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ATPase, class 1, type 8 A, member 2 (ATP8A2) is a P4-ATPase with a critical role in phospholipid translocation across the plasma membrane. Pathogenic variants in ATP8A2 are known to cause cerebellar ataxia, impaired intellectual development, and disequilibrium syndrome 4 (CAMRQ4) which is often associated with encephalopathy, global developmental delay, and severe motor deficits. Here, we present a family with two siblings born from a consanguineous, first-cousin union from Sudan presenting with global developmental delay, intellectual disability, spasticity, ataxia, nystagmus, and thin corpus callosum. Whole exome sequencing revealed a homozygous missense variant in the nucleotide binding domain of ATP8A2 (p.Leu538Pro) that results in near complete loss of protein expression. This is in line with other missense variants in the same domain leading to protein misfolding and loss of ATPase function. In addition, by performing diffusion-weighted imaging, we identified bilateral hyperintensities in the posterior limbs of the internal capsule suggesting possible microstructural changes in axon tracts that had not been appreciated before and could contribute to the sensorimotor deficits in these individuals., (© 2024. The Author(s).)

Biallelic mutations in Protein O-mannosyltransferase 1 (POMT1) are among the most common causes of a severe group of congenital muscular dystrophies (CMDs) known as dystroglycanopathies. POMT1 is a glycosyltransferase responsible for the attachment of a functional glycan mediating interactions between the transmembrane glycoprotein dystroglycan and its binding partners in the extracellular matrix (ECM). Disruptions in these cell-ECM interactions lead to multiple developmental defects causing brain and eye malformations in addition to CMD. Removing Pomt1 in the mouse leads to early embryonic death due to the essential role of dystroglycan during placental formation in rodents. Here, we characterized and validated a model of pomt1 loss of function in the zebrafish showing that developmental defects found in individuals affected by dystroglycanopathies can be recapitulated in the fish. We also discovered that pomt1 mRNA provided by the mother in the oocyte supports dystroglycan glycosylation during the first few weeks of development. Muscle disease, retinal synapse formation deficits, and axon guidance defects can only be uncovered during the first week post fertilization by generating knock-out embryos from knock-out mothers. Conversely, maternal pomt1 from heterozygous mothers was sufficient to sustain muscle, eye, and brain development only leading to loss of photoreceptor synapses at 30 days post fertilization. Our findings show that it is important to define the contribution of maternal mRNA while developing zebrafish models of dystroglycanopathies and that offspring generated from heterozygous and knock-out mothers can be used to differentiate the role of dystroglycan glycosylation in tissue formation and maintenance., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

One hurdle in the development of zebrafish models of human disease is the presence of multiple zebrafish orthologs resulting from whole genome duplication in teleosts. Mutations in inositol polyphosphate 5-phosphatase K (INPP5K) lead to a syndrome characterized by variable presentation of intellectual disability, brain abnormalities, cataracts, muscle disease, and short stature. INPP5K is a phosphatase acting at position 5 of phosphoinositides to control their homeostasis and is involved in insulin signaling, cytoskeletal regulation, and protein trafficking. Previously, our group and others have replicated the human phenotypes in zebrafish knockdown models by targeting both INPP5K orthologs inpp5ka and inpp5kb. Here, we show that inpp5ka is the more closely related orthologue to human INPP5K. While both inpp5ka and inpp5kb mRNA expression levels follow a similar trend in the developing head, eyes, and tail, inpp5ka is much more abundantly expressed in these tissues than inpp5kb. In situ hybridization revealed a similar trend, also showing unique localization of inpp5kb in the pineal gland and retina indicating different transcriptional regulation. We also found that inpp5kb has lost its catalytic activity against its preferred substrate, PtdIns(4,5)P 2 . Since most human mutations are missense changes disrupting phosphatase activity, we propose that loss of inpp5ka alone can be targeted to recapitulate the human presentation. In addition, we show that the function of inpp5kb has diverged from inpp5ka and may play a novel role in the zebrafish., (© 2023. The Author(s).)

