Reproducibility of toxicity across mode of toxic action in the Tetrahymena population growth impairment assay.

Toxicity data collected in a laboratory setting are the primary source of potency information used for regulatory, modeling, or risk assessment purposes. However, the relative reproducibility of such toxicity data is rarely discussed. This study investigated the reproducibility of growth impairment data for the freshwater ciliate Tetrahymena pyriformis exposed to a structurally diverse group of chemicals of varying hydrophobicity within different modes of toxic action, either non-covalent narcosis or covalent electro(nucleo)philicity. The proportions of chemicals representing each mode of toxic action, or mechanism of action within each mode, were not chosen to emulate the occurrence of manufactured chemicals or chemicals within the TETRATOX database. Chemicals for which prior toxicity data existed were re-tested and reproducibility was evaluated. The toxic potency values of the selected chemicals were largely reproducible after re-testing of the toxic potency, as 98% of the chemicals had re-test toxicity values within one log unit of the original potency value. To further scrutinize the reproducibility of toxicity values, differences between values were investigated by mode of toxic action. A stringent criterion for reproducibility was enforced, which dictated that the re-tested toxicity value must be encompassed by the fiducial interval (FI) of the original toxicity value and vice versa for the chemical to be considered reproducible. Toxicity values of 28 of the 50 re-tested chemicals conformed to the criterion set for reproducible values. Of the nonreproducible chemicals, seven were narcotics: four nonpolar or neutral narcotics and three other narcotics (e.g. polar narcotics). However, four of these seven narcotics did have toxicity values encompassed by one FI, but not the other FI. The remaining chemicals that did not have reproducible potency measurements were electro(nucleo)philic in nature. Certain toxicophores were highly represented among these chemicals. These included quinone derivatives, electron releasing amino and hydroxyl moieties, and electron withdrawing nitro substituents, often in tandem with strong leaving groups (i.e. halogens), and unsaturated alcohols. Lack of reproducibility was common among the chemicals that elicited toxicity after either abiotic or biotic transformation. There was no clear trend between hydrophobicity and lack of reproducibility. While data are limited, these results suggest that toxic potency values of chemicals acting via the electro(nucleo)philic mode of toxic action could be more susceptible to non-reproducibility. Ramifications of such lack of reproducibility could manifest in predictive toxicology models and their use in regulatory and risk assessment endeavors.

Correlation of Tetrahymena and Pimephales toxicity: evaluation of 100 additional compounds.

In the summary/recommendations for the Ecotoxicology Session of TestSmart--A Humane and Efficient Approach to Screening Information Data Sets (SIDS) Data Workshop, it was recommended that more population growth impairment data using Tetrahymena be generated and compared with available lethality data for the fathead minnow. To comply with this recommendation, 100 additional chemicals were tested in the ciliate assay. Toxicity values for the 96-h Pimephales promelas mortality assay (log[LC50(-1)]) and the 2-d Tetrahymena pyriformis growth assay (log[IGC50(-1)]) were compared. Each chemical was a priori assigned a mode of action. The majority of compounds were classified as either narcotics (n = 46) or direct-acting electro(nucleo)philes (n = 43), while 11 chemicals were listed as carboxylic acids, diesters, proelectrophiles, or weak acid respiratory uncouplers. Toxicities for narcotics showed an excellent relationship between endpoints with the coefficient of determination (r2) being 0.93. A weaker relationship, r2 = 0.78, was observed for the electro(nucleo)philes. The poorer fit for the covalent-reacting electro(nucleo)philes is attributed to differences in protocol, in particular, to test-medium composition and exposure scheme. Those chemicals whose potency is mediated by metabolism in fish (diesters and proelectrophiles) as well as the acids exhibited poor correlation between endpoints, with toxicity in the fish assay being greater than that predicted from the ciliate data. The regression analysis between endpoints, regardless of mode or mechanism of toxic action, yielded the model log(LC50(-1)) = 1.12(log[IGC50(-1)]) + 0.46, with n = 92, r2 = 0.82, s (root of the mean square error) = 0.87, F = 399, and p > F = 0.0001. A result for the present investigation supports earlier findings that, with noted exceptions, there is a strong relationship between toxicity potency as quantified by P. promelas mortality and T. pyriformis growth impairment.

Structure-toxicity relationships for selected halogenated aliphatic chemicals.

