Neurotoxicity screening of (illicit) drugs using novel methods for analysis of microelectrode array (MEA) recordings.

Annual prevalence of the use of common illicit drugs and new psychoactive substances (NPS) is high, despite the often limited knowledge on the health risks of these substances. Recently, cortical cultures grown on multi-well microelectrode arrays (mwMEAs) have been used for neurotoxicity screening of chemicals, pharmaceuticals, and toxins with a high sensitivity and specificity. However, the use of mwMEAs to investigate the effects of illicit drugs on neuronal activity is largely unexplored. We therefore first characterised the cortical cultures using immunocytochemistry and show the presence of astrocytes, glutamatergic and GABAergic neurons. Neuronal activity is concentration-dependently affected following exposure to six neurotransmitters (glutamate, GABA, serotonin, dopamine, acetylcholine and nicotine). Most neurotransmitters inhibit neuronal activity, although glutamate and acetylcholine transiently increase activity at specific concentrations. These transient effects are not detected when activity is determined during the entire 30min exposure window, potentially resulting in false-negative results. As expected, exposure to the GABAA-receptor antagonist bicuculline increases neuronal activity. Exposure to a positive allosteric modulator of the GABAA-receptor (diazepam) or to glutamate receptor antagonists (CNQX and MK-801) reduces neuronal activity. Further, we demonstrate that exposure to common drugs (3,4-methylenedioxymethamphetamine (MDMA) and amphetamine) and NPS (1-(3-chlorophenyl)piperazine (mCPP), 4-fluoroamphetamine (4-FA) and methoxetamine (MXE)) decreases neuronal activity. MXE most potently inhibits neuronal activity with an IC50 of 0.5µM, whereas 4-FA is least potent with an IC50 of 113µM. Our data demonstrate the importance of analysing neuronal activity within different time windows during exposure to prevent false-negative results. We also show that cortical cultures grown on mwMEAs can successfully be applied to investigate the effects of different (illicit) drugs on neuronal activity. Compared to investigating multiple single endpoints for neurotoxicity or neuromodulation, such as receptor activation or calcium channel function, mwMEAs can provide information on integrated aspects of drug-induced neurotoxicity more rapidly. Therefore, this approach could contribute to a faster insight in possible health risks and shorten the regulation process.

Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia.

Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event - leading to the preferential expression of VEGF-A165b over VEGF165a - prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy.

Left ventricular function in the post-infarct failing mouse heart by magnetic resonance imaging and conductance catheter: a comparative analysis.

AIM: Murine myocardial infarction (MI) models are increasingly used in heart failure studies. Magnetic resonance imaging (MRI) and pressure-volume loops by conductance catheter (CC) enable physiological phenotyping. We performed a comparative analysis of MRI vs. CC to assess left ventricular (LV) function in the failing mouse heart. METHODS: MI was created by LAD ligation. MRI (day 14) and CC (day 15) were used to determine LV end-diastolic volume (EDV), end-systolic volume (ESV) and ejection fraction (EF). RESULTS: Pooled data yielded moderate-to-strong linear correlations: EDV: R = 0.61; ESV: R = 0.72; EF: R = 0.81. We analysed three groups, no MI (sham, n = 10), small MI (<30% of LV, n = 14) and large MI (>30%, n = 20). Volumes and EF were consistently lower by CC than by MRI, but group differences were evident for both techniques. Receiver-operating characteristic analysis indicated good sensitivity and specificity for both techniques, with superior results for MRI. CONCLUSIONS: CC and MRI are highly valuable for evaluation of LV volume and function. MRI is recommended for longitudinal studies, accurate absolute volumes and anatomical information. Unique features of CC are its online signal with high temporal resolution, and advanced analysis of LV function and energetics.

Clinical validation of the "in silico" prediction of immunogenicity of a human recombinant therapeutic protein.

