Böttger T

CRYOPRESERVED PRIMARY HEPATOCYTES AS A CONSTANTLY AVAILABLE IN VITRO MODEL FOR THE EVALUATION OF HUMAN AND ANIMAL DRUG METABOLISM AND ENZYME INDUCTION

The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepalocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethylsulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and before cryopreservation, and (5) removal of unvital hepatocytes by Percoll centrifugation after thawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryopreserved by our standard procedure have a viability ≥ 80%. Metabolic capacity of cryopreserved hepatocytes determined by testosterone hydroxylation, 7-ethoxyresorufin-O-de-ethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transferase, sulfotransferase, and epoxide hydrolase activities is ≥60% of freshly isolated cells. Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the complex pattern of benzo[a]pyrene metabolites including phase II metabolites formed by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryopreserved hepatocyte cultures are required. We present a technique with cryopreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme activities of induced hepatocytes and solvent controls were smaller in the cryopreserved cells. In conclusion, cryopreserved hepatocytes held in suspension can be recommended for short-term metabolism or toxicity studies. Systems with cryopreserved hepatocyte cultures that could be applied for studies of enzyme induction are already in a state allowing practical application, but may be further optimized.

Factors Influencing Morbidity and Mortality after Pancreaticoduodenectomy: Critical Analysis of 221 Resections

n= 209) or total (n= 12) pancreaticoduodenectomy, in 12 cases combined with portal vein resection, was performed. Surgical complications were seen in 25%, but less than half of them were severe. General complications were seen in 18.5%. The 30- and 90-day mortality rates were 3.1%, and 5.7%, respectively. In a regression analysis the intraoperative blood loss, preoperative serum bilirubin, diameter of the pancreatic duct, and occurrence of surgical and nonsurgical complications had an independent influence on mortality. In addition to the experience of the surgeon in selecting the patients and his or her personal technical skills when performing a pancreaticoduodenectomy, better anticipation and management of postoperative complications is essential for improving the results of this operation.

Cultures with cryopreserved hepatocytes: applicability for studies of enzyme induction.

The use of hepatocyte cultures is well established for the study of drug-drug interactions. However, the major hindrance for the use of human hepatocyte cultures is that human hepatocytes are only occasionally available. This problem could be overcome by cryopreservation. Although cryopreserved hepatocytes have been recommended for short term applications in suspension, studies on induction of enzyme activity, requiring a more prolonged maintenance of cryopreserved hepatocytes in culture, represent a new field of research. In the present study, we established a technique that allows preparation of rat hepatocyte co-cultures, using cryopreserved hepatocytes. After incubation with phenobarbital (0.75 mM; 72 h) induction factors for the isoenzyme-dependent regio and stereoselective testosterone hydroxylations were 1.6, 2.2, 1.0, 2.1, 5.6, 2.4, 3.6, 4.5 and 0.9 for 2alpha-, 2beta-, 6alpha-, 6beta-, 7alpha-, 15beta-, 16alpha- and 16beta-hydroxytestosterone and 4-androsten-3,17 dione. Regarding induction factors of less than 2-fold, as questionable these induction factors were similar to those of cultures with freshly isolated hepatocytes and the induction pattern of the individual hydroxylation products was similar to the in vivo situation. In addition 3-methylcholanthrene (5 microM; 72 h) induced exclusively the formation of 7alpha-hydroxytestosterone (6.6-fold) in cultures with cryopreserved hepatocytes. This specificity also correlates to that obtained in rats. Although these induction factors were clearly satisfactory in cryopreserved cultures, the absolute activities of the main testosterone hydroxylation products were reduced when compared to fresh cultures. For instance, 6beta-hydroxytestosterone, the main metabolite in solvent controls was reduced to 79%, 7alpha-hydroxytestosterone, the main metabolite after induction with 3-MC, was reduced to 66% and 16beta-hydroxytestosterone, the main metabolite after induction with PB, was reduced to 52%. Similarly, EROD activity after induction with 3-methylcholanthrene in cryopreserved cultures was reduced to 62%, compared with that in fresh cultures. Although further optimization and validation is required, the data show that cytochrome P450 activities can clearly be induced in co-cultures of cryopreserved hepatocytes, in a fashion which for the investigated inducers, is similar to that in cultures from freshly isolated hepatocytes and similar to the in vivo situation.

