A perspective on In vitro developmental neurotoxicity test assay results: An expert panel review.

For decades, there has been increasing concern about the potential developmental neurotoxicity (DNT) associated with chemicals. Regulatory agencies have historically utilized standardized in vivo testing to evaluate DNT. Owing to considerations including higher-throughput screening for DNT, reduction in animal use, and potential cost efficiencies, the development of alternative new approach methods (NAMs) occurred; specifically, the advent of the DNT in vitro test battery (DNT IVB). SciPinion convened an expert panel to address specific questions related to the interpretation of in vitro DNT test data. The consensus of the expert panel was that the DNT IVB might be used during initial screening, but it is not presently a complete or surrogate approach to determine whether a chemical is a DNT in humans. By itself, the DNT IVB does not have the ability to capture nuances and complexity of the developing nervous system and associated outcomes including behavioral ontogeny, motor activity, sensory function, and learning/memory. Presently, such developmental landmarks cannot be adequately assessed in the DNT IVB or by other NAMs. The expert panel (all who serve as co-authors of this review) recommended that additional data generation and validation is required before the DNT IVB can be considered for application within global regulatory frameworks for decision-making.

Prevalence and antifungal susceptibility of Candida parapsilosis complex isolates collected from oral cavities of HIV-infected individuals.

At present, few data are available on the prevalence and antifungal susceptibility of Candida parapsilosis complex isolates from HIV-infected individuals. The C. parapsilosis complex comprises three species, C. parapsilosis sensu stricto, C. metapsilosis and C. orthopsilosis. Fifteen of 318 Candida isolates were identified as members of the C. parapsilosis complex by PCR and restriction fragment length polymorphism (RFLP). The prevalence of C. parapsilosis complex isolates was 4.7 %, 2.2 % being identified as C. parapsilosis sensu stricto and 2.5 % as C. metapsilosis, while no C. orthopsilosis was isolated. This is believed to be the first study that has identified isolates of C. metapsilosis obtained from the oral cavity of HIV-infected individuals. Antifungal susceptibility tests indicated that all the isolates were susceptible to amphotericin B (AMB), fluconazole (FLC), ketoconazole (KTC), itraconazole (ITC), voriconazole (VRC) and caspofungin (CASPO). Although isolates of C. parapsilosis sensu stricto and C. metapsilosis were susceptible to FLC, isolates of C. metapsilosis showed a tendency for higher MICs (≥1.0 µg ml(-1)). Based upon the frequency of candidiasis and the fact that certain isolates of the C. parapsilosis complex respond differently to FLC therapy, our data may be of therapeutic relevance with respect to susceptibility and potential resistance to specific antifungal agents. Our data suggest that C. metapsilosis can be a human commensal; its importance as a pathogen has yet to be confirmed.

Neurobehavioral alterations in rats exposed to acrylonitrile in drinking water.

This study was carried out on rodents, to explore the neurobehavioral effects of acrylonitrile (AN) administered in drinking water. Thirty, male, Sprague-Dawley rats were randomly divided into three groups: two exposure groups (50 and 200 ppm AN), and one control group (tap water without AN). Three tests, including the open field test, rotarod test and spatial water maze, were applied to evaluate locomotor activities, motor co-ordination and learning and memory, respectively, prior to initiation of the treatment, and at Week 4, 8 and 12 postexposure. There were no consistent changes in the open field test, except for locomotion and grooming episodes. In the rotarod test, AN significantly decreased the latencies to fall in a dose and time-dependent manner. In the spatial water maze test, rats exposed to AN for 12 weeks had significantly more training times and longer escape latencies than control animals. These findings indicate that oral exposure to AN induces neurobehavioral alterations in rats.

Drug-induced phospholipidosis: are there functional consequences?

Phospholipidosis induced by drugs with a cationic amphiphilic structure is a generalized condition in humans and animals that is characterized by an intracellular accumulation of phospholipids and the concurrent development of concentric lamellar bodies. The primary mechanism responsible for the development of phospholipidosis is an inhibition of lysosomal phospholipase activity by the drugs. While the biochemical and ultrastructural features of the condition have been well characterized, much less effort has been directed toward understanding whether the condition has adverse effects on the organism. While there are a few cationic amphiphilic drugs that have been reported to cause phospholipidosis in humans, the principal concern with this condition is in the pharmaceutical industry during preclinical testing. While this class of drugs should technically be referred to as cationic lipophilic, the term cationic amphiphilic is widely used and recognized in this field, and for this reason, the terminology cationic amphiphilic drugs (CADs) will be employed in this Minireview. The aim of this Minireview is to provide an evaluation of the state of knowledge on the functional consequences of CAD-induced phospholipidosis.

