Inhibition of O6-alkylguanine-DNA alkyltransferase in animal and human ovarian tumor cell lines by O6-benzylguanine and sensitization to BCNU

O⁶-Alkylguanine-DNA alkyltransferase (O⁶-AGT) activity in rat ovarian tumor lines O-342 and O-342/DDP was 103.4±18.4 and 240.9±40.2 fmol/mg protein, respectively; thus, cisplatin (DDP) resistance was paralleled by an increase in O⁶-AGT activity by a factor of approximately 2.3. The DDP-resistant line expressed a collateral resistance to BCNU. Both lines could be sensitized to BCNU by O⁶-BG, with sensitization factors of 6.0 and 2.1, respectively. In neither line did depletion of O⁶-AGT have any sensitizing effect towards DDP. In the human ovarian cancer lines SK-OV-3 and OAW 42, O⁶-AGT activity was 337.6±18.2 and 180.0±39.9 fmol/mg protein, respectively; in these lines depletion of O⁶-AGT activity by O⁶-BG treatment resulted in sensitization factors of 3.0 and 4.1, respectively. The increase in sensitivity of ovarian tumor cell lines against a chloroethylating agent by O⁶-AGT depletion and possible pharmacological advantages of regional (i.p.) administration of this combination might be beneficial in advanced ovarian cancer.Abbreviations BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) - CENU 2-chloroethylnitrosourea - DDP cisplatin ED₅₀, the effective dose required to inhibit colony formation or cell proliferation by 50% - O⁶-AGT O⁶-alkylguanine-DNA alkyltransferase - O⁶-BG O⁶-benzylguanine SF sensitization factor     

Accuracy of frozen section examination of testicular tumors of uncertain origin

INTRODUCTION: A total of 80-90% of all testicular masses are malignant germ cell tumors. Benign testicular lesions are recognized in approximately 10-20% enabling a testis-preserving surgery on the findings of frozen section examination (FSE). However, there are only sparse information with regard to the reliability of FSE in testicular tumors of uncertain dignity. Therefore, we retrospectively reviewed our experience concerning the reliability of FSE in primary testicular tumors by comparing each FSE result to the final diagnosis. PATIENTS AND METHODS: From 1974 to 2000, 354 patients were operated on a testicular tumor. During inguinal exploration and after clamping of the spermatic cord and appropriate dressing, a representative biopsy of the tumor was taken and sent for FSE. In case of malignancy radical orchiectomy was performed, in case of benign findings or in case of a germ cell tumor in a solitary testicle, the tumor was enucleated. Slides of FSE and the permanent sections were reviewed and compared with regard to the histological diagnosis and presence/absence of malignancy. RESULTS: Based on FSE, 317 tumors (89.5%) were found to be malignant ((100 seminomas (38.5%), 217 nonseminomas (61.5%)) and 37 tumors (10.5%) were benign (17 epidermoid cysts, 14 Leydig cell tumors, two cystadenomas, two simple cysts, two hemangiomas). Comparing FSE and definitive diagnosis, FSE correctly identified all malignant and benign lesions. There was a failure rate of 10 and 8% to differentiate seminomatous from nonseminomatous tumors and vice versa based on FSE, which, however, was irrelevant for the surgical management. Complications of the enucleations (n = 37) were: testicular atrophy in three cases, testicular hematoma in three cases, orchitis/epididymitis in one case. Not a single case disclosed a local relapse after a mean follow-up of 105 (12-240) months. CONCLUSIONS: Intraoperative FSE correctly identified all malignant and benign testicular masses including radical orchiectomy or organ-preserving surgery. Surgical management of testicular tumors based on FSE results is clinically practicable.     

Three-axis ellipsoidal fitting of videokeratoscopic height data after penetrating keratoplasty

PURPOSE: After penetrating keratoplasty corneal topography tends to be irregular and the fitting of spectacle glasses or contact lenses may be difficult. The purpose of this study was to demonstrate a mathematical method for approximation of discrete corneal topography height data with an ellipsoid for better appreciation of the clinical outcome after PK. PATIENTS AND METHODS: In 50 eyes (30 keratoconus, 20 Fuchs' dystrophy) penetrating keratoplasty was performed using nonmechanical trephination with the excimer laser 193 nm. Main outcome measures were objective corneal astigmatism (regular keratometry, corneal topography (TMS-1)), subjective refraction and best-corrected visual acuity (VA) in a fixed postoperative gate 3 and 12 month postoperatively and after suture removal. An approximation algorithm was applied for fitting a general ellipsoidal surface (not rotationally symmetric) to raw corneal topography height data. A set of parameters (meridional power, axis and asphericity) were calculated. The root mean square error (RMS) was determined between raw topography power data and the ellipsoidal model surface within an apical distance of 3 mm. The cylinder of subjective refraction was correlated with the keratometric readings, the Simulated Keratometry (SimK) of the topography system and the respective parameters of the model surface. RESULTS: The amount of the SimK cylinder yielded higher values than keratometry and the ellipsoidal fit; subjective refraction yielded the lowest value at each follow-up interval. The ellipsoidal fit showed the best correlation to the refractive cylinder at all follow-up stages (p = 0.04 at 3, p = 0.01 at 12 months and p = 0.002 after suture removal). The axis of the best ellipsoidal fit showed a significant correlation with the axis of the refractive cylinder at all follow-up intervals (p = 0.02 at 3 months, p = 0.01 before suture removal and p = 0.002 after suture removal). The axis of the keratometric cylinder showed a mild correlation at all follow-up examinations (p = 0.05 at 3 months, p = 0.02 before suture removal and p = 0.04 after suture). The cylinder of the topographic modeling system, however, showed a significant correlation with the refractive cylinder axis only after suture removal (p = 0.04). The paracentral corneal power of SimK (45.9D at 3 months, 44.4D at 12 months and 43.0D after suture removal) exceeded the respective values of conventional keratometry (43.1D at 3 months, 42.9D at 12 months and 41.7D after suture removal) and the ellipsoidal fit (43.3D at 3 months, 43.0D at 12 months and 41.8D after suture removal). The corneal asphericity from the ellipsoidal fit reached an approximately spherical shape in radial direction (A = 1.0) in the initial time period after penetrating keratoplasty, remained stable before suture removal and decreased significantly (p = 0.02) to a final value of A = 0.86 indicating a (normal) prolate shape of the cornea. The approximation error between the raw corneal topography height data and the best ellipsoidal fit model surface was nearly unchanged before suture removal (1.8 +/- 0.7 microm at 3 months and 1.9 +/- 1.1 microm at 12 months, p = 0.30) and decreased significantly to the examination after suture removal (0.9 +/- 0.5 microm, p = 0.01). CONCLUSIONS: The approximation of corneal topography height data with an ellipsoidal model surface renders reconstruction of clinically relevant corneal topography parameters including corneal asphericity. Even in markedly irregular corneal surfaces, such as after PK, the correlation of amount/axis of refractive cylinder with the model surface parameters is more accurate than with respective SimK values of corneal topography analysis.     

Superficial corneal effects of experimental nonmechanical penetrating keratoplasty using a Q-switched Er:YAG laser

PURPOSE: To assess thermal effects of Q-switched Er:YAG laser trephination to corneal epithelium and superficial stroma using different mask types and materials for experimental penetrating keratoplasty. METHODS: Laser trephination was performed in 20 freshly-enucleated porcine eyes (repetition rate 5 Hz, pulse energy 65 mJ, spot size 0.7 mm). We used flat, open-metal and ceramic masks for donor and recipient trephination placed directly onto the corneal surface. Main outcome measures as assessed by light microscopy after PAS staining of 8-microm paraffin sections included: extension of tissue thermal damage at the cut edge in the superficial and basal epithelial layers, the basement membrane and subepithelial stroma, and depth and width of epithelial/stromal involvement in the area of the donor mask contact. RESULTS: The thermal damage in the superficial epithelium was more pronounced in donor (mean extension 61.6 +/- 15.6 microm) than in recipient (29.4 +/- 24.9 microm, p= 0.05) trephination. In donor trephination, thermal damage zone of the superficial epithelial layer was significantly smaller with ceramic than with metal masks (21.0 +/- 23.0 versus 61.6 +/- 15.6 microm, p= 0.014). In contrast, differences at basal epithelial layer (p= 0.44), basement membrane (p= 0.79), and subepithelial stroma (p= 0.2) were not statistically significant. Superficial donor involvement of the cornea adjacent to the paracentral donor mask contact zone was seen neither with ceramic nor with metal masks. CONCLUSION: Superficial corneal alterations adjacent to the mask-cornea contact zone may be minimized by using the Er:YAG laser in a Q-switched mode. Ceramic masks, in contrast to metal masks, further reduce superficial thermal alterations at the cut edge.     

Risk factors for corneal allograft rejection: intermediate results of a prospective normal-risk keratoplasty study

PURPOSE: To analyze the incidence of and possible risk factors for endothelial corneal allograft rejection in a well-defined population following penetrating normal-risk keratoplasty. METHODS: Since 1996 a longitudinal prospective study has been conducted to analyze the results of normal-risk penetrating keratoplasty. All patients underwent a standardized protocol of follow-up treatment and examinations in our institution. Diagnosis of corneal endothelial rejection was based on slit-lamp biomicroscopy and laser flare photometry. Data were analyzed using a proportional hazard model for censored data (Cox model), and Kaplan-Meier survival curves. The following parameters were analyzed: age, gender, atopic dermatitis, dry eye symptoms of the recipient; surgeon, graft diameter, post-mortem time, storage time and graft preservation method; and duration of postoperative epithelial defects. RESULTS: Between 1996 and May 2001, 397 patients were recruited and followed with a median follow-up of 18 months. Episodes of endothelial graft rejection were observed in 22 patients (5.5%; 18 eyes with acute diffuse episodes and 4 eyes with chronic focal rejection episodes). In addition, 12 eyes (3%) showed isolated small keratic precipitates ("graft rejection suspects"). All but one graft regained clarity after topical and systemic steroid treatment. Most episodes occurred 11-18 months postoperatively. The percentage of grafts without any episode of endothelial allograft rejection was 95% after 12 months, 89% after 18 months, and 86.5% after 24 months. The following factors were associated with graft rejection: atopic dermatitis (P=0.021), clinically manifest tear insufficiency (P=0.007), and short duration of graft storage (P=0.008). No significant correlation was detected for the remainder of the analyzed factors (P>0.05). CONCLUSION: The incidence of episodes of corneal endothelial allograft rejection following normal-risk keratoplasty was 13.5% within the first two postoperative years. However, the frequency of irreversible immunologic graft failure (3 per thousand) was lower than reported in the literature. Patients should be regularly followed up for at least 18 months postoperatively. Patients with underlying atopic dermatitis or dry eyes should receive special ophthalmological care.     

Paraoxonase 1 Q192R (PON1-192) polymorphism is associated with reduced lipid peroxidation in R-allele-carrier but not in QQ homozygous elderly subjects on a tomato-rich diet

Summary. Background: The oxidative modification of LDL is considered to play a central role in the pathogenesis of atherosclerosis and coronary heart disease (CHD). Paraoxonase (PON1) protects LDL from oxidation and may therefore retard the developement of atherosclerosis. The PON1–192 polymorphism is associated with diminished PON1 concentrations and an increased risk for CHD in RR-allele subjects. Aim of the study: To investigate the effect of tomato juice consumption on PON1 activity and other parameters related to oxidative stress in healthy elderly subjects. Furthermore, the PON1–192 genotype has been determined in the volunteers in order to see whether possible treatment effects are related to the PON1–192 polymorphism. Methods: Fifty elderly subjects were randomly assigned to control (mineral water) or intervention group (tomato juice). Subjects of the tomato juice group consumed daily 330 mL tomato juice for 8 weeks. Antioxidant status was measured as LDL oxidation, plasma malondialdehyde, ferric reducing ability of plasma (FRAP) and PON1 activity. The PON1–192 polymorphism was determined by restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR). Plasma carotenoids were analyzed by HPLC. Results: Tomato juice consumption reduced LDL-oxidation and improved antioxidant status in R-allele carriers, but not in the QQ genotype group. PON1 activity increased irrespective of the genotype in both, control and intervention group. Conclusions: The changes in antioxidant status after tomato juice consumption seem to depend on the PON1–192 genotype. Healthy elderly, carrying the R-allele, could specificly reduce their higher cardiovascular risk by changing dietary habits. Received: 9 October 2002, Accepted: 28 October 2002 Correspondence to: Achim Bub     

Tissue-specific regulation of canine intestinal and hepatic phenol and morphine UDP-glucuronosyltransferases by beta-naphthoflavone in comparison with humans

UDP-glucuronosyltransferases (UGTs) are regulated in a species- and tissue-dependent manner by endogenous and environmental factors. The present study was undertaken to further our knowledge about regulation of UGTs in dogs, a species widely used in preclinical safety evaluation. beta-Naphthoflavone (BNF) was selected as a known aryl hydrocarbon receptor agonist and antioxidant-type inducer. The latter group of inducers is intensively investigated as dietary chemoprotectants against colon cancer. Dog UGTs were investigated in comparison with related human UGTs by examples, (i) expression of dog UGT1A6, the first sequenced dog phenol UGT, and (ii) morphine UGT activities, responsible for intestinal and hepatic first-pass metabolism of morphine. The following results were obtained: (i) dog UGT1A6 was found to be constitutively expressed in liver and marginally increased by BNF treatment. Expression was low in small intestine but ca. 6-fold higher in colon than for example in jejunum. Conjugation of 4-methylumbelliferone, one of the substrates of dog UGT1A6, was also enhanced 7-fold in colonic compared to jejunal microsomes. (ii) Compared to the corresponding human tissues, canine 3-O- and 6-O-morphine UGT activities were found to be >10-fold higher in dog liver and ca. 10-fold lower in small intestinal microsomes. Small intestinal morphine and 4-hydroxybiphenyl UGT activities appeared to be moderately (2- to 3-fold) induced by oral treatment with BNF. (iii) In contrast to dogs, morphine UGT activities were found to be similar in homogenates from human enterocytes and liver. The results suggest marked differences in tissue-specific regulation of canine vs. human hepatic and intestinal phenol or morphine UGTs.     

Antioxidative and myocardial protective effects of L-arginine in oxygen radical-induced injury of isolated perfused rat hearts

Oxygen-derived free radicals and oxidants (reactive oxygen intermediates, ROI) have been implicated in cardiovascular diseases. The protective role of nitric oxide (NO) against ROI-mediated tissue injury is not resolved. We tested the effects of exogenous NO, L- and D-arginine and a NO synthase inhibitor on electrolysis-induced cardiac injury and the generation of ROI by electrolysis. Superoxide dismutase (SOD) and catalase were used for comparison. Hearts ( n=7) from male rats (350+/-30 g) were perfused in vitro at 10 ml min(-1) g(-1), ROI generated by electrolysis of the perfusion medium (15 mA, 10 s), and cardiac function and the level of isoluminol-derived chemiluminescence in electrolysed perfusion medium documented for 15 min ( n=4). The ROI-induced maximal reduction of left ventricular developed pressure to 55+/-5% of baseline, and a 2.2+/-0.1-fold rise in coronary perfusion pressure 3 min after electrolysis, were prevented by SOD (50 U ml(-1)), catalase (100 U ml(-1)), S-nitroso- N-acetyl- D,L-penicillamine (SNAP, 100 nmol l(-1)); L-arginine (1 mmol l(-1)), N(G)-nitro- L-arginine (L-NNA, 200 micromol l(-1)) or D-arginine (1 mmol l(-1)). The effect of L-arginine was concentration dependent. In all cases, the beneficial effects were closely matched by a near-total reduction of ROI in the perfusion medium.We conclude that, besides mimicking or enhancing NO activity, L-arginine and donor-derived exogenous NO are cardioprotective by reducing ROI-mediated tissue injury. The protective effect of L-NNA and D-arginine implies that the protection results from a direct chemical interaction between the drug and the oxidizing species.     

Antibiotics for the treatment of onchocerciasis and other filarial infections

More effective drugs are needed for the treatment of human filarial diseases and the elimination of these infections as a public health problem. The drugs must either kill or sterilize adult worms. The relevant filariae, Onchocerca volvulus, Wuchereria bancofti and Brugia species, harbor rickettsial endoboacteria of the genus Wolbachia as symbionts. Animal experiments have shown that the elimination of these endobacteria causes inhibition of embryogenesis, and with Onchocerca ochengi a macrofilaricidal effect. Trials with human onchocerciasis patients using doxycydine demonstrated a long-term sterilizing activity, opening up a new strategy for the control of filarial infections. Indications of antiwolbachial therapy against onchocerciasis are discussed.     

Expression patterns of retinoblastoma protein in Parkinson disease

Cellular mechanisms implicated in Parkinson disease (PD) include oxidative stress, inflammatory response, excess dopamine, DNA damage, and loss of trophic support. These stimuli have been observed to induce changes in cell cycle proteins in several cell types. One of the key regulators of cell cycle progression is the retinoblastoma protein (pRb); therefore, we assessed the staining for pRb and its inactive hyperphosphorylated isoform, ppRb, in autopsy tissue from patients with PD. In PD we found abundant pRb staining in neuronal cytoplasm of the substantia nigra, mid-frontal cortex, and hippocampus by immunohistochemistry. In controls, pRb weakly stained nucleoli of neurons in the substantia nigra and exhibited no detectable staining in mid-frontal cortex and hippocampus. Staining for ppRb resulted in a shift from weak cytoplasmic staining in neurons from control cases to strong nuclear staining in PD cases, especially within the substantia nigra, mid-frontal cortex, and hippocampus. In the substantia nigra, ppRb also co-localized to Lewy bodies, which are a pathologic feature of PD. Lewy bodies are also found in diffuse Lewy body disease (DLBD) that do not consistently exhibit changes in pRb or ppRb. These results indicate that there are changes in pRb and its inactive phospho-isoform in neurons responding to neurodegenerative stimuli associated with PD.