Clinicopathologic Study for the Assessment of Resection for Ampullary Carcinoma

Abstract. In a prospective observational study including 34 patients with carcinoma of the ampulla of Vater, postoperative morbidity, mortality, and long-term survival were analyzed to determine the surgical procedure of choice. Surgically related postoperative complications were observed in 35.4% of patients after pancreatic resection. No patient died within the first 30 days postoperatively, and in-hospital mortality was 3%. Lymph node metastases were associated only with moderate or undifferentiated tumors larger than 0.6 cm in diameter that infiltrated beyond the ampulla of Vater. The median follow-up time was 4.3 years. The 5-year survival rate for the 31 patients undergoing radical resection was 62.7%. Multivariate analysis (including the covariates depth of tumor infiltration, lymph node metastases, and the ratio of metastatic to dissected lymph nodes) demonstrated that only this ratio exerted an independent influence on the prognosis (p= 0.001). The present series demonstrates that radical resection of ampullary cancer is the procedure of choice even in elderly patients. The most important factor influencing the survival rate is the extent of the lymph node dissection. The histopathologic investigation of our pancreatoduodenectomy specimens demonstrates clearly that local excision of ampullary cancer may be indicated only in high risk patients with a pT1, well differentiated tumor smaller than 0.6 cm in diameter.

Prognostic value of DNA analysis in colorectal carcinoma

Background. Reported experiences regarding the prognostic significance of DNA content in colorectal carcinoma have been a matter of controversy.

Diagnosing and Staging of Pancreatic Carcinoma – What Is Necessary?

The aim of the present prospective observational study was to diagnose and stage pancreatic carcinoma with a minimum of diagnostic procedures. Our experiences in 307 patients with a histologically confirmed pancreatic carcinoma show that for diagnosing pancreatic carcinoma sonography and computed tomography are sufficient in 95% of the cases. The combination of both has a sensitivity equal to that of endoscopic retrograde cholangiopancreatography (ERCP; 96.8 vs. 98.7%; n.s., χ2 test). ERCP is only indicated in cases with negative sonography and computed tomography, and suspicion of pancreatic cancer. For tumor staging, the routine performance of angiography cannot be recommended in view of the fact that although it provides greater sensitivity for the evaluation of an infiltration of the portal vein (80% for angiography vs. 22% for sonography or computed tomography), it is associated with a lower positive predictive value (56.4 vs. 68 and 72%) which results in a lower accuracy. Despite recent advantages in diagnostic technology, less than 50% of unresectable tumors were identified preoperatively at a 10% false-positive rate. The major reason for unresectability is infiltration into the mesenteric axis, which cannot be identified laparoscopically. Laparoscopy or percutaneous biopsy is recommended only in the presence of a tumor with suspicion of distant metastasis detected by radiological imaging and requiring histological confirmation. In conclusion, sonography and computed tomography as the only diagnostic images are sufficient for diagnosing and staging of pancreatic carcinoma in more than 95% of the patients. Only a small number of patients needs further diagnostic procedures.

Factors influencing survival after resection of pancreatic cancer : a DNA analysis and a histomorphologic study

Background. The influence of DNA content on prognosis in stomach cancer has been investigated rarely, and the results are controversial.

Cryotherapy for liver metastases.

