Meike Mevissen

Dose-dependent anticonvulsant and proconvulsant effects of nitric oxide synthase inhibitors on seizure threshold in a cortical stimulation model in rats.

In the central nervous system, nitric oxide (NO) is increasingly being considered as a trans-synaptic retrograde messenger, being involved for instance in cellular responses to stimulation of glutamate receptors of the NMDA subtype. Thus, compounds that modify NO production, such as NO synthase inhibitors, may provide a means of altering NMDA receptor function. The functional consequences of NO synthase inhibition are, however, complicated by the fact that NO not only serves as a messenger to activate guanylyl cyclase and so to raise cGMP in target cells in response to NMDA receptor stimulation but also to induce feedback inhibition of the NMDA receptor via a redox modulatory site on the receptor complex. This may explain the contrasting results obtained previously with NO synthase inhibitors in animal models of ischaemia and seizures. In the present study, we tried to resolve the reported discrepancies about the effects of NO synthase inhibitors in seizure models by studying such drugs at various doses in a novel model of cortical seizure threshold. In this model, the threshold for seizures in rats is determined at short time intervals by applying ramp-shaped electrical pulse-trains directly to the cerebral cortex, allowing one to determine the time course of anti- or proconvulsant drug effects in individual rats. Two NO synthase inhibitors, NG-nitro-L-arginine and NG-nitro-L-arginine methyl ester, were compared with a clinically effective antiepileptic drug, i.e. valproate.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo exposure of rats to a weak alternating magnetic field increases ornithine decarboxylase activity in the mammary gland by a similar extent as the carcinogen DMBA

Magnetic field (MF) exposure has been discussed in the process of tumor promotion as indicated by epidemiologic data as well as laboratory studies. However, the precise mechanisms of tumor promoting effects of MFs are unknown. Tumor promotion is often accompanied by an increase in the activity of the enzyme ornithine decarboxylase (ODC), i.e. a key enzyme in the biosynthesis of polyamines, which have roles in cell proliferation and control of gene expression. In the present work, we studied if exposure of female rats to a 50-Hz MF with a flux density of 50 microT influences ODC activity in different tissues, including the mamma. Rats were exposed for a period of 6 weeks either with or without oral administration of the chemical carcinogen DMBA and all data were compared with those from sham-exposed controls. Magnetic field exposure resulted in an approximate doubling of ODC in mammary tissue. A significant ODC increase was also seen in the spleen, but not in the liver, small intestine, bone marrow, and ear skin. The ODC increase produced by MF exposure in the mammae was of similar magnitude as that observed after treatment with DMBA. Combined treatment with MF and DMBA was not more effective in increasing ODC than treatment with DMBA alone, except for liver tissue. The present results on in vivo increases of ODC by MF exposure strengthen the hypothesis that weak 50-Hz MFs affect ODC activity and may thus function as a tumor-promoting or co-promoting agent.

Vaccination of mice with recombinant NcROP2 antigen reduces mortality and cerebral infection in mice infected with Neospora caninum tachyzoites

Rhoptry antigens are involved in a variety of cellular functions related to host cell invasion, formation of the parasitophorous vacuole and parasite-host cell interplay. The cDNA sequence of one of these antigens, NcROP2 was identified from Neospora caninum expressed sequence tags (ESTs), amplified by reverse transcription-PCR, expressed in Escherichia coli as a (His)(6)-tagged recombinant protein (recNcROP2) and purified over Ni(2+)-affinity chromatography. Both recNcROP2 and antibodies directed against recNcROP2 had a negative impact on N. caninum tachyzoite host cell invasion in vitro, indicating that this protein participates in the host cell entry process. Subsequently, the protective efficacy of NcROP2 as a potential vaccine candidate was evaluated in a C57BL/6 mouse cerebral disease model. Mice were vaccinated three times at 2-week intervals with recNcROP2 emulsified either in Freunds incomplete adjuvants (FIA) or saponin, and control groups were treated with adjuvants alone (adjuvants control) or PBS (infection control). Subsequently, mice were challenged with 2x10(6)N. caninum tachyzoites. Nine mice, all belonging to the infection control or adjuvants control groups, exhibited clinical signs of cerebral neosporosis and succumbed to infection, whilst no clinical signs were noted for recNcROP2-vaccinated mice. For all other animals, the experiment was terminated 35 days p.i. Cerebral parasite burdens were assessed by quantitative PCR in all mice, and were revealed to be significantly reduced in the recNcROP2-vaccinated mice. ELISA of sera revealed IgG1 to be elevated in recNcROP2-saponin vaccinated mice, whilst IgG2a was higher in recNcROP2-FIA vaccinated animals. This shows that, depending on the adjuvants used, vaccination with NcROP2 induces a protective Th-1- or Th-2-biased immune response against experimental N. caninum infection.

Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro.

Ketamine, a phencyclidine derivative, is used for induction of anesthesia, as an anesthetic drug for short term surgical interventions and in subanesthetic doses for postoperative pain relief. Ketamine undergoes extensive hepatic first-pass metabolism. Enantioselective capillary electrophoresis with multiple isomer sulfated β-cyclodextrin as chiral selector was used to identify cytochrome P450 enzymes involved in hepatic ketamine and norketamine biotransformation in vitro. The N-demethylation of ketamine to norketamine and subsequently the biotransformation of norketamine to other metabolites were studied via analysis of alkaline extracts of in vitro incubations of racemic ketamine and racemic norketamine with nine recombinantly expressed human cytochrome P450 enzymes and human liver microsomes. Norketamine was formed by CYP3A4, CYP2C19, CYP2B6, CYP2A6, CYP2D6 and CYP2C9, whereas CYP2B6 and CYP2A6 were identified to be the only enzymes which enable the hydroxylation of norketamine. The latter two enzymes produced metabolic patterns similar to those found in incubations with human liver microsomes. The kinetic data of ketamine N-demethylation with CYP3A4 and CYP2B6 were best described with the Michaelis-Menten model and the Hill equation, respectively. This is the first study elucidating the individual enzymes responsible for hydroxylation of norketamine. The obtained data suggest that in vitro biotransformation of ketamine and norketamine is stereoselective.

Linear relationship between flux density and tumor co-promoting effect of prolonged magnetic field exposure in a breast cancer model

Previous epidemiologic studies have suggested that exposure to 50- or 60-Hz (power-frequency) magnetic fields in occupational or residential environments may increase the risk of certain cancers, including breast cancer. However, in view of the methodological problems of epidemiological studies on associations between magnetic field exposures and increased incidence of cancers, laboratory studies are necessary to determine if 50/60-Hz magnetic fields are cancer promoters or can progress cancers. The objective of the present study was to characterize the relation, if any, between dose (i.e. flux density) of 50-Hz magnetic field exposure and tumor growth in a model of breast cancer in female rats. Mammary tumors were induced by the chemical carcinogen 7, 12-dimethylbenz[a]anthracene (DMBA). The dosing protocol of DMBA chosen for the magnetic field experiments induced mammary tumors in about 50% of sham-exposed control animals within 3 months after application. Per flux density, a group of 36-99 rats was exposed to a magnetic field of 50-Hz for 24 h/day 7 days/week; another group of 36-99 rats was sham-exposed under the same environmental conditions as the MF-exposed rats. The exposure chambers were identical for MF-exposed and sham-exposed animals. DMBA was administered orally at a dose of 5 mg per rat at the first day of exposure and at weekly intervals thereafter up to a total dose of 20 g per rat. Duration of MF- or sham-exposure was 91 days. At the end of the exposure period, the animals were sacrificed for examination of the number of mammary tumors. Four flux densities were studied in a total of 666 rats (including sham-exposed controls): 0.3-1 muT, 10 muT, 50 muT, and 100 muT. At autopsy, i.e. at the end of the 13 weeks period of MF-exposure, incidence of macroscopically visible mammary tumors was significantly enhanced in the experiment with 50 muT (25.5% above control) and 100 muT (50% above control). No increase in incidence of mammary tumors was seen in the experiment with 0.3-1 muT, while a 10% (non-significant) increase was determined in the experiment with 10 muT. Linear regression analysis of the data from the four experiments indicated a highly significant linear relation between flux density and increase in tumor incidence at time of autopsy. The correlation coefficient was 0.9944 (P < 0.01). The data demonstrate that long-term exposure of DMBA-treated female rats increases the growth of mammary tumors in a highly dose-related fashion.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of a low dose infusion of racemic and S-ketamine on the nociceptive withdrawal reflex in standing ponies

