Joint action of polycyclic aromatic hydrocarbons: predictive modeling of sublethal toxicity

Polycyclic aromatic hydrocarbons (PAHs) typically contaminate the environment as complex assemblages of different chemical compounds. Modeling approaches provide a means of estimating the toxicity of these PAH mixtures. In the present study, we tested the hypothesis that the joint effects of four PAHs: pyrene, phenanthrene, fluoranthene and naphthalene, on the growth rate of the crustacean Daphnia magna during sub-chronic exposure could be accurately predicted using a mathematical algorithm for concentration addition based upon the assumption that these PAHs impact growth by a common mode of action. Assessment of the individual toxicity of the four PAHs confirmed that these compounds elicited the common effect of retarding growth of daphnids at concentrations below those that were lethal to the organisms. Using the experimentally derived toxicity parameters for the individual chemicals, the toxicity of multiple mixtures of these four PAHs was modeled. These mixtures were based on concentrations reported in the environment and on equi-toxic concentrations. The effects of over 140 combinations of four mixture formulations on the growth rate of daphnids were experimentally determined and compared to model predictions. The concentration addition models tended to over predict the joint toxicity of these PAH mixtures and experimental data was better represented by an alternative model based upon the concept of independent joint action. Mixtures at environmentally relevant concentrations were predicted and experimentally demonstrated to have no effect on daphnid growth rates. Results indicate that PAHs elicit toxicity to daphnids by multiple mechanisms and demonstrate an appropriate modeling approach to assess the toxicity of these mixtures.     

Modification of the time-course of DMPEA toxicity in the mouse by centrally acting drugs

Survival time following the intraperitoneal injection of a lethal dose of dimethoxyphenylethyl ami ne (DMPEA) was found to be subject to modification by pretreatment with psychotropic drugs. A limited, but novel, classification of the tranquillizers and other psychotropic compounds may be possible according to whether they potentiate, antagonise or have no effect on the toxicity of DMPEA.     

A novel model integrated concentration addition with independent action for the prediction of toxicity of multi-component mixture

Concentration addition (CA) and independent action (IA) have been used to describe the mixture of components having similar and dissimilar mode of action (MOA), respectively. Environmentally relevant mixture does, however, not follow the strictly similar or dissimilar MOA. A novel model, which integrated CA with IA based on the multiple linear regression (ICIM), was proposed for predicting the toxicity of noninteractive mixture. The predictive power of the ICIM model was validated by data set 1 including 13 mixtures of nine components and data set 2 including six mixtures of six components. For data set 1, ten uniform design with fixed concentration ratio ray (UDCR) mixtures was used as a training set to build an ICIM model, and the model was used to predict the toxicity of the test set consisting of three equivalent-effect concentration ratio (EECR) mixtures. For data set 2, the ICIM model based on four UDCR mixtures was used to predict the remaining two EECR mixtures. It is concluded that the ICIM model shows a strong predictive power for the mixture toxicities in the two data sets, and its prediction is better than CA and IA where the two models deviate from the concentration-response data of the mixtures. Thus, ICIM model is a powerful tool to evaluate and predict mixture toxicity, and maybe offer an important approach in risk assessment of mixture toxicity.     

Modelling toxicity and mode of action of chemicals to analyse growth and emergence tests with the midge Chironomus riparius

We present a new growth test data analysis for toxicity tests with the midge Chironomus riparius. The analysis is based on mathematical models which proved to be able to predict growth and emergence of non-exposed organisms for diverse feeding levels or densities. Here, we adapt these models to account for toxicity. We distinguish between two modes of action of the compounds: decrease of feeding or increase of growth energy costs. The models are used to analyse growth data with organisms exposed to copper spiked artificial sediments. Both models provide a good fitting of the data in the case of feeding ad libitum, but only the growth costs model can account for effects of copper in the case of food limitation. We also show that the threshold of effects do not depend on the age (the no effect concentrations (NOEC) are 6, 7 and 9 mg/kg, respectively, for second, third and fourth instars larvae), but that, as soon as this threshold is exceeded, fourth instar larvae are less affected by copper than earlier larvae. Our models constitute a step towards a more biologically relevant analysis of standardized tests, which should facilitate both the understanding of the mechanisms of toxicity and the change of scale from the individual to the population.     