Abstract Cryotherapy is undergoing a renaissance in the treatment of nonresectable liver tumors. In a prospective case control study we assessed the morbidity, mortality, and efficacy of hepatic cryotherapy for liver metastases. Between January 1996 and September 1999 a total of 54 cryosurgical procedures were performed on 49 patients (median age 66 years, 21 women) with liver metastases. Patient, tumor, and operative details were recorded prospectively. Liver metastases originated from colorectal cancer (n=37), gastric cancer (n=3), renal cell carcinoma (n=2), and other primaries (n=7). Median follow-up was 13 months (1–32). The median number of liver metastases was 3 (range 1–10) with a median diameter of 3.9 cm (range 1.5–11). Twenty-one patients (43%) had cryoablation only, and 28 (57%) had liver resection in combination with cryoablation. One patient (2%) died within 30 postoperative days. Another 13 patients (27%) developed reversible complications. In 19 of 25 patients (76%) with preoperatively elevated serum CEA and colorectal metastases it returned to the normal range postoperatively. Twenty-eight patients (57%) developed tumor recurrence, eight of which with involvement of the cryosite. Overall median survival patients was 23 months, and survival in patients with colorectal metastases was 29 months. Hepatic cryotherapy is associated with tolerable morbidity and mortality. Efficacy is demonstrated by tumor marker results. Survival data are promising; however, long-term results must be provided to allow comparison with other treatment modalities.

Resistance factors in colon cancer tissue and the adjacent normal colon tissue: glutathione S-transferases α and π, glutathione and aldehyde dehydrogenase

Glutathione S-transferases (GST) alpha and pi, glutathione (GSH) and aldehyde dehydrogenase (ADH) were determined in colorectal cancer tissue specimens and in the adjacent normal colon tissue. The median contents in normal and cancer tissue were 8.1 (2.3-30.3) (5-95% quantiles) and 15.1 (5.3-50.3) microg/mg protein for GST pi (P = 0.035), 0.0 (0.0-1.4) and 0.4 (0.0-3.5) microg/mg protein for GST alpha (P = 0.019), 7.3 (1.3-22.7) and 5.6 (2.3-26.0) microg/mg protein for GSH (P = 0.171) and 30.8 (13.0-42.0) and 23.2 (9.0-32.9) microg/mg protein for ADH (P = 0.0017), respectively. Thus, the mean GST alpha and pi both significantly increased in colon cancer compared to the adjacent normal tissue, which underlines their importance as possible resistance factors. A highly significant correlation was obtained between the GSH content in colon cancer and normal tissue (P = 0.0017). Thus, the constitutive GSH expression seems to be maintained during tumor development. A similar correlation was obtained for ADH (P = 0.0075), but the median ADH was lower in cancer tissue compared to the adjacent normal tissue (P = 0.0017). Contrary to GSH and ADH, GST pi did not correlate between normal and colon cancer tissue. Whereas GSH and ADH correlated in normal colon tissue (P = 0.014), no significant correlation for GSH and ADH was observed in colon cancer tissue (P = 0.109). In conclusion, significant correlations between colon cancer and normal tissue were obtained, suggesting that the expression levels of these resistance factors are maintained during carcinogenesis in most patients.

DNA image cytometry. A prognostic tool in squamous cell carcinoma of the esophagus

In 45 patients who underwent an esophagus resection due to a squamous cell carcinoma, in addition to the TNM classification and usual morphologic criteria, the paraffin‐embedded material underwent deparaffinization, was rehydrated, and was mechanically and enzymatically processed into a single‐cell solution. For evaluating the DNA histogram this was analyzed with the help of automatic single‐cell cytophotometric study. The method, contrary to that of flow cytometric study, allows for the selective analysis of tumor cells due to the electronically, previously given selection criteria, whereas artifacts, stroma, and infection cells remain excluded from analysis. The multivariate analysis shows that the prognosis is only correlated with DNA content of the tumor cells. Patients with diploid tumors had a median survival time of 32 months as compared with the 22 months of patients with hypotriploid tumors, and 6.5 months with hypertriploid tumors. DNA cytometric analysis should be included when diagnosing patients with esophagus carcinoma.