Effects of interferons and hydrogen peroxide on CA3 pyramidal cells in rat hippocampal slice cultures.

In viral diseases of the CNS, both interferon-alpha/beta and interferon-gamma are produced intrathecally. At least some of the neurological symptoms associated with these diseases may be due to the effects of these cytokines. We have studied the actions of interferons on CA3 pyramidal cells in hippocampal slice cultures. Bath application of interferon-alpha/beta and interferon-gamma produced an excitatory effect on CA3 pyramidal cells and a decrease in evoked inhibitory postsynaptic potential amplitude, eventually leading to epileptiform bursting. These effects were slow in onset (several minutes), suggesting an indirect mechanism of action. Several lines of evidence suggest that the actions of interferons on pyramidal cells may at least in part be mediated by reactive oxygen intermediates, known to be released from non-neuronal cells: the effects of interferon on CA3 pyramidal cells were blocked by the free radical scavengers catalase and superoxide dismutase. Hydrogen peroxide reduced evoked inhibitory synaptic transmission, eventually leading to epileptiform bursting, thus mimicking several of the effects of interferons on pyramidal cells.

The concept of multispecies testing in industrial toxicology.

The multispecies approach in toxicity testing was originally motivated by the frequent findings of species differences in responsiveness to the pharmacological and acute toxic effects of chemicals. When the guidelines for repeated-dose toxicity experiments were developed in the early 1940s, the concept of using several species of animals was automatically included without careful scientific validation. In response to demands from the U.S. Food and Drug Administration and other national and international regulatory bodies, the protocols for acute and repeated-dose toxicity testing became highly formalized, and the requirement to conduct all studies in a rodent and a nonrodent species was established. With time, most guidelines also specified the species that had to be used, and the most common recommendation was to use the rat as the rodent and the dog as the nonrodent. In recent years, many reasons for differences in responsiveness of various animal species and man to the toxic effects of chemicals have been identified. This knowledge is now used extensively to assess contradictory findings in routine safety studies in the rodent and the nonrodent species. It is often possible to identify the species that appears to be more predictive for man than the other, and risk assessment for man is then based mainly on the findings in the more representative species. Since contrary toxicological findings in rodents and nonrodents occur frequently, one could propose to perform detailed scientific investigations prior to the selection of the species for toxicological investigations, as it has already been suggested by the working group of the European Society for the Study of Drug Toxicology in 1965 and is still an option mentioned in the currently valid EEC drug safety guidelines of 1983. For practical reasons such early investigations of biological properties and pharmacokinetic and metabolic characteristics of test chemicals in various laboratory animal species are hardly ever done prior to the initiation of safety studies. However, techniques are now developed with which species selection for toxicity testing can be made on the basis of rational scientific investigations.

Cardiotoxicity of vasodilators and positive inotropic/vasodilating drugs in dogs: an overview.

Standard toxicological studies in dogs using high doses of vasodilators and positive inotropic/vasodilating agents give rise to a species-specific cardiotoxicity. The reason may be the extreme sensitivity of the dog to the pharmacological effects of these drugs; exaggerated pharmacodynamic effects and prolonged disturbance of homeostasis mechanisms often are responsible for the observed organ lesions. An assessment of the toxicological relevance and the risk for patients taking the drugs at therapeutic doses cannot be made without taking into account their pathomechanisms and the pathophysiological basis of the exceptional reaction patterns occurring in dogs. A large series of vasodilating and positive inotropic agents are presented, their pharmacological properties are described, and toxicological effects in dogs are compared. In view of the poor correlation between the distinct cardiac lesions induced in dogs and a lack of comparable toxicity in humans, it appears desirable to reassess the adequacy of the standard toxicological approaches for these substances.

Decrease of epinephrine-induced arrhythmia threshold in ethanol exposed rats.

