Vasopressors for shock.

BACKGROUND: Besides reversing the underlying cause, the first line treatment for the symptoms of shock is usually the administration of intravenous fluids. If this method is not successful, vasopressors such as dopamine, dobutamine, adrenaline, noradrenaline and vasopressin are recommended. It is unclear if there is a vasopressor of choice, either for the treatment of particular forms of shock or for the treatment of shock in general. OBJECTIVES: To assess the efficacy of vasopressors for circulatory shock in critically ill patients. Our main aim was to assess whether particular vasopressors reduce overall mortality. We also intended to identify whether the choice of vasopressor influences outcomes such as length-of-stay in the intensive care unit and health-related quality of life. SEARCH STRATEGY: We searched MEDLINE, the Cochrane Central Register of Controlled Trials, EMBASE, PASCAL BioMed, CINAHL, BIOSIS, and PsychINFO:all from inception to November 2003; for randomized controlled trials. We also asked experts in the field and searched meta-registries for ongoing trials. SELECTION CRITERIA: We included randomized controlled trials comparing various vasopressors, vasopressors with placebo or vasopressors with intravenous fluids for the treatment of any kind of circulatory failure (shock). Mortality was the main outcome. DATA COLLECTION AND ANALYSIS: Two reviewers abstracted data independently. Disagreement between two reviewers was discussed and resolved with a third reviewer. We used random effects models for combining quantitative data. MAIN RESULTS: We identified eight randomized controlled trials. Reporting of methodological details was for many items not satisfactory: only two studies reported allocation concealment, and two that the outcome assessor was blind to the intervention. Two studies compared norepinephrine plus dobutamine with epinephrine alone in patients with septic shock (52 patients, relative risk of death 0.98, 95% confidence interval 0.57 to 1.67). Three studies compared norepinephrine with dopamine in patients with septic shock (62 patients, relative risk 0.88, 0.57 to 1.36). Two studies compared vasopressin with placebo in patients with septic shock (58 patients, relative risk 1.04, 0.06 to 19.33). One study compared terlipressin with norepinephrine in patients with refractory hypotension after general anaesthesia but there were no deaths (20 patients). REVIEWERS' CONCLUSIONS: The current available evidence is not suited to inform clinical practice. We were unable to determine whether a particular vasopressor is superior to other agents in the treatment of states of shock.

The role of liver transplantation in patients with Caroli's disease.

Caroli's disease, characterized by segmental or diffuse dilation of the intrahepatic biliary ducts, is a rare disease which is difficult to treat. The course of the disorder is characterized by recurrent episodes of cholangitis and hospital stays, with a consequent loss of quality-of-life and productive capacity, often ending in death due to uncontrolled infection. Endoscopic drainage of the bile duct, percutaneously or surgically, is palliative, and presents bad results in the follow-up of these patients. Orthotopic liver transplantation appears to be an effective curative option for the treatment of patients with Caroli's disease associated to complications. The authors present the course of two cases of this disease, associated with congenital fibrosis of the liver worsened by repeated episodes of cholangitis, submitted to orthotopic liver transplantation.

A comparison between topical and infiltrative bupivacaine and intravenous meperidine for postoperative analgesia after inguinal herniorrhaphy.

The purpose of the present study is to compare postoperative analgesia offered by the simple instillation of local anesthetic on the surgical wound, its infiltration with the same local anesthetic, and the use of an intravenous opioid. Sixty patients were divided into the three analgesia groups to be studied: instillation of local anesthetic (Group I), injection of local anesthetic (Group II), and intravenous opioid (Group III). The pain was quantified using the visual analogue scale. It was observed that there was better analgesia in Groups I and II during the first 6 hours postoperatively as compared with Group III (P < 0.0001). At the end of the 12 hours the three modes of analgesia proved comparable. However, after 24 hours there was better analgesic development in Group I, whereas Group II had greater postoperative morbidity. We conclude that the instillation of local anesthesia provides analgesia during the immediate postoperative period comparable to local infiltration using the same anesthetic. Both regional analgesia methods are more effective analgesics during the first 6 hours than are intravenous opioids. Furthermore the simple instillation of local anesthetic allows better analgesic evolution of the surgical wound after the first 24 hours considering the lower rate of resulting complications.

