Sparing the hippocampus and the hypothalamic- pituitary region during whole brain radiotherapy: a volumetric modulated arc therapy planning study.

BACKGROUND: Feasibility testing of a simultaneous sparing approach of hippocampus, hypothalamus and pituitary gland in patients undergoing whole-brain radiotherapy (WBRT) with and without a concomitant boost to metastatic sites. INTRODUCTION: Cognitive impairment and hormonal dysfunction are common side effects of cranial radiotherapy. A reduced dose application to the patho-physiologically involved functional brain areas, i.e. hippocampus, hypothalamus and pituitary gland, could reduce these common side effects. While hippocampal sparing is already a common practice to improve cognitive outcome, technical experience of additional combined sparing of the hypothalamus/pituitary gland (HT-P) is insufficient. METHODS: Twenty patients were included in the planning study. In 11 patients, a total dose of 36 Gy of WBRT (2 Gy per fraction) plus a simultaneous integrated boost (SIB) of 9 Gy (0.5 Gy per fraction, total dose: 45 Gy) to the brain metastases was applied. In 9 patients, prophylactic cranial irradiation (PCI) was simulated with a total dose of 30 Gy (2 Gy per fraction). In both patient cohorts, a sparing approach of the hippocampus and the HT-P area was simulated during WBRT. For all treatment plans, volumetric modulated arc therapy (VMAT) was used. Quality assurance included assessment of homogeneity, conformality and target coverage. RESULTS: The mean dose to the hippocampus and HT-P region was limited to less than 50% of the prescribed dose to the planning target volume (PTV) in all treatment plans. Dose homogeneity (HI) of the target volume was satisfying (median HI = 0.16 for WBRT+SIB and 0.1 for PCI) and target coverage (conformation number, CN) was not compromised (median CN = 0.82 for SIB and 0.86 for PCI). CONCLUSION: Simultaneous dose reduction to the hippocampus and the HT-P area did not compromise the PTV coverage in patients undergoing WBRT+SIB or PCI using VMAT. While the feasibility of the presented approach is promising, prospective neurologic, endocrine outcome and safety studies are required.

Genotoxic and mutagenic potential of camphorquinone in L5178/TK+/- mouse lymphoma cells.

OBJECTIVES: Camphorquinone (CQ) is the most important photoinitiator used in dental composite resins. Sparse data indicate a mutagenic potential of CQ. Therefore, it was aim of this study to evaluate the cytotoxicity, genotoxicity, and mutagenicity of CQ in L5178Y TK+/- mouse lymphoma cells. METHODS: L5178Y/TK+/- cells were exposed to different concentrations of non-irradiated CQ (0.25-2.5mM). Cytotoxicity was evaluated by propidium iodide assay, determination of suspension growth rate, relative total growth and the mitotic index. Intracellular levels of reactive oxygen/nitrogen species (ROS/RNS) were quantified by 2',7'-dichlorofluoresceine diacetate (DCFH-DA). Early induction of DNA strand breaks and oxidative DNA base lesions was assessed using the 8-hydroxyguanine DNA-glycosylase 1 (hOGG1)-modified alkaline comet assay, whereas mutagenicity of CQ was determined in the mouse lymphoma TK assay (MLA), according to OECD Guideline No. 490. RESULTS: CQ (0.5-2.5mM) induced concentration- and time-dependent inhibition of cell growth associated with increased ROS/RNS production, amounting to 2342%±1108% of controls after 90min at 2.5mM. Additionally, CQ concentration-dependently caused direct DNA-damage, i.e. formation of DNA strand breaks and 8-hydroxy-2'-deoxyguanosine. Whereas the MLA indicated lack of mutagenicity of CQ after a 4h of treatment, CQ concentration-dependently increased total mutant frequency (MF) after 24h (about 2-fold at 2.5mM). But, based on the global evaluation factor concept, increase in MF did not reach biologically relevance. SIGNIFICANCE: CQ induced concentration-dependent, cytotoxic and genotoxic effects in L5178Y/TK+/- cells, most likely due to oxidative stress, but without mediating obvious biological relevant mutagenicity.

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.

A comparative evaluation of ablations produced by high-frequency coagulation-, argon plasma coagulation-, and cryotherapy devices in porcine liver.

