Comparative toxicological pathology in mammals and fish: some examples with endocrine disrupters

Toxicologic pathology is a classical discipline in the toxicology arena, and despite various emerging techniques, still is a major cornerstone in the process of hazard identification and risk characterization. Most knowledge derives from laboratory animal studies and, to a lesser extent, human data. Currently interest is growing in applying toxicological pathology for lower animals, in particular fish as being the most developed aquatic genus. This is triggered by the interest in so-called endocrine disrupting chemicals (endocrine disrupters, EDCs), xenobiotics that interfere with the endocrine system and thus may affect reproduction and/or development, and for which pathology is an essential technique in general in vivo studies. As the aquatic ecosystem is a major recipient of pollutants, fish constitute an important potential target and can be used as a research and bio-monitoring tool. For this goal knowledge of the pathological responses of fish to EDCs is essential and therefore we have studied the responses of laboratory fish to a set of reference endocrine modulating chemicals. In this paper, such effects are compared with known response patterns in mammals, thereby accounting for the specific aspects of anatomy and physiology in fish.     

Yessotoxins: A toxicological overview

Yessotoxins (YTXs) are polyciclic ether compounds produced by phytoplanktonic dinoflagellates and accumulated in filter feeding shellfish. These toxins can be ingested by humans through contaminated seafood consumption. Initially, YTXs were classified as Diarrhetic Shellfish (DS) toxins but the biological activity of these compounds, which lack of diarrheogenic effects, differs from that of diarrheic toxins. Thus, YTXs have been recently classified as a separate group of algal toxins.Yessotoxin (YTX), homoyessotoxin and 45-hydroxy-homoyessotoxin are lethal after intraperitoneal injection to mice but not after single or repeated oral administration. The target organ seems to be the cardiac muscle cells, where these toxins induce light and electron microscopy ultrastructural changes not only after intraperitoneal injection, but also after oral exposure. On the other hand, di-desulfo-yessotoxin affects liver and pancreas, where it induces fatty degeneration. The mechanisms at the basis of the cardiac effects of YTX and homoyessotoxins are still not completely understood. No short term and chronic toxicity data are available as well as pharmacokinetic studies are lacking. Nevertheless, YTX is known to exert different in vitro activities, such as changes of intracellular calcium and cyclic AMP levels, alteration of cytoskeletal and adhesion molecules, caspases activation and opening of the permeability transition pore of mitochondria. This review reports the current knowledge on the in vivo toxicity and in vitro effects of these toxins.     

Nitrosative stress during infection-induced inflammation in fish: lessons from a host-parasite infection model

The inflammatory response should be considered a protective immune reaction of the host aimed at the removal of pathogens, sometimes irrespective of negative side-effects. In this review we discuss the differential contribution of macrophages and neutrophilic granulocytes to nitrosative stress in vivo and discuss how the timing and concentration of nitric oxide (NO·) are important factors determining the degree of nitrosative stress during parasite-induced inflammation. Infections of common carp (Cyprinus carpio) with the extracellular protozoan parasite Trypanoplasma borreli provide an excellent example of how adaptation and homeostasis are essential elements of the host-pathogen relationship. On the one hand, host-derived NO· interferes with clearance of IgM from the parasite surface and thus can be considered a protective immune reaction of the host. On the other hand, it is essential that the host limits the risks associated with the production of NO·, preventing suppressive effects on lymphocyte proliferation. We review, for both host and parasite, the role of oxygen and nitrogen radicals in the induction of nitrosative stress and the importance of antioxidant compounds for protection against these radicals. Finally, mediators of inflammation such as cytokines, chemokines or alarmins that are involved in the inflammatory response will be discussed in the context of the carp-T. borreli infection model.     

Method for determining cumulation factor in toxicological studies

A new method for determining the cumulation factor is proposed, which quantitatively describes the cumulative effect and allows the dosage regimes for repeated drug administration to be planned.     