Induction of CYP2C genes in human hepatocytes in primary culture.

The expression and inducibility of four CYP2C genes, including CYP2C8, -2C9, -2C18, and -2C19, was investigated in primary cultures of human hepatocytes. By the use of RNase protection assay and specific antibodies, each CYP2C mRNA and protein were quantified unequivocally. The four CYP2C mRNAs were expressed in human livers and cultured primary hepatocytes, but only the CYP2C18 protein was not detected. Compounds known to activate the pregnane X receptor (PXR) such as rifampicin, or the constitutively activated receptor (CAR) such as phenobarbital, induced CYP2C8, CYP2C9, and to a lesser extent CYP2C19 mRNAs and proteins. CYP2C18 mRNA was expressed but not inducible. The concentration dependence of CYP2C8 and CYP2C9 mRNAs in response to rifampicin and phenobarbital paralleled that of CYP3A4 and CYP2B6, the maximum accumulation being reached with 10 microM rifampicin and 100 microM phenobarbital. In contrast, dexamethasone produced maximum induction of CYP2C8 and CYP2C9 mRNAs at 0.1 microM while in these conditions neither CYP3A4 nor CYP2B6 was significantly induced. Moreover, the concentration dependence of CYP2C8 and CYP2C9 mRNAs in response to dexamethasone paralleled that of tyrosine aminotransferase. Furthermore, dexamethasone, which has been recently shown to up-regulate PXR and CAR expression through the glucocorticoid receptor, potentiated CYP2C8 and CYP2C9 mRNA induction in response to rifampicin and phenobarbital. Collectively, these results suggest the possible implication of at least three receptors in the regulation of CYP2C8 and CYP2C9 expression, i.e., glucocorticoid receptor, PXR, and/or CAR.

Acute hepatitis C complicated by pancreatitis: another extrahepatic manifestation of hepatitis C virus?

In hepatitis C virus (HCV) infection a number of extrahepatic manifestations have been described, generally caused by immune phenomena. Here we report a case of acute pancreatitis, detected during an acute hepatitis C infection, in an elderly female patient.

Comparative post-operative study of prostheses, with and without an anti-reflux valve system, in the palliative treatment of esophageal carcinoma.

BACKGROUND/AIMS: Palliative treatment of advanced esophageal carcinoma by esophageal tunnelization with a prosthesis allows immediate relief of dysphagia. However, the procedure is subject to a high rate of morbidity, including gastroesophageal reflux (GER) present in all patients with a prosthesis positioned through the gastroesophageal junction, resulting in complications (pyrosis, aspiration pneumonias, sleep disorders) and reduced quality of life in these patients who already have a lower rate of survival. In an attempt to reduce GER and its complications, the authors created a surgical prosthesis coupled to an anti-reflux valve system, comparing it to the use of an esophageal prosthesis without an anti-reflux valve mechanism. METHODOLOGY: Twenty-two patients were allocated to 2 tunnelization groups: esophageal prosthesis without an anti-reflux valve mechanism (group 1) and surgical prosthesis coupled to an anti-reflux valve system (group 2). The GER was quantified measuring esophageal-gastric pH, and using fluoroscopy, contrast radiographs and esophageal emptying scintigraphy. Initially, the pH of secretions in S1 (esophagus) and S2 (stomach) was determined using reagent strips after aspirating their contents with different syringes. First with the patient seated at rest in bed, later performing a Valsalva maneuver, deep breathing and forced coughing. The same procedure was performed with the patient in left lateral decubitus, right lateral decubitus, and dorsal decubitus with the head of the bed lowered to 20 degrees. After finishing these maneuvers, 15 ml of 1 molar acetic acid were infused through the catheter positioned in the antrum, and, after 5 min, S1 and S2 material sampling was repeated in the same positions as mentioned above. RESULTS: The pH values between the various positions and maneuvers performed in each group separately were not significantly different, but, if we compare the 2 groups, and the secretions obtained in S1 and S2, there was a significant difference in pH measures in all positions. In the patients in group 1, S1 presented a mean pH ranging from 2.87-3.62 in the initial measures, and between 2.17 and 3.5 after the infusion of 15 ml of 1 molar acetic acid. On the other hand, in group 2, the mean pH of S1 remained between 6.34 and 8.32 in the initial measures and between 4.99 and 7.33 in the presence of acid infusion. At the level of S2, the pH remained unchanged between 2 and 2.7, in both groups. CONCLUSIONS: The authors conclude that the association of an esophageal prosthesis with a valve system significantly reduces GER, as compared with its use alone. Furthermore, it allows marked reduction of the symptoms and resulting complications, and does not interfere clinically with esophageal emptying. It thus significantly improves the quality of life of these patients.