Confounding factors in toxicity testing.

The necessity for understanding normal human functions and the mechanisms which underlie dysfunction in these processes is essential in the promotion of a healthier lifestyle. To achieve this goal utilization of a suitable animal model is necessary in order to develop new pharmaceutical agents to alleviate diseases or chemicals to enhance the quality of life. It is incumbent upon investigators to choose a species in which pharmacokinetic principles are established and it is important that these phenomena resemble those of the humans. The choice of rats has specific advantages in that these rodents possess similar pharmacodynamic parameters to humans. Other advantages include availability, low cost, ease of breeding, and an extensive literature data-base to enable comparisons to present findings. However, in the interpretation of data from animals to humans, there are factors which need to be recognized as playing important roles in chemical-induced outcomes. The confounding factors include strain, supplier, age, gender, hormonal status and dietary intake. The aim of this article is to demonstrate that there are differences in the responsiveness of rat stock/strains to chemicals and that lack of consideration of confounding factors yields inappropriate conclusions regarding risk assessment for humans.

Ultraviolet radiation and reactive oxygen generation as inducers of keratinocyte apoptosis: protective role of tea polyphenols.

Ultraviolet A (UVA) radiation produces serious damage to skin, especially to dermis, but its damage to epidermis and responsible mechanisms are not fully understood. Studies were thus undertaken to investigate the effects of UVA or reactive oxygen species (ROS) on lipid peroxidation, cell cycle, and apoptosis in primary cultured rat keratinocytes and to determine the possible protective effects of tea polyphenols (TPP). UVA or ROS increased the release of plasma enzyme lactate dehydrogenase (LDH), and increased lipid peroxidation production (malondialdehyde, MDA), but decreased the activity of glutathione peroxidase (GSH-Px), indicating that UVA or ROS were cytostatic and peroxidizing to keratinocytes. TPP stabilized and protected cell membranes from ROS or UVA by inhibiting the release of LDH, lowering MDA levels, and increasing GSH-Px activity. Flow cytometry (FCM) analysis revealed that UVA or ROS decreased the proliferative index (PI); hence the cell growth was blocked in the S/G2 phase, with an increase in the percentage of apoptosis in primary keratinocytes. TPP modified the UVA or ROS-induced changes in PI and apoptosis. TPP may be useful to protect keratinocytes from UVA irradiation. In summary, these data demonstrated that UVA damage to skin keratinocytes in vitro was similar to that for ROS and that TPP protects against UVA-induced cytotoxicity by inhibiting lipid peroxidation and apoptosis.

In vitro chemopreventive effects of plant polysaccharides (Aloe barbadensis miller, Lentinus edodes, Ganoderma lucidum and Coriolus versicolor).

A plant polysaccharide, Aloe gel extract, was reported to have an inhibitory effect on benzo[a]pyrene (B[a]P)-DNA adduct formation in vitro and in vivo. Hence, chemopreventive effects of plant polysaccharides [Aloe barbadensis Miller (APS), Lentinus edodes (LPS), Ganoderma lucidum (GPS) and Coriolus versicolor (CPS)] were compared using in vitro short-term screening methods associated with both initiation and promotion processes in carcinogenesis. In B[a]P-DNA adduct formation, APS (180 micrograms/ml) was the most effective in inhibition of B[a]P binding to DNA in mouse liver cells. Oxidative DNA damage (by 8-hydroxydeoxyguanosine) was significantly decreased by APS (180 micrograms/ml) and CPS (180 micrograms/ml). In induction of glutathione S-transferase activity, GPS was found to be the most effective among plant polysaccharides. In screening anti-tumor promoting effects, APS (180 micrograms/ml) significantly inhibited phorbol myristic acetate (PMA)-induced ornithine decarboxylase activity in Balb/3T3 cells. In addition, APS significantly inhibited PMA-induced tyrosine kinase activity in human leukemic cells. APS and CPS significantly inhibited superoxide anion formation. These results suggest that some plant polysaccharides produced both anti-genotoxic and anti-tumor promoting activities in in vitro models and, therefore, might be considered as potential agents for cancer chemoprevention.

Effect of over-the-counter drugs on the unborn child: what is known and how should this influence prescribing?

