Serum markers of matrix turnover as predictors for the evolution of colorectal cancer metastasis under chemotherapy.

Connective tissue turnover plays a prominent role in tumour growth and metastasis. We followed serum levels of seven connective tissue parameters in 37 patients with colorectal cancer metastatic to the liver prior to and during chemotherapy. Serum samples with episodes of tumour control (n=112) showed an increase of matrix metalloproteinase-2 (MMP-2) (P

Neoadjuvant treatment with weekly high-dose 5-Fluorouracil as 24-hour infusion, folinic acid and oxaliplatin in patients with primary resectable liver metastases of colorectal cancer.

PURPOSE: To evaluate the efficacy and safety of neoadjuvant treatment comprising weekly high-dose 5-fluorouracil (5-FU) as a 24-hour infusion, folinic acid (FA) and biweekly oxaliplatin (L-OHP), followed by metastatic resection in patients with primarily resectable liver metastases of colorectal cancer (CRC). PATIENTS AND METHODS: 20 patients with primarily resectable liver metastases of CRC were enrolled in a prospective phase II study. On an outpatient basis, the patients received a treatment regimen comprising biweekly 85 mg/m(2) L-OHP in the form of a 2-hour intravenous infusion and 500 mg/m(2) FA as a 1- to 2-hour intravenous infusion, followed by 2,600 mg/m(2 ) 5-FU administered as a 24-hour intravenous infusion once weekly. A single treatment cycle comprised one infusion per week during a period of 6 weeks followed by a 2-week rest. Two cycles were administered, with a third being added when the treatment was well tolerated. Thereafter, curative resection of the liver metastases was attempted, and the patients were followed up. RESULTS: After neoadjuvant therapy, 2 of the original 20 patients showed complete remission (CR; 10%) and 18 patients partial remission (PR; 90%). As the main symptom of toxicity, diarrhea (CTC toxicity grade 3-4) was observed in 6 patients (30%), followed by vomiting in 3 patients (15%). The curative resectability rate was 80% (16 of 20). In 9 of 18 patients (50%) undergoing surgical intervention, mild postoperative complications, mainly wound healing disturbances (n = 5), occurred. No postoperative mortality was observed. Over a median follow-up of 23 months (12-38) 6 of 16 curatively resected patients developed distant metastases and 1 patient a local pelvic recurrence. The 2-year disease-free survival rate was 52% and the 2-year cancer-related survival rate 80%. CONCLUSION: The neoadjuvant treatment with weekly high-dose 5-FU in the form of a 24-hour infusion combined with FA and L-OHP is very effective and well tolerated. Surgical morbidity does not appear to be increased by the neoadjuvant treatment.

Multicenter field trial on possible health effects of toluene. I. Toluene body burdens in workers of the rotogravure industry.

Ambient air toluene concentrations as well as corresponding individual blood toluene levels were measured under conditions of a field trial, as basis for a correlation with possible acute effects. While the results of various psycho-physiological and medical evaluations after acute (Neubert et al., 2001) and long-term toluene exposure (Gericke et al., 2001) are published in accompanying papers, this publication deals with the exposure levels and body burdens characteristic of workers in the rotogravure industry in Germany at the time of the investigation (1993-1995). Besides providing some information on the exposure at various work-areas under occupational conditions, the correlation between a time-weighted average of the ambient air concentration with the corresponding blood toluene levels is analyzed. Limitations of such an attempt and possible pitfalls are discussed. In the largest field study so far performed on toluene exposure, 12 companies of the German rotogravure industry (and a total of 1528 volunteers) participated. Altogether, complete data sets, i.e. on both ambient air as well as blood toluene levels, were obtained from 1244 male and 124 female participants of the rotogravure industry with quite different degrees of toluene exposure. Rotogravure printers and their helpers were exposed to the highest toluene concentrations in ambient air. On the day of the evaluation, of 806 male volunteers within this group (of 1261 with verified exposure in air), 35 were exposed to a time-weighted average of 100 ppm (i.e. 375 mg/m(3)) or above, and 155 of the printers to concentrations between 50 and 100 ppm. Of the remaining 455 male participants of the rotogravure factories ('non-printers and helpers'), only three were exposed to toluene concentrations above 50 ppm. Only one of the 124 women working in the rotogravure factories was exposed to an average toluene concentrations above 100 mg/m(3) (i.e. 27 ppm). In 66 of the male volunteers toluene levels in blood of >850 microg/l were measured and 14 showed levels exceeding 1700 microg/l. When attempting to predict the resulting individual blood toluene levels from measurements of ambient air concentrations under field conditions, a considerable uncertainty is to be expected. We found a correlation coefficient of the regression curve of about 0.70, with numerous outliers (and a variation of the 12 factories between 0.52 and 0.88).