Subchronic toxicity study with ethylene-bis-(oxyethylene)-bis-(3-tert-butyl-4-hydroxy-5-methylhydro cin namate) in the cynomolgus monkey: lack of stimulation of the pituitary-thyroid-liver axis.

To evaluate the toxicological profile of the phenolic antioxidant ethylene-bis-(oxyethylene)-bis-(3-tert-butyl-4-hydroxy-5-methyl- hydrocinnamate) (EOC) in a non-human primate, male cynomolgus monkeys (Macaca fascicularis) were treated for 4 weeks by oral administration of 0, 200, or 1000 mg/kg body weight/day. Special attention was directed to parameters of the pituitary-thyroid-liver axis. Moderately increased liver weights and minimal to moderate hepatocellular hypertrophy were observed in treated animals. Otherwise, no treatment-related changes were detected in hematological, clinical chemistry, or urinalysis parameters or upon histopathological examination. Except for a slight induction of microsomal testosterone 16beta-hydroxylation, liver xenobiotic-metabolising enzyme activities and peroxisomal fatty acid beta-oxidation remained unchanged. Likewise, serum levels of thyroid stimulating hormone, thyroxine, 3,3',5-triiodothyronine and 3,3',5'-triiodothyronine as well as 5'-monodeiodinase type 1 mRNA levels in the liver, heart, cerebral cortex, and thyroid were found unchanged. The results demonstrate that, in the Cynomolgus monkey, EOC is only a very weak inducer of liver xenobiotic-metabolizing enzymes and has no effect on thyroid function. In contrast, upon feeding rats at dose levels up to 1000 ppm (equivalent to between 50 and 100 mg/kg body weight/day), EOC has been identified as a strong phenobarbital- and peroxisome proliferator-type inducer of hepatic xenobiotic-metabolizing enzymes, interfering with thyroid hormone homeostasis, causing thyroid follicular hypertrophy, and, upon chronic treatment, inducing thyroid gland follicular cell tumors (Thomas et al., 1995. In Toxicology of Industrial Compounds, pp. 319-339. Taylor and Francis). Thus, the results of this study with EOC in the cynomolgus monkey show that effects of xenobiotics on the pituitary-thyroid-liver axis as frequently observed in rodents can not necessarily be extrapolated to primates including man.

Alternative splicing produces transcripts encoding four variants of mouse G-protein-coupled receptor kinase 6.