Toxicity to the ciliate Tetrahymena pyriformis (log(IGC(50)(-1))) for 39 halogen-substituted alkanes, alkanols, and alkanitriles were obtained experimentally. Log(IGC(50)(-1)) along with the hydrophobic term, logK(ow) (1-octanol/water partition coefficient) and the electrophilic parameter, E(lumo) (the energy of the lowest unoccupied molecular orbital) were used to develop quantitative structure-activity relationships (QSARs). Two strong hydrophobic dependent relationships were obtained: one for the haloalkanes and a second for the haloalcohols. The relationship for the haloalkanes [log(IGC(50)(-1))=0.92 (logK(ow))-2.58; n=4, r(2)=0.993, s=0.063, f=276, Pr>f=0.0036] was not different from baseline toxicity. With the rejection of 1,3-dibromo-2-propanol as a statistical outlier, the relationship [log(IGC(50)(-1))=0.63(logK(ow))-1.18; n=19, r(2)=0.860, s=0.274, f=104, Pr>f=0.0001] was observed for the haloalcohols. No hydrophobicity-dependent model (r(2)=0.165) was observed for the halonitriles. However, an electrophilicity-dependent model [log(IGC(50)(-1))=-1.245(E(lumo))+0.73; n=15, r(2)=0.588, s=0.764, F=18.6, Pr>f=0.0009] was developed for the halonitriles. Additional analysis designed to examine surface-response modeling of all three chemical classes met with some success. Following rejection of statistical outliers, the plane [log(IGC(50)(-1))=0.60(logK(ow))-0.747(E(lumo))-0.37; n=34, r(2)=0.915, s=0.297, F=162, Pr>F=0.0001] was developed. The halogenated alcohols and nitriles tested all had observed toxicity in excess of non-reactive baseline toxicity (non-polar narcosis). This observation along with the complexity of the structure-toxicity relationships developed in this study suggests that the toxicity of haloalcohols and halonitriles is by multiple and/or mixed mechanisms of action which are electro(nucleo)philic in character.

Effects of lasalocid on coccidial infection and growth in young dairy calves.

Effects of lasalocid on coccidial infection and on calf growth were examined in 16 Holstein bull calves. Calves were assigned randomly to a 2 x 2 factorial arrangement of starter ration containing 0 or 40 mg of lasalocid/kg of starter, beginning when calves were 3 days old (SE = 0.046), and single oral inoculation with 0 or 30,000 sporulated oocysts (Eimeria bovis) at 28 days. Pelleted calf starter was fed ad libitum from day 1; milk replacer was fed at a rate of 3.6 kg/d until day 28. Mean daily gain, dry-matter intake, and body weight were increased in calves fed lasalocid and decreased in those inoculated with coccidia. Addition of lasalocid to the feed improved gains by 8% in uninoculated calves and by 50% in inoculated calves. Fecal oocyst numbers were reduced when lasalocid was fed to inoculated calves. Feces were more abnormal in calves inoculated with coccidia. Respiration rates, rectal temperatures, PCV, and serum sodium and potassium concentrations were unaffected by treatment. On the basis of findings in this study, lasalocid minimized effects of coccidial challenge inoculation and increased growth of calves.

Structure-toxicity relationships for aminoalkanols: a comparison with alkanols and alkanamines.

The relative toxicity (log IGC50(-1)) of 49 selected aliphatic amines and aminoalkanols was evaluated in the static Tetrahymena pyriformis population growth impairment assay. Excess toxicity, indicated by potency greater than predicted for non-polar narcotic alkanols, was associated with both classes of test chemicals. Moreover, the aminoalkanols were found to be more toxic than the corresponding alkanamines. A high quality 1-octanol/water partition coefficient (log K(ow)) dependent quantitative structure-activity relationship (QSAR), logIGC50(-1) = 0.78 (log K(ow)) - 1.42; r2 = 0.934, was developed for alkanamines. This QSAR represented the amine narcosis mechanism of toxic action. No quality QSAR was developed for the aminoalkanols. However, several structure-toxicity features were observed for this class of chemicals. Two-amino-1-hydroxy derivatives being more toxic than the corresponding derivatives, where the amino and hydroxy moieties were separated by methylene groups. Hydrocarbon branching next to the amino moiety resulted in decreased toxicity. Aminoalkanol alters lipid metabolism in T. pyriformis.

Structure-toxicity relationships for alkanones and alkenones.