INTRODUCTION: Hepatic resection is the only curative treatment option for primary or metastatic malignancies of the liver. Although R1 resections can also lead to prolonged survival, the main surgical goal is complete tumor resection (R0). To achieve this, additional treatment of the resection margin with ablation devices is discussed. Using a porcine in vivo model, we therefore analyzed the effect of different ablation devices on depth and completeness of hepatic parenchymal cell destruction. METHODS: Swabian-Hall strain pigs underwent ablation on the surface of the right, middle, or left liver lobe using seven different types of high-frequency (HF)-, cryotherapy (Cryo)-, or argon plasma coagulation (APC) devices. Penetration depth and volume were analyzed from histological sections. Severity of parenchymal cell destruction was assessed by a histomorphological score. RESULTS: The greatest penetration depth was achieved with Cryo (10.4 ± 1.7 mm), whereas HF and APC exhibited a smaller penetration depth. However, HF and APC compared to Cryo achieved complete destruction of the intralobular architecture and hepatocellular morphology depending on the application time and the adjusted power. CONCLUSION: HF, APC, and Cryo applied to the liver surface induce different parenchymal penetration depth and cell destruction. HF and APC are considered to be standard surgical instruments and therefore recommended as standard treatment, whereas Cryo may be used only if particularly deep penetration is required.

Do hospital treatments represent a 'teachable moment' for quitting smoking? A study from a stage-theoretical perspective.

Hospital treatments are assumed to be a 'teachable moment'. This phenomenon, however, is only poorly conceptualised and untested. A stage-theoretical perspective implies that a cueing event such as hospital treatments is a teachable moment if a stage progression, change of cognitions, or both occur. This concept is examined in a cross-sectional study by comparing smokers in two treatment settings, an emergency department (ED) and inpatient treatment after elective surgery, with smokers in a control setting. Setting differences were hypothesised in stage distribution, and levels of and stage differences in social-cognitive factors under control for possible confounders. Stage, social-cognitive factors and possible confounders were assessed in 185 ED smokers, 193 inpatient smokers and 290 control smokers. Compared to control smokers, ED and inpatient smokers were in higher stages; they perceived fewer risks and cons; inpatient smokers reported more concrete plans. Stage differences in self-efficacy among ED and inpatient smokers differed from those among control smokers, but the former corresponded more strongly to the theoretical stage assumptions. The results suggest that hospital treatments lead to a stage progression and change of corresponding cognitions, and thus represent a 'teachable moment'. Stage-matched interventions should be provided but consider differences in cognitions to be effective.

Evaluation of reproductive/developmental and repeated dose (subchronic) toxicity and cytogenetic effects in rats of a roofing asphalt fume condensate by nose-only inhalation.

A Type III Built-up Roofing Asphalt (BURA) fume condensate was evaluated for subchronic systemic toxicity and reproductive/developmental toxicity screening in Wistar rats, by OECD protocol 422 and OECD cytogenetic protocol 474. Animals were exposed by nose-only inhalation to target concentrations of 30, 100 and 300 mg/m³ total hydrocarbons (actual concentrations, 30.0, 100.1 and 297.3 mg/m³). The study was performed to assess potential hazards from asphalt fumes to which humans could be exposed during application. No adverse effects were seen for spermology, reproductive or developmental parameters or early postnatal development of offspring from day 1 to 4 postpartum. BURA fume condensate did not induce any significant increases in micronucleus frequency in polychromatic erythrocytes of rat bone marrow nor was neurobehavioral toxicity observed at any dose. Systemic effects were slight and seen at doses above those measured at work sites. The systemic NOAEC of 100 mg/m³ for males was based on decreased body weight gain, food consumption and increased absolute and relative lung wet weight correlated with slight histological changes in the lung, primarily adaptive in nature at 300 mg/m³. The female NOAEC of 30 mg/m³ was based on a statistically significant increase in relative wet lung weight at higher doses, correlated with slight histopathologic effects in the lungs at the highest dose. However, no increase in relative lung weight was seen in breeding females at 100 mg/m³.

Inhibitors of mdr1-dependent transport activity delay accumulation of the mdr1 substrate rhodamine 123 in primary rat hepatocyte cultures.