A family of protein kinases, termed G-protein-coupled receptor kinases (GRK1-6), is known to phosphorylate agonist-occupied G-protein-coupled receptors. We have identified mRNAs encoding four distinct mouse GRK6 isoforms (mGRK6), designated mGRK6-A through mGRK6-D. Mouse GRK6-B and mGRK6-C diverge from the known human GRK6 (577 residues) at residue 560 and are 13 residues longer and 16 residues shorter, respectively, than human GRK6, while mGRK6-A very likely represents the mouse equivalent of human GRK6. Mouse GRK6-D is identical to the other mGRK6 variants in the amino-terminal region, but comprises only 59 of the 263 amino acids of the putative catalytical domain. As mGRK6-D retains the region involved in interacting with activated receptors, but most likely lacks catalytic activity, this variant might represent a naturally occurring inhibitor of other GRKs. Analysis of the genomic organization of mGRK6 gene revealed that the four mRNAs are generated by alternative RNA splicing from a single approximately 14. 5-kb gene, made up of at least 17 exons and located on mouse chromosome 13. Similar to human GRK6, mGRK6-A contains three cysteine residues within its carboxyl-terminal region known to serve as substrates for palmitoylation. Mouse GRK6-B lacks these palmitoylation sites, but carries a basic carboxyl-terminus containing consensus sequences for phosphorylation by protein kinases C and cAMP/cGMP-dependent protein kinases. Mouse GRK6-C displays none of these motifs. Thus, mGRK6-A, mGRK6-B, and mGRK6-C are predicted to differ in terms of their regulation by carboxyl-terminal posttranslational modification. Analysis of mRNA expression revealed that the four mGRK6 mRNAs are differentially expressed in mouse tissues, suggesting that the four mGRK6 isoforms are involved in regulating tissue- or cell type-specific functions in vivo.

Pancreatic fistula after pancreaticoduodenectomy: a comparison between patients with periampullary tumors and chronic pancreatitis.

BACKGROUND/AIMS: Whether the frequency of anastomotic leak after pancreaticoduodenectomy for benign diseases is greater than for malignant conditions and whether fistula development is associated with surgical mortality remains controversial. The purpose of this study is to compare the incidence of anastomotic leak in patients operated on for chronic pancreatitis and periampullary tumors. METHODOLOGY: The authors retrospectively reviewed the charts of 67 patients (46 males, 21 females, mean age 47 years) submitted to pancreaticoduodenectomy for chronic pancreatitis and periampullary tumors between 1990 and 1996. RESULTS: In 44 patients with periampullary cancers, pancreatic fistula developed in 13 (29%) cases, and in 6 (26%) of the 23 patients with chronic pancreatitis (p>0.05). Of the 19 patients who developed this complication, 5 (26.3%) died, and in the remaining 48 cases, there was only one (2.1%) death (p<0.05). CONCLUSION: The frequency of pancreatic fistula after pancreaticoduodenectomy in patients with periampullary tumors and chronic pancreatitis is not different, but the presence of a fistula is strongly involved in postoperative mortality.

Repeat hepatectomy for colorectal liver metastases.

OBJECTIVE: The authors assess the long-term results of repeat hepatectomies for recurrent metastases of colorectal cancer and determine the factors that can predict survival. SUMMARY BACKGROUND DATA: Safer techniques of hepatic resection have allowed surgeons to consider repeat hepatectomy for colorectal metastases in an increasing number of patients. However, higher operative bleeding and increased morbidity have been reported after repeat hepatectomies, and the long-term benefit of these procedures needs to be evaluated. STUDY POPULATION: Sixty-four patients from a group of 243 patients resected for colorectal liver metastases were submitted to 83 repeat hepatectomies (64 second, 15 third, and 4 fourth hepatectomies). Combined extrahepatic surgery was performed in 21 (25%) of these 83 repeat hepatectomies. RESULTS: There was no intraoperative or postoperative mortality. Operative bleeding was not significantly increased in repeat hepatectomies as compared to first resections. Morbidity and duration of hospital stay were comparable to first hepatectomies. Overall and disease-free survival after a second hepatectomy were 60% and 42%, respectively, at 3 years and 41% and 26%, respectively, at 5 years. Factors of prognostic value on univariate analysis included the curative nature of first and second hepatectomies (p = 0.04 and p = 0.002, respectively), an interval between the two procedures of more than 1 year (p = 0.003), the number of recurrent tumors (p = 0.002), serum carcinoembryonic antigen levels (p = 0.03), and the presence of extrahepatic disease (p = 0.03). Only the curative nature of the second hepatectomy and an interval of more than 1 year between the two procedures were independently related to survival on multivariate analysis. CONCLUSIONS: Repeat hepatectomies can provide long-term survival rates similar to those of first hepatectomies, with no mortality and comparable morbidity. Combined extrahepatic surgery can be required to achieve tumor eradication. Repeat hepatectomies appear worthwhile when potentially curative.