Benzidine dihydrochloride: toxicological assessment in mice during chronic exposures

Although benzidine is recognized as a bladder carcinogen in humans and a liver carcinogen in laboratory animals, its toxicological effects appear to be extended to several other endpoints. This economically important chemical is the base for over 200 dyes and is used extensively in manufacturing. In a chronic lifespan study lasting 33 months, both sexes of F1 hybrid (genetically homogeneous) and monohybrid cross (genetically heterogeneous) mice from BALB/c male and C57BL/6 female crosses were exposed to benzidine dihydrochloride in their drinking water at concentrations of 0, 20, 30, 40, 60, 80, and 120 ppm for the females, and 0, 30, 40, 60, 80, 120, and 160 ppm for males. Animals were removed from the study when they were dead or moribund. In addition to hepatocellular carcinomas, there were several other toxicological end-points identified that appeared to be related to the administration of benzidine. Dose-response trends were noted for pigmentation of the spleen, hepatic cytological alterations, hyperplasia of the bile ducts, megakaryocytosis of the bone marrow, vacuolization of the brain, adenoma of the Harderian gland, atrophy of the ovaries, and angioma of the uterus. Also, dose-related effects were noted with respect to time to lung tumor and time to mortality due to reticulum-cell sarcomas.     

Stress-mediated decreases in brain-derived neurotrophic factor as potential confounding factor for acute tryptophan depletion-induced neurochemical effects

Acute tryptophan depletion (ATD) is extensively used to investigate the implication of serotonin (5-hydroxytryptamine; 5-HT) in the onset and treatment of depression and cognitive disorders. Brain-derived neurotrophic factor (BDNF) is strongly linked to the 5-HT system and plays an essential role in mood and memory processes. The present study investigated the effects of ATD upon BDNF in serum, hippocampus and prefrontal cortex in the rat to further explore the underlying mechanism of ATD.ATD significantly decreased peripheral tryptophan (TRP) levels and moderately interrupted 5-HT metabolism 4 h after administration of the nutritional mixture. Although no direct effects of ATD upon serum or brain BDNF concentrations were found, a stress-mediated, decrease in BDNF was observed in the prefrontal cortex. Moreover, brain TRP levels correlated positively with BDNF in both the prefrontal cortex and hippocampus. Thus, BDNF-mediated mechanisms due to ATD and/or its application stress might underlie ATD-induced neurochemical and behavioural alterations.     

Stress-induced hyperlocomotion as a confounding factor in anxiety and depression models in mice

Chronic stress is broadly used to model anxiety and depression. However, in chronic stress models, anxiety- and depression-like behaviors might be masked by unspecific effects of stress. We tested whether chronic stress in mice can induce unspecific changes in locomotion, and whether these changes interfere with the measurement of anxiety and forced-swimming behaviors. Also, we studied these latter behaviors in relation to the duration of stress, the lighting conditions during testing, and after the injection of diazepam. We employed a 4-week chronic stress paradigm, adopted from a model of stress-induced anhedonia and a 1-week subchronic stress, both consisting of rat exposure, restraint stress and tail suspension. Chronically stressed mice, tested under bright and moderate illumination, exhibited 'anxiolytic-like' behavior along with prolonged swimming and hyperactivity. These behaviors were not detectable under weak illumination or after the injection of diazepam (0.25 mg/kg). Instead, normal locomotion, increased anxiety and inhibited swimming were revealed under these conditions. Thus, chronic stress can induce hyperlocomotion in mice, which is triggered by acute stressors such as light, and interferes with the evaluation of anxiety and forced swimming. One week of stress did not change locomotion and forced swimming, and increased anxiety irrespective of illumination applied during testing. Our data can possibly explain previously reported contradictions in the behavioral testing of mice with chronic stress models of anxiety and depression.     

Tretinoin: A review of preclinical toxicological studies

Tretinoin (all-trans-retinoic acid) has shown efficacy in the treatment of acute promyelocytic leukemia. The preclinical toxicological profile of tretinoin was similar to that of other retinoids. The compound had a relatively low acute toxicity; repeated doses resulted in a substantial increase in toxicity that was a function of dose and duration of exposure. Bone fractures, elevated alkaline phosphatase, and testicular degeneration were produced by repeated doses of tretinoin. Teratogenicity was demonstrated in several species. Tretinoin was inactive in the Ames test but appeared to induce sister-chromatid exchanges in human diploid fibroblasts. Insufficient data are available to evaluate the carcinogenic potential of tretinoin. © 1993 wiley-Liss, Inc.     

