Cytogenotoxicity assessment of 3-(3,4-dihydroxyphenyl)-7,8-dihydroxycoumarin on HepG2/C3A cells and leukocytes.

3-(3,4-Dihydroxyphenyl)-7,8-dihydroxycoumarin is a newly synthesized coumarin derivative with a potent antioxidant effect. The aim of the present study is to investigate the safety of this compound, determining the in vitro cytotoxic and genotoxic in human peripheral blood mononuclear cells (PBMC) and in HepG2/C3A cells. Cell viability has been investigated by the trypan blue staining test and MTT assay and the genotoxicity by the comet assay and micronucleus test, using concentrations between 0.01 and 10 µg/ml. The compound proved to be noncytotoxic in both cell lines, at all tested concentrations, protecting the cells from the DNA damage. In addition, this molecule does not show clastogenic/aneugenic effects when performing the micronucleus test with cytokinesis blockade. Based on the obtained data, and the conditions of the experiments, we can conclude that the 3-(3,4-dihydroxyphenyl)-7,8-dihydroxycoumarin is a safe molecule up to a concentration of 10 µg/ml, which encourages further studies aiming to explore its potential as a drug candidate.

Evaluation of ThT augmentation and RLS inner filter effect caused by highly fluorescent coumarin derivative and establishing it as true inhibitor of amyloid fibrillation.

Screening of inhibitors that slow down or suppress amyloid fibrils formation relies on some simple but sensitive spectroscopy techniques. Thioflavin T (ThT) fluorescence assay is one of the most common, amyloid specific and sensitive method. However, if an inhibitor is itself fluorescent in the ThT fluorescence range, its screening becomes complicated and require complementary assays. One of such molecules, 6, 7-dihydroxycoumarin (6, 7-DHC, also known as aesculetin, esculetin, and cichorigenin) is fluorescent in the ThT emission range and absorbs in the ThT excitation range. Therefore, it can produce a subtractive effect attributed to primary inner filter effect and/or additive effect due to its self-fluorescence in ThT assay. Our study shows that 6, 7-DHC produces an additive effect in ThT fluorescence, which is minimized at high concentration of ThT and decrease in ThT fluorescence is solely due to its inhibitory effect against HSA fibrillation. These ThT fluorescence-based results are verified through other complementary assays, such as Rayleigh and dynamic light scattering and amyloid-specific Congo red binding assay. Furthermore, hydrophobicity reduction is studied through Nile red (NR) and kinetics through far-UV circular dichroism (far-UV CD) in place of the most commonly employed ThT assay owing to extremely high fluorescence of 6, 7-DHC during initial incubation period.

Icariside I specifically facilitates ATP or nigericin-induced NLRP3 inflammasome activation and causes idiosyncratic hepatotoxicity.