An association between cardiac arrhythmias and ethanol use has been observed for some time. The sympathetic nervous system presumably plays an important role in the manifestation of cardiovascular ethanol responses. Therefore, we investigated the effects of ethanol treatment on epinephrine-induced arrhythmias. Female Wistar rats received 10 vol% ethanol or 2.5% glucose (control group) in their drinking water for 45 days. In ether anesthetized animals of both groups epinephrine (10 micrograms/kg.min) was infused via a lateral tail vein. The threshold dose for arrhythmias after epinephrine infusion (mainly 2nd and 3rd degree AV-blocks) was reduced beginning 2 days after the start of the ethanol treatment and the incidence of AV-blocks during epinephrine infusion was increased. During the ethanol treatment the prohypertensive epinephrine effect was slightly increased. The reflex bradycardia was not changed after repeated epinephrine infusion by ethanol treatment, whereas it was nearly abolished in the control group. No blood ethanol could be detected during the time of epinephrine infusion (9-12 a.m.), but determinations at 11 p.m. yielded a concentration of 0.13 +/- 0.02 mg/g. The results show that the epinephrine-induced bradyarrhythmia threshold is reduced and the frequency of arrhythmic events is augmented in rats exposed to ethanol in the drinking fluid.

Neurotoxic effects of bismuth in vitro.

Although insoluble bismuth (Bi) salts are known to be neurotoxic, recently interest in oral Bi therapy has been renewed because of encouraging results obtained in the treatment of gastritis and peptic ulcer associated with Helicobacter pylori infection. For risk assessment of orally administered Bi preparations it is important to determine the minimal neurotoxic Bi concentration in the brain. This concentration was determined in cultures of brain, meninges and neuronal retina cells from embryonic chicks and in cultures of hippocampal slices from rats. Cytotoxicity, as assessed by neutral red uptake, thiazolylblue tetrazoliumbromide dehydrogenase activity and morphological criteria, occurred at Bi concentrations (about 10 mum) comparable with those that have been observed in humans and mice with Bi-induced encephalopathy. Regional differences of Bi effects on astrocytes, assessed by expression of glial fibrillary acidic protein, were observed in cell cultures of the embryonic chick brain. Measurement of expression of microtubular-associated protein type 2 indicated that astrocytes were much more sensitive to Bi than were nerve cells. Therefore it seems that astrocytes are an important target of Bi toxicity, and this would explain the reversibility of signs and symptoms of Bi encephalopathy on cessation of therapy. Acute exposure of cultured rat brain slices to Bi had no effect on the bioelectric activity of hippocampal pyramidal cells, whereas chronic exposure produced neuronal degeneration.

Predictive value of animal studies in toxicology.

The three main purposes of experimental toxicology are (1) determination of the toxicological spectrum in selected laboratory animal species; (2) extrapolation to other species and prediction of adverse effects in man; and (3) determination of safe levels of exposure. Toxicology has reached a satisfactory level of performance in identifying toxicity in animals, and experimental techniques are now available to characterize the toxicological potential of chemical substances in great detail. In many instances, extrapolation of toxicological findings in animals to man is possible. It must also be admitted that toxicological studies have, at times, failed to predict human toxicity. In many cases, this can be explained by biological differences between animals and man. A particularly difficult problem is the "low incidence responses" that occur only in especially sensitive individuals. They represent a challenge that can be met only be a determined research effort. Failure to predict toxic responses in man is sometimes also brought about by the toxicologists' own faults. It is essential, therefore, that we analyze the errors committed in the past and continuously evaluate and improve our performance. Society's demand for specific safe levels of exposure is difficult to meet. After a long period of confusion and confrontation, toxicology appears to be on the road to a truly science-based methodology for risk assessment.

Acute toxicity testing in the nonlethal dose range: a new approach.

A new modification of acute toxicity testing of chemicals in rats is presented. Instead of using death as the principal criterion of toxicity, it is based on a careful, standardized clinical assessment of toxic signs measured in the nonlethal dose range. Test compounds are administered to groups of rats at four dose levels, selected on the basis of pilot experiments. General indices of toxicity, i.e., body weight gain, food and water consumption, and body temperature, are recorded at regular intervals; activity in the home cage is monitored continuously with a newly developed passive infrared device; neurobehavioral dysfunctions are assessed repeatedly with a checklist; and routine hematology is done on the 4th day after administration of the test compounds. All measured signs of toxicity are scored in relation to the control groups so that the absolute magnitude of the score increases as a function of the deviation from the normal conditions of the animals. In order to take into consideration the course of intoxication and the rate of recovery, toxicity scores are added over the duration of the whole experiment. For each variable measured, the resulting total scores are converted into a relative point system, scaling from 0 to 10 points. These points are added to a single "total ToxScore" value for each dose group. A regression line is calculated for these total ToxScore points, and the dose giving 15 total ToxScore points is determined. Based on the results obtained with nine reference substances, a classification system is proposed that is comparable to that based on LD50 values. It is concluded that the proposed test procedure provides much more information on the signs of acute toxicity, the course of the intoxication, the slope of the dose-effect curve, and the rate of recovery than does the standard LD50 test. In addition, the degree of distress and suffering of the animals is reduced.