Background & Aims: The outbreak of COVID-19 has vastly increased the operational burden on healthcare systems worldwide. For patients with end-stage liver failure, liver transplantation is the only option. However, the strain on intensive care facilities caused by the pandemic is a major concern. There is an urgent need for ethical frameworks to balance the need for liver transplantation against the availability of national resources., Methods: We performed an international multicenter study of transplant centers to understand the evolution of policies for transplant prioritization in response to the pandemic in March 2020. To describe the ethical tension arising in this setting, we propose a novel ethical framework, the quadripartite equipoise (QE) score, that is applicable to liver transplantation in the context of limited national resources., Results: Seventeen large- and medium-sized liver transplant centers from 12 countries across 4 continents participated. Ten centers opted to limit transplant activity in response to the pandemic, favoring a "sickest-first" approach. Conversely, some larger centers opted to continue routine transplant activity in order to balance waiting list mortality. To model these and other ethical tensions, we computed a QE score using 4 factors - recipient outcome, donor/graft safety, waiting list mortality and healthcare resources - for 7 countries. The fluctuation of the QE score over time accurately reflects the dynamic changes in the ethical tensions surrounding transplant activity in a pandemic., Conclusions: This four-dimensional model of quadripartite equipoise addresses the ethical tensions in the current pandemic. It serves as a universally applicable framework to guide regulation of transplant activity in response to the increasing burden on healthcare systems., Lay Summary: There is an urgent need for ethical frameworks to balance the need for liver transplantation against the availability of national resources during the COVID-19 pandemic. We describe a four-dimensional model of quadripartite equipoise that models these ethical tensions and can guide the regulation of transplant activity in response to the increasing burden on healthcare systems., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Liver transplantation (LT) offers the best chance of survival in selected patients with hepatocellular carcinoma (HCC). Wait-list mortality or dropout due to tumor progression can be significant, and therefore, timely transplantation is critical. Liver grafts discarded by outside organ procurement organizations are a potential source of grafts for low Model for End-Stage Liver Disease tumor patients. The primary aim of this study was to assess the disease-free and overall survival of patients with HCC transplanted with imported liver grafts (ILGs). Review of all patients transplanted for HCC between June 2005 and December 2014 was performed. Data on demographics, survival, and HCC recurrence were analyzed. During this time period, 59 out of 190 (31%) recipients with HCC received ILG. Of these 59 grafts, 54 were imported from within the region and 5 were from national offers (outside the region). The mean cold ischemia time for local liver grafts (LLGs) was 4.1 ± 1.5 hours versus 5.1 ± 1.4 hours for ILG (P < 0.001). The 1-, 3-, and 5-year patient survival was 90%, 85%, and 83% and 85%, 80%, and 79% for LLG and ILG (P = 0.08), respectively. The observed disease recurrence rate for both LLG and ILG recipients was equivalent. The median wait-list time for HCC recipients was 43 days (range, 2-1167 days). In conclusion, with careful graft assessment, the use of ILGs results in comparable outcomes following LT and no increased risk of HCC recurrence. Use of ILGs maximizes the donor pool and results in a higher rate of transplantation for HCC recipients. Liver Transplantation 23 299-304 2017 AASLD., (© 2016 by the American Association for the Study of Liver Diseases.)

Background: Portal vein thrombosis (PVT) is relatively common among candidates for liver transplantation and can present significant intraoperative challenges. Depending on the extent of PVT, thromboendovenectomy (TEV), portal bypass, or systemic inflow may be required to restore portal inflow. While TEV is the most commonly used approach to restore anatomic portal inflow, portal vein injury and life-threatening hemorrhage are risks with this technique., Case Report: We present a salvage technique for managing portal vein injury during TEV using intraluminal balloon occlusion of the portal vein during portal vein repair and reconstruction. This alternative mode of bleeding control optimizes exposure to the retropancreatic space and avoids direct application of vascular clamps that can cause further injury to the vessel and surrounding tissue., Conclusion: Careful preoperative planning and anticipation of potential problems are essential for safe and effective management of complex PVT intraoperatively. The balloon-occlusion technique can facilitate safe and efficient repair of a portal vein injury during TEV for liver transplantation.