BACKGROUND: Epimedii Folium (EF) is commonly used for treating bone fractures and joint diseases, but the potential hepatotoxicity of EF limits its clinical application. Our previous study confirms that EF could lead to idiosyncratic drug-induced liver injury (IDILI) and hepatocyte apoptosis, but the mechanism remains unknown. Studies have shown that NLRP3 inflammasome plays an important role in the development of various inflammatory diseases such as IDILI. Specific stimulus-induced NLRP3 inflammasome activation may has been a key strategy for lead to liver injury. Therefore, main compounds derived from EF were chosen to test whether the ingredients in EF could activate the NLRP3 inflammasome and to induce IDILI. METHODS: Bone-marrow-derived macrophages (BMDMs) were treated with Icariside I, and then stimulated with inflammasome stimuli and assayed for the production of caspase-1 and interleukin 1ß (IL-1ß) and the release of lactate dehydrogenase (LDH). Determination of intracellular potassium, ASC oligomerization as well as reactive oxygen species (ROS) production were used to evaluate the stimulative mechanism of Icariside I on inflammasome activation. Mouse models of NLRP3 diseases were used to test whether Icariside I has hepatocyte apoptosis effects and promoted NLRP3 inflammasome activation in vivo. RESULTS: Icariside I specifically enhances NLRP3 inflammasome activation triggered by ATP or nigericin but not SiO2, poly(I:C) or cytosolic LPS. Additionally, Icariside I does not alter the activation of NLRC4 and AIM2 inflammasomes. Mechanically, Icariside I alone does not induce mitochondrial reactive oxygen species (mtROS), which is one of the critical upstream events of NLRP3 inflammasome activation; however, Icariside I increases mtROS production induced by ATP or nigericin but not SiO2. Importantly, Icariside I leads to liver injury and NLRP3 inflammasome activation in an LPS-mediated susceptibility mouse model of IDILI, but the effect of Icariside I is absent in the LPS-mediated mouse model pretreated with MCC950, which is used to mimic knockdown of NLRP3 inflammasome activation. CONCLUSIONS: Our study reveals that Icariside I specifically facilitates ATP or nigericin-induced NLRP3 inflammasome activation and causes idiosyncratic hepatotoxicity. The findings suggest that Icariside I or EF should be avoided in patients with diseases related to ATP or nigericin-induced NLRP3 inflammasome activation, which may be risk factors for IDILI. Video abstract.

Umbelliferone (7-hydroxycoumarin): A non-toxic antidiarrheal and antiulcerogenic coumarin.

Gastrointestinal diseases are very common problems; available treatments are very limited and come with a range of side effects. Coumarins are an extensive class of phenolic compounds that can be found in plants, fungi and bacteria. The 7-hydroxycoumarin, also known as umbelliferone (UMB), is a compound that comes from coumarin and has been showing biological activities in other studies. As of this scenario, the present study was designed to evaluate the acute oral toxicity, mutagenic, antidiarrheal, anti-bacterial, and antiulcerogenic effects, and antioxidant capacity of UMB. An investigation was conducted through the hippocratic screening method and through histopathological analysis in animals to evaluate the effects of acute oral administration of a dose of 50, 100 and 200 mg/kg of UMB. A micronucleus test on peripheral blood of Swiss mice, which were orally treated with three doses (50, 100 and 200 mg/kg), was conducted to evaluate mutagenic activities. The antiulcerogenic activity was accomplished through the ethanol-induced damage method. Antidiarrheal activities were tested for inducing diarrhea with castor oil and evaluating intestinal transit duration; additionally, the antimicrobial effect against some enteropathogenic bacteria was analyzed. Finally, the antioxidant capability was determined by the capacity of the UMB sample to kidnap the stable radical 2,2-diphenyl-1-picrylhydrazyl. Of the evaluated doses, signs of toxicity after acute administration of the compound were not observed. UMB presented antiulcerogenic activity (100 and 200 mg/kg), which was explained because of its antioxidant capacity. A gastro protective effect was similar to the positive control, and the UMB was able to significantly reduce intestinal transit, and also diarrheal symptoms. Furthermore, UMB had an anti-bacterial effect with minimum inhibitory concentration fluctuating between 62.5 and 1000 µg/mL. Based on these findings, we can suggest that UMB has important biological activities in vivo and in vitro and is not toxic under the evaluated circumstances, which demonstrates its large potential for pharmacological use.

Transmission blocking sugar baits for the control of Leishmania development inside sand flies using environmentally friendly beta-glycosides and their aglycones.