P-glycoproteins (P-gps) encoded by mdr1 (multidrug resistance) genes mediate extrusion of numerous lipophilic xeno- and endobiotics through the plasma membrane. Rhodamine 123 (Rh123), a fluorescent dye which is accumulated by mitochondria, is a mdr1 substrate and a well-established tool to study mdr1 transport activity. Inhibitors of mdr1-dependent transport such as verapamil or cyclosporin A have been found to decrease Rh123 efflux from mdr1-expressing cells. Mdr1b gene expression increases with time in primary rat hepatocyte culture. In hepatocytes cultured for 4 days and expressing high levels of P-gp, intracellular Rh123 accumulation was enhanced in the presence of mdr1 inhibitors (cyclosporin A, 8 and 80 microM, verapamil, 8 and 80 microM, or triton X-100, 8 microM). Surprisingly, in hepatocytes expressing low levels of P-gp (after 1 day of culture), time-dependent Rh123 accumulation was not enhanced, but delayed by cyclosporin A, verapamil or triton X-100. In these cells orthovanadate (50 microM), an inhibitor of P-glycoprotein ATPase activity, suppressed Rh123 accumulation, while tetraethylammonium (200 microM), an organic cation transporter (OCT) substrate, had no effect. The paradoxical delay in Rh123 accumulation by verapamil and cyclosporin A occurred eventhough these compounds decreased dye extrusion from Rh123 pre-loaded cells. These observations suggest that a hitherto unknown mechanism which is sensitive to modulators of mdr1-activity contributes to Rh123 uptake or accumulation in primary rat hepatocytes.

Expression and functional activity of P-glycoprotein in cultured hepatocytes from Oncorhynchus mykiss.

P-glycoproteins encoded by multidrug resistance 1 (mdr1) genes are ATP-dependent transporters located in the plasma membrane that mediate the extrusion of hydrophobic compounds from the cell. Using cultured isolated rainbow trout hepatocytes, we characterized an mdr1-like transport mechanism of the teleost liver. Immunoblots with the monoclonal antibody C219, which recognizes a conserved epitope of P-glycoproteins, revealed the presence of immunoreactive protein(s) of 165 kDa in trout liver and cultured hepatocytes. In trout liver sections, the immunohistochemistry with C219 stained bile canalicular structures. Compounds known to interfere with mdr1-dependent transport (verapamil, vinblastine, doxorubicin, cyclosporin A, and vanadate) all increased the accumulation of rhodamine 123 by hepatocytes. Verapamil, vinblastine, and cyclosporin A decreased the efflux of rhodamine 123 from hepatocytes preloaded with rhodamine 123. By contrast, the substrate of the canalicular cation transporter tetraethylammonium and the inhibitor of the multidrug resistance-associated protein MK571 had no effect on rhodamine 123 transport. The results demonstrate the presence of an mdr1-like transport system in the teleost liver and suggest its function in biliary excretion.

Physiological oxygen tensions modulate expression of the mdr1b multidrug-resistance gene in primary rat hepatocyte cultures.

P-Glycoprotein transporters encoded by mdr1 (multidrug resistance) genes mediate extrusion of an array of lipophilic xenobiotics from the cell. In rat liver, mdr transcripts have been shown to be expressed mainly in hepatocytes of the periportal region. Since gradients in oxygen tension (pO(2)) may contribute towards zonated gene expression, the influence of arterial and venous pO(2) on mRNA expression of the mdr1b isoform was examined in primary rat hepatocytes cultured for up to 3 days. Maximal mdr1b mRNA levels (100%) were observed under arterial pO(2) after 72 h, whereas less than half-maximal mRNA levels (40%) were attained under venous pO(2). Accordingly, expression of mdr protein and extrusion of the mdr1 substrate rhodamine 123 were maximal under arterial pO(2) and reduced under venous pO(2). Oxygen-dependent modulation of mdr1b mRNA expression was prevented by actinomycin D, indicating transcriptional regulation. Inhibition of haem synthesis by 25 microM CoCl(2) blocked mdr1b mRNA expression under both oxygen tensions, whereas 80 microM desferrioxamine abolished modulation by O(2). Haem (10 microM) increased mdr1b mRNA levels under arterial and venous pO(2). In hepatocytes treated with 50 microM H(2)O(2), mdr1b mRNA expression was elevated by about 1.6-fold at venous pO(2) and 1.5-fold at arterial pO(2). These results support the conclusion that haem proteins are crucial for modulation of mdr1b mRNA expression by O(2) in hepatocyte cultures and that reactive oxygen species may participate in O(2)-dependent signal transduction. Furthermore, the present study suggests that oxygen might be a critical modulator for zonated secretion of mdr1 substrates into the bile.

Contribution of mdr1b-type P-glycoprotein to okadaic acid resistance in rat pituitary GH3 cells.