OBJECTIVE To investigate the effect of plasma concentrations obtained by a low dose constant rate infusion (CRI) of racemic ketamine or S-ketamine on the nociceptive withdrawal reflex (NWR) in standing ponies. STUDY DESIGN Prospective, blinded, cross-over study. ANIMALS Six healthy 5-year-old Shetland ponies. METHODS Ponies received either 0.6 mg kg(-1) racemic ketamine (group RS) or 0.3 mg kg(-1) S-ketamine (group S) intravenously (IV), followed by a CRI of 20 microg kg(-1)minute(-1) racemic ketamine (group RS) or 10 microg kg(-1)minute(-1) S-ketamine (group S) for 59 minutes. The NWR was evoked by transcutaneous electrical stimulation of a peripheral nerve before drug administration, 15 and 45 minutes after the start of the bolus injection and 15 minutes after the end of the CRI. Electromyographic responses were recorded and analysed. Arterial blood was collected before stimulation and plasma concentrations of ketamine and norketamine were measured enantioselectively using capillary electrophoresis. Ponies were video recorded and monitored to assess drug effects on behaviour, heart rate (HR), mean arterial blood pressure (MAP) and respiratory rate. RESULTS The NWR was significantly depressed in group RS at plasma concentrations between 20 and 25 ng mL(-1) of each enantiomer. In group S, no significant NWR depression could be observed; plasma concentrations of S-ketamine (9-15 ng mL(-1)) were lower, compared to S-ketamine concentrations in group RS, although this difference was not statistically significant. Minor changes in behaviour, HR and MAP only occurred within the first 5-10 minutes after bolus drug administration in both groups. CONCLUSION Antinociceptive activity in standing ponies, demonstrated as a depression of the NWR, could only be detected after treatment with racemic ketamine. S-ketamine may have lacked this effect as a result of lower plasma concentrations, a more rapid metabolism or a lower potency of S-ketamine in Equidae so further investigation is necessary.

Complex effects of long-term 50 Hz magnetic field exposure in vivo on immune functions in female Sprague-Dawley rats depend on duration of exposure.

In previous studies we have demonstrated that 50 Hz, 100 microT magnetic field (MF) exposure of female Sprague-Dawley rats for 13 weeks significantly enhances the development and growth of mammary tumors in a breast cancer model. The present study was designed to test the hypothesis that, at least in part, the tumor (co)promoting effect of MF exposure is due to MF effects on the immune surveillance system, which is of critical importance in protecting an organism against the development and growth of tumors. For this purpose, female Sprague-Dawley rats of the same age as in the mammary tumor experiments were continuously exposed for different periods (2, 4, 8, and 13 weeks) to a 50 Hz, 100 microT MF. Control groups were sham-exposed simultaneously. Following the different exposure periods, splenic lymphocytes were cultured and the proliferative responses to the T-cell-selective mitogen concanavalin A (Con A) and the B-cell-selective pokeweed mitogen (PWM) were determined. Furthermore, the production of interleukin-1 (IL-1) was determined in the splenocyte cultures. The mitogenic responsiveness of T cells was markedly enhanced after 2 weeks of MF exposure, suggesting a co-mitogenic action of MF. A significant, but less marked increase in T-cell mitogenesis was seen after 4 weeks of MF exposure, whereas no difference from sham controls was determined after 8 weeks, indicating adaptation or tolerance to this effect of MF exposure. Following 13 weeks of MF exposure, a significant decrease in the mitogenic responsiveness of lymphocytes to Con A was obtained. This triphasic alteration in T-cell function (i.e., activation, tolerance, and suppression) during prolonged MF exposure resembles alterations observed during chronic administration of mild stressors, substantiating the hypothesis that cells respond to MF in the same way as they do to other environmental stresses. In contrast to T cells, the mitogenic responsiveness of B cells and IL-1 production of PWM-stimulated cells were not altered during MF exposure. The data demonstrate that MF in vivo exposure of female rats induces complex effects on the mitogenic responsiveness of T cells, which may lead to impaired immune surveillance after long-term exposure.

Enantioselective CE analysis of hepatic ketamine metabolism in different species in vitro.