BACKGROUND: The sand fly Lutzomyia longipalpis is the main vector of American visceral leishmaniasis, a disease caused by parasites of the genus Leishmania. Adults of this insect feed on blood (females only) or sugar from plant sources, but their digestion of carbohydrates is poorly studied. Beta-glycosides as esculin and amygdalin are plant compounds and release toxic compounds as esculetin and mandelonitrile when hydrolyzed. Beta-glucosidase and trehalase are essential enzymes in sand fly metabolism and participate in sugar digestion. It is therefore possible that the toxic portions of these glycosides, released during digestion, affect sand fly physiology and the development of Leishmania. RESULTS: We tested the oral administration to sand flies of amygdalin, esculin, mandelonitrile, and esculetin in the sugar meal. These compounds significantly decreased the longevity of Lutzomyia longipalpis females and males. Lutzomyia longipalpis adults have significant hydrolytic activities against esculin and feeding on this compound cause changes in trehalase and ß-glucosidase activities. Female trehalase activity is inhibited in vitro by esculin. Esculin is naturally fluorescent, so its ingestion may be detected and quantified in whole insects or tissue samples stored in methanol. Mandelonitrile neither affected the amount of sugar ingested by sand flies nor showed repellent activity. Our results show that mandelonitrile significantly reduces the viability of L. amazonensis, L. braziliensis, L. infantum and L. mexicana, in a concentration-dependent manner. Esculetin caused a similar effect, reducing the number of L. infantum and L. mexicana. Female L. longipalpis fed on mandelonitrile had a reduction in the number of parasites and prevalence of infection after seven days of infection with L. mexicana, either by counting in a Neubauer chamber or by qPCR assays. CONCLUSIONS: Glycosides have significant effects on L. longipalpis longevity and metabolism and also affect the development of parasites in culture and inside the insect. These observations might help to conceptualize new vector control strategies using transmission blocking sugar baits.

Molecular Engineering of α-Substituted Acrylate Ester Template for Efficient Fluorescence Probe of Hydrogen Polysulfides.

In this article, hydrogen polysulfide (H2Sn)-mediated Michael addition/cyclization cascade reactions toward acrylate ester analogues were exploited and utilized to construct novel and robust H2Sn-specific fluorescence probe for the first time. Through rational molecular engineering of the α-substituted acrylate ester template, the optimal candidate probe FP-CF3 containing trifluoromethyl-substituted acrylate ester group as recognition unit and 3-benzothiazol-7-hydroxycoumarin dye BHC as signal reporter can highly selectively detect H2Sn over other reactive sulfur species, especially biothiols including cysteine (Cys) and homocysteine (Hcy)/glutathione (GSH), with a rapid and significant turn-on fluorescence response (less than 60 s for response time and over 44-fold for signal-to-background ratio). The fast response and high selectivity of FP-CF3 for H2Sn could be attributed to a kinetically and spatially favored pentacyclic addition produced by the dual nucleophilic reaction of H2Sn with the CF3-substituted acrylate group. The big off-on fluorescence response is due to the pentacyclic intermediate results in the release of the highly fluorescent BHC. Moreover, it has been successfully applied in imaging of endogenous H2Sn fluctuation in living cells.

Phytotoxicity of umbelliferone and its analogs: Structure-activity relationships and action mechanisms.

Two coumarins, umbelliferone and daphnoretin, were isolated from roots of Stellera chamaejasme L; the former had been identified as one of the main allelochemicals in our previous studies. Both of them have the skeleton of 7-hydroxycoumarin, but showed different phytotoxic effects. Umbelliferone and its analogs were then prepared to investigate the structure-activity relationship of hydroxycoumarins and screened for phytotoxicity. The inhibitory effects varied observably in response to the coumarin derivatives, especially umbelliferone (1), 7-hydroxy-4-methylcoumarin (3) and coumarin (10) displayed strong inhibition of lettuce and two field weeds, Setaria viridis and Amaranthus retroflexus, and compounds 11 and 12 also exhibited phytotoxic activity with species specificity. The number and location of hydroxyl groups were importantly responsible for the phytotoxicity. A C7 hydroxyl group was considered to be a potentially active site and methyl substitution at the C4 position contributed significantly to the activity. The phytotoxic mechanism was briefly studied with umbelliferone by evaluating the reactive oxygen species (ROS) and chlorophylls level in lettuce seedlings. The results showed that umbelliferone induced the accumulation of ROS in the root tip and significantly decreased the chlorophyll content in the leaves. Thus, a ROS-mediated regulation pathway and the inhibition of photosynthesis were definitely involved in the phytotoxicity of umbelliferone.