Okadaic acid as well as other, structurally different, inhibitors of serine/threonine phosphatases 1 and 2A induce apoptosis in pituitary GH3 cells. Incubation with stepwise raised concentrations of okadaic acid resulted in the isolation of cells that were increasingly less sensitive to the cytotoxic effect of this agent. After about 18 months cells were selected that survived at 300 nM okadaic acid, which is about 30 times the initially lethal concentration. This study revealed that a major pharmacokinetic mechanism underlying cell survival was the development of a P-glycoprotein-mediated multidrug resistance (MDR) phenotype. The increase in mRNA levels of the mdr1b P-glycoprotein isoform correlated with the extent of drug resistance. Functional assays revealed that increasing drug resistance was paralleled by a decreased accumulation of rhodamine 123, a fluorescent dye which is a substrate of mdr1-mediated efflux activity. Resistance could be abolished by structurally different chemosensitizers of P-glycoprotein function like verapamil and reserpine but not by the leukotriene receptor antagonist MK571 which is a modulator of the multidrug resistance-associated protein (MRP). Okadaic acid resistance included cross-resistance to other cytotoxic agents that are substrates of mdr1-type P-glycoproteins, like doxorubicin and actinomycin D, but not to non-substrates of mdr1, e.g. cytosine arabinoside. Thus, functional as well as biochemical features support the conclusion that okadaic acid is a substrate of the mdr1-mediated efflux activity in rat pituitary GH3 cells. Maintenance of resistance after withdrawal of okadaic acid as well as metaphase spreads of 100 nM okadaic acid-resistant cells suggested a stable MDR genotype without indications for the occurrence of extrachromosomal amplifications, e.g. double minute chromosomes.

Reactive oxygen species participate in mdr1b mRNA and P-glycoprotein overexpression in primary rat hepatocyte cultures.

P-glycoproteins encoded by multidrug resistance type 1 (mdr1) genes mediate ATP-dependent efflux of numerous lipophilic xenobiotics, including several anticancer drugs, from cells. Overexpression of mdr1-type transporters in tumour cells contributes to a multidrug resistance phenotype. Several factors shown to induce mdr1 overexpression (UV irradiation, epidermal growth factor, tumour necrosis factor alpha, doxorubicin) have been associated with the generation of reactive oxygen species (ROS). In the present study, primary rat hepatocyte cultures that exhibit time-dependent overexpression of the mdr1b gene were used as a model system to investigate whether ROS might participate in the regulation of intrinsic mdr1b overexpression. Addition of H2O2 to the culture medium resulted in a significant increase in mdrlb mRNA and P-glycoprotein after 3 days of culture, with maximal (approximately 2-fold) induction being observed with 0.5-1 mM H2O2. Furthermore, H2O2 led to activation of poly(ADP-ribose) polymerase, a nuclear enzyme activated by DNA strand breaks, indicating that ROS reached the nuclear compartment. Thus, extracellularly applied H2O2 elicited intracellular effects. Treatment of rat hepatocytes with the catalase inhibitor 3-amino-1,2,4-triazole (2-4 mM for 72 h or 10 mM for 1 h following the hepatocyte attachment period) also led to an up-regulation of mdrlb mRNA and P-glycoprotein expression. Conversely, antioxidants (1 mM ascorbate, 10 mM mannitol, 2% dimethyl sulphoxide, 10 mM N-acetylcysteine) markedly suppressed intrinsic mdr1b mRNA and P-glycoprotein overexpression. Intracellular steady-state levels of the mdrl substrate rhodamine 123, determined as parameter of mdr1-type transport activity, indicated that mdr1-dependent efflux was increased in hepatocytes pretreated with H2O2 or aminotriazole and decreased in antioxidant-treated cells. The induction of mdr1b mRNA and of functionally active mdr1-type P-glycoproteins by elevation in intracellular ROS levels and the repression of intrinsic mdrlb mRNA and P-glycoprotein overexpression by antioxidant compounds support the conclusion that the expression of the mdr1b P-glycoprotein is regulated in a redox-sensitive manner.

Induction of mdr1b mRNA and P-glycoprotein expression by tumor necrosis factor alpha in primary rat hepatocyte cultures.