Multicenter field trial on possible health effects of toluene. II. Cross-sectional evaluation of acute low-level exposure.

Data on possible acute effects of today's relevant low-level exposure to toluene are contradictory, and information on possible effects of exposure under occupational conditions is largely lacking. In a controlled, multi-center, blinded field trial, effects possibly associated with acute toluene exposure were evaluated in workers of 12 German rotogravure factories. Medical examinations (inquiries on subjective symptoms, and standard tests of psycho-physiological and psycho-motor functions) were performed on almost 1500 volunteers, of whom 1290 were toluene-exposed (1178 men and 112 women), and about 200 participants served as references (157 men and 37 women), but the main aim of the trial was to reveal dose-response relationships. All volunteers were of the morning work-shift (6 h exposure). Both individual ambient air concentrations (time-weighted average) during the work-shift, as well as blood toluene concentrations after the work-shift were measured. Therefore, the medical data could for the first time be correlated with the actual individual body burden (blood toluene level) at the time of testing. In order to largely exclude confounding by chronic toluene exposure, kinetic measurements as well as the psycho-physiological and psycho-motoric tests were performed before and after the work-shift. Except for minor statistical deviations, neither convincing dose-dependent acute effects could be demonstrated with regression analyses in male volunteers at the exposure levels evaluated, nor were significant differences found when applying group statistics (highly toluene-exposed group versus volunteers with negligible exposure). Due to the rather large number of participants, the predictive power of the study is high, especially when compared with previous publications. In two psycho-physiological tests, a few more female volunteers with quite low toluene body burdens (<340 microg/l blood) showed relatively low scores when compared with participants of the reference group. Although evidence for a medical relevance is meager, the small numbers of participants, in both the exposure and the reference groups, hamper a reliable interpretation of the results concerning exposure levels above 85 microg toluene/l blood, and it is difficult to take confounding factors adequately into account. For the end points evaluated and under occupational conditions, neither blood toluene levels of 850 to 1700 microg/l (in the highest exposure group [EXPO-IV] with 56 participants), as measured 1/2 (+/-1/2) h after the work-shift, nor ambient air concentrations (time-weighted average over 6 h) between 50 and 100 ppm (188-375 mg/m(3)) were convincingly associated with alterations in psycho-physiological and psycho-motoric performances or increased the frequency of subjective complaints in male volunteers. For higher dose ranges of toluene exposure (i.e. >1700 microg toluene/l blood [or >100 ppm in ambient air]), our data set is too small for far reaching conclusions. Our data are insufficient for conclusions on a possibly higher susceptibility to toluene of some female workers. Results of kinetic studies and possible effects of long-term exposure are discussed in two accompanying publications (Neubert et al., 2001; Gericke et al., 2001).

Multicenter field trial on possible health effects of toluene. III. Evaluation of effects after long-term exposure.

