Multiple blood-brain barrier transport mechanisms limit bumetanide accumulation, and therapeutic potential, in the mammalian brain.

There is accumulating evidence that bumetanide, which has been used over decades as a potent loop diuretic, also exerts effects on brain disorders, including autism, neonatal seizures, and epilepsy, which are not related to its effects on the kidney but rather mediated by inhibition of the neuronal Na-K-Cl cotransporter isoform NKCC1. However, following systemic administration, brain levels of bumetanide are typically below those needed to inhibit NKCC1, which critically limits its clinical use for treating brain disorders. Recently, active efflux transport at the blood-brain barrier (BBB) has been suggested as a process involved in the low brain:plasma ratio of bumetanide, but it is presently not clear which transporters are involved. Understanding the processes explaining the poor brain penetration of bumetanide is needed for developing strategies to improve the brain delivery of this drug. In the present study, we administered probenecid and more selective inhibitors of active transport carriers at the BBB directly into the brain of mice to minimize the contribution of peripheral effects on the brain penetration of bumetanide. Furthermore, in vitro experiments with mouse organic anion transporter 3 (Oat3)-overexpressing Chinese hamster ovary cells were performed to study the interaction of bumetanide, bumetanide derivatives, and several known inhibitors of Oats on Oat3-mediated transport. The in vivo experiments demonstrated that the uptake and efflux of bumetanide at the BBB is much more complex than previously thought. It seems that both restricted passive diffusion and active efflux transport, mediated by Oat3 but also organic anion-transporting polypeptide (Oatp) Oatp1a4 and multidrug resistance protein 4 explain the extremely low brain concentrations that are achieved after systemic administration of bumetanide, limiting the use of this drug for targeting abnormal expression of neuronal NKCC1 in brain diseases.

Comparative dosimetry for children and rodents exposed to extremely low-frequency magnetic fields.

We describe a method to correlate E-fields induced by exposure to extremely low frequency magnetic fields in laboratory mice and rats during in vivo experiments to those induced in children. Four different approaches of mapping relative dose rates between humans and rodents are herein proposed and analyzed. Based on these mapping methods and volume averaging guidelines published by the International Commission on Non-Ionizing Radiation Protection (ICNRP) in 2010, maximum and median induced field values for whole body and for tissues of children and rodents were evaluated and compared. Median induced electric fields in children younger than 10 years old are in the range 5.9-8.5 V/m per T (±0.4 dB). Maximum induced electric fields, generally in the skin, are between 48 V/m and 228 V/m per T (±4 dB). To achieve induced electric fields of comparable magnitude in rodents, external magnetic field must be increased by a factor of 4.0 (±2.6 dB) for rats and 7.4 (±1.8 dB) for mice. Meanwhile, to achieve comparable magnetic field dose in rodents, ratio is close to one. These induced field dose rates for children and rodents can be used to quantifiably compare experimental data from in vivo studies with data on exposure of children from epidemiological studies, such as for leukemia. Bioelectromagnetics. 37:310-322, 2016. © 2016 Wiley Periodicals, Inc.

Extremely low-frequency magnetic fields and risk of childhood leukemia: A risk assessment by the ARIMMORA consortium.

Exposure to extremely low-frequency magnetic fields (ELF-MF) was evaluated in an International Agency for Research on Cancer (IARC) Monographs as "possibly carcinogenic to humans" in 2001, based on increased childhood leukemia risk observed in epidemiological studies. We conducted a hazard assessment using available scientific evidence published before March 2015, with inclusion of new research findings from the Advanced Research on Interaction Mechanisms of electroMagnetic exposures with Organisms for Risk Assessment (ARIMMORA) project. The IARC Monograph evaluation scheme was applied to hazard identification. In ARIMMORA for the first time, a transgenic mouse model was used to mimic the most common childhood leukemia: new pathogenic mechanisms were indicated, but more data are needed to draw definitive conclusions. Although experiments in different animal strains showed exposure-related decreases of CD8+ T-cells, a role in carcinogenesis must be further established. No direct damage of DNA by exposure was observed. Overall in the literature, there is limited evidence of carcinogenicity in humans and inadequate evidence of carcinogenicity in experimental animals, with only weak supporting evidence from mechanistic studies. New exposure data from ARIMMORA confirmed that if the association is nevertheless causal, up to 2% of childhood leukemias in Europe, as previously estimated, may be attributable to ELF-MF. In summary, ARIMMORA concludes that the relationship between ELF-MF and childhood leukemia remains consistent with possible carcinogenicity in humans. While this scientific uncertainty is dissatisfactory for science and public health, new mechanistic insight from ARIMMORA experiments points to future research that could provide a step-change in future assessments. Bioelectromagnetics. 37:183-189, 2016. © 2016 Wiley Periodicals, Inc.