[Alternative methods, the present and future]. / Les méthodes alternatives, présent et avenir.

Alternative methods are already widely used in fundamental biological research. Among 6,649 papers given at the FASEB 1989 meeting, nearly 60 p. 100 deal with experimental data obtained without the use of live animals. Alternative methods are less frequently utilized in industrial Toxicology. The reason of this discrepancy is discussed as well as the efforts made to reduce the numbers of animals for acute toxicity studies (LD 50) and cutaneous and eye tolerance. Present scientific developments as well as the possible agreement by regulatory agencies of the newly developed tests are presented. In spite of the increasing acceptance of alternative methods, large numbers of animals are still used. The reasons for this state of affairs are analysed, as well as the efforts which are made to encourage research for the replacement of animals in programs traditionally based on animal experimentation.

Alternatives to animal experimentation: developing in-vitro methods and changing legislation.

Despite recent changes in legislation in several countries and general reduction in the use of animals in biomedical research, the impatience of antivivisectionists to see reductions in animal experimentation shows no signs of abating. Gerhard Zbinden analyses the reasons for this continuing dissatisfaction, arguing that real progress has been made in biomedical research, but that the complexities of developing internationally recognized regulations constitute a barrier to rapid change in product safety testing methods.

Effects of recombinant human alpha-interferon in a rodent cardiotoxicity model.

Recombinant human alpha-interferon infused intravenously into rats at doses of 10(6) (1st infusion) and 10(5) IU (2nd and 3rd infusion) produced marginal evidence of liver damage but no serious toxicity. During the 2nd and 3rd infusions an increased incidence of cardiac arrhythmias of various types was observed. In 2 rats electrocardiographic evidence of myocardial ischemia was noted. No changes in myocardial structure were demonstrated by light and electron microscopy. With the high dose the decrease in body temperature resulting from anesthesia was significantly reduced. Antibodies to interferon were demonstrated in the majority of the animals.

Safety evaluation of biotechnology products.

Preclinical safety studies with biotechnology products are not only performed for regulatory purposes, but should first and foremost provide information about the potential toxic effects in patients. The initial toxicological experience, using standard testing procedures developed for drugs of small molecular weight, often gave disappointing results, and the development of antibodies against the heterologous products cast doubt upon the validity of the testing approach. In order to assess the safety of new biotechnology products, compounds must be looked at on a case by case basis. Exaggerated pharmacodynamic effects are often responsible for the major toxicological problems. For some compounds, 'intrinsic toxicity', i.e. adverse effects due to the molecule per se, may play a role. With others, 'biological toxicity', i.e. the activation of physiological processes such as antigen-antibody interaction, release of mediators and cytokines, or initiation of the arachidonic acid-prostaglandin cascade, may be the cause of the observed adverse effects. Examples are given that show the importance of a good understanding of the biological mechanisms of action of toxicity observed in animals and in patients.

[Neurobehavioral toxicology]. / Neiropovedencheskaia toksikologiia.

The various experimental approaches to detect and characterize neurotoxic chemicals, i.e. pathomorphology, electrophysiology, neurobiochemistry and ethology, are discussed. In recent years neurobehavioral techniques have been used widely in toxicology because of their non-invasiveness and considerable discriminative power. Advantages and disadvantages of neurobehavioral techniques are discussed. Two approaches, namely development of animal models that imitate human neurobehavioral disorders and diseases, and measurement of basic elements of behavior are presented. Examples are given to demonstrate how modern computerized behavioral techniques are able to assess various basic elements of behavior. Through microanalysis of the data detailed information on behavioral effects of various neurotoxins can be obtained. These concepts are demonstrated with experiments using ethanol, caffeine and methylmercury as reference substances. Many more chemical substances must be tested with these techniques to obtain the necessary information for predictive conclusions for humans.