Antibodies elicited by protein therapeutics can cause serious side effects in humans. We studied immunogenicity of a recombinant fusion protein (FPX) consisting of two identical, biologically active, peptides attached to human Fc fragment. EpiMatrix, an in silico epitope-mapping tool, predicted promiscuous T-cell epitope(s) within the 14-amino-acid carboxy-terminal region of the peptide portion of FPX. On administration of FPX in 76 healthy human subjects, 37% developed antibodies after a single injection. A memory T-cell response against the above carboxy-terminus of the peptide was observed in antibody-positive but not in antibody-negative subjects. Promiscuity of the predicted T-cell epitope(s) was confirmed by representation of all common HLA alleles in antibody-positive subjects. As predicted by EpiMatrix, HLA haplotype DRB1*0701/1501 was associated with the highest T-cell and antibody response. In conclusion, in silico prediction can be successfully used to identify Class II restricted T-cell epitopes within therapeutic proteins and predict immunogenicity thereof in humans.

Malformations in offspring of diabetic rats: morphometric analysis of neural crest-derived organs and effects of maternal vitamin E treatment.

BACKGROUND: We have previously reported on a malformation-prone Sprague-Dawley rat substrain (U), which presents a high frequency of micrognathia in the offspring of diabetic mothers. This malformation is related to impaired development of the cranial neural crest cells (NCC); the defect may be prevented by antioxidative treatment of the mother. METHODS: We have therefore investigated whether fetuses of diabetic rats display other malformations associated with altered cranial NCC development and whether maternal vitamin E supplementation may affect such malformations. RESULTS: Fetuses of diabetic rats showed low-set external ears, severely malformed Meckel's cartilage, small thyroid and thymus, and absence of parathyroid glands. Cardiac anomalies were frequently observed, including rightward displacement of the aorta, double outlet right ventricle (DORV), persistent truncus arteriosus (PTA) combined with ventricular septal defects due to a malaligned outlet septum. The malformations in the outflow tract included abnormalities of the great arteries; right-sided aortic arch/descending aorta, and double aortic arches. These defects tended to occur together within individual fetuses. Maternal dietary treatment with 2% vitamin E markedly reduced the severity of the malformations. CONCLUSIONS: The phenotypic appearance of these defects is strikingly similar to the DiGeorge anomaly in humans, which has been found in children of diabetic mothers together with an overrepresentation of PTA and DORV. The malformations associated with defective NCC development in the offspring of diabetic U rats show several morphological similarities to those in humans; hence the teratogenic mechanisms may be similar and accessible for study.

Rearrangement of O-cinnamoyltaxicin I to a novel C-13 spiro-taxane.

During the large-scale synthesis of an O-cinnamoyltaxicin I acetonide, an intermediate for the semisynthesis of 7-deoxypaclitaxel derivatives, side-product 3 was formed via a vinylogous retro-aldol reaction and a long-range hydride shift from O-cinnamoyltaxicin I (1) under alkaline reaction conditions. Compound 3 has two hemi-acetal bridges at C-1,C-9 and C-10,C-13. Compound 4 was formed from side-product 3 under acidic reaction conditions and is the first C-13 spiro-taxane described in the literature. This spiro-taxane has two acetal bridges between C-1, C-13 and C-10,C-13.

8-Hydroxydeoxyguanosine in DNA from leukocytes of healthy adults: relationship with cigarette smoking, environmental tobacco smoke, alcohol and coffee consumption.

