Rama S. Dwivedi

Almost total conversion of pancreas to liver in the adult rat: A reliable model to study transdifferentiation

Study of transdifferentiation provides an excellent opportunity to investigate various factors and mechanisms involved in repression of activated genes and derepression of inactivated genes. Here we describe a highly reproducible in vivo model, in which hepatocytes are induced in the pancreas of adult rats that were maintained on copper-deficient diet containing a relatively non-toxic copper-chelating agent, triethylenetetramine tetrahydrochloride (0.6% w/w) for 7-9 weeks and then returned to normal rat chow. This dietary manipulation resulted in almost complete loss of pancreatic acinar cells at the end of copper-depletion regimen, and in the development of multiple foci of hepatocytes during recovery phase. In some animals, liver cells occupied more than 60% of pancreatic volume within 6-8 weeks of recovery. Northern blot analysis of total RNA obtained from the pancreas of these rats revealed the expression of albumin mRNA. Albumin was demonstrated in these pancreatic hepatocytes by immunofluorescence. The advantages of this model over the previously described models are: a) low mortality (10%), b) depletion of acinar cells, and c) development of multiple foci of hepatocytes in 100% of rats.

Pancreatic hepatocytes : an in vivo model for cell lineage in pancreas of adult rat

Multiple foci of hepatocytes differentiate in the pancreas of adult rats subjected to a copper depletion-repletion regimen. Copper deficiency for seven to nine weeks causes an irreversible depletion of over 80% of the acinar cells in the pancreas. When transferred to a normal diet, these rats exhibit only a minimal and spotty acinar cell recovery. This disruption of tissue organization appears to trigger a profound change in cellular commitment, which leads to hepatocyte differentiation in the “oval cells” in the periductal interstitium and the epithelial cells lining the small pancreatic ductules. Pancreatic hepatocytes express several liver-specific genes including albumin, a2u-globulin, carbamoylphosphate synthetase-I, and urate oxidase. Both carbamoylphosphate synthetase-I and glutamine synthetase, the ammonia-metabolizing enzymes, are expressed by all pancreatic hepatocytes; in liver, these are expressed by different populations of hepatocytes. The magnitude of hepatocyte differentiation in this model should facilitate studies on the molecular events regulating changes in cell lineage or differentiation commitment within the pancreas.

Induction of peroxisome proliferation and hepatic tumours in C57BL/6N mice by ciprofibrate, a hypolipidaemic compound.

The hepatic effects of ciprofibrate, a potent peroxisome proliferator, were evaluated in male C57BL/6N mice, a mouse strain with very low incidence of spontaneous liver tumour development. Dietary feeding of ciprofibrate (0.0125% or 0.025% w/w) for 2 weeks resulted in a marked proliferation of peroxisomes (9-fold increase) and several-fold increase (8- to 10-fold) in the activity of peroxisomal beta-oxidation enzymes. Feeding ciprofibrate at 0.025% concentration for 15 months followed by a 0.0125% for 6 months led to the development of hepatic adenomas in 8/14 (57%) and hepatocellular carcinomas (HCC) in 3/14 (21%) mice. In mice given 0.0125% ciprofibrate for 18 months 5 of 8 (62%) and 3 of 8 (37%) developed adenomas and HCC respectively. Similar to the findings observed in rats, both the adenomas and HCC were negative for gamma-glutamyltranspeptidase. These results in C57BL/6N mice of hepatocarcinogenic effect of ciprofibrate, a non-genotoxic chemical, indicate that peroxisome proliferation can be used as a reliable parameter to evaluate the carcinogenicity of hypolipidaemic compounds.

Comparison of the peroxisome proliferator-induced pleiotropic response in the liver of nine strains of mice.

We have investigated the hepatic effect of ciprofibrate, a potent peroxisomal proliferator, in 9 strains of mice to ascertain whether all strains show similar peroxisome proliferation or if there are any that are resistant to the induction of peroxisome proliferation. Dietary feeding of ciprofibrate at 2 concentrations (0.0125% or 0.025% w/w) for 2 weeks resulted in a significant increase in liver weight (170 to 200%) and a 7- to 11-fold increase in volume density of peroxisomes. Catalase and peroxisomal β-oxidation enzymes increased by 1.7- to 2.7- and 1.9- to 9.3-fold, respectively, over the controls. SDS-polyacrylamide slab gel electrophoresis of post-nuclear fractions of livers showed a marked increase in 80,000-mol. wt. polypeptide. Immunocytochemical studies, as expected, revealed higher levels of PBE. Ciprofibrate treatment also induced hepatic DNA synthesis in all strains as determined by [3H]thymidine incorporation and autoradiography. Dot blot analysis of total RNA from livers of ciprofibrate-treated mice (5 strains) showed a significant increase in peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (PBE) mRNA. When the 9 strains were ranked for each parameter, CBA/Ca was the least responsive mouse strain and the B6C3F1 was the most responsive. However, the results of this study indicate that there is no significant interstrain difference in rankings across strains to ciprofibrate-induced hepatic pleiotropic response.