In rotogravure industry, contributing considerably to mass color printing of catalogues and magazines, toluene is still extensively used as paint solvent, and many printers have been exposed to this chemical for several decades. Information on adverse health effects associated with long-term toluene exposure is still controversial. In a multi-center study, adverse health effects possibly associated with long-term toluene exposure were evaluated. In 12 rotogravure factories, 1226 male volunteers were recruited, and sufficient information on exposure and on medical data was compiled for about 1077 of them. Evaluations included: physical examination, standard tests of psycho-physiological and psycho-motoric performances, self-report of subjective symptoms, and data on a variety of laboratory blood tests. The medical data were correlated with the length (months) of toluene exposure, and a rough estimate of the extent of exposure (i.e. highly exposed printers and their helpers versus employees working at locations with low or negligible toluene exposure). A small reference group (n=109) was selected from companies of the paper industry. When linear regression curves were calculated (test results versus duration of exposure), extremely low overall coefficients of determination (r(2) adj.) of a few percent were estimated within the data clouds, with sometimes statistically significant P-values. Closer analyses revealed a strong influence of the confounding factor age, no clustering of abnormal values of highly toluene-exposed volunteers, and the vast majority or all values of the highly and long-term toluene-exposed participants staying within the reference ranges. Thus, no medical relevance of P-values <0.05 could be recognized in this evaluation, and there may have been some border-line deviations or results largely occurring by chance in the large trial. In a small cluster of the many rotogravure printers toluene-exposed for more than 20 years, the highest systolic blood pressure values of the study were found, but many possible confounding factors were not taken into account. Data on acute exposure and possible effects are presented in accompanying papers (Neubert et al., 2001a, Neubert et al., 2001b). Restricting the conclusions to the end points evaluated as well as the apparent limitations of the evaluation, no evidence was found that long-term occupational toluene exposure extending over several decades in the rotogravure industry in the Western part of Germany was convincingly associated with chronic adverse health effects or convincingly altered surrogate markers in still working male volunteers. Several peculiarities and pitfalls arising when interpreting medical data associated with such a type of environmental exposure must be considered. Reversibility of alterations previously induced at higher levels of toluene-exposure, as well as a healthy workers effect, cannot be excluded for some of the medical end points evaluated.

[Prevention and therapy of diabetic foot syndrome. Preventing complications]. / Prävention und Therapie des diabetischen Fusssyndroms. So vermeiden Sie Komplikationen.

Selective prevention and stage-orientated treatment of the diabetic foot syndrome helps to avoid late complications. A prerequisite is good blood sugar control, early use of ACE inhibitors, CSE inhibitors and--based on the vascular situation, with consideration given to indications--platelet aggregation inhibitors. Regular inspection of the feet must be taken seriously. Every diabetic should be taught the rules of behavior in young years. The primary physician is responsible for coordinating further treatment and early referral to specialized foot clinics. In the event of an infection, specific treatment of the usually polymicrobial infection should be initiated. Long-term antibiotic treatment is not to be recommended. Wherever necessary, the calculated antibiotic therapy must be corrected on the basis of the results of antibiotic sensitivity testing of swabs taken from deep tissue (better: exudate from the floor of the wound. In the case of superinfected ulcers, wound debridement by the specialist surgeon is considered a must. Also, late stages of diabetic foot can be successfully managed in specialized centers by "foot-relieving" measures, revascularization, arthrodesis, etc.

Dy-EOB-DTPA: tolerance and pharmacokinetics in healthy volunteers and preliminary liver imaging in patients.