Marked differences in the effect of antiepileptic and cytostatic drugs on the functionality of P-glycoprotein in human and rat brain capillary endothelial cell lines.

PURPOSE: The expression of P-glycoprotein (Pgp) is increased in brain capillary endothelial cells (BCECs) of patients with pharmacoresistant epilepsy. This may restrict the penetration of antiepileptic drugs (AEDs) into the brain. However, the mechanisms underlying increased Pgp expression in epilepsy patients are not known. One possibility is that AEDs induce the expression and functionality of Pgp in BCECs. Several older AEDs that induce human cytochrome P450 enzymes also induce Pgp in hepatocytes and enterocytes, but whether this extends to Pgp at the human BBB and to newer AEDs is not known. METHODS: This prompted us to study the effects of various old and new AEDs on Pgp functionality in the human BCEC line, hCMEC/D3, using the rhodamine 123 (Rho123) efflux assay. For comparison, experiments were performed in two rat BCEC lines, RBE4 and GPNT, and primary cultures of rat and pig BCECs. Furthermore, known Pgp inducers, such as dexamethasone and several cytostatic drugs, were included in our experiments. RESULTS: Under control conditions, GPNT cells exhibited the highest and RBE4 the lowest Pgp expression and Rho123 efflux, while intermediate values were determined in hCMEC/D3. Known Pgp inducers increased Rho123 efflux in all cell lines, but marked inter-cell line differences in effect size were observed. Of the various AEDs examined, only carbamazepine (100 µM) moderately increased Pgp functionality in hCMEC/D3, while valproate (300 µM) inhibited Pgp. CONCLUSIONS: These data do not indicate that treatment with AEDs causes a clinically relevant induction in Pgp functionality in BCECs that form the BBB.

(R)-[(11)C]verapamil is selectively transported by murine and human P-glycoprotein at the blood-brain barrier, and not by MRP1 and BCRP.

INTRODUCTION: Positron emission tomography (PET) with [(11)C]verapamil, either in racemic form or in form of the (R)-enantiomer, has been used to measure the functional activity of the adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) at the blood-brain barrier (BBB). There is some evidence in literature that verapamil inhibits two other ABC transporters expressed at the BBB, i.e. multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP). However, previous data were obtained with micromolar concentrations of verapamil and do not necessarily reflect the transporter selectivity of verapamil at nanomolar concentrations, which are relevant for PET experiments. The aim of this study was to assess the selectivity of verapamil, in nanomolar concentrations, for Pgp over MRP1 and BCRP. METHODS: Concentration equilibrium transport assays were performed with [(3)H]verapamil (5 nM) in cell lines expressing murine or human Pgp, human MRP1, and murine Bcrp1 or human BCRP. Paired PET scans were performed with (R)-[(11)C]verapamil in female FVB/N (wild-type), Mrp1((-/-)), Mdr1a/b((-/-)), Bcrp1((-/-)) and Mdr1a/b((-/-))Bcrp1((-/-)) mice, before and after Pgp inhibition with 15 mg/kg tariquidar. RESULTS: In vitro transport experiments exclusively showed directed transport of [(3)H]verapamil in Mdr1a- and MDR1-overexpressing cells which could be inhibited by tariquidar (0.5µM). In PET scans acquired before tariquidar administration, brain-to-blood ratio (Kb,brain) of (R)-[(11)C]verapamil was low in wild-type (1.3 ± 0.1), Mrp1((-/-)) (1.4 ± 0.1) and Bcrp1((-/-)) mice (1.8 ± 0.1) and high in Mdr1a/b((-/-)) (6.9 ± 0.8) and Mdr1a/b((-/-))Bcrp1((-/-)) mice (7.9 ± 0.5). In PET scans after tariquidar administration, Kb,brain was significantly increased in Pgp-expressing mice (wild-type: 5.0 ± 0.3-fold, Mrp1((-/-)): 3.2 ± 0.6-fold, Bcrp1((-/-)): 4.3 ± 0.1-fold) but not in Pgp knockout mice (Mdr1a/b((-/-)) and Mdr1a/b((-/-))Bcrp1((-/-))). CONCLUSION: Our combined in vitro and in vivo data demonstrate that verapamil, in nanomolar concentrations, is selectively transported by Pgp and not by MRP1 and BCRP at the BBB, which supports the use of (R)-[(11)C]verapamil or racemic [(11)C]verapamil as PET tracers of cerebral Pgp function.