The developing organism is unique in its responsiveness to drugs. The predictability of therapeutic effectiveness and safety of drugs in pregnancy using the adult as a model for pharmacokinetics and pharmacodynamics can result in grave consequences in the fetus. There exists a general misconception that since over-the-counter (OTC) drugs are readily available, these pharmaceutical agents can be viewed as safe to use by adults. Ingestion of OTC preparations during pregnancy results in placental transfer and accumulation of these drugs in the fetus. As the fetus lacks the ability to handle pharmaceutical agents, since renal function, metabolic pathways, etc. are not fully developed, drug exposure in utero may produce deleterious effects in the fetus but not the mother. Clinicians are aware of drug-induced effects on the fetus and have dramatically reduced the use of prescription drugs during pregnancy. However, the use of self-medication (OTC) has significantly increased during pregnancy through extensive, effective advertising by the pharmaceutical industry and lack of sufficient data indicating an OTC effect on the fetus. However, the consequences of OTC drug use need to be established, since these compounds continue to be used extensively, especially during pregnancy.

Diet and rat strain as factors in nervous system function and influence of confounders.

The necessity for understanding normal human cognitive processes and behavior, and the mechanisms which result in dysfunction in these processes are dependent on utilization of a suitable animal model. In order to develop pharmaceutical agents to alleviate mental disturbances and enable the individual to cope within the norms of society, it is incumbent upon investigators to choose a species in which pharmacokinetic principles are established and resemble those of humans. The choice of rats in cognition research studies has specific advantages in that these animals possess similar pharmacodynamic parameters to humans. Further advantages include availability, low cost, ease of breeding, maintenance and an extensive literature database which enable comparisons to present findings. However, there are substantial differences in the performance of various rat strains in tasks of learning, memory, attention, and responses to stress or drugs. In addition to rat strain, quantity of food also exerts profound consequences on animal behavior. The aim of this review is to demonstrate that there are differences in the central nervous system responsiveness of rat strains to chemical and these could be related to factors such as source of supplier, type and quantity of feed, or season of the year. It is also evident that the genotype differs amongst strains and this may be responsible for the observed differences in CNS sensitivity to chemicals. Strain differences must be identified and taken into consideration in interpretation of assessment of neurobehavioral functions. It is also incumbent upon the investigators to utilize healthy (diet-controlled) animal models.

Bioavailability in rats of bound pesticide residues from tolerant or susceptible varieties of soybean and canola treated with metribuzin or atrazine.

Susceptible or tolerant varieties of soybean treated with metribuzin, or canola treated with atrazine were grown in a controlled environment. Shoots and fruits were harvested at maturity and extracted exhaustively with methanol. The extracted materials containing bound 14C residues were subsequently fed to rats for two days. The elimination of 14C in urine and feces was monitored for 4 days and the distribution of radioactivity in liver, kidney and heart was studied. Higher 14C residues in urine were present in animals fed fruits compared to shoots of soybean or canola of both susceptible and tolerant varieties. The bound atrazine residues from the pods of Atr Tower (tolerant variety of canola) were more bioavailable than Tower (susceptible variety of canola). Bioavailability of bound atrazine from the shoots of canola in both varieties was very low. In soybean fed animals, bound metribuzin derived 14C from the susceptible variety (Maple Amber) was more bioavailable than from the resistant variety (Maple Arrow). However, feeding the animals with susceptible or tolerant varieties of soybean or canola containing bound residues of 14C metribuzin or 14C atrazine for two days did not result in the accumulation of radioactivity in the body organs studied. Thus our data show that the bioavailability of these bound pesticides was dependent on the type of plant parts ingested and the variety of plant species.

An evaluation of possible mechanisms underlying amiodarone-induced pulmonary toxicity.

The effectiveness of amiodarone in the treatment of cardiac arrhythmias is limited due to the development of pulmonary toxicity. Although the biochemical and morphologic characteristics associated with amiodarone-induced pulmonary toxicity (AIPT) are well-defined, the mechanisms underlying this disorder remain unknown. This review focuses on proposed mechanisms of AIPT, in particular (i) direct cellular damage; (ii) the role of phospholipidosis; (iii) the correlation between drug burden and toxicity; (iv) the role of the immune system; (v) the generation of oxidants; (vi) changes in membrane properties; and (vii) miscellaneous biochemical considerations. Additional discussion of the role of amiodarone's primary metabolite, desethylamiodarone, in AIPT and the involvement of preexisting lung dysfunction in the susceptibility to AIPT is included. With a clearer understanding of the possible contributions of these mechanisms to AIPT, it may be possible to develop strategies to alleviate toxicity and prolong the usefulness of amiodarone in the treatment of cardiac arrhythmias.