This article presents a miniature shock wave driven micro-jet generator to deliver liquid drugs into human skin, to a controlled depth, with minimal invasion. The device can release the vaccine/drug to the depth of dermal blood vessels, without breaching much of the microcirculation system of dermis. The drug delivery technique is needle-free, which can reduce pain, trauma, and contamination besides minimal dosage and systemic exposure. The device can also be used to deliver liquid or colloidal drugs into soft tissues in human. The mechanical analyses of the device were carried out by analyzing the strength of the impulse of the shock wave, measuring the velocity of the generated jet and capturing the pressure exerted by the jet on the target. The penetrating ability of the jet was investigated by delivering it into sample of human skin and gelatin slabs. Theoretical analyses were carried out on the physics of the delivery and the predicted results had a close agreement with the experimental observations. The development can offer an important cost-effective solution to needle-free health care worldwide. Biotechnol. Bioeng. 2016;113: 2507-2512. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Objectives: Isolated intrahepatic recurrence is noted in up to 40% of patients following curative liver resection for colorectal liver metastases (CLM). The aims of this study were to analyse the outcomes of repeat hepatectomy for recurrent CLM and to identify factors predicting survival., Methods: Data for all liver resections for CLM carried out at one centre between 1998 and 2011 were analysed., Results: A total of 1027 liver resections were performed for CLM. Of these, 58 were repeat liver resections performed in 53 patients. Median time intervals were 10.5 months between the primary resection and first hepatectomy, and 15.4 months between the first and repeat hepatectomies. The median tumour size was 3.0 cm and the median number of tumours was one. Six patients had a positive margin (R1) resection following first hepatectomy. There were no perioperative deaths. Significant complications included transient liver dysfunction in one and bile leak in two patients. Rates of 1-, 3- and 5-year overall survival following repeat liver resection were 85%, 61% and 52%, respectively, at a median follow-up of 23 months. R1 resection at first hepatectomy (P = 0.002), a shorter time interval between the first and second hepatectomies (P = 0.02) and the presence of extrahepatic disease (P = 0.02) were associated with significantly worse overall survival., Conclusions: Repeat resection of CLM is safe and can achieve longterm survival in carefully selected patients. A preoperative knowledge of poor prognostic factors helps to facilitate better patient selection., (© 2013 International Hepato-Pancreato-Biliary Association.)

Background: Spontaneous liver bleeding (SLB) is a rare but potentially fatal complication. In contrast to the East, various benign pathologies are the source of SLB in the West. An accurate diagnosis and a timely implementation of appropriate treatment are crucial in the management of these patients. The present study presents a large Western experience of SLB from a specialist liver centre., Methods: A retrospective analysis of patients presented with SLB between January 1995 and January 2011., Results: Sixty-seven patients had SLB, 44 (66%) were female and the median age at presentation was 47 years. Abrupt onset upper abdominal pain was the presenting symptom in 65 (97%) patients. The aetiology for SLB was hepatic adenoma in 27 (40%), hepatocellular carcinoma (HCC) in 17 (25%) and various other liver pathologies in the rest. Emergency treatment included a conservative approach in 42 (64%), DSA and embolization in 6 (9%), a laparotomy and packing in 6 (9%) and a liver resection in 11 (16%) patients. Eleven (16%) patients had further planned treatments. Seven (10%) died during the same admission but the mortality was highest in patients with HELLP syndrome. At a median follow-up of 54 months all patients with benign disease are alive. The 1-, 3- and 5-year survival of patients with HCC was 59%, 35% and 17%, respectively., Conclusion: SLB is a life-threatening complication of various underlying conditions and may represent their first manifestation. The management should include initial haemostasis followed by appropriate staging investigations to provide a definitive treatment for each individual patient., (© 2012 International Hepato-Pancreato-Biliary Association.)