Tariquidar and elacridar are dose-dependently transported by P-glycoprotein and Bcrp at the blood-brain barrier: a small-animal positron emission tomography and in vitro study.

Elacridar (ELC) and tariquidar (TQD) are generally thought to be nontransported inhibitors of P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), but recent data indicate that they may also be substrates of these multidrug transporters (MDTs). The present study was designed to investigate potential transport of ELC and TQD by MDTs at the blood-brain barrier at tracer doses as used in positron emission tomography (PET) studies. We performed PET scans with carbon-11-labeled ELC and TQD before and after MDT inhibition in wild-type and transporter-knockout mice as well as in in vitro transport assays in MDT-overexpressing cells. Brain entrance of [(11)C]ELC and [(11)C]TQD administered in nanomolar tracer doses was found to be limited by Pgp- and Bcrp1-mediated efflux at the mouse blood-brain barrier. At higher, MDT-inhibitory doses, i.e., 15 mg/kg for TQD and 5 mg/kg for ELC, brain activity uptake of [(11)C]ELC at 25 minutes after tracer injection was 5.8 ± 0.3, 2.1 ± 0.2, and 7.5 ± 1.0-fold higher in wild-type, Mdr1a/b((-/-),()) and Bcrp1((-/-)) mice, respectively, but remained unchanged in Mdr1a/b((-/-))Bcrp1((-/-)) mice. Activity uptake of [(11)C]TQD was 2.8 ± 0.2 and 6.8 ± 0.4-fold higher in wild-type and Bcrp1((-/-)) mice, but remained unchanged in Mdr1a/b((-/-)) and Mdr1a/b((-/-))Bcrp1((-/-)) mice. Consistent with the in vivo findings, in vitro uptake assays in Pgp- and Bcrp1-overexpressing cell lines confirmed low intracellular accumulation of ELC and TQD at nanomolar concentrations and increased uptake at micromolar concentrations. As this study shows that microdoses can behave pharmacokinetically differently from MDT-inhibitory doses if a compound interacts with MDTs, conclusions from microdose studies should be drawn carefully.

Effects of 50 Hz magnetic field exposure on the stress marker α-amylase in the rat mammary gland.

PURPOSE: Concerns about adverse health effects of environmental exposure to 50/60 Hz magnetic fields (MF) have initiated numerous studies on laboratory animals with varying outcomes. Previously, we reported that rat strains responded differently to MF regarding mammary cell proliferation and tumor development indicating that (epi)genetic factors might influence MF effects in the breast tissue, yet without any identified mechanism. In the present study, α-amylase, recently introduced as a stress marker in humans, was investigated in the mammary gland of Fischer 344 (F344) and Lewis rats, two strains with distinct stress sensitivity. MATERIALS AND METHODS: F344 rats were sham- and MF-exposed (50 Hz, 100 µT) for different time periods, Lewis rats for two weeks. For comparison, diethylstilbestrol was administered at single or repeated doses. RESULTS: α-Amylase activity was significantly enhanced in the F344 mammary glands after 2 and 4 weeks of MF, whereas no reproducible effects were observed in Lewis rats. Diethylstilbestrol increased the α-amylase after repeated dosing. CONCLUSIONS: Although α-amylase represents a difficult parameter in animal studies because of its stress sensitivity, it should be considered for investigations in humans and cell cultures as a biomarker for MF susceptibility and a target to examine possible MF mechanisms since α-amylase affects cell growth.

Gene expression in the mammary gland tissue of female Fischer 344 and Lewis rats after magnetic field exposure (50 Hz, 100 µT) for 2 weeks.

PURPOSE: The issue of whether exposure to environmental power-frequency magnetic fields (MF) has impact on breast cancer development still remains equivocal. Previously, we observed rat strain differences in the MF response of breast tissue, so that the genetic background plays a role in MF effects. The present experiment aimed to elucidate candidate genes involved in MF effects by comparison of MF-susceptible Fischer 344 (F344) rats and MF-insensitive Lewis rats. MATERIALS AND METHODS: Female F344 and Lewis rats were exposed to MF (50 Hz, 100 µT) for two weeks, and a whole genome microarray analysis in the mammary gland tissue was performed. RESULTS: A remarkably decreased α-amylase gene expression, decreases in carbonic anhydrase 6 and lactoperoxidase, both relevant for pH regulation, and an increased gene expression of cystatin E/M, a tumor suppressor, were observed in MF-exposed F344, but not in Lewis rats. CONCLUSION: The MF-exposed F344 breast tissue showed alterations in gene expression, which were absent in Lewis and may therefore be involved in the MF-susceptibility of F344. Notably α-amylase might serve as a promising target to study MF effects, because first experiments indicate that MF exposure alters the functionality of this enzyme in breast tissue.

