Few of the more than 65,000 chemicals listed in the Environmental Protection Agency (EPA) inventory have been tested for neurotoxicity. The nervous system may be especially vulnerable to toxicants because many compounds can cross the blood-brain barrier and induce irreversible damage. Additionally, the young, the elderly, and other sensitive populations may be particularly susceptible to neurotoxic injury. The EPA has developed guidelines including neurobehavioral, neuropathological, and neurochemical tests for the identification of possible neurotoxicants. In the present review, tests included in the current EPA guidelines for neurotoxicity testing are described and evaluated. The main benefit of the tests is that regulators are familiar with them, thus facilitating interpretation. Additionally, validation data on these tests are available for many known neurotoxicants. These factors make it difficult to introduce new methods that may include in vitro and other techniques. The current in vivo tests can be costly and prolonged and can involve the use of many laboratory animals, making them inappropriate for generalized use on existing chemicals. It is suggested that alternative tests be incorporated for screening of large numbers of chemicals and that testing priority be given to chemicals on the basis of structure/ activity relationships, lipophilicity, bioaccumulation, and extent of exposure.
The experimental techniques used for the evaluation of the gastrointestinal side-effects of drugs in animals are discussed. Special emphasis is given to the estimation of fecal blood loss by means of 51Cr-labeled erythrocytes which permits the accurate quantitative determination of the total drug-induced GI damage.
All currently known antiarrhythmic agents can induce or worsen arrhythmias. Inappropriate dosage selection, mistakenly based on pharmacokinetic data from “normal” subjects, may result in adverse reactions when an antiarrhythmic drug is given to patients. Unexpected variations in drug clearance can increase plasma concentration of antiarrhythmic agents and aggravate arrhythmias. Changes in the rate of drug metabolism by the liver, e.g., due to cessation of alcohol or drugs that induce hepatic metabolism, can reduce drug clearance, making a previously well-tolerated dose toxic. Another possible explanation for adverse drug reactions is nonlinear protein binding. Recently, genetic determinants of drug metabolism have been identified as explanations of interindividual variations in drug responsiveness. Finally, the interactions of antiarrhythmic agents may also lead to aggravation of arrhythmias. A better understanding of the pharmacology of antiarrhythmic agents can reduce, if not prevent, the occurrence of potentially lethal proarrhythmic events.
Rat liver microsomes have been immobilized in a membrane by gelatin entrapment. The resulting membranes can be attached to an oxygen electrode to provide a sensor for several compounds. NADPH and NADH are determined by utilizing the liver microsomal NADPH oxidase activity, which is, at least in part, due to the cytochrome P-450 system. The calibration graph for NADPH is linear up to 1 mM. A multi-enzyme system localized in liver microsomes allows the determination of 20–800 μM glucose-6-phosphate. The non-enzymatic lipid peroxidation in liver microsomes, which is induced by ascorbate, allows 0.5–2.5 mM ascorbate to be determined.
In order to understand some of the haemostatic mechanisms in rats for the interpretation of toxicological data, basic haemostatic parameters with a special emphasis on platelet functions were first measured in vitro. The results of reactions of rat platelets to many aggregating agents suggest that only ADP may be a consistently significant aggregator. The search for physiologic aggregators revealed ADP to be available from erythrocytes. Adhesion reaction also required ADP. Collagen was not considered to be essential for either reaction. Aggregation and adhesion were probably both reversible in flowing blood, while irreversible thrombi were formed in blood at rest ex vivo. Blood coagulation parameters determined revealed that the intrinsic pathway may be more important than the extrinsic one. The rate of intrinsic coagulation reaction was rapid, and plasma coagulation appeared to be of primary importance while the influence of platelet aggregation was minor. A simple model of rat haemostatic mechanism is proposed based on these results. Additionally, to define the relative contribution of platelets versus other cellular and plasma coagulation in vivo, rats were administered antiplatelet drugs (ticlopidine, suprofen and clopidogrel) and an anticoagulant (warfarin) intraperitoneally. Bleeding times (BTs) were significantly increased in all treated groups. ADP-induced platelet aggregations were significantly depressed by the administration of the three antiplatelet drugs, while kaolon-activated partial thromboplastin time and prothrombin time were greatly increased in the warfarin-treated rats. The increase in BT may be due to the inhibition of platelet activity or blood coagulation defect in rats given antiplatelet drugs or warfarin, respectively. These results suggest that platelets play a key role in haemostasis in the rat. Two possible explanations of the disparity between in vitro and in vivo results may be that functional tests used here are not adequate to cover the properties of rat platelets or that mechanisms leading to the formation of platelet thrombi in rats are ADP-dependent adhesion and ADP-induced aggregation.