The relative toxicity (log IGC-1(50)) of 54 selected alkanones, both aliphatic and aromatic, as well as, alkenones and alkynones was evaluated in the static Tetrahymena pyriformis population growth assay. Excess toxicity, an indicator of bioreactivity, was associated only with the alpha-beta unsaturated alkenones and alkynones. Moreover, the alkynones were found to be more toxic than corresponding alkenones. A high quality 1-octanol/water partition coefficient (log Kow) dependent structure-toxicity relationship, log IGC-1(50) = 0.86 (log Kow) - 2.27; r2 = 0.955, was developed for alkanones. This QSAR represented the nonpolar narcosis mechanism of toxic action. Toxicity of alkenones was predicted by the highest-occupied-molecular-orbital energy (HOMO), log IGC-1(50) = -3.474 (HOMO) -35.357; r2 = 0.897, and the difference between HOMO and the lowest-unoccupied-molecular-orbital energy (LUMO), log IGC-1(50) = -3.559 (HOMO-LUMO gap) - 36.106; r2 = 0.903. The alpha-beta unsaturated ketones are considered soft electrophiles. Moreover, the toxicity of the aliphatic alkanones and alkenones was predicted by log Kow and LUMO, log IGC-1(50) = 0.69 (log Kow) - 2.55 (LUMO) + 0.05; r2 = 0.852.

Structure-toxicity relationships for aliphatic compounds encompassing a variety of mechanisms of toxic action to Vibrio fischeri.

QSARs based upon the logarithm of the octanol-water partition coefficient, log P, and energy of the lowest unoccupied molecular orbital, ELUMO were developed to model the toxicity of aliphatic compounds to the marine bacterium Vibrio fischeri. Statistically robust, hydrophobic-dependent QSARs were found for chloroalcohols and haloacetonitriles. Modelling of the toxicity of the haloesters and the diones required the use of terms to describe both hydrophobicity and electrophilicity. The differences in intercepts, slopes, and fit of these models suggest different electrophilic mechanisms occur between classes, as well as within the diones and haloesters. In order to model globally the toxicity of aliphatic compounds to V. fischeri, all the data determined in this study were combined with those determined previously for alkanones, alkanals, and alkenals. A highly predictive two-parameter QSAR [pT15 = 0.760(log P) - 0.625(ELUMO) - 0.466; n = 63, s = 0.462, r2 = 0.846, F = 171, Pr > F = 0.0001] was developed for the combined data that models across classes and is independent of mechanisms of action. The toxicity of these compounds to V. fischeri compares well to the toxicity (50% population growth inhibition) to the ciliate Tetrahymena pyriformis (r2 = 0.850).

Population growth impairment of sulfur-containing compounds to Tetrahymena pyriformis.

A series of 37 aliphatic and aromatic sulfur-containing compounds were evaluated in 2-day Tetrahymena pyriformis population growth impairment assay. The results indicate that, except for select compounds, the in-ring sulfur-containing compounds, sulfates, sulfites, thiols, sulfones, and sulfoxides model as neutral and noncovalent-reacting narcotics. Abiotic loss due to volatility appears to interfere in accurate model prediction because actual toxicity was less than predicted. Vinyl sulfones and sulfoxides are more toxic than predicted using neutral narcosis. Tetrahymena exposed to methyl vinyl sulfone exhibits a direct relationship between the exposure concentration and the generation times with no lag phase in growth. As such, these population growth kinetics mimic those reported for hydrophilic neutral narcotics (i.e., ethanol and acetone). Tetrahymena exposed to phenyl vinyl sulfone exhibit a long concentration-dependent lag phase, which is followed by population growth at rates not different from controls. These population growth kinetics are similar but more dramatic than those reported for hydrophobic neutral narcotics (i.e., butylbenzene and 2-decanone). These results are useful in that they imply that sulfur-containing compounds for the most part act as simple narcotics. Therefore, their toxicity can be modeled with simple structure-toxicity relationships without much fear of underestimation of potency.

Changes in blood glucose, nonesterified fatty acids, and ketones in response to weaning and feed intake in young calves.

Effects of weaning age on blood glucose, ketones, and nonesterified fatty acids were examined using 16 Holstein heifer calves. Animals entered the study at 6 +/- 3.5 d of age and were fed 1.8 kg of milk twice daily to 28 (early weaning) or 56 d (late weaning) and a commercial pelleted calf starter from 0 (early) or 28 d (late). Blood was sampled once weekly for 14 wk and analyzed for beta-hydroxybutyrate and acetoacetate. Plasma was analyzed for glucose and nonesterified fatty acids. Blood beta-hydroxybutyrate increased with increasing grain intake and was greater during wk 0 to 4 and 5 to 8 in calves weaned early than in those weaned late. Blood acetoacetate followed trends similar to beta-hydroxybutyrate and averaged 23.8 and 16.1 microM in calves weaned early and late, respectively. Plasma glucose and nonesterified fatty acids declined with age and were lower during 5 to 8 wk in calves weaned early. Data suggest that ketone concentrations resulted from alimentary ketogenesis, which increased rapidly after weaning.