RATIONALE AND OBJECTIVES: To investigate the tolerance and pharmacokinetics of the new liver-specific x-ray contrast agent Dy-EOB-DTPA [(4S)-4-(4-ethoxybenzyl)-3,6,9-tris(carboxylatomethyl)-3,6,9-triazaundecanedioic acid, dysprosium (Dy) complex, disodium salt] in healthy volunteers and to obtain preliminary imaging data by abdominal spiral computed tomography (CT) in tumor patients with liver metastases. METHODS: A total of 40 healthy male volunteers received 10-minute intravenous infusions of 0.05, 0.1, 0.25, 0.375, or 0.5 mmol/kg Dy-EOB-DTPA (n = 6 per dose group) or placebo (n = 10). Blood, urine, and feces were sampled for Dy measurements by inductively coupled plasma atomic emission spectrometry (ICP-AES) and for the detection of possible metabolites by high-performance liquid chromatography analysis with ICP-AES detection. Safety parameters were determined before, during, and after the study. Two patients with suspected liver metastases first received 120 mL of iopromide (300 mg iodine/mL; approximately 0.6 mmol/kg) and, 24 or 72 hours later, Dy-EOB-DTPA at a dose of 0.25 mmol/kg. Computed tomography images were obtained 50 seconds after iopromide administration and before and 90 minutes after Dy-EOB-DTPA administration. RESULTS: Dysprosium-EOB-DTPA was well tolerated. At the higher doses (0.375 and 0.5 mmol/kg), there was a slight increase in side effect intensity. In general, nausea, headache, and paresthesia mainly were reported as mild to moderate adverse events. Laboratory parameters did not exceed the normal range. Electrocardiographic, vital sign, or hemodynamic parameters were not affected by contrast agent administration. The terminal half-life of elimination of Dy-EOB-DTPA was approximately 1.5 hours, total clearance was 2 to 3 mL x min(-1) x kg(-1), and the renal clearance was approximately 1.5 mL x min(-1) x kg(-1). There was a significant dose dependence for the following parameters: maximal concentration in blood, terminal half-life, mean residence time, total clearance, urinary excretion, and fecal excretion. The volume of distribution in the steady state and renal clearance were not dependent on dose. In the blood and urine, no metabolites of Dy-EOB-DTPA could be detected. In the tumor patients, CT scanning after Dy-EOB-DTPA injection increased the number of detected metastases from 27 (plain scan) to 40 (iopromide) and then to 41 (Dy-EOB-DTPA) in patient No. 1 and from 1 (plain scan and iopromide) to 3 (Dy-EOB-DTPA) in patient No. 2. CONCLUSIONS: Dysprosium-EOB-DTPA was shown to be a well-tolerated liver-specific contrast agent. Its pharmacokinetic profile is characterized by a terminal half-life of approximately 1.5 hours. There are indications of saturation of liver uptake at the highest dose level of 0.5 mmol/kg. In comparison with plain scans and scans performed after iodinated contrast agent administration, Dy-EOB-DTPA seems to increase the number of detectable liver lesions.

CEA and CA 19-9 measurement as a monitoring parameter in metastatic colorectal cancer (CRC) under palliative first-line chemotherapy with weekly 24-hour infusion of high-dose 5-fluorouracil (5-FU) and folinic acid (FA).

BACKGROUND: There have been contradictory reports on the benefit of CEA and CA 19-9 measurements in patients with metastatic colorectal cancer using palliative chemotherapy. The object of this study was to examine the diagnostic accuracy of monitoring of palliative chemotherapy by means of CEA and CA 19-9. PATIENTS AND METHODS: The tumour markers CEA and CA 19-9 were subjected to serial measurement in patients with metastatic colorectal cancer (n = 90) using palliative first-line chemotherapy with weekly 24-hour infusion of high-dose 5-FU with FA and were compared with objective response according to WHO criteria. 85 patients could be evaluated. 43 patients (51%) initially had elevated CEA (> or = 10 ng/ml) and 33 patients (39%) elevated CA19-9 (> or = 50 IE/ml). In 24 patients (28%), both markers were initially increased. With CEA positive patients, 143 cycles of chemotherapy were carried out, which showed the following response in the various cycles: 21 episodes with progressions (ePD), 69 episodes with no change (eNC), 53 episodes with partial/complete remission (ePR/eCR). With CA 19-9 positive patients, 100 cycles of chemotherapy were carried out with the following results: 21 episodes with progressions (ePD), 48 episodes with eNC, and 31 episodes with ePR/eCR. RESULTS: A CEA rise by at least 50% differentiated between ePD versus eNC/ePR/eCR with a sensitivity of 76% and specificity of 90%. With CEA decreases of at least 30% in 99% of these patient episodes (78 of 79), a tumour progression could be excluded. Patients with an initial drop in CEA after the first cycle of chemotherapy of at least 50% of the initial level had a significantly higher probability of achieving an ePR/eCR in further therapy (relative risk 2.9; P = 0.002). With an CA 19-9 increase of at least 30%, a sensitivity progression of 62% and a specifity of 90% could be calculated. A CA 19-9 decrease of at least 60% excludes a progression in 95% of the patient episodes. CONCLUSIONS: A CEA or CA 19-9 rise is only conditionally appropriate for recording progressions. A progression however, can be excluded with falling levels with high diagnostic accuracy, in which CEA offers a greater degree of certainty than CA 19-9. With a drop in CEA 79 of 143 (= 55%) of the CT scans could be saved, in which case 78 of 79 patient episodes (99%) were correctly assessed as 'no progression'. In patients with an increased CEA and CA 19-9 the CEA determination is sufficient for the further monitoring. A confirmation of these results by multicenter trials can result in a considerable decrease of monitoring costs for palliative treatment.