Acute toxicity of organic chemicals to Gammarus pulex correlates with sensitivity of Daphnia magna across most modes of action

We investigated the sensitivity of the freshwater crustacean amphipod Gammarus pulex towards organic xenobiotic compounds in comparison to the sensitivity of the crustacean cladoceran Daphnia magna. In addition we studied the influence of the chemical's mode of action on the relationship between the sensitivity of G. pulex and that of D. magna. We tested the acute toxicity of twelve compounds (Malathion, Aldicarb, Carbofuran, 2,4-dichloroaniline, 2,4-dichlorophenol, 1,2,3-trichlorobenzene, 4,6-dinitro-o-cresol, 2,4,5-trichlorophenol, Ethylacrylate, 4-nitrobenzyl-chloride, Sea-nine, Imidacloprid) with different modes of action and physicochemical properties towards the freshwater amphipod G. pulex in laboratory experiments. Additional toxicity data was collected from the peer-reviewed literature and databases (data pairs for 44 chemicals in total). The chemicals were assigned to seven mode of action groups. The relationship between the sensitivity of G. pulex (48h-LC50s and 96h-LC50s) and that of D. magna (48h-EC50s) was investigated using regression analysis and correlation plots. G. pulex is two to three orders of magnitude more sensitive towards neonicotinoids than D. magna (P=0.0046, n=3). For organophosphates we found that D. magna is more sensitive than G. pulex by approximately a factor of six (P=0.0256, n=6). There was no significant difference between the sensitivity of D. magna and that of G. pulex in any of the other mode of action groups; however chemicals with the same mode of action grouped together in the same area of the correlation plot. Without the neonicotinoids 75% of all G. pulex toxicity data were within one order of magnitude of the D. magna data and 100% within two orders of magnitude. The regressions with all data and with all data minus neonicotinoids were both significant linear relationships with slopes around one and intercept around zero. Thus, G. pulex is generally equally sensitive towards organic xenobiotics as D. magna.     

Enhancement of developmental toxicity effects of chemicals by gestational stress. A review

Risk assessment of developmental toxicants is almost exclusively based on single chemicals studied in animals under controlled experimental conditions, as to reduce stress. Although humans may be exposed simultaneously to numerous hazards, little is known about the interaction of prenatal chemical exposures with other factors, such as maternal stress, itself a modifier of fetal development. Gestational stress has been hypothesized to enhance the developmental toxicity of chemicals. This review identified 36 animal studies investigating if maternal stress may enhance the effects of prenatal chemical exposure, and evaluated the presented hypothesis. Studies of a broad range of chemicals and developmental endpoints support the notion, that maternal stress is able to enhance the effects of developmental toxicants, although stress mitigated chemically induced effects in a few cases. Maternal stress most often enhanced chemical developmental toxicity at dose levels associated with severe maternal toxicity or where test agents were already above threshold for effect. Thus, LOAEL(chemical) was generally similar to LOAEL(chemical+stress), although not necessarily for the same endpoint. It should be noted that the database contained a limited number of studies, and only a single high dose level was applied in most studies, rendering establishment of NOAELs for combined exposures impossible. Furthermore, for some compounds, the margin between human exposure levels and the LOAEL(chemical+stress) seems small. Future studies are recommended to investigate compounds, for which maternal stress was already proven as an enhancer, at lower dose levels. Interactive response seems to depend on stressor severity and timing of chemical exposure relative to maternal stress which should be further scrutinized.     

Methods for the assessment of the toxicity of environmental chemicals to earthworms

In view of the impending publication of standards for earthworm toxicity testing by the Commission of the European Communities, a review has been made of the recent literature on earthworm toxicology. Relevant studies are reviewed from the standpoints of methods used, reproducibility of results, and ability to extrapolate laboratory results to field situations. Eisenia foetida, a commonly used test species, is much less sensitive to agricultural chemicals than other, native earthworms and is of doubtful utility for extrapolating laboratory data to field conditions, but when native soil organisms are used, such extrapolations show good general agreement. Standardization of test conditions and broadening of the data base are encouraged.     