Ketamine, an injectable anesthetic and analgesic consisting of a racemic mixture of S‐and R‐ketamine, is routinely used in veterinary and human medicine. Nevertheless, metabolism and pharmacokinetics of ketamine have not been characterized sufficiently in most animal species. An enantioselective CE assay for ketamine and its metabolites in microsomal preparations is described. Racemic ketamine was incubated with pooled microsomes from humans, horses and dogs over a 3 h time interval with frequent sample collection. CE data revealed that ketamine is metabolized enantioselectively to norketamine (NK), dehydronorketamine and three hydroxylated NK metabolites in all three species. The metabolic patterns formed differ in production rates of the metabolites and in stereoselectivity of the hydroxylated NK metabolites. In vitro pharmacokinetics of ketamine N‐demethylation were established by incubating ten different concentrations of racemic ketamine and the single enantiomers of ketamine for 8 min and data modeling was based on Michaelis–Menten kinetics. These data revealed a reduced intrinsic clearance of the S‐enantiomer in the racemic mixture compared with the single S‐enantiomer in human microsomes, no difference in equine microsomes and the opposite effect in canine microsomes. The findings indicate species differences with possible relevance for the use of single S‐ketamine versus racemic ketamine in the clinic.

In vitro evaluation of differences in phase 1 metabolism of ketamine and other analgesics among humans, horses, and dogs.

OBJECTIVE To investigate cytochrome P450 (CYP) enzymes involved in metabolism of racemic and S-ketamine in various species and to evaluate metabolic interactions of other analgesics with ketamine. SAMPLE POPULATION Human, equine, and canine liver microsomes. PROCEDURES An analgesic was concurrently incubated with luminogenic substrates specific for CYP 3A4 or CYP 2C9 and liver microsomes. The luminescence signal was detected and compared with the signal for negative control samples. Ketamine and norketamine enantiomers were determined by use of capillary electrophoresis. RESULTS A concentration-dependent decrease in luminescence signal was detected for ibuprofen and diclofenac in the assay for CYP 2C9 in human and equine liver microsomes but not in the assay for CYP 3A4 and methadone or xylazine in any of the species. Coincubation of methadone or xylazine with ketamine resulted in a decrease in norketamine formation in equine and canine liver microsomes but not in human liver microsomes. In all species, norketamine formation was not affected by ibuprofen, but diclofenac reduced norketamine formation in human liver microsomes. A higher rate of metabolism was detected for S-ketamine in equine liver microsomes, compared with the rate for the S-enantiomer in the racemic mixture when incubated with any of the analgesics investigated. CONCLUSIONS AND CLINICAL RELEVANCE Enzymes of the CYP 3A4 family and orthologs of CYP 2C9 were involved in ketamine metabolism in horses, dogs, and humans. Methadone and xylazine inhibited in vitro metabolism of ketamine. Therefore, higher concentrations and diminished clearance of ketamine may cause adverse effects when administered concurrently with other analgesics.

CE provides evidence of the stereoselective hydroxylation of norketamine in equines

CE with multiple isomer sulfated‐CD as selector was used for the simultaneous analysis of the stereoisomers of ketamine, norketamine, 5,6‐dehydronorketamine and hydroxylated metabolites of norketamine in liquid/liquid extracts of (i) in vitro incubations with ketamine or norketamine and equine liver microsomes and (ii) plasma and urine of ponies receiving a target‐controlled infusion of ketamine under isoflurane anesthesia. Hydroxynorketamine metabolites with the hydroxy group at the cyclohexanone ring could be shown to be formed stereoselectively both in vitro and in vivo. Due to the lack of standard compounds, urinary extracts were fractionated by HPLC followed by characterization of the collected fractions with CE and LC‐MSn with 0.7 mmu mass discrimination. Comparison of LC‐MSn data obtained with the fractions, an in vitro microsomal sample, and both pony urine and hydrolyzed pony urine led to the identification of four hydroxylated norketamine metabolites with hydroxylation at the cyclohexanone ring, two with hydroxylation at the aromatic ring and four hydroxylated metabolites of ketamine. Due to the lower detection sensitivity, only the four hydroxynorketamine metabolites with hydroxylation at the cyclohexanone ring were observed by CE. The data suggest that demethylation of ketamine followed by hydroxylation of norketamine at the cyclohexanone ring is the major metabolic pathway in equine species and that the ketamine metabolism is highly stereoselective.