Hypoglycemic sensorimotor polyneuropathy associated with insulinoma.

Hypoglycemia-induced peripheral neuropathy due to insulinoma is unusual and, as far as we know, has previously been reported in only 34 patients. In this case report, we describe the clinical features, electrophysiological features, and pathological findings in a 37-year-old patient with polyneuropathy from repeated hypoglycemic episodes over a 9-year period that related to an insulinoma. The literature is discussed. The reported case is of special interest because the peripheral neuropathy led to the correct diagnosis.

In the search for specific inhibitors of human 11beta-hydroxysteroid-dehydrogenases (11beta-HSDs): chenodeoxycholic acid selectively inhibits 11beta-HSD-I.

OBJECTIVE: Selective inhibitors of 11beta-hydroxysteroid-dehydrogenase type I may be of therapeutical interest for two reasons: i) 9alpha-Fluorinated 11-dehydrosteroids like 11-dehydro-dexamethasone (DH-D) are rapidly activated by human kidney 11beta-hydroxysteroid-dehydrogenase type II (11beta-HSD-II) to dexamethasone (D). If the same reaction by hepatic 11beta-HSD-I could be selectively inhibited, DH-D could be used for selective renal immunosuppressive therapy. ii) Reduction of cortisone to cortisol in the liver may increase insulin resistance in type 2 diabetes mellitus, and inhibition of the enzyme may lead to a decrease in gluconeogenesis. Therefore, we characterized the metabolism of DH-D by human hepatic 11beta-HSD-I and tried to find a selective inhibitor of this isoenzyme. METHODS: For kinetic analysis of 11beta-HSD-I, we used microsomes prepared from unaffected parts of liver segments, resected because of hepatocarcinoma or metastatic disease. For inhibition experiments, we also tested 11beta-HSD-II activity with human kidney cortex microsomes. The inhibitory potency of several compounds was evaluated for oxidation and reduction in concentrations from 10(-9) to 10(-5)mol/l. RESULTS: Whereas D was not oxidized by human liver microsomes at all, cortisol was oxidized to cortisone with a maximum velocity (V(max)) of 95pmol/mg per min. The reduction of DH-D to D (V(max)=742pmol/mg per min, Michaelis--Menten constant (K(m))=1.6 micromol/l) was faster than that of cortisone to cortisol (V(max)=187pmol/mg per min). All reactions tested in liver microsomes showed the characteristics of 11beta-HSD-I: K(m) values in the micromolar range, preferred cosubstrate NADP(H), no product inhibition. Of the substances tested for inhibition of 11beta-HSD-I and -II, chenodeoxycholic acid was the only one that selectively inhibited 11beta-HSD-I (IC(50) for reduction: 2.8x10(-6)mol/l, IC(50) for oxidation: 4.4x10(-6)mol/l), whereas ketoconazole preferentially inhibited oxidation and reduction reactions catalyzed by 11beta-HSD-II. Metyrapone, which is reduced to metyrapol by hepatic 11beta-HSD-I, inhibited steroid reductase activity of 11beta-HSD-I and -II and oxidative activity of 11beta-HSD-II. These findings can be explained by substrate competition for reductase reactions and by product inhibition of the oxidation, which is a well-known characteristic of 11beta-HSD-II. CONCLUSIONS: Our in vitro results may offer a new concept for renal glucocorticoid targeting. Oral administration of small amounts of DH-D (low substrate affinity for 11beta-HSD-I) in combination with chenodeoxycholic acid (selective inhibition of 11beta-HSD-I) may prevent hepatic first pass reduction of DH-D, thus allowing selective activation of DH-D to D by the high affinity 11beta-HSD-II in the kidney. Moreover, selective inhibitors of the hepatic 11beta-HSD-I, like chenodeoxycholic acid, may become useful in the therapy of patients with hepatic insulin resistance including diabetes mellitus type II, because cortisol enhances gluconeogenesis.