Background: Early detection of pancreatic fistula (PF) may improve the outcome after pancreaticoduodenectomy, and exclusion of PF may allow earlier drain removal and accelerate recovery. The aim of the present study was to evaluate the relationship between drain fluid amylase on the first postoperative day (DFA(1)) and PF., Patients and Methods: This work was designed as a prospective study and included patients undergoing pancreaticoduodenectomy in a single center. For each patient, DFA was measured on the first and fifth postoperative days, and PF was defined by drainage of amylase-rich fluid on the fifth postoperative day (DFA(5) >300 U/l). A cut-off value of DFA(1) was derived, which yielded sensitivity and negative predictive value of 100% for predicting a PF., Results: A total of 70 patients (47% male) who underwent pancreaticoduodenectomy (Whipple procedure: 37; pylorus-preserving procedure: 33) between April 2009 and March 2010 were included. Nine of those patients developed a PF (grade A-2; B-5; C-2). There were two postoperative deaths (3%). The DFA(1) value significantly correlated with DFA(5) (Spearman rank coefficient 0.68; p < 0.0001). The median DFA(1) of patients with a PF (6,205; range 357-23,391) was significantly higher than in patients without a PF (69; range 5-5,180; p = 0.01; unpaired t test). No patient with a PF had a DFA(1) ≤350 U/l, compared to 48/61 patients (79%) without a PF. Using 350 U/l as a cut-off, a low DFA(1) excluded a PF with a sensitivity, specificity, positive and negative predictive values of 100, 79, 41, and 100%, respectively., Conclusions: Drain fluid amylase on the DFA(1) after pancreaticoduodenectomy stratifies patients according to likelihood of developing a PF.

Renal dysfunction is common in patients awaiting liver transplantation (LT) and affects outcome following LT. Combined liver and kidney transplantation (CLKT) has been proposed as effective treatment for patients with chronic diseases of both organs, some with hepatorenal syndrome and for liver-based metabolic diseases affecting kidney. This study is undertaken to analyze results of CLKT at a single center. Of 2690 LTs performed between 1992 and 2007, there were 39 CLKTs; most common indications were metabolic, cirrhosis and polycystic disease. With follow-up of up to 170 months, 11 died (overall survival 71.8%); one-, five-, and 10-yr patient and liver graft survival is 77%, 73.7%, and 73.7%, respectively, and kidney graft survival is 77%, 70%, and 70%, respectively. Survival among metabolic group (78.6%) appeared to be better than non-metabolic group (68%); however, this difference was not significant (p = 0.39). Fifteen surviving patients (53.6%) have mild/moderate renal impairment (creatinine > or = 125 micromol/L). None has severe renal failure (serum creatinine > or = 250 micromol/L) or end-stage renal disease requiring hemodialysis. CLKT has good results in selected groups of patients. It provides protection to kidney allograft in liver-based metabolic diseases affecting kidney. The rate of acute rejection episodes of kidney is low. Significant proportion develops long-term mild/moderate renal dysfunction. Careful attention to immunosuppression to minimize nephrotoxicity may help.