Extracellular vesicles in toxicological studies: key roles in communication between environmental stress and adverse outcomes

External stressors, especially environmental toxicants can disturb biological homeostasis and thus lead to adverse health effects. However, there is limited understanding of how cells directly exposed to stressors transmit the signals to cells indirectly in contact with stressors. Extracellular vesicles (EVs) are receiving increasing attention as signal transductors between various types of cells in organisms. Cargo in EVs, including RNAs, proteins, lipids, and other signal molecules can be transferred between cells and become critical determining factors of intercellular communication. EVs can be a powerful mediator of environmental stimuli. It has been shown that external stressors reshape the secretion of EVs, modify the composition of EVs, and thus influence the mediating function of EVs. These abnormal EVs can lead to dysfunction of recipient cells, and even the pathogenesis of diseases. In this review, we first summarized current knowledge about the responses of EVs to external stimuli, including chemicals and chemical mixtures. Then we explained how these altered EVs regulate signal pathways in recipient cells, thus mediating physio-pathological responses in detail. The most up-to-date evidence from molecular, cellular, animal and human levels was synthesized to systematically address the mediating roles of EVs. EVs can be regarded as a bridge to link external stressors and internal response. Further toxicological and molecular epidemiological studies are expected to provide further insight into the roles of EVs in toxicology. The gaps in the engulfment of toxicants into EVs are listed as the priority to be solved in future studies.     

Tetrabromobisphenol A induced oxidative stress and genotoxicity in fish Channa punctatus

Tetrabromobisphenol A (TBBPA) is the most widely used brominated flame retardant and its increased use in common products such as plastics, electronic equipment, etc., has raised concern about its ecotoxicity. The present study was conducted to investigate the oxidative stress and genotoxic potential of TBBPA on fresh water fish Channa punctatus by measuring malondialdehyde level and DNA damage, respectively. Fish were exposed to 5.09?mg/l (1/2 of LC₅₀) of TBBPA along with positive (acetone) and negative controls (water) for 96?h. The blood samples were collected at 24, 48, 72 and 96?h post exposure. The results of the study showed significantly increased oxidative stress and DNA damage in the exposed groups as compared to controls. The effect of duration is also found to be significant. The findings of the study would be helpful in risk assessment of TBBPA-induced oxidative stress and genotoxicity among aquatic organisms.     

Contamination is a frequent confounding factor in toxicology studies with anthraquinone and related compounds

Anthraquinone (AQ) (9,10-anthracenedione) is an important compound in commerce. Many structurally related AQ derivatives are medicinal natural plant products. Examples include 1-hydroxyanthraquinone (1-OH-AQ) and 2-hydroxyanthraquinone (2-OH-AQ), which are also metabolites of AQ. Some commercial AQ is produced by the oxidation of anthracene (AQ-OX). In the recent past, the anthracene used was distilled from coal tar and different lots of derived AQ often contained polycyclic aromatic hydrocarbon contaminants, particularly 9-nitroanthracene (9-NA). Many toxicology studies on AQ used contaminated anthracene-derived AQ-OX, including a National Toxicology Program (NTP) 2-year cancer bioassay that reported a weak to modest increase in tumors in the kidney and bladder of male and female F344/N rats and in the livers of male and female B6C3F1 mice. The AQ-OX used in that bioassay was mutagenic and contained 9-NA and other contaminants. In contrast, purified AQ is not genotoxic. The purpose of this paper is to provide additional information to help iterpret the NTP cancer bioassay. This paper describes a quantitative analytical study of the NTP anthracene-derived AQ-OX test material, and presents the results of mutagenicity studies with the 1-OH-AQ and 2-OH-AQ metabolites and the primary contaminant 9-NA. Purified 1-OH-AQ and 2-OH-AQ exhibited only weak mutagenic activity in selected strains of tester bacteria and required S9. Literature reports of potent mutagenic activity for 1-OH-AQ and 2-OH-AQ in bacteria minus S9 are, once again, very likely the result of the presence of contaminants in the test samples. Weak activity and limited production of the 1-OH-AQ and 2-OH-AQ metabolites are possible reasons that AQ fails to exhibit activity in numerous genotoxicity assays. 9-NA was mutagenic in tester strains TA98 and TA100 minus S9. This pattern of activity is consistent with that seen with the contaminated AQ-OX used in the NTP bioassay. Analysis of all the mutagenicity and analytical data, however, indicates that the mutagenic contamination in the NTP bioassay probably resides with compounds in addition to 9-NA. 9-NA exhibited potent mutagenic activity in the L5178Y mammalian cell mutagenicity assay in the presence of S9. The positive response was primarily associated with an increase in small colony mutants suggesting a predominance of a clastogenic mechanism. Quantitative mutagenicity and carcinogenicity potency estimates indicate that it is plausible that the contaminants alone in the NTP AQ-OX bioassay could have been responsible for all of the observed carcinogenic activity. Although AQ-OX is no longer commercially used in the United States, many of the reported genotoxicity and carcinogenicity results in the literature for AQ and AQ derivative compounds must be viewed with caution.     