Metabolism of synthetic corticosteroids by 11 beta-hydroxysteroid-dehydrogenases in man.

The presence of an 11 beta-hydroxyl group is essential for the anti-inflammatory and immunosuppressive effects of glucocorticoids. Interconversion of the 11 beta-hydroxyl into the corresponding 11 beta-keto group and vice versa by 11 beta-hydroxysteroid-dehydrogenase (11 beta-HSD) may thus play a pivotal role in the efficacy of these steroids. Therefore, we have compared the metabolism of the endogenous glucocorticoid cortisol (F) with that of synthetic 9 alpha-fluorinated steroids by 11 beta-HSDs in humans in vivo and in vitro. Whereas 30% of the free steroids in urine after oral administration of 5 mg of F is F itself and 70% the inactive keto-product cortisone (E), the urinary excretion of an identical dose of oral 9 alpha-fluorocortisol (9 alpha FF) is 90% 9 alpha FF and 10% inactive 9 alpha-fluorocortisone (9 alpha FE). Kidney slices similarly convert F much faster to E than 9 alpha FF to 9 alpha FE; conversely, renal 11 beta-reduction of 9 alpha FE to 9 alpha FF is much more effective than that of E to F. Kinetic analyses in human kidney cortex microsomes prove that the preference of fluorinated steroids for reduction in human kidney slices is catalyzed by 11 beta-HSD type II: the NADH-dependent conversion of 11-dehydro-dexamethasone (DH-D), another fluorinated steroid, to dexamethasone (D) is very effective (high affinity, high Vmax), whereas reduction of E to F is very slow. In human liver microsomes (11 beta-HSD type I), nonfluorinated (E) and fluorinated 11-dehydrosteroids (DH-D) are both reduced to their corresponding active 11-hydroxyderivatives but with a Michaelis-Menten constant about 20-fold higher than for kidney microsomes (11 beta-HSD-II). Our results suggest that the decreased renal 11 beta-oxidation of 9 alpha-fluorinated steroids may offer pharmacokinetic advantages for renal immunosuppression. Furthermore, administration of fluorinated 11-dehydrosteroids is a new and exciting idea in glucocorticoid therapy in that small amounts of oral DH-D may pass the liver largely unmetabolized (11 beta-HSD-I has low affinity for such steroids) and may then be activated to D by high-affinity 11 beta-HSD-II, thus allowing selective immunosuppression in organs expressing 11 beta-HSD-II (kidney and colon).

Metabolism of dexamethasone in the human kidney: nicotinamide adenine dinucleotide-dependent 11beta-reduction.

