damage was the centrilobular loss of hepatocellular glycogen, a feature that remained obvious until the pathological changes resolved. Fasting of mice prior to APAP overdose led to similar, though more severe changes, whereby cell death was almost exclusively via necrosis with a variably intense centrilobular neutrophil infiltration. At 15 hpd, histology showed that the damage had subsided in fed CD-1 mice, in association with increased proliferation and evidence of full regeneration including complete glycogen reconstitution at 24 hpd. In C57BL/6J mice, there was prolonged evidence of centrilobular hepatocyte degeneration and death, by both apoptosis and coagulative necrosis, which declined but was still present at 36 hpd. In fasted mice of both strains, there was evidence of substantial ongoing hepatocyte death and neutrophil recruitment until the end of the study at 24 and 36 hpd, respectively, together with delayed hepatocyte proliferation. The extent of the inflammatory response after APAP-induced DILI and the regenerative capacity of the liver were assessed via the quantitative assessment of the transcription of the cytokines TNF-a, IL-6 and IL-10, and the transcription factor NF-kB and cell cycle protein, cyclin-D1 in the liver and, for TNF-a and IL-6, the spleen, using RT-qPCR. Fed animals showed early upregulation of hepatic TNF-a, obvious at 5 hpd in CD-1 and at 10 hpd in C57BL/6J mice, with levels dropping thereafter. In fasted mice, a further increase was seen after these time points, correlating also with the more pronounced neutrophil influx. Splenic TNF-a transcription seemed to follow the hepatic transcription alongside serum protein levels that were continuously elevated at the later time points, suggesting systemic effect. Fed mice, and in particular CD-1 mice, showed higher hepatic and splenic IL-6, IL-10, NF-kB and cyclin-D1 mRNA levels than fasted mice, correlating with effective liver regeneration. In fasted mice of both strains, the transcription of these markers started to increase only towards the end of the study period. The quantitative assessment of hepatocyte proliferation, based on the in situ expression of PCNA, confirmed the earlier onset and faster liver regeneration in fed animals, with an earlier onset also in fed CD-1 mice than in fed C57BL/6J mice. In conclusion, the results of the present study confirm circadian rhythms for body weight and hepatic GSH and ATP content, features that could interfere with study outcomes. They also provide definite evidence that fasting prior to APAP overdose does not only modulate the resulting liver injury, but has further effects, i.e. the induction of an inflammatory response and a delay in the subsequent liver regeneration. Finally, they confirm that the inbred C57BL/6J mice are more susceptible to APAP-induced DILI when fed prior to dosing, but show that both strains react in a similar way when dosed after a period of fasting.
Supervisor: Kipar, Anja ; Antoine, Daniel ; Stewart, James Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral