Dose-response relationship of temozolomide, determined by the Pig-a, comet, and micronucleus assay.

Temozolomide (TMZ), a monofunctional alkylating agent, was selected as a model compound to determine its quantitative genotoxic dose-response relationship in different tissues (blood, liver, and jejunum) and endpoints [Pig-a-, comet-, and micronucleus assay (MNT)] in male rats. TMZ was administered p.o. over 5 consecutive days (day 1-5), followed by a treatment-free period of 50 days (day 6-56) and a final administration prior to necropsy (day 57-59). TMZ showed a dose-dependent increase in DNA damage in all interrogated endpoints. A statistically significant increase in Pig-a mutant phenotypes was observed on day 44 starting at 7.5 mg/kg/day for mutant reticulocytes (for RETCD59-) and at 3.75 mg/kg/day for mutant red blood cells (RBCCD59-), respectively. In addition, a statistically significant increase in cytogenetic damage, as measured by micronucleated reticulocytes, was observed starting at 3.75 mg/kg/day on day 3 and 1.5 mg/kg/day on day 59. DNA strand breaks, as detected by the comet assay, showed a dose-dependent and statistically significant increase in liver, blood, and jejunum starting at doses of 3.75, 3.75, and 7.5 mg/kg/day, respectively. The dose-response relationships of the Pig-a, MNT, and comet data were analyzed for possible points of departure (PoD) using the benchmark-dose (BMD) software PROAST with different critical effect sizes (CES) (BMD0.1, BMD0.5, BMD1, and BMD1SD). Overall, PoD values show a high concordance between different tissues and endpoints, underlining the suitability of this experimental design to explore quantitative dose-response relationships in a variety of different tissues and endpoints, while minimizing animal use.

Nuclear trafficking of EGFR by Vps34 represses Arf expression to promote lung tumor cell survival.

Epidermal growth factor receptor (EGFR) is a cell surface receptor that has an essential role in cell proliferation and survival, and overexpression of EGFR is a common feature of human cancers. In Non-small-cell lung cancer (NSCLC), activating mutations of EGFR have also been described. We recently showed that mutant EGFR-L858R inhibits the expression of the p14ARF tumor-suppressor protein to promote cell survival. In this study, we defined the molecular bases by which EGFR controls Arf expression. Using various lung tumor models, we showed that EGF stimulation inhibits Arf transcription by a mechanism involving the nuclear transport and recruitment of EGFR to the Arf promoter. We unraveled the vesicular trafficking protein Vps34 as a mediator of EGFR nuclear trafficking and showed that its neutralization prevents the accumulation of EGFR to the Arf promoter in response to ligand activation. Finally, in lung tumor cells that carry mutant EGFR-L858R, we demonstrated that inhibition of Vps34 using small interfering RNA restrains nuclear EGFR location and restores Arf expression leading to apoptosis. These findings identify the Arf tumor suppressor as a new transcriptional target of nuclear EGFR and highlight Vps34 as an important regulator of the nuclear EGFR/Arf survival pathway. As a whole, they provide a mechanistic explanation to the inverse correlation between nuclear expression of EGFR and overall survival in NSCLC patients.

Assessment of mechanisms driving non-linear dose-response relationships in genotoxicity testing.

In genetic toxicology, risk assessment has traditionally adopted linear dose-responses for any compound that causes genotoxic effects. Increasing evidence of non-linear dose-responses, however, suggests potential cellular tolerance to low levels of many genotoxicants with diverse modes of action. Such putative non-linear dose-responses need to be substantiated by strong mechanistic data that identifies the mechanisms responsible for the tolerance to low doses. This can be achieved by experimental demonstration of cytoprotective mechanisms and by providing experimental support for the existence of tolerance mechanisms against low dose effects. By highlighting key experiments into low dose mechanisms, this review aims to clarify which mechanistic data are required to support the use of non-linear dose-response models in risk assessment. Such key experiments are presented and discussed for alkylating agents, oxidants, particulate matter, nucleoside analogues, topoisomerase inhibitors and aneugens and exemplify the use of gene knockout models or transgenic models as well as chemical modulators of key effectors of relevant pathways and their impact on dose-response relationships. In vitro studies are particularly valuable to elucidate mechanisms of low-dose protection or lack thereof, while in vivo experiments are most appropriate for deriving a safe dose. In order to evaluate the existence of non-linear dose-response relationships for genotoxicants, we suggest that careful attention should be given to the mode of genotoxic action, relevant biomarkers of exposure, as well as to the existence and impact of potential cytoprotective mechanisms like detoxifying metabolism and DNA repair.

Influence of experimental conditions on data variability in the liver comet assay.

The in vivo comet assay has increasingly been used for regulatory genotoxicity testing in recent years. While it has been demonstrated that the experimental execution of the assay, for example, electrophoresis or scoring, can have a strong impact on the results; little is known on how initial steps, that is, from tissue sampling during necropsy up to slide preparation, can influence the comet assay results. Therefore, we investigated which of the multitude of steps in processing the liver for the comet assay are most critical. All together eight parameters were assessed by using liver samples of untreated animals. In addition, two of those parameters (temperature and storage time of liver before embedding into agarose) were further investigated in animals given a single oral dose of ethyl methanesulfonate at dose levels of 50, 100, and 200 mg/kg, 3 hr prior to necropsy. The results showed that sample cooling emerged as the predominant influence factor, whereas variations in other elements of the procedure (e.g., size of the liver piece sampled, time needed to process the liver tissue post-mortem, agarose temperature, or time of lysis) seem to be of little relevance. Storing of liver samples of up to 6 hr under cooled conditions did not cause an increase in tail intensity. In contrast, storing the tissue at room temperature, resulted in a considerable time-dependent increase in comet parameters.

Effects of ρ-Da1a a peptidic α(1) (A) -adrenoceptor antagonist in human isolated prostatic adenoma and anaesthetized rats.

BACKGROUND AND PURPOSE: ρ-Da1a, a 65 amino-acid peptide, has subnanomolar affinity and high selectivity for the human α(1) (A) -adrenoceptor subtype. The purpose of this study was to characterize the pharmacological effects of ρ-Da1a on prostatic function, both in vivo and in vitro. EXPERIMENTAL APPROACH: ρ-Da1a was tested as an antagonist of adrenaline-induced effects on COS cells transfected with the human α(1) (A) -adrenoceptor as well as on human isolated prostatic adenoma obtained from patients suffering from benign prostatic hyperplasia. Moreover, we compared the effects of ρ-Da1a and tamsulosin on phenylephrine (PHE)-induced increases in intra-urethral (IUP) and arterial pressures (AP) in anaesthetized rats, following i.v. or p.o. administration. KEY RESULTS: On COS cells expressing human α(1) (A) -adrenoceptors and on human prostatic strips, ρ-Da1a inhibited adrenaline- and noradrenaline-induced effects. In anaesthetized rats, ρ-Da1a and tamsulosin administered i.v. 30 min before PHE significantly antagonized the effects of PHE on IUP. The pK(B) values for tamsulosin and ρ-Da1a for this effect were similar. With regards to AP, ρ-Da1a only reduced the effect of PHE on AP at the lowest dose tested (10 µg·kg(-1) ), whereas tamsulosin significantly reduced PHE effects at doses between 10 and 150 µg·kg(-1) . CONCLUSIONS AND IMPLICATIONS: ρ-Da1a exhibited a relevant effect on IUP and a small effect on AP. In contrast, tamsulosin antagonized the effects of PHE on both IUP and AP. We conclude that ρ-Da1a is more uroselective than tamsulosin. ρ-Da1a is the most selective peptidic antagonist for α(1A) -adenoceptors identified to date and could be a new treatment for various urological diseases.

Acetylcholine stimulates release of endothelium-derived relaxing factor in coronary arteries of human organ donors.

OBJECTIVE: To determine whether cholinergic stimulation of human coronary endothelial cells induces the release of endothelium-derived relaxing factor (EDRF), denuded human coronary artery rings or denuded rabbit aorta rings were either directly perfused or superfused by the effluent of intact or denuded human coronary artery segments in a perfusion bioassay system. DESIGN: Coronary vessels were dissected from hearts of organ donors and prepared for use as donor segments and detector rings in the perfusion bioassay system. Segments were continuously perfused by an oxygenated Krebs-Ringer solution. Rings were suspended between two stirrups, one of which was connected to an isometric force transducer. They were either directly perfused (no tissue had been previously perfused by the same Krebs-Ringer) or superfused by the effluent of an endothelium-intact or -denuded coronary segment. Human rings were contracted with prostaglandin F2 alpha (2 x 10(-6) M) and rabbit rings were contracted with phenylephrine (10(-6) M). RESULTS: Tension in detector tissue did not change during superfusion with effluent from endothelium-denuded or -intact human coronary artery segments, showing that there was no basal release of EDRF/nitric oxide (NO) in this preparation. Acetylcholine (ACh) (10(-6) or 10(-5) M) added to the perfusate increased the tension of the precontracted endothelium-denuded human rings directly perfused (50 +/- 32%) but not of the precontracted rabbit rings, showing that ACh is a potent agonist of human coronary vascular smooth muscle. ACh added to the coronary segment in the endothelium-intact superfusion mode had no effect in 33% or induced a small relaxation (8.8 +/- 1.6%) in 66% of the preparations. Hemoglobin (10(-5) M), a scavenger of EDRF/NO, caused a further increase in tension (32 +/- 15%). Adding calcium ionophore (A 23187;10(-5) M), which stimulates EDRF/NO release through a nonreceptor-mediated mechanism, to the coronary artery segment induced a relaxation in the endothelium-perfused detector rings which did not relax to ACh (30 +/- 2%); this relaxation was reversed by methylene blue (10(-5) M), a blocker of soluble guanylate cyclase. NO endothelin, a potent vasoconstrictor released by the endothelial cells, was detected in the effluent of intact coronary vessels. CONCLUSION: The results suggest that in two-thirds of the six preparations studied, cholinergic stimulation can release EDRF from human donor coronary artery endothelial cells.

Halothane 1.5 MAC, isoflurane 1.5 MAC, and the contractile responses of coronary arteries obtained from human hearts.

Active vasoconstriction of epicardial coronary arteries can cause myocardial ischemia in patients with coronary artery disease. Relief of vasoconstriction can improve blood flow to the heart. The purpose of this study was to determine if 1.5 MAC halothane and 1.5 MAC isoflurane would each attenuate contractions evoked by three putative mediators of coronary constriction in coronary arteries removed from the hearts of human beings. Hearts were obtained in the operating room from five patients undergoing cardiac transplantation and from six brain-dead patients undergoing organ donation procedures. Coronary arteries were dissected free, cut into rings, and studied in organ chambers. Endothelium-dependent relaxations to 10(-6) M bradykinin were examined; they indicated a variable degree of endothelial dysfunction in vessels used in the experiments. Contractile responses to 40 mM KCl were tested and were used as control contractions. Contractions evoked by serotonin, histamine, and prostaglandin F2 alpha were measured and were expressed as a percent of contractile responses evoked by 40 mM KCl. Halothane depressed the agonist-induced contractions. Maximal contractile responses to serotonin were 130% +/- 28% in untreated rings and 63% +/- 10% in rings exposed to halothane (P less than 0.03). Responses to histamine were 183% +/- 46% untreated and 121% +/- 26% during halothane administration (P less than 0.05), and responses to prostaglandin F2 alpha were 227% +/- 42% untreated and 148% +/- 18% with halothane (P less than 0.05). Isoflurane had no effect on contractions. The results demonstrate that 1.5 MAC halothane, but not 1.5 MAC isoflurane, attenuates contractile responses evoked by putative mediators of coronary vasoconstriction in coronary arteries removed from the hearts of human beings.

Direct delivery of medication into a brain tumor through multiple chronically implanted catheters.

A recurrent malignant glioma was treated with cisplatin delivered directly into the tumor. The drug was delivered via 68 small catheters distributed evenly throughout the tumor area. Each catheter was connected to an osmotic minipump that delivered 0.5 microliter of drug solution each hour for 10 days. There were no side effects from the catheter implantation, chemotherapy, or catheter removal. Although the chemotherapy halted progression of the tumor, it recurred, and the patient died 6 months after treatment.