8-Hydroxydeoxyguanosine (8-OHdG) has been widely used as a biomarker of oxidative DNA damage in both animal and human studies. However, controversial data exist on the relationship between 8-OHdG formation and age, sex and tobacco smoking in humans, while few or no data are available on other exposures such as environmental tobacco smoke, alcohol, coffee and tea consumption. We investigated the level of 8-OHdG in DNA from peripheral leukocytes among 102 healthy adults living in Brescia province, North Italy, aged 25-45 (mean: 35.2 years), of which 51 were males. 8-OHdG levels expressed as a ratio to total deoxyguanosine (8-OHdG/106 dG) in DNA showed wide interindividual variation, the highest value (63.8) being 6. 2-fold greater than the lowest (10.3). Current smokers showed lower mean 8-OHdG values than subjects who never smoked (29.3 and 34.0, respectively, p<0.05), and an inverse relationship was found between 8-OHdG and lifetime smoking, which was independent of age, sex and body mass index. An inverse relationship was also found with coffee drinking while no association was observed with alcohol and tea consumption, exposure to environmental tobacco smoke and use of vitamins in all subjects, and with use of oral contraceptives in females. The inverse relationship between smoking status and 8-OHdG levels could be explained by the presence of efficient repair processes for the oxidative damage induced by smoking. In this study, the smokers were relatively young (77% were less than 40 years) and only 7% smoked 30 or more cigarettes a day. In conclusion, it would appear that 8-OHdG levels in leukocytes may not provide a sensitive marker of exposure to tobacco smoking.

Survey of the management of third stage of labour in The Netherlands.

UNLABELLED: The standard practice during the third stage of labour of Dutch midwives and obstetricians was elucidated by a questionnaire mailed to all Dutch midwives and obstetricians. Prophylactic oxytocics in the third stage are used as a routine by 55% of the obstetricians and only 10% of the midwives. Oxytocin is the drug of first choice. CONCLUSION: Routine use of prophylactic oxytocics in the third stage is not the standard practice in the Netherlands. Obstetricians are much more likely to use prophylaxis than midwives.

Effects of a dietary load of acid or base on changes induced by lactose in rats.

Feeding lactose or other slowly digestible carbohydrates to adult mammals may induce a variety of effects including hyperplasia and neoplasia. The most fundamental effect probably is the increased production in the large intestine of short-chain fatty acids (SCFA) resulting from increased fermentation of carbohydrate residues. To find out whether the increased production of these acidic compounds is involved in the induction of certain alterations caused by low-digestibility carbohydrates, the modifying effects of an acidifying (NH4Cl) or an alkalizing (KHCO3) diet supplement on lactose-induced changes in rats were studied. Three groups of 50 rats per sex were fed a 20% lactose diet unsupplemented or supplemented with 1% NH4Cl or 2% KHCO3, for at most 2.5 yr. One control group was fed the basal diet which contained wheat starch instead of lactose. Feeding lactose resulted in wet faecal pellets, reduced pH of the faeces, higher intake of food and water, lower body weights, increased caecal weights and fewer deaths. These effects were not significantly modified by NH4Cl or KHCO3. Feeding lactose increased urinary calcium levels, the effect being enhanced by NH4Cl and reduced by KHCO3. Lactose also tended to increase blood values of alkaline phosphatase and to decrease those for bicarbonate and base excess. These tendencies were generally more marked with NH4Cl, and less marked or absent with KHCO3. In addition, rats fed lactose showed decreased severity of nephrosis, increased mineralization and hyperplasia of the renal pelvic epithelium, and relatively high incidences of Leydig cell hyperplasia and neoplasia. NH4Cl supplementation was associated with a relatively small number of single and multiple tumours, with decreased incidences of hyperplasia and mineralization of the renal pelvis epithelium and with a markedly reduced incidence of proliferative changes in the adrenal medulla. With the KHCO3 supplement the incidences of Leydig cell proliferation and of bladder tumours were relatively high. These findings, in particular the differences between the diet groups in urinary calcium levels and possibly also the variations in blood levels of alkaline phosphatase, bicarbonate and base excess, suggest that the acidic end products of carbohydrate fermentation (SCFA) act as an acid load on the body.

[Allergic contact eczema due to 'tea tree' oil]. / Allergisch contacteczeem voor 'tea tree'-olie.