In vitro assessment of mutagenic and genotoxic effects of coumarin derivatives 6,7-dihydroxycoumarin and 4-methylesculetin.

Coumarins are naturally occurring compounds, widely distributed throughout the plant kingdom (Plantae), and possess important pharmacological properties, including inhibition of oxidative stress. In this context, newly synthesized coumarin compounds are being produced due to their potent antioxidant activities. Therefore, the aim of the present study was to determine the in vitro cytotoxic, mutagenic, and genotoxic effects of 6,7-dihydroxycoumarin (6,7-HC) and 4-methylesculetin (4-ME) using the Salmonella/microsome test and in cultured human lymphocytes the comet assay and micronucleus test. The three coumarin derivatives concentrations evaluated in comet and MN assays were 2, 8, and 32 µg/mL, selected through a preliminary trypan blue-staining assay. In the Ames test, the 5 concentrations tested were 62.5, 125, 250, 500, and 750 µg/plate. Positive (methyl methane-sulfonate, MMS) and negative (dimethyl sulfoxide, DMSO) control groups were also included in the analysis. Our results showed that 4-ME induced greater cytotoxicity at high concentrations than 6,7-HC. In addition, both compounds were not mutagenic in the Ames test and not genotoxic or clastogenic/aneugenic in cultured human lymphocytes.

Inducible protection of human astrocytoma 1321N1 cells against hydrogen peroxide and aldehyde toxicity by 7-hydroxycoumarin is associated with the upregulation of aldo-keto reductases.

Reactive oxygen species (ROS) and consequent aldehydic lipid peroxidation products have been identified as significant in the progression of neurodegenerative diseases such as Alzheimer's and Parkinson's. Understanding and enhancing endogenous cellular protection against oxidants and aldehydes is therefore of interest in developing strategies to combat these diseases. In this study the role of the aldo-keto reductases AKR7A2 and AKR1C3 in protecting human astrocytoma 1321N1 cells against oxidant and aldehyde toxicity was investigated using siRNA gene silencing. Results show that both enzymes are responsible for part of the intrinsic protection against aldehydes and oxidants. Treating cells with sub-lethal concentrations of oxidant or aldehyde stress or with the natural coumarin 7-hydroxycoumarin (umbelliferone) revealed that endogenous resistance to aldehydes and oxidants can be induced significantly. The basis of the inducible protection by 7-hydroxycoumarin was shown to be associated with induction of the aldo-keto reductases AKR7A2 (1.5-fold) and AKR1C (3-fold), and this inducible protection was sufficient to overcome siRNA silencing of AKR1C3. These results indicate the importance of AKR family members in the detoxication of aldehydes, and also show that the natural phytochemical 7-hydroxycoumarin is a potential therapeutic candidate for neurodegenerative diseases.

Induction of apoptosis by esculetin in human leukemia U937 cells: roles of Bcl-2 and extracellular-regulated kinase signaling.

In the present study, we reported that apoptosis induced by esculetin, a phenolic compound with apoptotic activity in cancer cells, was markedly blocked by Bcl-2-overexpression, but restored by HA14-1, a small-molecule Bcl-2 inhibitor, in human leukemic U937 cells. The combined use of esculetin and HA14-1 effectively induced Bid cleavage and loss of mitochondrial membrane potential (MMP, Deltapsi(m)) leading to the activation of caspases and cleavage of poly(ADP-ribose) polymerase (PARP) in Bcl-2-overexpressing (U937/Bcl-2) cells. Combined treatment with esculetin and HA14-1 upregulated the expression of death receptor 4 (DR4), and activation of extracellular-regulated kinase (ERK) in a time-dependent manner. In addition, esculetin and HA14-1-mediated apoptosis was reduced by ERK inhibitors through inhibition of DR4 expression, suggesting that the synergistic effect was at least partially mediated through ERK-dependent induction of DR4 expression. The results indicate that HA14-1-induced reversal of the anti-apoptotic effect of Bcl-2 confers apoptosis sensitivity to esculetin by a mitochondrial amplification step and through the ERK-dependent induction of DR4 expression in U937/Bcl-2 cells. Thus, HA14-1 reversal of Bcl-2-mediated esculetin resistance suggests a novel strategy for increasing esculetin sensitivity in Bcl-2-overexpressing leukemia cells.