Background: Spontaneous rupture of hepatocellular carcinoma (HCC) is a life-threatening presentation, with an incidence of <3% of HCC patients in Western countries. The reported overall mortality is < or =50% in Asian countries, where the incidence is 12% to 14%. The aim of this study was to report a single center's experience of patients with ruptured HCC during a 11-year period., Methods: A retrospective review was performed of all patients who presented with ruptured HCC between 1995 and 2005. Data on clinical features, treatment strategies, and survival outcomes were collected. Statistical methods included univariate analysis and Kaplan-Meier survival estimates with log-rank test., Results: A cohort of 21 patients (15 male and 6 female) was identified. Fourteen (66.6%) patients had histologic evidence of underlying cirrhosis, ad the median age at presentation was 68 years (interquartile range [IQR] 61 to 69). Ten of these patients (71.4%) were hemodynamically unstable at presentation. The mean tumor size was 8.5 cm (range 3 to 13), and there was multifocal disease in 6 (42.8%) patients. The etiology of cirrhosis was hepatitis B infection in 3, hepatitis C in 3, alcohol in 4, and cryptogenic in 4 patients. Initial bleeding control was attempted by transarterial embolization (TAE) in 7 (50%) and by emergency surgery in 7 patients (50%). Four of the operations were performed at referring hospitals, and 3 were performed at our institution. Two patients (14.2%) underwent palliative treatment only. Definitive treatment included resection at emergency surgery in 1, staged hepatectomy in 1, and transarterial chemoembolization in 2 patients. There were 7 patients who were noncirrhotic and had a median age of 51 years (IQR 42 to 60). Of these, 6 (87.5%) were hemodynamically unstable at presentation. Mean tumor size was 9 cm (range 6 to 18) and confined to right lobe in all patients. Primary hemostasis was successfully achieved by TAE in 2 and perihepatic packing in 1 patient. Definitive treatment was provided by emergency hepatectomy in 4 and staged hepatectomy in 3 patients. Patients with cirrhosis (n = 14) had a median survival rate of <30 days. Child-Pugh score at presentation (median 7, IQR 5 to 8) correlated strongly with overall survival (P <.0001). Median survival for noncirrhotic patients was 20 months (IQR 2 to 31). One patient without cirrhosis survived for 122 months without disease recurrence., Conclusions: Spontaneous rupture of HCC is an uncommon presentation in Western countries. Primary hemostasis, followed by emergency or staged hepatic resection, is the treatment of choice. Median survival in patients initially treated with surgery was better than that observed in patients who underwent initial TAE, although this was not statistically significant. Patients who had no underlying liver disease had better prognosis than those who had cirrhosis.

Background: Chylous ascites (CA) following pancreatico-duodenectomy (PD) is a rare complication secondary to disruption of the lymphatics during extended retroperitoneal lymph node dissection. The majority of cases do not develop CA, possibly due to patency of the proximal thoracic duct and good collaterals. CA may be due to a consequence of occult obstruction of the proximal thoracic duct by malignant infiltration or tumor embolus. This study was to report the incidence of CA and its outcomes of management., Methods: A retrospective search of our liver database was performed using the key words "pancreatico-duodenectomy", "chylous ascites" from January 2000 to December 2005. The medical records of CA patients and their management and outcome were reviewed., Results: In 138 patients who had undergone PD in our centre for pancreatic malignancy, 3 were identified with CA and managed by abdominal paracentesis. CA resolved in 2 patients with low fat medium chain triglyceride diet alone and 1 patient had total parenteral nutrition (TPN) for persistent CA. Resolution of CA occurred in these 3 patients at a median follow-up of 4 weeks (range 4-12 weeks). Histologically, resected specimen confirmed pancreatic adenocarcinoma in all the patients. Two patients developed loco-regional recurrences at a median follow up of 8 months (range 6-10 months). And the other was currently disease free at a 10-month follow up., Conclusions: CA as an uncommon postoperative complication requires frequent paracentesis, prolonged hospital stay, and delayed adjuvant chemotherapy. CA is treated with low fat medium chain triglyceride diet or occasionally TPN is required.