Population growth kinetics and bulk membrane lipid alterations in Tetrahymena pyriformis: exposure to pentachlorophenol.

This study describes effects of exposure of the freshwater ciliate Tetrahymena pyriformis to the "classic" weak acid respiratory uncoupler pentachlorophenol (PCP) on the population growth kinetics and membrane lipid profiles. The assessment of growth kinetics of naive populations exposed to PCP, at concentrations eliciting < 50% growth inhibition, showed generation times of exposed cultures similar to generation times of controls but preceded by a short lag phase (< 2 h). Assessment of exposed cultures exhibiting > 50% growth inhibition revealed generation times that increased with increasing concentrations of toxicant. In addition, the relative percentages of selected fatty acid methyl esters (FAMEs) in both pellicle and mitochondrial membranes were examined. Upon exposure to PCP the relative percentages of FAMEs 12:0, 14:0, 16:0, 16:1, and 18:0 did not change. However, with exposure to PCP a decrease was observed for FAMEs 15:0 and 17:0. Conversely, with PCP exposure there was an increase in FAME 18:1. A comparison of these results with those elicited upon exposure to the model narcotic 1-octanol reveals marked differences in both growth kinetics and fatty acid shifts.

Parametrization of electrophilicity for the prediction of the toxicity of aromatic compounds.

The aim of this study was to determine which descriptor best parametrized the electrophilicity of aromatic compounds with regard to their acute toxicity. To achieve this, toxicity data for 203 substituted aromatic compounds containing a nitro- or cyano group were evaluated in the 40-h Tetrahymena pyriformis population growth impairment assay. Quantitative structure-activity relationships (QSARs) were developed relating toxic potency [log(IGC(50)(-1))] with hydrophobicity quantified by the 1-octanol/water partition coefficient (log P) and electrophilic reactivity quantified by the molecular orbital parameters, either the energy of the lowest unoccupied molecular orbital (E(LUMO)) or maximum acceptor superdelocalizability (A(max)) was developed. For the full data set, E(LUMO) and A(max) were collinear (r = 0.87). A comparison of the QSARs [log(IGC(50)(-1)) = 0.40 log P - 0.94E(LUMO) - 1.27; n = 203, r(2) = 0.60, s = 0.49, F = 151] and [log(IGC(50)(-1)) = 0.37 log P + 13.1A(max) - 4.30; n = 203, r(2) = 0.70, s = 0.42, F = 237] reveals A(max) to be the better electrophilic parameter for modeling these data. Analysis of outliers indicates a preponderance of 4-subsituted nitrophenols and nitroanilines. Smaller datasets (51 and 102 compounds) selected in order to reduce the collinearity between A(max) and E(LUMO) were also evaluated. Results indicate A(max) to be the superior descriptor of electrophilicity for the purpose of toxicological QSARs for aromatic compounds. Development of QSARs using partial least-squares yielded similar results.

Bioavailability, biodegradation, and acclimation of Tetrahymena pyriformis to 1-octanol.

Previous work has indicated that the ubiquitous freshwater ciliate Tetrahymena pyriformis acclimates to the presence of hydrophobic chemicals acting by nonpolar narcosis. Four explanations have been identified to explain this apparent acclimation: (1) genetic adaptation occurs resulting in a resistant population, (2) T. pyriformis quickly biodegrades hydrophobic chemicals resulting in a perceived acclimation response, (3) hydrophobic chemicals are not bioavailable, and (4) T. pyriformis contain an endogenous biochemical adaptation system which can quickly cause cellular changes resulting in acclimation. Results of biodegradation experiments indicated that the total extractable 1-octanol did not change over the duration of the experiments. Bioavailability experiments were performed using the solid-phase microextraction technique. Although there is a decrease in freely available concentrations of 1-octanol over a 2.5 log unit range of Tetrahymena population density, the freely available concentration is constant for the population densities used for population growth experiments. Genetic change is highly unlikely since acclimation occurs in less than the time required for one population division. It is hypothesized that the acclimation response seen in Tetrahymena results from partitioning of the chemical into the membrane followed by active changes in the membrane structure to restore homeostasis.