[Value of left cholangiojejunostomy in palliative treatment of malignant obstruction of the hepatic hilus]. / La valeur de la cholangio-jéjunostomie gauche dans le traitement palliatif de l'obstruction maligne du hile hépatique.

The aim of this study is to analyse the results of the intrahepatic cholangiojejunostomy in the duct of segment III for malignant hilar obstruction performed in a group of 17 consecutive patients. An index for life quality, named comfort index, allowing the numeric comparison of the results was evaluated. An important reduction of the jaundice in 100% of the cases that were discharged from the hospital was observed. The hospital mortality was 11.8% with a comfort index of 81.9%. These results allow the following conclusions: 1) the employed technique represent an effective method for the solution of the cholestasis caused by unresectable tumors of the hepatic hilus; 2) the comfort index of 81.9% showed that the method is suitable as a palliative treatment of malignant hilar biliary obstruction.

The significance of mouse liver tumor formation for carcinogenic risk assessment: results and conclusions from a survey of ten years of testing by the agrochemical industry.

A survey was performed on the results of 138 carcinogenicity studies conducted in various mouse strains by the agrochemical industry over the period 1983-1993. Data for liver tumor incidence, liver weight, and histopathology were collected along with data on genotoxicity. Studies were judged positive or negative for liver tumor formation on the basis of apparent dose response, malignancy, and difference from historical control values using a weight of evidence approach. Thirty-seven studies were judged to be positive for liver tumorigenicity in one or both sexes. There was no evidence showing an influence of the mouse strain and the duration of the study on the proportion of positive studies. Although 8 of the chemicals tested in the 138 studies were positive in the Ames test, only one of these was judged positive for carcinogenicity. Only 6 of the 37 positive chemicals had any other reported positive genotoxicity findings. A clear relationship between hepatomegaly at 1 year after exposure and a positive tumorigenic outcome at 18 months or 2 years after exposure was demonstrated. Whereas the average relative liver weight of top dose animals was 110% of control in negative studies, it was 150% in positive studies. Likewise, very few negative studies demonstrated significant pathological findings after 1 year, whereas the majority of positive studies had significant liver pathology. The implications of these findings for extrapolation to humans are discussed.

Resection of nonresectable liver metastases from colorectal cancer after neoadjuvant chemotherapy.

OBJECTIVE: The authors discuss the technique and evaluate the results of an aggressive surgical approach in patients with primarily unresectable colorectal liver metastases that were downstaged by chronomodulated chemotherapy. BACKGROUND: Resection is the best treatment of colorectal liver metastases, but it may be achieved in only 10% of patients. In the remaining 90%, survival is poor, even after partial response to chemotherapy. Little is known about the results of curative hepatectomy in patients whose metastases are downstaged by chemotherapy. PATIENTS AND METHODS: Fifty-three patients with colorectal liver metastases initially unresectable because of ill located (8), large (8), multinodular (24) lesions, or because of extrahepatic disease (13) were downstaged by a systemic chronomodulated chemotherapy associating 5-fluorouracil, folinic acid and Oxaliplatin to the point that operation could be performed. This consisted of a major hepatectomy in 37 patients and a minor resection in 16. Associated procedures (including 5 two-stage hepatectomies and 3 pulmonary resections) were performed in 25 patients. RESULTS: There was no operative mortality. Complications occurred in 14 patients. The cumulative 3- and 5-year survival rates were 54% and 40% (according to the type of lesions: ill-located, 75% and 48%; large, 62% and 62%; multinodular, 54% and 40%; extrahepatic, 43% and 14%). Hepatic recurrence (34 patients, 64%) was amenable to repeat surgery in 15 cases. CONCLUSIONS: Liver resection may be achieved in some previously unresectable patients with the help of an effective chemotherapy. The benefit in survival seems comparable to that obtained with primary liver resection (40% at 5 years). This therapeutic strategy involves a multimodal approach, including repeat hepatectomies and extrahepatic surgery.