In four patients, three women aged 45, 29 and 52 years and a man aged 45 years, allergic contact dermatitis due to 'tea tree' oil was diagnosed. The case of the man was published before. 'Tea tree' oils are essential oils distilled from the leaves of myrtaceous trees and shrubs occurring in Australia and South-East Asia. The 'tea tree' oil available in the Netherlands is distilled from the Melaleuca alternifolia and mainly contains eucalyptol. Eucalyptol is probably the most important allergen.

Comparison of cereal-based diet with purified diet by short-term feeding studies in rats, mice and hamsters, with emphasis on toxicity characteristics.

Animal diets used in toxicity studies are prepared either from natural ingredients (cereal-based diet) or from more refined products (purified diet). The type of diet may influence both the outcome of the study and the values obtained with the various parameters in test and control animals. To detect the parameters sensitive to changes in diet composition, short-term (4-wk) studies were conducted in rats, mice and hamsters fed either a cereal-based diet or the AIN-76A purified diet supplemented with vitamins and minerals at the highest recommended levels for each of the species used. Although the purified diet was more palatable to rats and showed a higher protein quality, growth rate and food intake were generally slightly higher with the cereal-based diet in each of the species examined. The haematological values of the two diet groups were generally comparable. On the cereal-based diet the production of faeces was considerably higher than on the purified diet and was accompanied by a higher weight of the caecum. These findings were attributed to the relatively high level and mixed composition of the fibre fraction in the cereal-based diet. Blood levels of cholesterol and phospholipids were clearly lower on the cereal-based diet than on the purified diet. Because the differences were probably due to the level and composition of the fibre fraction, they support the suggestion to replace the 5% cellulose of the AIN-76A diet by a higher level of a more composite but well defined source of dietary fibre.

Effects of high levels of brussels sprouts in the diet of rats.

To examine the possible harmful effects of feeding Brussels sprouts to rats, groups of 10 male, weanling rats received the non-dehydrated vegetable in moist diets at levels providing 2.5-30% of the dry matter for 4 wk. A first study comprised test diets with 15 and 30% of the dry matter as uncooked or cooked Brussels sprouts and control diets without and with 0.2% potassium thiocyanate (KSCN) for comparison. The second study comprised diets with 0, 2.5, 5, 10 and 20% of the cooked vegetable and diets with 0 and 20% of the cooked vegetable with extra iodine. Diets with the uncooked vegetable contained considerably less intact glucosinolates than did diets with the cooked product, probably as a result of more extensive enzymatic degradation in the uncooked product. Growth depression and decreased food intake, not accompanied by decreased food efficiency, occurred in rats fed 10% or more dry matter as Brussels sprouts. These findings were less marked with the cooked than with the uncooked vegetable, probably because of unpalatability. Decreased levels of blood haemoglobin and plasma thyroxin were found with 15% or more Brussels sprouts. Prothrombin times were increased if 2.5% or more was fed. Thyroid stimulating hormone was increased by feeding potassium thiocyanate, but not by feeding the vegetable. Increased kidney weights and impaired kidney function not accompanied by microscopic renal changes were observed in rats fed 10% or more Brussels sprouts. Increased liver weights, which occurred from the 5% level, were accompanied by microscopic hepatic changes only at feeding levels from 10% of the cooked vegetable. 'Morphological activation' of the thyroid was increased with 10% or more of the cooked vegetable and with 0.2% KSCN. Iodine supplementation of the diets did not influence the results obtained with the vegetable. These studies indicated that 2.5% Brussels sprouts dry matter in the diet was not without effect, and that the thyroid characteristics were less sensitive to Brussels sprouts than were other criteria examined.

Chronic toxicity and carcinogenicity study of isomalt in rats and mice.