Nonsteroidal anti-inflammatory drug (NSAID)-induced apoptosis is considered to be an important mechanism in the antineoplastic effects and damage produced by the drugs in the gastrointestinal tract. In this study, two different gastric cancer cell lines, MKN28 (mutant-type p53) and AGS (wild-type p53), were compared as to growth inhibition, apoptosis, and cell cycle and apoptosis-related gene expression in response to indomethacin treatment. Cell growth was measured by MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Apoptosis was characterized by acridine orange staining and DNA fragmentation, and cell cycle kinetics by flow cytometry. The mRNA and protein levels of p53, p21waf1/cip1, and c-myc were determined by Northern and Western blotting. The results showed that indomethacin initiated growth inhibition and apoptosis in both cell lines without cell cycle shifting. AGS cells were more sensitive to growth inhibitory activity and apoptosis of indomethacin than MKN28 cells. In MKN28 cells, the levels of p53, p21waf1/cip1, and c-myc mRNA remained unchanged over the 24-hr treatment with indomethacin, but the p53 protein level was elevated after 4 hr. There was no change in the p21waf1/cip1 and c-myc protein levels in the MKN28 cells. In AGS cells, a progressive increase in c-myc mRNA and protein levels was noted, while p53 and p21waf1/cip1 remained unchanged. It can be concluded that wild-type p53 and/or up-regulation of c-myc is associated with indomethacin-mediated differential apoptosis in gastric epithelial cells.
The literature reviewing data on the relationship between anticonvulsant drugs and cognitive abilities has been reviewed. The number of studies that have been completed is small, and many of the drugs used in the management of epileptic patients have not been assessed at all. It is concluded from the available literature that phenytoin, phenobarbitone, and primidone all effect cognitive abilities. Phenytoin and possibly primidone seem to be involved in the production of insidious deterioration of the mental state characterised by a progressive fall in the IQ, sometimes resulting in a picture of encephalopathy. The possible mechanisms of the production of these abnormalities has been discussed, in particular the relationship to folic acid and monoamine metabolism.
Opening of plasmalemmal K+ channels leads to cellular hyper-polarization which, in excitable tissues possessing voltage-dependent Ca2+ channels, prevents the opening of such channels and thus prevents excitation. In the last few years an increasing number of compounds have been identified which elicit their effects by opening K+ channels, preferentially in smooth muscle, but also in other excitable tissues. These include the novel benzopyrans cromakalim and SDZ PCO 400 and also well known antihypertensives like minoxidil sulphate, diazoxide and pinacidil. After a short overview of the various families of K+ channel openers (KCOs), their basic pharmacological properties including inhibition by the sulfonylureas (like glibenclamide) will be presented. The therapeutic potential of these compounds in the cardiovascular field (as antihypertensives and, in particular, as antiischemic agents in heart and skeletal muscle), and in asthma (where they reverse established airway hyperreactivity) will also be discussed.Despite recent progress in our understanding of the mechanism of action of the KCOs, major questions remain open, e.g. concerning the type of K+ channel(s) opened by these compounds and the mechanism(s) of vasorelaxation. An improved understanding of the action of these compounds at the molecular level is instrumental for the design of novel and more specific KCOs.