Defibrillatory action of glibenclamide is independent from ATP-sensitive K+ channels and free radicals

This study investigated whether glibenclamide exerts a defibrillatory action and if this action is mediated by a blockade of ATP-sensitive K+ channels (K(ATP)) or by an anti-free radical mechanism. Aerobically perfused isolated rat hearts were subjected to 10 min of pacing-induced ventricular fibrillation (VF) followed by 10 min of perfusion without pacing (post-VF period), in the presence of solvent (controls), 1 microM K(ATP) blocker glibenclamide, 10 microM K(ATP) opener cromakalim, and their combination, respectively. In controls, pacing-induced VF caused a significant deterioration in cardiac function in the post-VF period. Spontaneous defibrillation was 42%. Glibenclamide improved post-VF cardiac function and resulted in 100% (P < 0.05) spontaneous defibrillation. Cromakalim did not significantly affect post-VF cardiac function and the incidence of spontaneous defibrillation as compared with controls. The combination of the compounds improved cardiac function and resulted in 83% (P < 0.05) spontaneous defibrillation. In separate experiments, 2,5-dihydroxybenzoic acid formation in the perfusate as a marker of hydroxyl radical formation was measured by high-performance liquid chromatography and cardiac superoxide production was assessed by lucigenin-enhanced chemiluminescence during pacing-induced VF. Glibenclamide did not affect hydroxyl radical generation or myocardial superoxide content during VF. The conclusion is that glibenclamide exerts a defibrillatory action and improves post-VF cardiac function in rat hearts and these effects are independent from K(ATP) and free radicals.     

The anti-inflammatory action of danazol and leuprorelin acetate depot on endometriosis is CRH independent

Corticotropin-releasing hormone (CRH) is a hypothalamic neuropeptide involved in the neuroendocrine response to stress, also playing a role in cell mediated immune functions. The aim of this study was to determine the circulating in the serum CRH levels in women with endometriosis and investigate the effect of the routinely 6-month administered treatment of danazol or leuprorelin acetate depot on these hormonal levels. Serum CRH levels were not significantly different in women with endometriosis and in the control group. The 6-month danazol or leuprorelin treatment had no effect on the levels of CRH. Three months after danazol treatment CRH levels were significantly lower (p < 0.005) than those before treatment. In contrast, after treatment with leuprorelin, CRH levels were significantly higher (p < 0.001). Our results suggest that endometriosis is not associated with CRH and that danazol as well as leuprorelin acetate depot have no effect on these levels during the treatment-specific period. However, they both showed significant fluctuations after the administration of these compounds ceased.     

Protective action of propionyl-L-carnitine on toxicity induced by hyperbaric oxygen

The protective effect of propionyl-L-carnitine against hyperbaric oxygen toxicity was studied by in vivo experiments on mice. The treatment reduced both the percentage of animals with convulsions and the death rate. Tissue signs of toxicity and pulmonary weight increase were less marked in treated animals than in controls. Results may indicate that propionyl-L-carnitine needs to build up to critical levels in cells and mitochondria before its metabolic effects can be fully felt.     

Freshly prepared rat hepatocytes used in screening the toxicity of blue-green algal blooms

The acute toxicity of extracts of blue-green algae was tested in freshly prepared rat hepatocytes in suspension. The results were compared with the traditional in vivo mouse bioassay. Sixty samples of natural algal blooms from freshwater lakes in Norway, Sweden, and Finland and 14 samples cultured in the laboratory were tested. The mouse bioassay revealed hepatotoxins in a large number of the algae, while neurotoxins were not found. Acute hepatotoxicity in vitro was scored by measurement of leakage of the enzyme lactate dehydrogenase (LDH) from damaged cells and of morphological changes of the cells. The correlation coefficients between mouse toxicity and LDH, mouse toxicity and morphological cell damage, and between LDH and morphological cell damage were 0.812, 0.735, and 0.882, respectively. Consequently, the rat hepatocyte toxicity test seems to be well suited for screening blooms of blue-green algae for the presence of hepatotoxins.     

The influence of modes of action and physicochemical properties of chemicals on the correlation between in vitro and acute fish toxicity data

New EU legislation is providing an impetus for research aimed at replacing acute fish toxicity testing with in vitro alternatives. In line with such research, the objective of this study was to determine what factors influence the correlation between in vitro and fish toxicity data. Basal cytotoxicity (IC₅₀) and acute toxicity data from fathead minnow (LC₅₀) of 82 industrial organic chemicals were obtained from the Halle Registry of Cytotoxicity and the US EPA Fathead Minnow Database. A good correlation between IC₅₀ with LC₅₀ data was found (r 0.84). Yet, IC₅₀ data were less sensitive than LC₅₀ data by an order of magnitude. Using multiple regression analysis, the octanol–water partition coefficient (K_OW) and the Henry’s Law Constant (H) were found to significantly explain the low absolute sensitivity. The mode of action (MOA) of the chemical was found to significantly explain the general variation in the log IC₅₀/log LC₅₀ regression line. These results support the notion that (a) the bioavailability of hydrophobic (high K_OW) and volatile (high H) chemicals is significantly lower in in vitro assays than in the fish bioassay and (b) multiple cell types and endpoints should be included to mimic the modes of action possible in the whole organism.     