The chronic toxicity and possible carcinogenicity of the sugar replacer isomalt was studied in Wistar rats and Swiss mice. Groups of 50 animals of each sex were fed 0, 2.5, 5 or 10% isomalt in the diet for nearly 2.5 yr (rats) or 2 yr (mice). Control groups received either basal diet with 10% maize starch or basal diet with 10% sucrose. Additional groups of ten rats/sex were fed the same diets and were killed after 1 yr. Isomalt and sucrose were included in the diet at the expense of maize starch. Administration of isomalt was started, in rats, in utero, and in mice, at weaning age. Feeding isomalt did not affect the appearance or behaviour of rats or mice, nor did it cause diarrhoea. Mortality rate was unaffected. Body weights of rats and mice fed 10% isomalt were generally slightly lower than those of controls. Periodic examinations of rats for haematological criteria, clinical chemistry of the blood, urine composition and kidney function did not reveal any changes of toxicological significance. Periodic haematological examinations of mice were likewise negative. Caecal enlargement was observed in rats and mice of the high-dose group, but the microscopic structure of the caecal wall was unaffected. An increased number of treated male and female rats showed hyperplasia of the urothelium in the renal pelvis accompanied by mineralization, whereas the number of females showing corticomedullary mineralization was decreased in the treated groups. The incidence, type or location of neoplasia provided no evidence of a carcinogenic potential of isomalt. Feeding 10% sucrose did not induce significant differences compared with the controls fed 10% maize starch, whereas isomalt at levels of up to 10% produced some of the changes that are common to rats fed high levels of poorly digestible carbohydrates.

Induction of hyperplasia in the bladder epithelium of rats by a dietary excess of acid or base: implications for toxicity/carcinogenicity testing.

In previous studies we observed an increased incidence of hyperplasia in the epithelium of the urinary bladder of rats fed cereal-based stock diet supplemented with 6% monosodium glutamate (MSG) for 3 months. Hyperplasia was not enhanced, however, when 6% MSG was fed in a purified casein diet. Further studies have been conducted to identify the dietary factor that caused the different response with the two diets. Feeding MSG had a marked alkalizing effect on the urine. Rats fed purified diet produced urine of higher acidity than did those fed stock diet, a finding attributed to the greater excess of base in the stock diet. When diets with a considerable excess of cations were fed, urinary pH showed a characteristic pattern of widely differing values during a 24-hr period, with high values (pH greater than or equal to 8] for several hours of darkness, when food intake was high, declining during the day to a minimum at the end of the light period. Hyperplasia of the bladder epithelium was induced not only by feeding MSG, but also by feeding 5% of the alkalizing salt KHCO3, both in purified diet and in stock diet. The epithelial response to an alkalizing substance was prevented by simultaneous feeding of the acidifying salt NH4Cl. These findings indicate that the bladder changes induced by MSG are attributable to its alkalizing properties rather than to MSG per se. Moderate to severe hyperplasia of the bladder epithelium was induced also by feeding 5% NH4Cl in purified diet, a procedure accompanied by a further lowering of urinary pH. These findings showed that hyperplasia of the bladder epithelium of rats can be induced both by acidifying and by alkalizing the urine through manipulation of the acid-base balance of the basal diet. There is thus a possibility that, in carcinogenicity studies, administration of compounds to rats in the form of a salt may lead to erroneous conclusions.

Some effects of injected hyoscine butylbromide: a versatile class experiment in human pharmacology.

1 An experiment with hyoscine butylbromide has been used to demonstrate how drug effects differ in intensity and time course after intravenous (20 mg) and subcutaneous (40 mg) injection with and without massage of the injection site. It also demonstrates how objective and subjective observations may be related, and gives students practice in the interpretation of experimental data. 2 The experiment takes about 2 h, and the effects of the drug have worn off completely after about 3 hours. 3 The structure of the discussion of the experiment and its results with the students are described to draw attention to the questions that can be asked of the data. The results obtained with 41 subjects are summarized to illustrate this discussion. 4 Various ways of modifying the experiment to suit other teaching objectives are mentioned. The need to consult the local Ethics Committee before undertaking experiments on students is emphasized.