Biological relevance and consequences of chemical- or metal-induced DNA cross-linking.

A vast number of chemicals are known to induce mutagenesis and/or carcinogenesis in mammals. Although disruption of cellular nuclear material resulting ultimately in mutagenesis/carcinogenesis can be accomplished by various mechanisms, the search for biomarkers of chemical-induced toxicity continues. This review focuses on the ability of certain metals or chemicals to bind to DNA in a cross-link fashion in whole animal as well as under in vitro conditions. The methodologies currently used to determine DNA cross-linking are described. The biological relevance of the presence of chemical- or metal-induced DNA cross-linking as a measure of carcinogenesis in humans is still under debate, as there is no clear correlation between the disease and the DNA cross-link reaction.

Bioavailability of bound pesticide residues and potential toxicologic consequences - an update.

The growing demand for enhanced food productivity to meet the needs of the global population has led farmers to use sophisticated agricultural technology in which pesticides play a crucial role. Pesticide use has a positive and dramatic impact on agricultural production through protection of crops against insects, pests, and disease, but every effort must be made to ensure that application is safe and, more importantly, to assure safety for human and environmental health. Since our initial review (1), global usage of pesticides has increased and knowledge regarding the biological significance of bound pesticide residues has expanded. The fact that more reports are appearing in the literature signifies an increased awareness of the presence of bound pesticide residues and indicates that a greater number of scientists are attempting to establish the potential toxicologic consequences of this pesticides residue fraction. Rico (2), in a review in 1990, concluded that covalently bound residues in edible animal tissues were not carcinogenic, were not readily bioavailable, and hence produced little, if any, adverse effects. Whether this conclusion is applicable to the presence and consequences of bound pesticide residues in plants and food commodities remains unanswered. Thus, the aim of this updated review is not simply to list studies on bioavailability of bound pesticide residues in grains subsequent to the review in 1992 (1) but also to establish the toxicological impact of this chemical fraction on health.

Role of rat strain in the differential sensitivity to pharmaceutical agents and naturally occurring substances.

The development of drugs to combat diseases, chemicals to improve food production, or compounds to enhance the quality of life necessitates, by law, the use of laboratory animals to test their safety. In order to simulate the human condition it is necessary to choose a species in which pharmacokinetic and toxicokinetic mechanisms are established and resemble those of humans. The advantages of the use of the rat in drug and chemical toxicity testing include (a) metabolic pathway similarities to humans; (b) numerous similar anatomical and physiological characteristics; (c) a large database, which is extremely important for comparative purposes; and (d) the ease of breeding and maintenance of animals at relatively low cost. However, the choice of rat can be complicated, especially when over 200 different strains of rat are known to exist. The aim of this review is to summarize genetically determined differences in the responsiveness of rat strains to drugs and naturally occurring chemicals and to show that susceptibility is dependent on the target organ sensitivities, which may also be strain dependent. It is suggested that detailed studies of strain differences may help to clarify toxic mechanisms. Such studies are usually best conducted using inbred strains in which the genetic characteristics have been fixed, rather than in outbred stocks in which individual samples of animals may differ, the phenotype is variable, and the stocks are subject to substantial genetic drift. The fact that strains may differ also needs to be taken into account in assessing the potential hazard of the chemical, particularly when a study involves only a single strain and therefore provides no assessment of likely strain variation.

Comparative evaluation of amiodarone-induced phospholipidosis and drug accumulation in Fischer-344 and Sprague-Dawley rats.

Amiodarone (AD) and its major metabolite, desethylamiodarone (desethylAD), are both phospholipogenic. The present study was undertaken to evaluate the comparative susceptibilities of male Fischer-344 and Sprague-Dawley rats to AD-induced phospholipidosis in alveolar macrophages (AMs), liver and kidney tissue and the concomitant accumulation of AD and desethylAD in these cells, tissues and plasma. Rats were administered AD (100 mg/kg/day, p.o.) for 1 week. Plasma concentrations of AD and desethylAD were approximately 4- and 12-fold higher, respectively, in Fischer-344s compared to Sprague-Dawleys 24 h after the last dose. AD and desethylAD levels in AMs were approximately 12- and 25-fold higher, respectively, in Fischer-344s than Sprague-Dawleys. In the liver and kidney, levels of both compounds were also significantly higher in Fischer-344s than Sprague-Dawleys. Ultrastructural features indicative of phospholipidosis were not observed consistently in any tissue except AMs from treated Fischer-344s. AM total phospholipid increased nearly 5-fold in Fischer-344s, while Sprague-Dawleys showed no increase over control. AMs from both strains incubated with 10 microM AD or desethylAD in vitro were not significantly different in their accumulation of the compounds. When incubated with AD or desethylAD, the lysosomal phospholipases A1 partially purified from AMs of both strains were equally sensitive to inhibition as measured by the drug concentration giving 50% inhibition in activity (IC50). The results of this study indicate that at the same administered dose, AD and desethylAD, accumulate to higher tissue levels and are more phospholipogenic in male Fischer-344 rats than in male Sprague-Dawley rats. The basis for the high susceptibility of Fischer-344 rats to AM-induced phospholipidosis is unknown at present but appears not to be related to biochemical or cellular features of the AMs.