Background: Laparoscopic cholecystectomy (LC) is the operation of choice for removal of the gallbladder. Unrecognized bile duct injuries present with biliary peritonitis and systemic sepsis. Bile has been shown to cause damage to the vascular wall and therefore delay the healing of injured arteries leading to pseudoaneurysm formation. Failure to deal with bile leak and secondary infection may result in pseudoaneurysm formation. This study was to report the incidence and outcomes of pseudoaneurysm in patients with bile leak following LC referred to our hospital., Methods: A retrospective analysis of our prospectively maintained liver database using key words pseudoaneurysm, bile leak and bile duct injury following laparoscopic cholecystectomy from January 2000 to December 2005 was performed., Results: A total of 86 cases were referred with bile duct injury and bile leak following LC and of these, 4 patients (4.5%) developed hepatic artery pseudoaneurysm (HAP) presenting with haemobilia in 3 and massive intra-abdominal bleed in 1. Selective visceral angiography confirmed pseudoaneurysm of the right hepatic artery in 2 cases, cystic artery stump in one and an intact but ectatic hepatic artery with surgical clips closely applied to the right hepatic artery at the origin of the cystic artery in the fourth case. Effective hemostasis was achieved in 3 patients with coil embolization and the fourth patient required emergency laparotomy for severe bleeding and hemodynamic instability due to a ruptured right hepatic artery. Of the 3 patients treated with coil embolization, 2 developed late strictures of the common hepatic duct (CHD) requiring hepatico-jejunostomy and one developed a stricture of left hepatic duct. All the 4 patients are alive at a median follow up of 17 months (range 1 to 65) with normal liver function tests., Conclusions: HAP is a rare and potentially life-threatening complication of LC. Biloma and subsequent infection are reported to be associated with pseudoaneurysm formation. Late duct stricture is common either due to unrecognized injury at LC or secondary to ischemia after embolization.

Hepatoblastoma (HB) is a rare germ cell tumour of childhood usually presenting with progressive abdominal distention. However, presentation as acute abdomen is a rare occurrence and is secondary to spontaneous rupture. This presentation carries high mortality. To our knowledge, six cases of ruptured hepatoblastoma have previously been reported, although the long-term outcome has not been clear. We report a case of ruptured HB who was managed by initial control of haemorrhage by laparotomy followed by chemotherapy with high-risk hepatoblastoma protocol as per SIOPEL 2 (cisplatin, carboplatin and doxorubicin) and a staged hepatectomy 5 months later. Patient is currently disease free at 6-year follow-up. Staged hepatectomy after initial control of haemorrhage does not preclude a curative resection.

Background: Transcatheter arterial chemoembolization (TACE) is a recommended first line therapy for unresectable hepatocellular carcinoma (HCC). Serious complications such as neutropenic sepsis and hepatic decompensation are well known, but rupture of HCC following TACE is a rare and potentially fatal complication. The aim of this study was to identify the incidence of ruptured HCC following TACE and the associated risk factors., Methods: A retrospective analysis was performed using our liver database with key words "chemoembolization", "ruptured HCC" covering the patients who received chemoembolization from January 1995 to December 2005. There were no exclusions., Results: A total of 294 patients received chemoembolization in 530 sessions during the 10-year period. Of these, 2 ruptured following treatment (incidence 0.68%). The mean age was 65 years and the interval between the treatment and rupture was 2 and 24 days. The common factors were male sex, large tumor size (range 11-13 cm), and exophytic tumor growth. One patient died 2 days after rupture with hepatic decompensation while the second is alive after a 6-month follow up without tumor recurrence., Conclusions: Ruptured HCC following TACE is a rare but serious complication. Large tumor size, male sex, and exophytic growth of tumor may be predisposing factors for rupture.

