Thesis (Ph.D.)--Boston University / The liver has been well established as being one of the most important organs involved in the metabolism of adrenocortical steroids. Liver perfusion, incubations with liver slices, homogenates and purified liver preparations all effect extensive changes on the steroid nucleus. In order to develop a step-wise scheme for the in vitro metabolism of a typical adrenocortical steroid and to localize enzyme systems effecting single metabolic transformations, 11-deoxycortisol was incubated with various rat liver homogenate fractions and the conversion products isolated and identified. The liver fractions were obtained by differential centrifugation of liver homogenates. This procedure afforded a 6000 x g supernatant, a 6000 x g residue (corresponding to mitochondria in sedimentation properties), a 78,000 x g residue (corresponding to microsoaes in sedimentation properties) and a 78.000 x g particulate free supernatant. Each tissue preparation was incubated with 11-deoxycortisol at 37° c. for two hours with flasks open to the atmosphere. A tissue to steroid ratio of approximately 1000:1, based on the initial weight of the liver, was employed [TRUNCATED].

Acute liver failure confers a high risk of death, with liver transplantation offering the only effective therapy for life-threatening cases. Hepatic macrophages are crucial for innate immune integrity and effective hepatocyte proliferation. The macrophage may therefore present a novel therapeutic target to enhance regeneration following acute liver injury. In this thesis I describe the development and use of mouse models of liver injury including partial hepatectomy, partial hepatectomy plus chronic liver injury and paracetamol intoxication. I show the development of liver function assays in these models including quantification of hepatic clearance of indocyanine green by fluorescent imaging and assessment of hepatic phagocytic capacity using fluorescent microbeads. I then describe macrophage based therapeutic interventions in mouse models of liver injury. Firstly the direct administration of bone marrow derived macrophages in partial hepatectomy plus chronic liver injury. I then tested the administration of macrophage colony stimulating factor in mouse models of partial hepatectomy, partial hepatectomy plus chronic liver injury and paracetamol intoxication, describing the phenotype and exploring mechanisms of action. Collaborating with others I assessed serum CSF1 levels in humans with liver injury due to partial hepatectomy or paracetamol intoxication. I show that in acute liver failure a high serum CSF1 level is predictive of survival, indicating a new mechanistic biomarker.

The effects of di-2 ethylhexyl phthalate (DEHP) over periods of 3 days to 9 months were examined in both male and female rats using biochemical, histological and ultrastructural techniques. Responses occurred in a characteristic order. Initial effects included : changes in the. distribution of lipid in the liver, proliferation of hepatic peroxisomes and induction of laurate hydroxylases. More slowly developing changes were (1) hypertrophy of the hepatocytes, (2) centrilobular loss of glycogen and (3) a fall in glucose-6-phosphatase activity. After 9 months of treatment, accumulation of lipid-loaded lysosomes were observed. All these changes were observed in rats treated with 1000 or 200 mg/kg/day of DEHP, but rats treated with 50 mg/kg/day of DEHP did not show the later changes. In addition to these sustained alterations, two transient changes were observed. Male rats, treated with 1000 mg/kg/day of DEHP showed changes in the biliary system as shown by electron microscopy, by examination of bile flow, bile enzymes and proteins. Furthermore, a burst of mitosis occurred immediately after commencement of treatment of DEHP, the magnitude and time of which was dose-dependent. Changes in female rats were qualitatively similar, but quantitatively smaller, than in male rats. Mature male rats treated for 3 or 13 days with either DEHP or the hypolipidaemic drugs fenofibrate or clofibrate, showed similar changes to young rats with the exception of the mitotic burst which did not occur in these animals. The initial short term effects of DEHP and the straight chain analogues, di-n-octyl phthalate (DN0P) and di-n- hexyl phthalate (DNHP) in vivo and their respective esters, MEHP and MNHP, were reproduced in cultured hepatocytes. There was induction of peroxisomal enzymes in response to treatment with DEHP or MEHP but little or no induction after treatment with DNOP, MNOP or MNHP. Accumulation of lipid was seen after 24 hours of treatment With MEHP, MNOP and MNHP. However, the mitotic burst was not reproduced in cultured hepatocytes treated with MEHP, neither was there a fall in glucose-6-phosphatase activity. There was no increase in H[2]O[2] production either in vitro in cultured hepatocytes treated with MEHP, or in vivo as measured by catalase compound I in perfused liver from rats treated with DEHP in the diet. There was no evidence for mutagenic activity of DEHP or MEHP in the Ames Test. Treatment of isolated hepatocytes with MEHP, straight chain phthalate esters or clofibrate, resulted in early marked pertubations in lipid metabolism, namely, an increase in production of neutral fats and generally in fatty acid oxidation. This may explain the increased storage of lipid, in the liver. There was a marked difference in response between hepatocytes isolated from fed and fasted rats, the latter being more sensitive to all compounds. Indeed with fed rats there was, in some cases, a slight decrease in fatty acid oxidation. The effects on lipid metabolism were observed at concentrations which produced peroxisome proliferation in cultured cells.