Strain as a determinant factor in the differential responsiveness of rats to chemicals.

The beneficial effects derived from the use of chemicals in agriculture, energy production, transportation, pharmaceuticals, and other products that improve the quality of life are clearly established. However, continued exposure to these chemicals is only advantageous in conditions where the benefit far outweighs toxic manifestations. By law, determination of risk of toxicity necessitates the use of laboratory animals to establish whether chemical exposure is safe for humans. To simulate the human condition, it is incumbent upon investigators to choose a species in which pharmacokinetic and toxicokinetic principles are established and resemble those of humans. Some of the advantages to the use of rat in chemical toxicity testing include (a) similarities in metabolism, anatomy, and physiological parameters to humans; (b) the short life span, especially for carcinogenesis study; (c) the availability, ease of breeding, and maintenance at a relatively low cost; and (d) the existence of a large database to enable comparison of present to reported literature findings. However, the choice of rat can be complicated by several factors such as sex, age, and nutrition, but especially strain, where currently there are over 200 different strains of rat known to exist. The aim of this review is to demonstrate that there are differences in the responsiveness of rat strains to chemicals and that the susceptibility observed is dependent on the tissue examined. It is evident that the genotype differs among strains, and this may be responsible for differences in sensitivities to chemicals. Awareness of strain as a factor in susceptibility to toxicant action needs to be taken into account in interpretation of relevance of risk of toxicity for humans.

Pharmacokinetics, metabolism and toxicity of the plant-derived photoxin alpha-terthienyl.

The plant-derived insecticide, alpha-terthienyl was prepared by synthesis as 3',4'-di[14C]-alpha-terthienyl for pharmacokinetic and metabolism studies. When administered orally to rats at a single dose of 50 mg/kg, excretion of the labelled material was maximal one day after administration and declined to no measurable quantities by day 4. Two metabolites [1,4-di(2'-thienyl)1,4-butadione and 2-2'-bithiophene-5-carboxylic acid] and trace quantities of the parent material were isolated from the urine and chemically identified. These represent the first metabolites of alpha-terthienyl identified. In pilot acute and subacute trials, unlabelled alpha-terthienyl was non-toxic when administered orally to rats as the "ready to use" formulation (0.1% active ingredient). The pure compound had an LD50 of 110 mg/kg when administered intraperitoneally to rats. The results suggest that there is considerable separation with respect to mosquito larvae and non-target mammal sensitivity.

In vivo and in vitro reversibility of chlorphentermine-induced phospholipidosis in rat alveolar macrophages.

Chlorphentermine (CP) is a cationic, amphiphilic drug (CAD) that has been studied widely for its ability to induce phospholipidosis, a disorder characterized by excessive accumulation of cellular phospholipid and ultrastructural development of lysosomal lamellar bodies (LLBs) in the cell. The accumulation of inducing drug correlates with increasing phospholipids. In the present study, we examined the reversibility of this disorder in rat alveolar macrophages (AMs) following a 7-day treatment (30 mg/kg/day, ip). The reversibility of phospholipidosis was examined under in vivo conditions and under in vitro conditions in cell cultures for a period of up to 12 days. There was a marked reduction in cellular CP levels and phospholipid content after 4 days of recovery, both in vivo and in vitro; however, there was no indication of significant loss of LLBs. Beyond this time point, ultrastructural recovery from phospholipidosis lagged behind the biochemical recovery temporally and was somewhat less rapid in vitro than in vivo. By 12 days of recovery, AMs from both groups had recovered biochemically, but a moderate level of LLBs was still present in some AMs in the in vitro recovery group. The results of this study indicate that there are more similarities than differences when comparing the recovery of phospholipidotic cells in vitro to that occurring in vivo. We conclude that the use of cell cultures may prove valuable in studying the reversibility of CAD-induced phospholipidosis.