Eicosanoids mediate melanotic nodulation reactions to viral infection in larvae of the parasitic wasp, Pimpla turionellae.

Nodulation is the quantitatively predominant insect cellular immune function activated in response to bacterial, fungal and some viral infections. We posed the hypothesis that parasitoid insects express melanotic nodulation reactions to viral challenge and that eicosanoids mediate these reactions. Treating fifth-instar larvae of the ichneumonid endoparasitoid Pimpla turionellae with Bovine Herpes Simplex Virus-1 (BHSV-1) induced nodulation reactions in a challenge dose-dependent manner. Experimental larvae treated with the cyclooxygenase inhibitor, indomethacin, the lipoxygenase inhibitor, esculetin, and the phospholipase A2 inhibitor, dexamethasone, resulted in severely impaired nodulation reactions to our standard BHSV-1 challenge dose. The immunoinhibitory influence of dexamethasone was reversed in larvae reared on culture medium amended with arachidonic acid, the fatty acid precursor of eicosanoid biosynthesis. Larvae that had been reared on media amended with indomethacin, esculetin, or dexamethasone were also compromised in their nodulation reactions to viral challenge. The influence of the orally administered pharmaceutical was expressed in a dose-dependent manner. Finally, wasp larvae reared in the presence of indomethacin and dexamethasone expressed significantly decreased levels of phenoloxidase activity in response to viral challenge. These findings draw attention to the idea that endoparasitoid insects express cellular immune reactions to viral challenge; they also support our hypothesis that eicosanoids mediate nodulation reactions to viral challenge in these highly specialized insects.

A study of the role of cell cycle events mediating the action of coumarin derivatives in human malignant melanoma cells.

6-Nitro-7-hydroxycoumarin (6-NO2-7-OHC) and 3,6,8-trinitro-7-hydroxycoumarin (3,6,8-NO2-7-OHC) have previously been shown to be potent and selective anti-proliferative agents to the human skin cell line, SK-MEL-31. Here, we investigate the reversibility of their cytotoxicity, along with their effects on DNA synthesis and cell cycle events. Comparative studies were carried out using the main metabolite of coumarin in man, 7-hydroxycoumarin (7-OHC). 6-NO2-7-OHC and 3,6,8-NO2-7-OHC, were found to be irreversible cytotoxic agents, unlike 7-OHC. All three derivatives inhibited DNA synthesis, but 7-OHC was only nitro-derivatives which acted in an irreversible manner. Flow cytometric studies demonstrated that both nitro-derivatives caused a dose- and time-dependant S phase accumulation. 7-OHC exerted a similar effect, but appeared to be less potent. Finally, the two nitro-derivatives caused a dose-dependant inhibition of the S phase regulatory protein, cyclin A. Consequently, these and other nitro-derivatives of 7-OHC may represent novel therapeutic agents for the treatment of malignant melanoma as they are capable of selective and irreversible cytotoxicity.

Evaluation of the developmental toxicities of coumarin, 4-hydroxycoumarin, and 7-hydroxycoumarin using FETAX.