During the years 1974 and 1975 only 20 per cent of published reports on the effects of drugs on behavioral responses utilized mammals other than rats and mice Of these, 36 per cent were cats, 12 per cent rhesus monkeys, 6 per cent dogs, 5 per cent squirrel monkeys and 4 per cent other species of subhuman primates Drug dlsposmon and metabohsm has not been as extensively studied in the cat as in subhuman primates and dogs, although data are available on the in v lvo metabohsm of such neurologically active compounds as dlphenylhydantom, ketamme, papaverme, tryptophan and some of the benzodlazeplnes. Differences in response to drugs can arise from persistence of the active drug m the ammal, or from formation of unique metabohtes which may be pharmacologically active. They can also arise from deficiencies in or different act~wttes of enzymes concerned m their metabohsm, and they can be due to specific effects in a mammahan species Comparisons between reported blologlcal half-hves (tv2's) of drugs m plasma m various specms of animals zs often ddlicult on account of varying routes of admm|strat|on and dosages used. Mellett 0969) showed an mterspecies relat|onsh|p for cyclophospham|de occurring when the dosage x tm v0as plotted against area under the curve on a log scale. Certain d|fferences m neurological and behavioral effect have been shown to occur I/I ammal specles as a result of drfferences in metabolism..
The deficient secretory phenotype of Chinese hamster ovary (CHO) cells is a major limitation for high-level production of biopharmaceuticals, particularly for those with complex molecular architectures and post-translational modifications. To improve CHO cell secretory capacity, we recently engineered CHO cell hosts to overexpress BLIMP1 (CHOB), in a cell engineering strategy that transformed the cellular machinery and led to significantly higher product yields and cell-specific productivities for different rproteins. Here, as a follow-up to our previous study, we developed new CHO cell hosts that co-overexpress BLIMP1 and XBP1s ( CHOBX ), two transcription factors that together drive the professional secretory function of antibody-producing plasma cells. We found that the CHOBX cells presented an improved performance over that of CHOB cells, with better product yields and cell-specific productivities for a recombinant IgG1 and a ‘difficult-to-express’ EPO-Fc fusion protein. These improvements in the CHOBX-derived cell lines resulted from a series of physiological and metabolic changes due to the synergetic co-expression of BLIMP1 and XBP1s. Firstly, cells presented an inhibited cell growth and arrested cell cycle in G1/G0 phase, features that were directly linked to BLIMP1 expression levels. Secondly, cells increased protein translation (both overall and recombinant protein), expanded the endoplasmic reticulum and improved their capacity to secrete protein more effectively. Lastly, cells showed a metabolic profile favouring energy production, with a pronounced lactate switch and increased consumption of amino acids. This study highlights the value of transcription factors for reprogramming CHO cells towards a desired phenotype, offering the potential to engineer cells with new functionalities for enhanced manufacturing of recombinant therapeutic proteins.
Antipsychotic drugs (APDs) have a variety of important therapeutic applications for neuropsychiatric disorders. However, they are routinely prescribed off-label across all age categories, a controversial practice given their potential for producing metabolic and extrapyramidal side effects. Evidence also suggests that chronic treatment with some APDs may lead to impairments in cognition and decreases in brain volume, although these findings are controversial. The purpose of the studies described here was to evaluate one of the most commonly prescribed APDs, quetiapine, for chronic effects on recognition memory, brain-derived neurotrophic factor (BDNF), its precursor proBDNF, as well as relevant downstream signaling molecules that are known to influence neuronal plasticity and cognition. Multiple cohorts of adult rats were treated with quetiapine (25.0 mg/kg/day) for 30 or 90 days in their drinking water then evaluated for drug effects on motor function in a catalepsy assessment, recognition memory in a spontaneous novel object recognition (NOR) task, and BDNF-related signaling molecules in the post mortem hippocampus via Western Blot. The results indicated that oral quetiapine at a dose that did not induce catalepsy, led to time-dependent impairments in NOR performance, increases in the proBDNF/BDNF ratio, and decreases in Akt and CREB phosphorylation in the hippocampus. These results indicate that chronic treatment with quetiapine has the potential to adversely affect recognition memory and neurotrophin-related signaling molecules that support synaptic plasticity and cognitive function. Given the widespread use this APD across multiple conditions and patient populations, such long-term effects observed in animals should be considered.