Mammalian liver exhibits expression of members of the family of multidrug resistance (mdr) transporters (P-glycoproteins). P-glycoprotein isoforms encoded by mdr1 genes participate in extrusion of an array of xenobiotics into the bile. Induction of mdr1b mRNA expression has been shown to occur in rat hepatocytes in response to hepatotrophic growth factors. As the cytokine tumor necrosis factor alpha (TNF-alpha) is known to exert a direct mitogenic effect on hepatocytes, its influence on mdr1b expression was investigated. In primary rat hepatocytes cultured in the absence of TNF-alpha, a time-dependent increase in basal expression of mdr1b mRNA and in immunodetectable P-glycoprotein was observed. In cells treated with TNF-alpha (4,000 U/ml) for 3 days, expression of mdr1b mRNA and of immunodetectable P-glycoprotein was induced approximately twofold. Moreover, intracellular steady-state levels of the mdr1 substrate rhodamine 123 were decreased in cells pretreated with TNF-alpha in comparison to controls, indicating an increase in functional transporter(s) mediating dye extrusion. Treatment of hepatocytes with antioxidants (1 mM ascorbic acid and 2% dimethyl sulfoxide) for 3 days markedly suppressed mdr1b mRNA and P-glycoprotein expression both in cells cultured in the presence of TNF-alpha and in the absence of the cytokine, but did not fully abolish mdr1b mRNA induction by TNF-alpha, supporting the notion that reactive oxygen species participate in regulation of basal mdr1b gene expression during hepatocyte culture. In conclusion, the present data indicate that by inducing mdr1b expression in hepatocytes, TNF-alpha may affect the capacity of the liver for extrusion or detoxification of endogenous or xenobiotic mdr1 substrates.

Characterization of two pituitary GH3 cell sublines partially resistant to apoptosis induction by okadaic acid.

Pituitary GH3 cells die by apoptosis when treated with okadaic acid, a specific inhibitor of ser/thr phosphatases. Incubations starting at concentrations of 5 and 12.5 nM followed by stepwise rises resulted in two populations (the S1 and S2 sublines) that proliferated at initially lethal 30 nM. Cells were partially resistant to higher concentrations of okadaic acid and its derivative methyl okadaate. Toxicity of the structurally distinct inhibitors cantharidic acid and calyculin A was differently affected in the two resistant lines. The enhanced expression of the P-glycoprotein was one mechanism of resistance in S1 and S2. Resistance was reversed completely (S1) or partially (S2) by the addition of verapamil. In addition, phosphatase activity, presumably PP2A, was increased in S2. Therefore, pharmacokinetic and pharmacodynamic mechanisms can protect pituitary GH3 cells from apoptotic cell death by okadaic acid.

Modulation of P-glycoprotein and mdr1b mRNA expression by growth factors in primary rat hepatocyte culture.

P-glycoproteins encoded by members of the mdr gene family function as membrane-situated transport proteins, isoforms of which are involved in conferring a form of multidrug resistance by participating in secretion of various xenobiotics. In primary rat hepatocytes maintained in serum-free culture, accumulation of immunodetectable P-glycoprotein and mdr1b mRNA occurred in a time-dependent manner and was accompanied by a substantial decrease in retention of the mdr1 substrate rhodamine 123. However, incubation of cells with epidermal growth factor (EGF) or with insulin-like growth factor I (IGF-I) markedly enhanced time-dependent accumulation of P-glycoprotein and mdr1b mRNA. Furthermore, EGF-treated cells exhibited decreased intracellular rhodamine 123 retention, an effect partially inhibited by the chemosensitizer verapamil. These data suggest that an increase in (a) functional transporter(s) eliciting transport of mdr1 substrates occurs under EGF.

[Postnatal weight gain in albino mice under various light regimens]. / Untersuchungen über die postnatale Gewichtszunahme der Albinomaus unter verschiedenen Lichtregimen.

The following experiments on male and female mice of the strain AB Jena Halle were designed to study the effect of various light regimens on the body weight during the postnatal development from birth to 60 days of age. Characteristic phases of the increase in weight appear in both cyclic illumination (12L:12D, 50 lux) and constant light (24 L, 50 lux). These phases are dependent upon the wavelength and the effective period of light. Such a phase is pronounced in the puberal period in which was found a relationship between the body weight and the wavelength. At the end of the experiments, remarkable low body weights appear in red exposition in both sexes, specially in the females. Continuous white or coloured light effects, in principle, a more distinctive sec-dimorphism of the body weight than 12L:12D, because the increase of the body weight of the males is higher in 24L: as in 12L: 12D. Female mice did not show such a reaction. It follows from the results that various light regimens and various light qualities influence the postnatal increase of body weight.