Recently, two distinct isoenzymes of 11beta-hydroxysteroid-dehydrogenase (11beta-HSD) have been cloned and characterized in several species: The isoenzyme 11beta-HSD-I is widely distributed, bidirectional, prefers NADP(H) and has a low substrate affinity. The isoenzyme 11beta-HSD-II seems to exclusively oxidize physiological glucocorticoids, uses NAD as cosubstrate, has high substrate affinity, and is only found in mineralocorticoid target tissues and the placenta. Synthetic steroids fluorinated in position 9, however, are rapidly reduced by human kidney cortex slices. We attempted to find out which isoenzyme is responsible for this unexpected reductase activity. We studied the 11beta-HSD activity towards cortisol (F)/cortisone (E) and dexamethasone (D)/11-dehydro-dexamethasone (DH-D) in microsomes prepared from human kidney cortex. For the reaction E to F (not for DH-D to D!), glucose-6-phosphate and glucose-6-phosphate-dehydrogenase had to be added as a NADH/NADPH-regenerating system. Oxidation of F to E: NAD was the exclusively used cosubstrate; the affinity [Michael's constant (Km) for F = 25.5 nmol/L] and the maximum velocity (Vmax = 22.9 nmol/mg/min) were high. Reduction of E to F: Without the NADH/NADPH-regenerating system, this reaction was very slow. With this system, the Km value for E was in the nanomolar range (80.6 nmol/L) and the Vmax value was very low (0.88 nmol/mg/min). The reaction was clearly NADH-preferring. For the steroid pair F/E, the quotient Vmax(oxidation)/Vmax(reduction) (=26) demonstrates an equilibrium far on the 11-keto side. Oxidation of D to DH-D: With NAD as the only used cosubstrate, the kinetic analysis is compatible with the existence of two different NAD-dependent isoenzymes: Km for D = 327 nmol/L, Vmax = 53.5 nmol/mg/min and Km for D = 81.2 nmol/L; Vmax = 20.4 nmol/mg/min. Reduction of DH-D to D: The maximum velocity was higher than that of all other reactions tested: Vmax = 226.0 nmol/mg/min. The reaction was exclusively NADH-dependent; the Km value for DH-D was 68.4 nmol/L. For D/DH-D, the ratio Vmax(oxidation)/Vmax(reduction) was 0.24, demonstrating a shift to reductase activity with the reaction equilibrium far on the 11-hydroxy side. The reaction F to E was inhibited by E, DH-D, and D in a concentration-dependent manner. In conclusion, the cosubstrate dependence, the Km value of the oxidation of F and the product inhibition are in good correspondence with data for the cloned human 11beta-HSD-II. The NADH-dependent 11beta-reduction of E and especially of DH-D are inconsistent with the dogma of an unidirectional 11beta-HSD-II. The preference of D for the reductase reaction in human kidney slices is probably caused by the fluor atom in position 9, is catalyzed by 11beta-HSD-II, and leads to an activation of 11-DH-D to D in the human kidney.

The metabolism of 9 alpha-fluorinated steroids in the human kidney.

UNLABELLED: We compared the renal metabolism of 9 alpha-fluorinated steroids with that of the unfluorinated, endogenous steroid cortisol (F). By defining kinetic variables, we characterized isoenzyme activities of 11 beta-hydroxysteroid-dehydrogenase (11 beta-HSD). METHODS: I) In human kidney slices, we studied the conversion of 9 alpha-fluoro-cortisol (FF) and F to their oxo-products (and vice versa). II) In human kidney microsomes, we performed the kinetic analysis of 11 beta-HSD activity for the steroid pairs F/cortisone (E) and dexamethasone (D)/11-dehydro-dexamethasone (DH-D). RESULTS: I) In kidney slices, FF is very weakly oxidized to 9 alpha-fluorocortisone (FE), while the reduction of FE to FF is very effective. In contrast, E is hardly reduced to F, but F is strongly inactivated to E. II) Enzyme kinetics in kidney microsomes: 1a) Oxidation of F to E: exclusively NAD-dependent; K(m) = 25.5 nmol/L. b) Reduction of E to F: clearly NADH-preferring; K(m) = 81 nmol/L; Vmax(oxidation) /Vmax(reduction) (F/E) = 26.2a) Oxidation of Dtto DH-D: exclusively NAD-dependent; K(m) = 81 nmol/L b) Reduction of DH-D to D: exclusively NADH-dependent; K(m) = 68 nmol/L; Vmax(oxidation)/Vmax(reduction) (D/DH-D) = 0.09. Thus, the equilibrium of FF/FE in human kidney slices is far on the biologically active hydroxy-side. This shift, induced by the 9-fluorination, gives a good explanation for its mineralocorticoid potency. The cosubstrate dependence and the K(m)-value of the oxidation of F are similar to those of the cloned human 11 beta-HSD-II. For the first time, we could show a NADH-dependent reduction of E to F. Moreover, we found that the preference of D/DH-D for the reductase reaction (see the quotients Vmax(oxidation)/Vmax(reduction)) is due toaa NADH-dependent enzyme (probably 11 beta-HSD-II). These results provide strong evidence against the "dogma" of an "unidirectional" 11 beta-HSD-II.