MDR1 hypermethylation contributes to the progression of neuroblastoma.

MDR1 hypermethylation plays an important role in pathogenesis and progression of neuroblastoma tumors. This hypothesis was tested by studying the methylation status of MDR1 gene promoter in neureoblastoma biopsy specimens during the progression of tumor from stage-1 to stage-4. Results of our findings demonstrate an inverse correlation between the methylation status of MDR1 promoter and MDR1 expression during the progression of disease from stage-1 to stage-4 as determined by methylation specific PCR (MSP) and RT/PCR analyses. The results of the RT/PCR and MSP analyses were validated by quantitative Real Time PCR analysis. Data from present study suggest that transcriptional inactivation of MDR1 gene due to increased MDR1 promoter methylation may be a contributing factor in pathogenesis and progression of neuroblastoma tumors, and may be used in designing an effective treatment therapy to neuroblastoma patients.

Invasive aspergillosis causing small bowel infarction in a patient of carcinoma breast undergoing chemotherapy.

Background To report a 45 year old lady presenting with proximal jejunal gangrene due to invasive Aspergillosis. The patient was undergoing adjuvant chemotherapy for advance carcinoma of breast (Stage IV). Methods The patient was referred to our surgical emergency for acute abdominal symptoms for 6 hours. Histopathology revealed bowel wall necrosis and vascular invasion by Aspergillus Fumigatus. Postoperative recovery was uneventful and the patient received Amphotericin-B (1 mg/kg/day) for invasive aspergillosis. Invasive pulmonary aspergillosis was confirmed by isolating Aspergillus Fumigatus from bronchoalveolar lavage and by a positive circulating galactomannan test (ELISA Assay). Results Detailed history revealed dry cough and two episodes of haemoptesis for 2 weeks. Haemogram and counts revealed anemia and neutropenia. Plain X – ray of the abdomen showed multiple air fluid levels and ultrasound of the abdomen revealed distended bowel loops. On exploration small bowel was found to be gangrenous. The patient was successfully managed by supportive treatment and conventional intravenous Amphotericin-B for 2 weeks. The lady was discharged one week after completion of antifungal therapy and one month later she underwent toilet mastectomy. The lady came to follow up for 1 year and she is currently under hormone therapy. Conclusion With the emergence of new and powerful immunosuppressive, anticancer drugs and potent antibiotics the survival of transplant and critically ill patients has remarkably increased but it has shown a significant rise in the incidence of invasive opportunistic fungal infections. We conclude hat the diagnosis of invasive gastrointestinal aspergillosis may be considered in a neutropenic patient with acute abdominal symptoms.

Lead exposure alters the drug metabolic activity and the homeostasis of essential metal ions in the lenticular system of the rat.

Potential lead exposure to the eyes as a result of the use of traditional cosmetic Kohl in Asia, Africa and the Middle East has been a subject of recent debate to the scientific community. In continuation of our earlier work we therefore examine in the present study, the drug metabolic activity and the homeostasis of essential metal ions in the lenticular system of adult rats exposed to long term low level lead (lead acetate 0.1% w/v). The results of our investigation demonstrate that long term low level lead exposure impaired the phase I & phase II metabolic activity of the lenticular system when assessed by aminopyrine demethylase, benzo[a]pyrene hydroxylase, aniline hydroxylase and UDP glucuronyl transferase (UDPGT), glutathione S-transferase (GST), respectively. A more pronounced decrease (55%) in GST was noticed compared to UDPGT, aminopyrene demethylase, benzo[a]-pyrene hydroxylase and aniline hydroxylase (20-30%). Increased lead concentration in the lenticular system of the rats as monitored by atomic absorption spectroscopy resulted in a significant decrease (15-35%) in the levels of Ca, Cu, Zn and Fe, along with a progressive loss in body weight. Respective increase in blood lead level was also monitored parallel to increase in lenticular lead concentration at different time points in lead treated rats. The present investigation, therefore, demonstrates that long term low level lead exposure to rats results in a profound impairment in the homeostasis of essential metal ions, lenticular drug metabolizing enzymatic activity and significant loss in body weight when compared to untreated control rats. Whether such a decrease in these functions reflects an inhibition of protein synthesis at transcriptional/post transcriptional levels or gene regulation at molecular level remains to be established.