Objectives: Non-Hodgkin lymphoma predominantly involving the pancreas is a rare tumor and accounts for less than 0.7% of all pancreatic malignancies and 1% of extranodal lymphomas. Diagnosis of primary pancreatic lymphoma can be difficult because it may mimic carcinoma. The principal aims of this review were to highlight the difficulties encountered in making a diagnosis and to identify the role of surgery., Methods: A PubMed search was conducted using the following terms: primary pancreatic lymphoma and non-Hodgkin lymphoma of the pancreas. Additional references were sourced from key articles., Results: A total of 89 reported cases of pancreatic lymphoma between 1951 and 2005 were reviewed. An accurate preoperative diagnosis of primary pancreatic lymphoma is not always possible. A complete response rate of 100% and a long-term survival rate of 94% have been reported with surgery and adjuvant chemotherapy when compared with a 5-year survival rate of less than 50% and an overall 3-year disease-free survival rate of 44% with current chemotherapy, radiotherapy, or combined methods., Conclusion: Pancreaticoduodenectomy may have a therapeutic role in association with chemotherapy.

Despite decades of research, the etiology of type 1 diabetes mellitus (DM) is unknown. Several risk factors have been associated with type 1 DM, including viral infections, genetic predisposition, nutritional factors, and chemicals. Several investigators hypothesize that the etiologies of type 1 DM result from a complex interaction of genetic and environmental factors. In this paper we review the epidemiologic data linking enteroviruses to type 1 DM and discuss potential mechanisms of pathogenesis.

P-Glycoprotein the multidrug resistance (mdr) efflux transporter is encoded by class 1 mdr genes (mdr1) in humans and rodent species. In rat liver and in rat hepatocytes in primary culture, expression of mdr1 genes can be induced with the carcinogenic aromatic amine 2-acetylaminofluorene (2-AAF). As a consequence, increased P-glycoprotein levels led to an accelerated efflux of vinblastine from the hepatocytes and to resistance towards vinblastine-mediated cytotoxicity. N-Hydroxylation, an obligatory initial step in the activation of 2-AAF into electrophilic DNA-binding metabolites is catalyzed predominantly by cytochrome P450 (CYP)1A2, an isozyme present in normal rat liver. In rat hepatocytes in primary culture, mdr1 induction with 2-AAF could be inhibited by addition of the CYP1A-inhibitor alpha-naphthoflavone, indicating the requirement for metabolic conversion of 2-AAF to act as an inducer of mdr1 gene expression. Both N-hydroxy-2-AAF and the mutagenic 2-AAF derivative N-acetoxy-2-AAF (AAAF) were more potent than 2-AAF as mdr1 inducers. mdr1 induction also decreased when deacetylation of AAAF, which strongly accelerates its conversion into a mutagen, was inhibited with paraoxon. Furthermore, rat liver epithelial cells stably transfected with mouse CYP1A2 showed inducibility of mdr1 gene expression with 2-AAF, whereas the parental cell line, which is devoid of CYP1A2 activity, did not. These findings indicate that electrophilic metabolites formed during 2-AAF or AAAF metabolism are responsible for mdr1 induction in rat hepatocytes. The increased mdr1 gene expression may reflect an adaptive cellular response to electrophiles which includes enhanced synthesis of P-glycoprotein aimed to protect the cell from further damage.

We expressed mouse cytochrome P1-450 and P3-450 using recombinant vaccinia virus gene expression system in HeLa cells that were devoid of significant basal levels of P-450. HeLa cells were infected with the recombinant vaccinia virus containing either mouse cytochrome P1-450 or P3-450 cDNA, and the cell lysates were analyzed for the kinetics of P-450 enzyme activity and protein expression at the same time. 7-Ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activities were measured as an expression of the cytochrome P-450 enzyme activities. Both cell lines began to express these enzyme activities as early as 12h after infection. The activities increased linearly up to the 24 h time point, and were kept for 36 h. Western immunoblot analysis showed that these cytochrome P-450 proteins were detected at 16 h and reached maximum quantity at 24 h after infection. These data showed a good correlation between cytochrome P-450 enzyme activity and protein concentration throughout the process of P-450 gene expression by vaccinia virus vector, suggesting a complete formation of cytochrome P-450 holoenzyme from the early stage of the protein expression.