Stereotactic administration of intratumoral chronic chemotherapy of recurrent malignant gliomas.

There is a continuous search for more efficient treatment of malignant cerebral gliomas. The work of Penn and Croin demonstrates clearly that survival of rats having experimental gliomas is significantly increased by intratumoral chemotherapy. It has been shown that chronic depth electrodes for the investigation of epileptic patients is a reliable and safe method. Using this proven technology originally developed by Talairach, Szikla and co-workers we have developed catheters for intratumoral chemotherapy. Three patients have been treated. It is premature to pretend that this is a treatment which will lengthen the patient's life. Nevertheless, the feasibility of the technique, its precision and safety are assured. Following this course of treatment, we have seen no deteriorations, no side effects, no bone marrow depressions, and no sign of toxicity. Chemical analysis has shown that cisplatin was delivered to the tumor.

Extracranial and intracranial meningiomas.

Meningiomas constitute about 18% of primary intracranial tumors, and they are rarely observed in patients under 30 years of age. In this study, we present the case of a 17-year-old patient who was first seen for a right cervical mass. Further investigation revealed that a meningioma of the posterior cranial fossa had invaded the temporal bone, the infratemporal fossa and the right parapharyngeal region and was at the origin of that cervical mass. This intra- and extracranial meningioma was completely resected in two stages with very satisfactory results. We have reviewed the literature pertaining to the extracranial extensions of meningiomas originating in the posterior fossa.

Lobulated fibers in neuromuscular diseases.

Lobulated fibers have been observed in biopsies of 13 patients with various neuromuscular disorders including limb-girdle muscular dystrophy (3), distal myopathy (2), scapuloperoneal muscular dystrophy (2), congenital myopathy, Kugelberg-Welander syndrome, hypothyroidism, steroid myopathy, osteomalacia and systemic lupus erythematosus (on steroids). In all cases there were fibers characterised by small subsarcolemmal triangular aggregates or more diffuse collections extending into the interior of the muscle fiber. These were strongly reactive with oxidative enzymes, acid phosphatase, periodic acid-Schiff (PAS), Verhoeff-van Gieson (VVG) and also stained red with the Gomori trichrome technique. In 5 cases core-like fibers were also seen. Morphometric analysis of the NADH-tetrazolium reductase (NADH-TR) preparations in 11 cases showed atrophy of the lobulated fibers. Ultrastructural studies of lobulated fibers disclosed large peripheral mitochondrial aggregates and focal areas with Z-line streaming and disrupted myofibrils. We consider this structural change of the muscle fiber as a reflection of muscle cell disruption and suggest that they may progress from lobulated fibers to more atrophic core-like fibers.

Rhabdomyosarcoma of the ovary: ultrastructural study of a case and review of literature.

Pure rhabdomyosarcomas are extremely rare and lethal primary neoplasms of the ovary. The available literature revealed 14 such cases. This report describes the light and electron microscopic findings of another case. At the electron microscopic level, rhabdomyogenesis recapitulates skeletal muscle differentiation during early fetal life. Rhabdomyoblasts in the ovary may arise from either the uncommitted stromal fibroblasts or from fibroblasts of endometriotic stroma. The age of patients at diagnosis ranged between 13 months and 86 years with 60% of women aged 40 years and older. About 50% of the cases were diagnosed with disease beyond the ovary, and survival ranged between 18 days and 15 months after diagnosis. Aggressive combination therapy including surgery, radiation, and chemotherapy appears to be the only hope for improved survival.

Ovarian Sertoli-Leydig cell tumor with rhabdomyosarcoma: an ultrastructural study.

The clinical and light and electron microscopic findings of a moderately differentiated, virilizing, Sertoli-Leydig cell tumor (SLT) with pleomorphic rhabdomyosarcoma of the ovary are presented. The tumor recapitulates the primitive embryonal testis and rhabdomyogenesis, respectively. The natural history, including pathogenesis, of this peculiar and rare tumor is discussed in the light of the pertinent literature on SLT and ovarian rhabdomyosarcoma. It seems that when rhabdomyosarcoma is a significant or predominant component of SLT, as occurred in this case, the prognosis is poor and is that of rhabdomyosarcoma in general.