Salivary α-amylase exhibits antiproliferative effects in primary cell cultures of rat mammary epithelial cells and human breast cancer cells.

BACKGROUND: Breast cancer is one of the most diagnosed cancers in females, frequently with fatal outcome, so that new strategies for modulating cell proliferation in the mammary tissue are urgently needed. There is some, as yet inconclusive evidence that α-amylase may constitute a novel candidate for affecting cellular growth. METHODS: The present investigation aimed to examine if salivary α-amylase, an enzyme well known for the metabolism of starch and recently introduced as a stress marker, is able to exert antiproliferative effects on the growth of mammary gland epithelial cells. For this purpose, primary epithelial cultures of breast tissue from two different inbred rat strains, Fischer 344 (F344) and Lewis, as well as breast tumor cells of human origin were used. Treatment with human salivary α-amylase was performed once daily for 2 days followed by cell counting (trypan blue assay) to determine alterations in cell numbers. Cell senescence after α-amylase treatment was assessed by ß-galactosidase assay. Endogenous α-amylase was detected in cells from F344 and Lewis by immunofluorescence. RESULTS: Salivary α-amylase treatment in vitro significantly decreased the proliferation of primary cells from F344 and Lewis rats in a concentration-dependent manner. Noticeably, the sensitivity towards α-amylase was significantly higher in Lewis cells with stronger impact on cell growth after 5 and 50 U/ml compared to F344 cells. An antiproliferative effect of α-amylase was also determined in mammary tumor cells of human origin, but this effect varied depending on the donor, age, and type of the cells. CONCLUSIONS: The results presented here indicate for the first time that salivary α-amylase affects cell growth in rat mammary epithelial cells and in breast tumor cells of human origin. Thus, α-amylase may be considered a novel, promising target for balancing cellular growth, which may provide an interesting tool for tumor prophylaxis and treatment.

Do ATP-binding cassette transporters cause pharmacoresistance in epilepsy? Problems and approaches in determining which antiepileptic drugs are affected.

Resistance to multiple antiepileptic drugs (AEDs) is a common problem in epilepsy, affecting at least 30% of patients. One prominent hypothesis to explain this resistance suggests an inadequate penetration or excess efflux of AEDs across the blood - brain barrier (BBB) as a result of overexpressed efflux transporters such as P-glycoprotein (Pgp), the encoded product of the multidrug resistance- 1 (MDR1, ABCB1) gene. Pgp and MDR1 are markedly increased in epileptogenic brain tissue of patients with AED-resistant partial epilepsy and following seizures in rodent models of partial epilepsy. In rodent models, AED-resistant rats exhibit higher Pgp levels than responsive animals; increased Pgp expression is associated with lower brain levels of AEDs; and, most importantly, co-administration of Pgp inhibitors reverses AED resistance. Thus, it is reasonable to conclude that Pgp plays a significant role in mediating resistance to AEDs in rodent models of epilepsy - however, whether this phenomenon extends to at least some human refractory epilepsy remains unclear, particularly because it is still a matter of debate which AEDs, if any, are transported by human Pgp. The difficulty in determining which AEDs are substrates of human Pgp is mainly a consequence of the fact that AEDs are highly permeable compounds, which are not easily identified as Pgp substrates in in vitro models of the BBB, such as monolayer (Transwell(®)) efflux assays. By using a modified assay (concentration equilibrium transport assay; CETA), which minimizes the influence of high transcellular permeability, two groups have recently demonstrated that several major AEDs are transported by human Pgp. Importantly, it was demonstrated in these studies that Pgp-mediated transport highly depends on the AED concentration and may not be identified if concentrations below or above the therapeutic range are used. In addition to the efflux transporters, seizure-induced alterations in BBB integrity and activity of drug metabolizing enzymes (CYPs) affect the brain uptake of AEDs. For translating these findings to the clinical arena, in vivo imaging studies using positron emission tomography (PET) with (11)C-labelled AEDs in epileptic patients are under way.

Impact of seizure activity on free extracellular phenytoin concentrations in amygdala-kindled rats.