Relationship between TGF alpha-induced DNA synthesis and prostaglandin synthesis in human HaCaT keratinocytes.

The relationship between transforming growth factor alpha (TGF alpha)-induced cell proliferation and prostaglandin synthesis was investigated using growth-arrested human keratinocytes of the HaCaT line. Depending on the TGF alpha concentration, the stimulation of DNA synthesis (5-fold) was found to be either insensitive (at < 10 ng/ml TGF alpha) or sensitive (at > or = 20 ng/ml TGF alpha) to inhibition by both indomethacin, an inhibitor of prostaglandin synthases (PGHS) 1 and 2 and the PGHS 2-specific inhibitor NS-398. Indomethacin-effected inhibition did not correlate with cytotoxicity and was restricted to a narrow time window after growth factor administration. The indomethacin- and NS-398-sensitive mitogenic effect of TGF alpha correlated with an early increase of arachidonic acid release and prostaglandin (PGE2, PGF2alpha) synthesis, whereas the PGHS inhibitor-insensitive TGF alpha effect did not. TGF alpha-induced prostaglandin synthesis was due to a growth factor-induced PGHS-2 activity as indicated by its suppression by NS-398. However, attempts to overcome the PGHS inhibitor-dependent suppression of TGF alpha-induced DNA synthesis by adding prostaglandins (E1, E2, F2alpha, G2) to the cultures proved to be unsuccessful. Thus, TGF alpha-induced synthesis of prostaglandins seems not to be involved in the mediation of the mitogenic effect of the growth factor on human keratinocytes in culture.

Stimulatory effect of thapsigargin, a non-TPA-type tumor promoter, on arachidonic acid metabolism in the murine keratinocyte line HEL30 and on epidermal cell proliferation in vivo as compared to the effects of phorbol ester TPA.

The effect of thapsigargin (Tg), a non-12-O-tetradecanoylphorbol-13-acetate (TPA) type skin tumor promoter, on arachidonic acid and prostaglandin E2 (PGE2) formation in HEL30 keratinocytes and on epidermal DNA synthesis in vitro and in vivo (mouse skin) was investigated and compared with that of the phorbol ester TPA. On a molar basis Tg was 30-fold more potent in inducing the arachidonic acid/PGE2 release than TPA. Applied together, the two agents showed a strong synergistic action. The response critically depended on the presence of Ca2+ in the extracellular medium. While the TPA-induced release was mediated by protein kinase C (PKC) the Tg-induced release was not. In contrast to TPA (1 microM), which is a stimulator of HEL30 DNA synthesis, Tg (0.1-1 microM) inhibited DNA labeling in vitro due to a pronounced cytotoxic effect. TPA did not exhibit such an effect. In vivo both agents were practically equipotent in inducing epidermal DNA synthesis and hyperplasia with TPA having an approximately 10-fold higher irritating potential than Tg. It is concluded that the hyperplasiogenic and tumor-promoting effect of Tg in vivo is due to cytotoxicity causing cell death and regenerative hyperproliferation. Thus, Tg-induced skin tumor promotion seems to resemble tumor promotion by mechanical skin wounding, whereas TPA evokes a more specific, i.e. PKC-mediated response. Since despite these mechanistic differences both agents induce an immediate release of arachidonic acid/PGE2 in keratinocytes, this response may be considered to provide an in vitro parameter for irritancy and tumor promotion.