Hepatocellular carcinoma (HCC) is a devastating malignancy originating from hepatocytes. There is an urgent need for novel therapeutic approaches. Currently explored gene therapy systems have not yet achieved significant survival benefits. The aim of this thesis was the development and evaluation of novel genetic approaches to this malignancy.

CYP3A4 is the most abundant P450 enzyme expressed in the human liver and it is responsible for the metabolism of approximately 50% of all clinically administrated drugs. The CYP3A4 gene is transcriptionally regulated by xenobiotics and previous work has demonstrated the first 300bp of proximal promoter to be the minimal requirement for such activation. Several nuclear receptors (CAR, SXR) have been shown to be involved in the induction of the CYP3A4 gene. The aim of this work was to further delineate the molecular basis of CYP3A4 gene expression. In vitro DNase I footprinting was carried out using HepG2 nuclear extracts to map the sites of DNA-protein interaction within the -301/+7 region of the CYP3A4 gene. Putative protein assignment for these sites using in silico analysis revealed the potential binding of transcription factors previously shown to be involved in the regulation of other CYP genes (Sp1, HNF3 and C/EBP?) at the identified protein-DNA interaction sites. These regulatory regions were then disrupted by mutagenesis and their functional effect assessed by transient transfections of reporter gene plasmids into HuH7 hepatoma cells. Statistically significant reductions of reporter gene expression were observed when putative C/EBPa and HNF sites were altered, in both the basal and rifampicin (SXR ligand) induced states. This finding suggests the involvement of proteins binding at these sites in the regulation of the CYP3A4 gene expression. An examination of the CYP3A4 promoter (-1056/+7) from 11 human DNA samples exhibiting a 14.3 fold variability in CYP3A mediated metabolism failed to show the presence of mutations. Protein-DNA interaction analysis were carried out within the newly identified CYP3A4*IE and CYP3A4*1F alleles as well as the CYP3A4 *1B allele. The results implicate the Spl transcription factor in the regulation of the CYP3A4 gene, albeit at a more distal binding site. The findings described in this thesis suggest a substantial involvement of transcription factors other than SXR/CAR in expression of CYP3A4. Because of the polymorphic expression of several liver- and hormone-dependent transcription factors their role in CYP3A4 regulation must be taken into account to understand drug-induction mechanisms and assess variability in inter-individual drug response.

No description available.

No description available.