Access of antiepileptic drugs (AEDs) to the epileptic focus region is considered to be influenced by seizure-associated changes in blood-brain barrier (BBB) function and blood flow. Enhanced leakiness of the BBB has been reported as a consequence of seizure activity, and this is controversially discussed to either favor accumulation of AEDs in epileptic tissue or to limit free extracellular concentrations of AEDs due to enhanced protein extravasation. On the other hand, multidrug transporter overexpression has been described following seizure activity, which can limit brain penetration of AEDs in brain regions involved in seizure generation and spread. Aim of the present study was to determine, how these complex alterations at the BBB influence penetration of a standard AED to the site of seizure initiation. Microdialysis experiments were performed in amygdala-kindled rats and in electrode-implanted, non-kindled rats with the microdialysis probe located directly adjacent to the stimulation-recording depth electrode. Penetration of the AED phenytoin to the extracellular fluid in the focus region was investigated at different time points in relation to seizure activity elicited in kindled rats. Integrity of the BBB was determined by Evans blue. Access of phenytoin to the amygdala proved to be comparable in non-kindled, electrode-implanted control rats and in kindled rats 2 h or fourteen days following a single generalized seizure. When a single generalized seizure was elicited 10 min following phenytoin administration, average phenytoin brain dialysate levels were significantly lower (up to 45%) than those of control animals. During a self-sustained status epilepticus, phenytoin access to the site of seizure initiation tended to be lower in the early phase following drug administration, but reached control level 2 h later. The data clearly demonstrate that seizure-induced alterations in BBB integrity and function do not increase extracellular brain levels of phenytoin in affected brain regions, but rather tend to decrease the free concentration of phenytoin in the extracellular compartment.

Evaluation of transport of common antiepileptic drugs by human multidrug resistance-associated proteins (MRP1, 2 and 5) that are overexpressed in pharmacoresistant epilepsy.

Resistance to antiepileptic drugs (AEDs) is one of the most serious problems in the treatment of epilepsy. Accumulating experimental evidence suggests that increased expression of the drug efflux transporter P-glycoprotein (Pgp) at the blood-brain barrier may be involved in the mechanisms leading to AED resistance. In addition to Pgp, increased expression of several multidrug resistance-associated proteins (MRPs) has been determined in epileptogenic brain regions of patients with pharmacoresistant epilepsy. However, it is not known whether AEDs are substrates for MRPs. In the present experiments, we evaluated whether common AEDs are transported by human MRPs (MRP1, 2 and 5) that are overexpressed in AED resistant epilepsy. For this purpose, we used a highly sensitive assay (concentration equilibrium transport assay; CETA) in polarized kidney cell lines (LLC, MDCKII) transfected with human MRPs. The assay was validated by known MRP substrates, including calcein-AM (MRP1), vinblastine (MRP2) and chloromethylfluorescein diacetate (CMFDA; MRP5). The directional transport determined with these drugs in MRP-transfected cell lines could be blocked with the MRP inhibitor MK571. However, in contrast to transport of known MRP substrates, none of the common AEDs (carbamazepine, valproate, levetiracetam, phenytoin, lamotrigine and phenobarbital) used in this study was transported by MRP1, MRP2 or MRP5. A basolateral-to-apical transport of valproate, which could be inhibited by MK571 and probenecid, was determined in LLC cells (both wildtype and transfected), but the specific transporter involved was not identified. The data indicate that common AEDs are not substrates for human MRP1, MRP2 or MRP5, at least in the in vitro models used in this study.

Exposure to antiepileptic drugs does not alter the functionality of P-glycoprotein in brain capillary endothelial and kidney cell lines.

Several major antiepileptic drugs, including carbamazepine, phenytoin and phenobarbital, induce xenobiotic metabolizing enzymes via activation of nuclear receptors, including pregnane X receptor (NR1I2) and constitutive androstane receptor (NR1I3). Via activation of these xenobiotic sensors, antiepileptic drugs may also induce the expression of efflux transporters such as P-glycoprotein (Pgp) in different tissues, including intestine, liver, kidney and brain. Increased expression of Pgp in brain capillary endothelial cells, which form the blood-brain barrier, could limit the penetration of antiepileptic drugs into the brain and therefore decrease their therapeutic efficacy. As a consequence, it is important to know whether antiepileptic drugs alter the expression or functionality of Pgp in endothelial cells. In the present study, we studied the effects of exposure to phenobarbital, phenytoin and carbamazepine on Pgp expression and functionality in the rat brain endothelial cell line GPNT. For comparison with drug effects on endothelial cells, a dog kidney cell line (MDCK II) was used. Furthermore, several known Pgp inducers (dexamethasone, doxorubicin, and rifampicin) were included in the study. Functionality of Pgp was determined by uptake assays, using known Pgp substrates (digoxin and vinblastine) and transport inhibitors (tariquidar, MK571). In GPNT cells, exposure to dexamethasone increased Pgp functionality, while antiepileptic drug exposure at clinically relevant concentrations did not exert any significant induction of Pgp expression or function. Similarly, antiepileptic drug exposure did not affect Pgp in MDCK cells. The lack of antiepileptic drugs to induce Pgp in brain capillary endothelial cells and kidney cells is in contrast to their known effect on Pgp expression in hepatic and intestinal cells, substantiating tissue differences in the regulation of Pgp.