Irreversible cytoskeletal disarrangement is independent of caspase activation during in vitro azaspiracid toxicity in human neuroblastoma cells

Azaspiracid-1 (AZA-1) is a marine toxin discovered in 1995. Besides damage to several tissues in vivo, AZA-1 has been shown to cause cytotoxicity in a number of cell lines and alterations in actin cytoskeleton and cell morphology. We studied the reversibility of AZA-1-induced morphological changes in human neuroblastoma cells and their dependence on caspases and signaling pathways involved in cytoskeleton regulation. Morphological/cytoskeletal changes were clearly observed by confocal microscopy 24h after the addition of toxin, without recovery upon toxin removal. Interestingly, 2min of incubation with AZA-1 was enough for the cytoskeleton to be altered 24-48h later. The activation of caspases by AZA-1 was studied next using a fluorescent caspase inhibitor. A cell population with activated caspases was observed after 48h of exposure to the toxin, but not at 24h. Two fragments and a stereoisomer of AZA-1 were tested to analyze structure-activity relationship. Only ABCD-epi-AZA-1 was active with a similar effect to AZA-1. Additionally, regarding the involvement of apoptosis/cytoskeleton signaling in AZA-1-induced morphological effects, inhibition of caspases with Z-VAD-FMK did not affect AZA-1-induced cytoskeletal changes, suggesting, together with the activation kinetics, that caspases are not responsible for AZA-1-elicited morphological changes. Modulation of PKA, PKC, PI3K, Erk, p38MAPK, glutathione and microtubules with inhibitors/activators did not inhibit AZA-1-induced actin cytoskeleton rearrangement. The JNK inhibitor SP600125 seemed to slightly diminish AZA-1 effects, however due to the effects of the drug by itself the involvement of JNK in AZA-1 toxicity needs further investigation. The results suggest that AZA-1 binds irreversibly to its cellular target, needing moieties located in the ABCDE and FGHI rings of the molecule. Cytotoxicity of AZA-1 has been previously described without reference to the type of cell death, we report that AZA-1 induces the activation of caspases, commonly used as an early marker of apoptosis, and that these proteases are not responsible for AZA-1-induced cytoskeleton disarragement in human neuroblastoma cells.     

The effect of thyroid hormones on the action of some centrally acting drugs

1. The effect of administration of thyroxine or thyroidectomy on the pharmacological action of (+)-amphetamine, caffeine, hexobarbitone and morphine was determined in rats or mice.2. Locomotor activity induced by (+)-amphetamine or caffeine was increased by hyperthyroidism and decreased by hypothyroidism.3. The LD50s of (+)-amphetamine and caffeine in hyperthyroid rats were 1/30 and 2/5 that of control rats. With each drug, the LD50 regression lines in hyperthyroid and control rats were not parallel, suggesting that hyperthyroidism modifies the mechanism of the toxic effects. Hypothyroidism reduced toxicity to (+)-amphetamine.4. Hexobarbitone sleeping time was prolonged in hyperthyroid male rats, but was shortened in hyperthyroid female rats. In control rats, sleeping time was approximately four times as long in females as it was in males. Ethinyloestradiol treatment and castration also prolonged sleeping time in male rats. No further prolongation was produced by combined administration of thyroxine and ethinyloestradiol, but thyroxine further prolonged the sleeping time of castrated rats indicating that its mode of action in producing these changes is not mediated via sex hormones.5. In contrast to rats, a sex difference in the duration of action of hexobarbitone was not found in mice. Thyroxine prolonged sleeping time equally in each sex.6. Analgesia induced by morphine in mice was unaffected by hyperthyroidism. No increase in sedative or ;Straub tail' activity could be detected, but toxicity was increased when higher doses of morphine were used.7. The mechanism by which thyroid hormones produce these changes in sensitivity to centrally acting drugs is discussed. It is suggested that the effects of thyroxine vary according to whether the mode of action of the drug or its metabolism is modified.     