An investigation was undertaken to study the kinetics and possible cellular control mechanisms for imipramine HCl metabolism in the isolated perfused rat liver. The isotope ¹⁴C-imipramine was used and quantification was done by liquid scintillation counting. Analysis for imipramine (IMI), desmethylimipramine (DMI), free hydroxy (OH), glucuronide (G) and N-oxide (N-0) metabolites was done on the perfusate, bile and liver. The rate of IMI metabolism was found to be dependent on two major enzymatic routes, N-demethylation (formation of DMI) and aromatic hydroxylation (formation of G, OH) of imipramine and one minor enzymatic route, N-oxidation (N-O). The rate of aromatic hydroxylation of IMI was found to be inhibited after thirty minutes, with IMI concentration 2 X 10 ⁻⁵M. This inhibition of aromatic hydroxylation could not be detected if the perfusate half-life for IMI (t½=18.5 minutes) was the only parameter monitored. After incubation periods of fifteen, thirty and sixty 80 per cent and the remainder of IMI was in the perfusate. The dose of IMI was varied (0.5 X 10⁻⁵ M, 1 X 10⁻⁵ M and 2 X 10⁻⁵ M) for metabolism by the perfused rat liver. The incubation time was kept constant at fifteen minutes. The rate of imipramine metabolism (formation of DMI and GOH) followed first order kinetics when the dose of IMI was 0.5 X 10⁻⁵ M or 1 X 10⁻⁵ M. Increasing the dose of IMI to 2 X 10⁻⁵ M slightly suppressed the formation of DMI and the formation of GOH followed zero order kinetics. It was found that the endogenous DMI formed from IMI metabolism inhibited the formation of GOH after fifteen minutes and thirty minutes of IMI metabolism as shown by the following results. DMI (1.65, 3.33, 6.66 or 13.32 X 10⁻⁶ M) was preincubated prior to addition of IMI. DMI (1.65 or 3.33 X 10⁻⁶ M) was found to specifically inhibit aromatic hydroxylation of IMI. Higher concentration of DMI (6.66 or 13.32 X 10⁻⁶ M) inhibited the formation of GOH and DMI. Ethyl alcohol (1 mM) preincubated prior to addition of 1 X 10⁻⁵ M of IMI specifically inhibited DMI formation. No inhibition of GOH occurred. Ethyl alcohol (1 mM) caused inhibition of formation of DMI from IMI metabolism when the dose of IMI was 2 X 10⁻⁵ M. The incubation time for IMI metabolism was fifteen and sixty minutes. With this decrease of DMI formation, the formation of GOH increased after fifteen or sixty minutes of incubation time. From these experiments it was concluded that suppression of aromatic hydroxylation of imipramine was due to the formation of endogenous DMI formed from IMI metabolism. Optimal conditions were found to study possible cellular control mechanisms for IMI metabolism in the isolated perfused rat liver. The dose of IMI was 1 X 10⁻⁵ M and the incubation time was fifteen minutes. Dibutyryl cyclic AMP (2 X 10⁻⁶ M) caused inhibition of IMI metabolism. DMI formation was inhibited 28 per cent while GOH formation was inhibited 29 per cent. NADPH (1.1 X 10⁻⁶ M) or NADH (1.3 X 10⁻⁶ M) was found to inhibit imipramine metabolism. GOH and DMI were both inhibited. Succinic acid (1.6 X 10⁻³ M) was found to inhibit DMI formation but not GOH. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Introduction: COVID-19 affects the liver, causing alteration in liver biochemistry tests such as aspartate transferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP), total bilirubin and albumin. Objective: To determine the prevalence of alteration in liver functions tests and associated factors for severity among Peruvian COVID-19 patients. Materials and methods: A descriptive, retrospective and cross-sectional study was performed in 4 public hospitals in Peru. Patients admitted to hospitalization wards and intensive care units with a diagnosis COVID-19 were enrolled. The evaluation of AST, ALT, ALP, total bilirubin and albumin was performed. Associations with demographic and medical data were assessed. Results: 1,100 patients were enrolled, of which 81.7% had altered liver function tests. Only 2.8% of the patients had cirrhosis and 2.1% hepatitis B/C virus. AST and ALT were altered at admission in 64.7% and 63.7%, of the patients respectively. Factors associated with liver injury were: being female OR=0.53 (95% CI: 0.39-0.73; p<0.01), dyslipidemia OR=1.72 (95% CI: 1.10-2.70; p=0.01), previous medication OR=1.56 (95% CI: 1.12 -2.16, p<0.01) and fever OR=1.43 (95% CI: 1.03-1.199, p=0.03). Disease severity was associated with levels of AST and ALT (p<0.01). Patients taking self-medication OR=1.56 (95% CI: 1.12-2.16; p<0.01) and paracetamol OR= 1.41 (95% CI:1.01-1.98; p=0.04) had higher risk of liver injury. Meanwhile, corticosteroids OR=0.55 (95% CI: 0.38-0.78; p<0.01) and enoxaparin OR=0.53 (95% CI: 0.35- 0.81; p<0.01) were protective factors. Conclusions: Peruvian patients with COVID- teration in liver function tests, high levels of AST and ALT were related to disease severity. / Introducción: La COVID-19 afecta al hígado, provocando alteración en las pruebas de función hepática como aspartato aminotransferasa (AST), alanina aminotransferasa (ALT), fosfatasa alcalina (FA), bilirrubina total y albúmina. Objetivo: Determinar la prevalencia de alteración en las pruebas de función hepática y su asociación con la severidad en pacientes peruanos con COVID-19. Materiales y métodos: Se realizó un estudio descriptivo, retrospectivo y transversal en 4 hospitales públicos del Perú. Se incluyeron pacientes admitidos en hospitalización y unidades de cuidados intensivos con diagnóstico de COVID-19. Se realizó la evaluación de AST, ALT, FA, bilirrubina total y albúmina. Se evaluaron las asociaciones con datos demográficos y médicos. Resultados: Se incluyeron 1,100 pacientes, de los cuales el 81,7% presentaba alteraciones en las pruebas de función hepática. Solo el 2,8% de los pacientes tenía cirrosis y el 2,1% infección por virus de la hepatitis B / C. Se encontraron niveles alterados de AST y ALT al ingreso en el 64,7% y 63,7% de los pacientes, respectivamente. Los factores asociados con alteración en pruebas de función hepáticas fueron: ser mujer OR = 0,53 (IC 95%: 0,39-0,73; p <0,01), dislipidemia OR=1,72 (IC 95%: 1,10-2,70; p=0,01), uso de medicación previa OR = 1,56 (IC del 95%: 1,12 -2,16, p <0,01) y fiebre OR = 1,43 (IC del 95%: 1,03-1,199, p = 0,03). La gravedad de la enfermedad se asoció con los niveles de AST y ALT (p <0,01). Los pacientes que se automedicaban OR = 1,56 (IC 95%: 1,12-2,16; p <0,01) y tomaban paracetamol OR = 1,41 (IC 95%: 1,01-1,98; p =0,04) tenían mayor riesgo de injuria hepática. Mientras tanto, los corticosteroides OR=0,55 (IC del 95%: 0,38-0,78; p <0,01) y la enoxaparina OR=0,53 (IC del 95%: 0,35-0,81; p <0,01) fueron factores protectores. Conclusiones: los pacientes peruanos con COVID-19 presentaron alta prevalencia de alte hepática, niveles elevados de AST y ALT se relacionaron con la gravedad de la enfermedad.