The developmental toxicities of coumarin and hydroxycoumarin metabolites were evaluated using FETAX. Young X. laevis embryos were exposed to coumarin, 4-hydroxycoumarin, and 7-hydroxycoumarin in each of two separate concentration-response experiments with and without an exogenous metabolic activation system (MAS) and/or inhibited MAS. The MAS was treated with carbon monoxide (CO), cimetidine (CIM), or ellipticine (ELL) to selectively modulate cytochrome P-450 activity. The MAS was also treated with cyclohexene oxide (CHO) to selectively modulate epoxide hydrolase activity. Without the MAS or inhibited MAS, coumarin and 7-hydroxycoumarin were nearly equitoxic, whereas 4-hydroxycoumarin was nearly 2-fold less developmentally toxic than coumarin on an equimolar basis. Addition of the MAS and CIM-MAS increased the developmental toxicities of coumarin and, particularly, 4-hydroxycoumarin. Addition of the CHO-MAS greatly increased the developmental toxicity of coumarin and, especially, 4-hydroxycoumarin. Addition of the ELL- or CO-inhibited MAS did not increase the developmental toxicity of coumarin. However, addition of the intact MAS did not alter the developmental toxicity of 7-hydroxycoumarin. Results from these studies suggested that P-450; specifically ELL-inhibited P-450 (arylhydrocarbon hydroxylase) may have been responsible for increasing the developmental toxicity of coumarin. Furthermore, the increased toxicity of coumarin or 4-hydroxycoumarin following co-incubation with CHO-treated microsomes indicated that highly toxic epoxide intermediates may be produced from oxidative P-450 metabolism and that epoxide hydrolase may play a role in detoxification of the reactive intermediates.

Effects of selected organophosphate insecticides on serum cholinesterase isoenzyme patterns in the rat.

The serum cholinesterase (ChE) isoenzyme in rats shows 6 bands after polyacrylamide gradient gel electrophoresis. The effects of organophosphates (fenthion, chlorpyrifos, diazinon, bromophos, propaphos, haloxon, and DFP) on serum ChE isoenzyme bands were studied in 32 male and 32 female 6-w-old Sprague-Dawley rats. Each organophosphate was randomly administered to 4 male and 4 female rats. Blood samples were collected from the abdominal aorta under halothane anesthesia 6 h after dosing. The isoenzyme patterns were determined simultaneously with erythrocyte and serum ChE activities. Changes were observed in all 6 bands of the serum ChE isoenzymes after administration of fenthion, chlorpyrifos and propaphos. Diazinon had no influence on band 6, and DFP and bromophos had no influence on band 5. Haloxon did not effect any of the serum ChE isoenzyme bands. Serum ChE was most suppressed by fenthion, followed by DFP, bromophos, chlorpyrifos, propaphos and diazinon in that order of effect. Serum ChE activity was not suppressed by haloxon. Erythrocyte ChE activity was suppressed by every organophosphate. This experiment demonstrated a correlation between the organophosphate suppression of serum ChE activity and the concentration of serum ChE isoenzyme band 6.

Metabolism and toxicity of coumarin on cultured human, rat, mouse and rabbit hepatocytes.

We compared the cytotoxic effect of coumarin and its derivatives, 7-hydroxycoumarin (7-OHC), 4-hydroxycoumarin (4-OHC), o-hydroxyphenyl acetic acid (OHPAA) and o-coumaric acid (CA), on cultured hepatocytes from human, rat, mouse and rabbit liver. At 10(-5) and 5 x 10(-5) M, coumarin and its derivatives did not give rise to any signs of toxicity on cultured hepatocytes of the four species. At 10(-4) M, coumarin, but not its derivatives, induced release of lactate dehydrogenase (LDH) into the medium, especially in rat hepatocyte cultures. Intracellular LDH activities were correspondingly reduced. The cytotoxic effect of coumarin in cultured rat hepatocytes was evidenced on morphological examination and from the results of the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium (MTT) reduction test. At higher concentrations (5 x 10(-4) M), 7-OHC and CA were also found to be cytotoxic in cultured rat hepatocytes. The cytotoxic effect of coumarin (5 x 10(-4) M) was decreased in the presence of SKF 525-A, a cytochrome P450 inhibitor. Interspecies comparisons showed that rat hepatocytes were the most sensitive to the toxicity of coumarin and its derivatives, whereas human hepatocytes were the most resistant. Our results suggest that the cytotoxicity of coumarin is metabolism and species-dependent. Thus, the rat may not be a suitable model for evaluating the pharmacological hazards of coumarin in humans.