Several major antiepileptic drugs are substrates for human P-glycoprotein.

One of the current hypotheses of pharmacoresistant epilepsy proposes that transport of antiepileptic drugs (AEDs) by drug efflux transporters such as P-glycoprotein (Pgp) at the blood-brain barrier may play a significant role in pharmacoresistance in epilepsy by extruding AEDs from their intended site of action. However, several recent in vitro studies using cell lines that overexpress efflux transporters indicate that human Pgp may not transport AEDs to any relevant extent. In this respect it has to be considered that most AEDs are highly permeable, so that conventional bi-directional transport assays as used in these previous studies may fail to identify AEDs as Pgp substrates, particularly if these drugs are not high-affinity substrates for Pgp. In the present study, we used a modified transport assay that allows evaluating active transport independently of the passive permeability component. In this concentration equilibrium transport assay (CETA), the drug is initially added at identical concentration to both sides of a polarized, Pgp-overexpressing cell monolayer instead of applying the drug to either the apical or basolateral side for studying bi-directional transport. Direct comparison of the conventional bi-directional (concentration gradient) assay with the CETA, using MDR1-transfected LLC cells, demonstrated that CETA, but not the conventional assay, identified phenytoin and phenobarbital as substrates of human Pgp. Furthermore, directional transport was determined for lamotrigine and levetiracetam, but not carbamazepine. Transport of AEDs could be completely or partially (>50%) inhibited by the selective Pgp inhibitor, tariquidar. However, transport of phenobarbital and levetiracetam was also inhibited by MK571, which preferentially blocks transport by multidrug resistance transporters (MRPs), indicating that, in addition to Pgp, these AEDs are substrates of MRPs. The present study provides the first direct evidence that several AEDS are substrates of human Pgp, thus further substantiating the transporter hypothesis of pharmacoresistant epilepsy.

Exposure of Fischer 344 rats to a weak power frequency magnetic field facilitates mammary tumorigenesis in the DMBA model of breast cancer.

The possibility that long-term exposure to relatively weak power frequency magnetic fields (MFs) emanating from the generation, transmission and use of electricity could increase the risk of breast cancer is a matter of ongoing debate. Laboratory studies using well-defined exposure conditions are useful to examine whether exposure to MF affects mammary tumorigenesis. Previous studies from different laboratories using the 7,12-dimethylbenz[a]anthracene (DMBA) model of breast cancer in female Sprague-Dawley (SD) rats have been inconclusive, which has been related to differences in MF sensitivity between SD substrains used in these studies. When we compared the effects of MF exposure on cell proliferation in the mammary gland of various outbred and inbred rat strains, Fischer 344 was the only inbred strain that exhibited a marked increase in cell proliferation. Based on these data, we suggested that MF exposure should significantly facilitate development and growth of mammary tumors in Fischer 344 rats, which was tested in the present study. Groups of 108 DMBA-treated rats were either MF exposed (100 muT, 50 Hz) or sham exposed for 26 weeks. MF exposure significantly facilitated mammary tumorigenesis. The incidence of rats with grossly recorded, histologically verified adenocarcinomas was increased by 45% (P = 0.0095). The most pronounced MF effect on tumor incidence was seen in the cranial inguinal complexes (L/R5). These data indicate that Fischer 344 rats are a suitable inbred strain to study the mechanisms underlying the effects of MF exposure on mammary tumorigenesis.

Prophylactic treatment with levetiracetam after status epilepticus: lack of effect on epileptogenesis, neuronal damage, and behavioral alterations in rats.