Habituation of tactile startle is altered by drugs acting on serotonin-2 receptors

Ligand binding studies indicate that multiple serotonin (5-HT) binding sites exist in the brain. To relate these putative receptor subtypes to startle reactivity and habituation, compounds with varying specificities for 5iHT1 and 5-HT2 binding sites were administered to rats prior to the presentation of 201 startling tactile stimuli. The 5-HT2 antagonists cyproheptadine, cinanserin, ritanserin, and ketanserin increased the rate of tactile startle habituation without affecting initial levels of reactivity. The 5-HT1A agonists 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, and 5-methoxy-N,N-dimethyltryptamine, the 5-HT1B agonist m-trifluoromethylphenylpiperazine, and the 5-HT2 agonist quipazine affected startle reactivity rather than having specific effects on habituation. The effects of the exogenous 5-HT2 antagonists were consistent with the effects of manipulations of endogenous 5-HT. Specifically, the serotonin depleting agents parachlorophenylalanine and parachloroamphetamine accelerated startle habituation. Conversely, the serotonin reuptake inhibitor fluoxetine decreased startle habituation. These findings support the hypothesis that serotonergic systems modulate the habituation of tactile startle via actions at 5-HT2 receptors.     

Milrinone inhibits sympathetic-mediated tachycardia by a postjunctional action independent of cyclic AMP.

In pithed rats with stimulated sympathetic outflow, the phosphodiesterase inhibitor milrinone (0.3 mg/kg, i.v.) decreased the peak tachycardiac response produced by both sympathetic nerve stimulation (15 s at 0.5-3 Hz) and norepinephrine administration (0.3-5 micrograms/kg, i.v.). However, another phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 0.5 mg/kg, i.v.) had no effect on the peak tachycardic response to sympathetic stimulation. Similarly, in isolated rat atria, milrinone (9 mumol/L) inhibited the tachycardia produced by norepinephrine, whereas IBMX (1 mumol/L) had no effect. The inhibitory effect of milrinone on sympathetic responses was not due to changes in norepinephrine release since milrinone (9 mumol/L) increased norepinephrine release in isolated rat atria incubated with [3H]norepinephrine. When the duration of the tachycardia (rather than the peak tachycardic response) produced by sympathetic nerve stimulation was measured, it was found to be prolonged by both milrinone and IBMX, suggesting that in this case cyclic AMP was involved. Furthermore, in contrast to its inhibitory effects on norepinephrine-induced tachycardia in rat atria, milrinone enhanced the tachycardia produced by the adenylate cyclase activator forskolin. These results suggest that milrinone has complex actions on sympathetic control of heart rate and that beta-adrenoceptor tachycardia occurs by mechanisms dependent on and independent of cyclic AMP.     

A comparison of the acute toxicity of chemicals to fish, rats and mice

The acute toxicity of chemicals to rainbow trout, as shown by intraperitoneal injections (IP LD50), oral dosing (oral LD50) and aqueous exposure (LC50) was compared with published values for IP LD50S and oral LD50S of mice and rats. The method of comparison was by simple linear regression analyses of log-transformed data, modified to recognize that X (fish toxicity) was neither fixed nor measured without error. Within-species comparisons demonstrated very strong linear correlations (r = 0.866-0.998) between IP and oral LD50S. Variability was least for the fish data since it was all generated in one laboratory. Comparisons between species of IP and oral LD50S gave correlation coefficients ranging from 0.59 to 0.95 with the majority over 0.80. Correlations were best (r = 0.83-0.94) between fish LD50S and rat and mice IP LD50S. Correlations were poorest between fish and mammalian oral LD50S (r = 0.59-0.66) because the sample sizes and the ranges of values were very small. In all cases, the slopes were close to, or equalled, 1.0. Comparisons of fish LC50S to fish or mammalian LD50S were not as successful. Correlation coefficients ranged from 0.19 to 0.83. Presumably the cause was the aqueous exposure. Interactions of the chemicals with water (e.g. dissociation) and with lipid membranes (partitioning) should cause considerable variations in uptake efficiency. However, adjustments of LC50S for dissociation constants and partition coefficients did not improve these correlations, probably because there were few chemicals for which all data were available. These comparisons demonstrate a potential for a wider use of surrogate species in toxicity testing and for adapting existing data from mammalian toxicology to aquatic hazard assessments.