[Internal pancreatic fistula: surgical treatment]. / Fistule pancréatique interne: traitement chirurgical.

The authors present 13 cases of internal pancreatic fistula, of which 11 were secondary to a chronic pancreatitis and two were caused by an abdominal trauma. Beside the clinical picture, the diagnosis was anticipated by the high amylase levels present in the fluid obtained by paracentesis or thoracocentesis. The diagnosis was confirmed by the radiological analysis of the pancreatic duct system, when an endoscopic retrograde pancreatography was performed in seven patients, one pancreatography was carried out during surgery in five cases, and one patient underwent an injection of hydrosoluble contrast in the pleural cavity. The treatment was a latero-lateral pancreaticojejunoanastomosis in five cases, associated with a corpora-caudal pancreatectomy in four patients; a cephalic duodenopancreatectomy was performed in one case. Two patients underwent a cystoenteroanastomosis, while the option chosen in the last four cases was an external drainage. One patient refused to undergo surgical treatment. Operation mortality was null. The conclusion was that an adequate surgical treatment results in the occlusion of the internal pancreatic fistula and, furthermore, allows for the definitive resolution of underlying pancreatic affection.

Bromopropylate: induction of hepatic cytochromes P450 and absence of covalent binding to DNA in mouse liver.

Oral administration of benzilic acid ester-based acaricide bromopropylate at daily doses of 3, 15, 100, and 300 mg/kg body wt to young adult male Tif:MAGf mice for 14 days caused slightly increased liver weights in the high-dose group. A dose-dependent increase of the microsomal cytochrome P450 content was accompanied by elevated ethoxycoumarin O-deethylase, ethoxyresorufin O-deethylase, pentoxyresorufin O-depentylase, and total testosterone hydroxylase activities. When compared with mice treated in parallel with the model compounds for hepatic xenobiotic metabolizing enzyme induction, phenobarbitone, and 3-methylcholanthrene, the enzyme activity changes observed with bromopropylate largely equalled those expressed in phenobarbitone-treated mice. Immunochemical studies with monoclonal antibodies against rat liver cytochrome P450 isoenzymes of the gene families 1A, 2B, 3A, and 4A confirmed that bromopropylate is a phenobarbitone-type inducer in the mouse liver. Titration of liver microsomal suspensions with bromopropylate yielded Type I substrate binding spectra. The specific amplitude was increased 1.5-fold when microsomes from bromopropylate-treated mice (300 mg/kg body wt) were used instead of control microsomes, indicating the induction of cytochromes P450 catalyzing the oxidative metabolism of the test compound. Single oral administration of 300 mg/kg body wt [14C]bromopropylate to male mice, without or following pretreatment for 14 days with 300 mg/kg body wt unlabeled bromopropylate, gave no indication for DNA binding of the test compound in the liver. This excludes a genotoxic potential via covalent DNA modification. The results suggest that, in analogy to phenobarbitone, bromopropylate acts as a tumor promotor rather than a tumor initiator in the mouse liver.

Oxygen tension, insulin, and glucagon affect the preservation and induction of cytochrome P450 isoforms in cultured rat hepatocytes.