Levetiracetam (LEV) is a structurally novel antiepileptic drug (AED) which has demonstrated a broad spectrum of anticonvulsant activities both in experimental and clinical studies. Previous experiments in the kindling model suggested that LEV, in addition to its seizure-suppressing activity, may possess antiepileptogenic or disease-modifying activity. In the present study, we evaluated this possibility by using a rat model in which epilepsy with spontaneous recurrent seizures (SRS), behavioral alterations, and hippocampal damages develop after a status epilepticus (SE) induced by sustained electrical stimulation of the basal amygdala. Two experimental protocols were used. In the first protocol, LEV treatment was started 24h after onset of electrical amygdala stimulation without prior termination of the SE. In the second protocol, the SE was interrupted after 4h by diazepam, immediately followed by onset of treatment with LEV. Treatment with LEV was continued for 8 weeks (experiment #1) or 5 weeks (experiment #2) after SE, using continuous drug administration via osmotic minipumps. The occurrence of SRS was recorded during and after treatment. In addition, the rats were tested in a battery of behavioral tests, including the elevated-plus maze and the Morris water maze. Finally, the brains of the animals were analyzed for histological lesions in the hippocampal formation. With the experimental protocols chosen for these experiments, LEV did not exert antiepileptogenic or neuroprotective activity. Furthermore, the behavioral alterations, e.g., behavioral hyperexcitability and learning deficits, in epileptic rats were not affected by treatment with LEV after SE. These data do not support the idea that administration of LEV after SE prevents or reduces the long-term alterations developing after such brain insult in rats.

Auditory and vestibular defects and behavioral alterations after neonatal administration of streptomycin to Lewis rats: Similarities and differences to the circling (ci2/ci2) Lewis rat mutant.

The clinical usefulness of aminoglycoside antibiotics is limited by their ototoxicity. In rodents, damage to the inner ear is often associated with rotational behavior and locomotor hyperactivity reminiscent of such behaviors resulting from an imbalance of forebrain dopamine systems. Based on previous observations in the circling (ci2/ci2) Lewis (LEW) rat mutant, a spontaneous mutation leading to hair cell loss, deafness, impairment of vestibular functions, lateralized circling, hyperactivity and alterations in the nigrostriatal dopamine system, we have recently hypothesized that vestibular defects during postnatal development, independent of whether induced or inherited, lead to secondary changes in the dopaminergic system within the basal ganglia, which would be a likely explanation for the typical behavioral phenotype seen in such models. In the present study, we directly compared the phenotype induced by streptomycin in LEW rats with that of the ci2 LEW rat mutant. For this purpose, we treated neonatal LEW rats over 3 weeks by streptomycin, which induced bilateral degeneration of cochlear and vestibular hair cells. Following this treatment period, the behavioral syndrome of the streptomycin-treated animals, including the lateralized rotational behavior, was almost indistinguishable from that of ci2 mutant rats. However, in contrast to the ci2 mutant rat, all alterations, except the hearing loss, were only transient, disappearing between 7 and 24 weeks following treatment. In conclusion, in line with our hypothesis, vestibular defects induced in normal LEW rats led to the same phenotypic behavior as the inherited vestibular defect of ci2 mutant rats. However, with increasing time for recovery, adaptation to the vestibular impairment developed in streptomycin-treated rats, while all deficits persisted in the mutant animals. At least in part, the transient nature of the abnormal behaviors resulting from treatment with streptomycin could be explained by adaptation to the vestibular impairment by the use of visual cues, which is not possible in ci2 rats because of progressive retinal degeneration in these mutants. Although further experiments are needed to prove this hypothesis, the present study shows that direct comparisons between these two models serve to understand the mechanisms underlying the complex behavioral phenotype in rodents with vestibular defects and how these defects are compensated.

Valproic acid is not a substrate for P-glycoprotein or multidrug resistance proteins 1 and 2 in a number of in vitro and in vivo transport assays.

The antiepileptic drug valproic acid (VPA) is widely used in the treatment of epilepsy, bipolar disorders, and migraine. However, rather high doses are required for the clinical effects of VPA, which is due to its relatively inefficient delivery to the brain. The poor brain distribution of VPA is thought to reflect an asymmetric transport system at the blood-brain barrier (BBB). Based on recent data from in vitro experiments, multidrug resistance proteins (MRPs) have been proposed to be involved in the efflux transport of VPA at the BBB. In the present study, we used different experimental in vitro and in vivo strategies to evaluate whether VPA is a substrate for MRPs or the efflux transporter P-glycoprotein (Pgp). In contrast to known Pgp or MRP substrates, such as cyclosporin A or vinblastine, no directional transport of VPA was observed in cell monolayer efflux assays using the kidney cell lines Madin Darby canine kidney II and LLC-PK1, which had been transfected with either human or mouse cDNAs for the genes encoding Pgp, MRP1, or MRP2. Likewise, no indication for efflux transport of VPA was obtained in a rat microdialysis model, using inhibitors of either Pgp or MRPs. Furthermore, a significant role of MRP2 in brain efflux of VPA was excluded by using MRP2-deficient rats. Our data do not support the hypothesis that MRP1 or MRP2 is involved in the efflux of VPA from the brain. Thus, the molecular identity of the putative transporter(s) mediating the active efflux of VPA from the brain remains to be elucidated.