Toxicity of coumarin (1,2-benzopyrone) on human peripheral blood mononuclear cells and human and murine bone marrow progenitor stem cells.

Coumarin (1,2-benzopyrone), in combination with cimetidine, has been subjected to separate clinical trials for the treatment of advanced renal cell carcinoma, malignant melanoma, and non-small cell lung cancer. While objective tumor regressions were observed only in renal carcinoma, no symptomatic or organ dysfunction toxicity was observed in any of the trials. The purpose of the present in vitro study was to determine the concentrations of coumarin and 7-hydroxycoumarin (7-HC) that would be toxic to human peripheral blood mononuclear cells (PB-MNC) and human and murine bone marrow (GM) progenitor stem cells. Coumarin was nontoxic for PB-MNC in concentrations up to 100 micrograms/ml. Concentrations of coumarin or 7-HC greater than or equal to 200 micrograms/ml produced significant suppression of human marrow GM stem cell activity. Coumarin greater than or equal to 25 micrograms/ml produced suppression of murine marrow GM stem cell activity. Differences in human and murine marrow sensitivity probably reflect interspecies differences in metabolism of coumarin. Correlations between toxic concentrations in vitro and maximally achievable serum concentration in vivo in humans await the results of further clinical trials.

Anti-inflammatory and peripheral analgesic activity of esculetin in vivo.

Esculetin determined a reduction in oedema and granulocyte infiltration in the Croton oil ear test in vivo. The drug was able to inhibit Acethylcholine-writhing test and showed a LD50 of 1450 mg/kg i.p. and greater than 2000 mg/kg by mouth. In our experimental conditions, esculetin has an anti-inflammatory and analgesic effect.

Haloxon-induced delayed neurotoxicity: effect of plasma A (aryl) esterase activity on severity of lesions in sheep.

A markedly different response to the delayed neurotoxic effects of a single dose of 400 or 800 mg/kg of the organophosphorus anthelmintic haloxon was observed in two populations of sheep. Animals with a gentically determined low level of activity of the plasma enzyme A (aryl) esterase developed clinical signs of delayed neurotoxicity within one month. Lesions relating to degeneration of myelinated nerve fibers were seen in brain stem, spinal cord, and on occasion peripheral nerve. The incidence of clinical signs, and severity of lesions as determined by semiquantitative morphological study of the spinal cord, were greater in animals given the higher dose. Neither clinical signs nor lesions relating to organophosphate-induced delayed neurotoxicity were seen in sheep with high plasma activity of A esterase.

Consideration of dose levels for delayed neurotoxicity testing in hens: the relationship of neurotoxic dosages to acute LD50 values.

The oral LD50 of a compound in hens does not reliably predict the neurotoxic dose of that compound. The oral LD50 in rats does not predict either the oral LD50 in hens or the neurotoxic dose in hens, so there is no justification for using the rat value to predict the neurotoxic dosage in hens. A strategy is presented for a stepwise approach to neurotoxicity testing in hens.

Somatosensory-evoked response of ataxic Angora goats in suspected haloxon-delayed neurotoxicity.

Somatosensory-evoked responses (SER) were recorded in 3 Angora goats with moderate posterior paresis. The cause of posterior paresis was unknown. The goats had recently been given haloxon and had also been grazing in a pasture with carpetweed (Kallstroemia hirsutissima). Haloxon and carpetweed have each been reported to cause posterior paresis in livestock. An electrical stimulus applied subcutaneously in the pelvic limb of the affected goats failed to evoke an SER at the cerebral cortex. Electrical stimulation in the thoracic limb evoked a normal-appearing SER. The absence of SER from pelvic limb stimulation indicated a functional impairment of the somatosensory pathways.