Patients with liver cirrhosis are susceptible to fungal infections. Due to low sensitivity of culture-based methods, we applied a real-time PCR assay targeting the 18S rRNA gene in combination with direct sequencing and terminal-restriction fragment length polymorphism (T-RFLP) in order to establish a novel tool to detect fungal DNA and to quantify and differentiate Candida DNA, also in polyfungal specimens. In total, 281 samples (blood n=135, ascites n=92, duodenal fluid n=54) from 135 patients with liver cirrhosis and 52 samples (blood n=26, duodenal fluid n=26) from 26 control patients were collected prospectively. Candida DNA was quantified in all samples. Standard microbiological culture was performed for comparison. Blood and ascites samples, irrespective of the patient cohort, showed a method-independent low fungal detection rate of approximately 1%, and the Candida DNA content level did not exceed 3.0x101 copies ml-1 in any sample. In contrast, in duodenal fluid of patients with liver cirrhosis high fungal detection rates were discovered by using both PCR- and culture-based techniques (81.5% vs. 66.7%; p=0.123) and the median level of Candida DNA was 3.8x105 copies ml-1 (2.3x102-6.3x109). In cirrhosis and controls, fungal positive culture results were confirmed by PCR in 96% and an additional amount of 44% of culture negative duodenal samples were PCR positive. Using T-RFLP analysis in duodenal samples, overall 85% of results from microbial culture were confirmed and in 75% of culture-negative but PCR-positive samples additional Candida species could be identified. In conclusion, PCR-based methods and subsequent differentiation of Candida DNA might offer a quick approach to identifying Candida species without prior cultivation.