Pesticides in cannabis: A review of analytical and toxicological considerations

A review of the literature surrounding the use, analysis, and detection of pesticide material for cannabis cultivation is presented. The use of pesticides in crop cultivation is not new, and cannabis crops are no exception. Studies have found that the use of these are common and that high levels of the pesticides are transferred into the cannabis smoke. The most common pesticide classes associated with cannabis are insecticides, acaricides, and fungicides. Over 350 different pesticide products may be used on cannabis materials and of these, 16 pesticides and three plant growth regulators (PGR) are considered to be the main candidates. Many of the pesticides found in cannabis samples destined for consumption are classed as moderately hazardous by the World Health Organization. Analytical methods for pesticide detection on cannabis are being developed with a view to implementing quality control of cannabis, where it is legal, before being sold. However, no standardized protocol exists. The pesticide levels found in the cannabis samples tested were generally low (less than μg/g), these results do not, however, provide information on chronic low‐dose adverse effects of pesticides in relation to cannabis consumption. Currently no research exists on the toxicity of pyrolyzed pesticides in humans from smoking cannabis. More studies are needed to further understand this potentially harmful health threat.     

Physiological alterations during pregnancy: Impact on toxicokinetics

The physiological changes that occur in the alimentary, cardiovascular, pulmonary, and renal organ systems during pregnancy are designed to increase availability of nutrients to and remove wastes from the fetus. Although this is a general requirement, not all animals use the same strategies to meet these goals. These physiological adaptations will impact on toxicokinetics and may alter toxicodynamics. Absorption, distribution, metabolism, transfer between maternal and fetal compartments, and elimination will change for many xenobiotics during pregnancy. The changes in body weight, total body water, plasma proteins, body fat, and cardiac output will alter the distribution of many xenobiotics (Hytten and Leitch, 1971; Hytten and Chamberlain, 1980; Mattison, 1986). As the toxicokinetic parameters change across species, it is important to understand their impact on chemicals associated with maternal, placental, and fetal toxicity for appropriate cross-species extrapolation.     

A unifying concept for assessing toxicological interactions: changes in slope.

Robust statistical methods are important to the evaluation of toxicological interactions (i.e., departures from additivity) among chemicals in a mixture. However, different concepts of joint toxic action as applied to the statistical analysis of chemical mixture toxicology data or as used in environmental risk assessment often appear to conflict with one another. A unifying approach for application of statistical methodology in chemical mixture toxicology research is based on consideration of change(s) in slope. If the slope of the dose-response curve of one chemical does not change in the presence of other chemicals, then there is no interaction between the first chemical and the others. Conversely, if the rate of change in the response with respect to dose of the first chemical changes in the presence of the other chemicals, then an interaction is said to exist. This concept of zero interaction is equivalent to the usual approach taken in additivity models in the statistical literature. In these additivity models, the rate of change in the response as a function of the i(th) chemical does not change in the presence of other chemicals in a mixture. It is important to note that Berenbaum's (1985, J. Theor. Biol. 114, 413-431) general and fundamental definition of additivity does not require the chemicals in the mixture to have a common toxic mode of action nor to have similarly shaped dose response curves. We show an algebraic equivalence between these statistical additivity models and the definition of additivity given by Berenbaum.     

First exposure in man: toxicological considerations

The safety of a volunteer/patient who participate in the very first human trial relies on data from animal experimentation and on the design of the trial. Recommendations on the type and extent of preclinical safety studies that should be conducted prior to first dose in man have been developed by the International Conference on Harmonisation, and the European Committee for Proprietary Medicinal Products. These recommendations include studies designed to characterise local tolerance and general toxicity of the drug candidate as well as its genotoxic potential and ability to interfere with reproduction. For trials which can be categorised as low dose PK screening trials and trials with products where rodent and non-rodent (primarily dog) models do not show any biological response (e.g. some biotechnology-derived hormones and cytokines) other testing paradigms should be used. The present recommendations for preclinical testing have had an important impact on the documented impressive safety record of phase I clinical trials. In this spirit we extend our warmest and sincerest thanks to Professor Jens S. Schou for his long and deep engagement in European and International harmonisation of preclinical test recommendations. His efforts have had a substantial impact on the present testing recommendations, which are of obvious benefit to the safety of the patient.