The role of oxygen tension, insulin, and glucagon on the preservation and induction of cytochrome P450 isoenzyme activities and contents was investigated in rat hepatocytes cultured for 4 days on crude liver membrane fractions at 4 or 13% O2. At 13% O2, three out of six immunochemically analyzed P450 isoenzymes were significantly higher than in 4% O2. Exposure to phenobarbital (PB) from Days 1 to 4 dose dependently increased the protein content and decreased the albumin secretion in 13% O2 cultures only. The maximal induction of P450 isoenzymes CYP2B1/2B2 (20- to 25-fold) and CYP2C6 (6-fold) were found at 0.75 mM PB at both oxygen tensions. In contrast, the highest induction of CYP1A1/1A2 (3-fold), of CYP3A (2-fold), and EROD activity were found with 3 mM PB in 4% O2 cultures. CYP2B-dependent testosterone hydroxylation at positions 16 alpha/beta was elevated to a greater extent in 13% O2 cultures (96-fold at 0.75 mM PB) compared to 4% O2 cultures (42-fold). This activity was affected by the insulin concentrations and the insulin:glucagon ratio. With decreasing insulin concentration (100 to 1 nM) or with increasing insulin:glucagon ratios (1:100-1:0.1), the enzyme activity increased preferentially in 4% O2 cultures. The results of these investigations demonstrate that different tissue oxygen tension modulates the responsiveness of the cultured hepatocytes to the glucoregulatory hormones insulin and glucagon and this modulation results in a altered activity of cytochrome P450 isoforms.

Crude liver membrane fractions and extracellular matrix components as substrata regulate differentially the preservation and inducibility of cytochrome P-450 isoenzymes in cultured rat hepatocytes.

The influence of cell-substrata interactions on the preservation of basal or in-vivo-induced microsomal cytochrome P-450 isoenzyme contents in cultured rat hepatocytes and on the adaptive responses after exposure to phenobarbital or 3-methylcholanthrene in vitro, was investigated. Hepatocytes from untreated or phenobarbital-treated rats were cultured in serum-free, aprotinin-supplemented culture medium in 96-well microtiter plates coated with collagen type I (COL), laminin, fibronectin or crude liver membrane fractions/collagen type I (CMF/COL). Basal cell functions were characterized by measuring the total protein content and lactate dehydrogenase release. The relative contributions of CYP1A1/2, CYP2B1/2, CYP2C6, CYP2C11, CYP3A and CYP4A isoenzymes were determined with ELISA using monoclonal antibodies raised against purified cytochromes P-450 from rat liver microsomes. The characterization of the CMF revealed that contaminations with mitochondria, nuclei and lysosomes are relatively low. Among these, membranes derived from the endoplasmic reticulum appeared to be the major organelle contaminant of the CMF. The matrix components laminin, fibronectin and collagen type IV were found in appreciable amounts. Hepatocytes from untreated rats, cultured for up to nine days on CMF/COL-coated plates, retained their relative cytochrome P-450 contents at 1.5-3-fold higher levels when compared to cells cultured on COL, fibronectin or laminin. Similarly, hepatocytes from phenobarbital-treated rats preserved the contents of barbiturate-inducible CYP2B1/2 and CYP3A proteins best when cultured on CMF/COL. After exposure of hepatocytes cultured on CMF/COL to phenobarbital from days 3-6, CYP3A proteins were enhanced more than twofold and CYP2B1/2, depending on the exposure level, increased 1.3-6-fold. After exposure to 3-methylcholanthrene, a threefold increase of CYP1A proteins was found in CMF/COL and laminin cultures. These results indicate that CMF/COL, as a substratum in rat hepatocyte cultures, regulates gene expression of cytochromes P-450 isoenzymes for up to 9 days and provides a matrix which enables the cells to respond qualitatively similar to the response observed in different zones of the liver. This activity cannot be replaced by single-matrix components.

Immunoelectron microscopic localization of cytochrome P-450 isoenzyme CYP4A1 in liver, ileum and kidney of nafenopin treated male rats.

A monoclonal antibody raised against and specific for cytochrome P-450 isoenzyme CYP4A1 was used to investigate the subcellular distribution of this enzyme in the liver, kidney and ileum of nafenopin treated rats by means of immunoelectron microscopy. In the liver and kidney, labelling was restricted to peroxisomes and mitochondria of hepatocytes and proximal tubular epithelial cells whereas in ileum, immunolabelling was exclusively detected in mitochondria of absorptive cells.