Differences in the transport of the antiepileptic drugs phenytoin, levetiracetam and carbamazepine by human and mouse P-glycoprotein.

In view of the important role of P-glycoprotein (Pgp) and other drug efflux transporters for drug distribution and resistance, the identification of compounds as substrates of Pgp-mediated transport is one of the key issues in drug discovery and development, particularly for compounds acting on the central nervous system. In vitro transport assays with Pgp-transfected kidney cell lines are widely used to evaluate the potential of compounds to act as Pgp substrates or inhibitors. Furthermore, such cell lines are also frequently utilized as a substitute for more labor-intensive in vitro or in vivo models of the blood-brain barrier (BBB). Overexpression of Pgp or members of the multidrug resistance protein (MRP) family at the BBB has been implicated in the mechanisms underlying resistance to antiepileptic drugs (AEDs) in patients with epilepsy. Therefore, it is important to know which AEDs are substrates for Pgp or MRPs. In the present study, we used monolayers of polarized MDCKII dog kidney or LLC-PK1 pig kidney cells transfected with cDNA containing either human MDR1, MRP2 or mouse mdr1a and mdr1b sequences to measure the directional transport of AEDs. Cyclosporin A (CsA) and vinblastine were used as reference standards for Pgp and MRP2, respectively. The AEDs phenytoin and levetiracetam were directionally transported by mouse but not human Pgp, whereas CsA was transported by both types of Pgp. Carbamazepine was not transported by any type of Pgp and did not inhibit the transport of CsA. In contrast to vinblastine, none of the AEDs was transported by MRP2 in transfected kidney cells. The data indicate that substrate recognition or transport efficacy by Pgp differs between human and mouse for certain AEDs. Such species differences, which are certainly not restricted to human and mouse, may explain, at least in part, the controversial data which have been previously reported for AED transport by Pgp in preparations from different species. However, because transport efficacy of efflux transporters such as Pgp or MRP2 may not only differ between species but also between tissues, the present data do not exclude that the AEDs examined are weak substrates of Pgp or MRP2 at the human BBB.

Power frequency magnetic fields increase cell proliferation in the mammary gland of female Fischer 344 rats but not various other rat strains or substrains.

Epidemiological data have raised concerns about the relationship between exposure to power frequency magnetic fields (MFs) and breast cancer. We have shown previously that 50-Hz MFs at microtesla flux densities enhance mammary gland tumor development and growth in the 7,12-dimethylbenz[a]anthracene (DMBA) model of breast cancer in female Sprague-Dawley (SD) rats. We also demonstrated that MF exposure results in an enhanced proliferative activity of the mammary epithelium of SD rats, which is a likely explanation for the cocarcinogenic or tumor-promoting effects of MF exposure in the DMBA model. Comparison of different SD substrains indicated that the genetic background plays a pivotal role in these effects of MF exposure. This prompted us to compare the effects of MF exposure (100 microT, 2 weeks) on cell proliferation in the mammary gland in eight different strains and substrains of outbred and inbred rats. Proliferation of epithelial cells in the mammary tissue and adjacent skin was examined by labeling proliferating cells with bromodeoxyuridine (BrdU). In addition to the MF-sensitive SD substrain (SD1) previously used in our experiments, Fischer 344 rats were the only strain in which MF exposure significantly enhanced BrdU labeling in the mammary epithelium, indicating a marked increase in cell proliferation. The MF-induced increase in BrdU labeling in Fischer 344 rats was similar to that seen after DMBA application. Furthermore, whole mount analysis of mammary tissue from Fischer 344 rats demonstrated that MF exposure increased the number of terminal end buds, i.e. the site of origin of mammary carcinomas. By comparison with MF-insensitive inbred rat strains, Fischer 344 rats may serve to evaluate the genetic factors underlying sensitivity to cocarcinogenic or tumor-promoting effects of MF exposure.