Comparative study on the metabolism of the ergot alkaloids ergocristine, ergocryptine, ergotamine, and ergovaline in equine and human S9 fractions and equine liver preparations.

1. Ergopeptine alkaloids like ergovaline and ergotamine are suspected to be associated with fescue toxicosis and ergotism in horses. Information on the metabolism of ergot alkaloids is scarce, especially in horses, but needed for toxicological analysis of these drugs in urine/feces of affected horses. The aim of this study was to investigate the metabolism of ergovaline, ergotamine, ergocristine, and ergocryptine in horses and comparison to humans. 2. Supernatants of alkaloid incubations with equine and human liver S9 fractions were analyzed by reversed-phase liquid-chromatography coupled to high-resolution tandem mass spectrometry with full scan and MS2 acquisition. Metabolite structures were postulated based on their MS2 spectra in comparison to those of the parent alkaloids. All compounds were extensively metabolized yielding nor-, N-oxide, hydroxy and dihydro-diole metabolites with largely overlapping patterns in equine and human liver S9 fractions. However, some metabolic steps e.g. the formation of 8'-hydroxy metabolites were unique for human metabolism, while formation of the 13/14-hydroxy and 13,14-dihydro-diol metabolites were unique for equine metabolism. Incubations with equine whole liver preparations yielded less metabolites than the S9 fractions. 3. The acquired data can be used to develop metabolite-based screenings for these alkaloids, which will likely extend their detection windows in urine/feces from affected horses.

Microbiomic comparison of the intestine of the earthworm Eisenia fetida fed ergovaline.

Tall fescue toxicosis and ergot alkaloids cost U.S. livestock producers approximately one billion dollars in annual livestock production loss annually. Ergovaline (EV) is the tall fescue alkaloid primarily responsible for clinical disease in livestock. Since native ruminal microorganisms have not been attributed to the detoxification of EV, finding detoxifying microbes from other environments is desirable. One possible source for potential microorganisms that can degrade EV is the anaerobic gut of the earthworm, Eisenia fetida. This study describes a comparative microbial analysis of earthworm digestive tracts receiving 10,000 ppb EV (E+ treatment) when compared with a control treatment with no detectable amounts of EV (E- treatment). An HPLC assay determined a 25% loss of EV from the E+ treatment was microbial in nature. A community microbiomic approach of constructing 16S-rRNA gene clone libraries was used to compare the microbes affected by the two treatments. RDPII tools such as Classifier and Libcompare were used in the analysis of 16S sequences. DOTUR analysis was used to examine the richness and diversity of the two microbial populations in these experiments. The results indicate there are few significant differences in the microbial community structure between the two microbiomes.

Ergot alkaloid mycotoxins: physiological effects, metabolism and distribution of the residual toxin in mice.

The complex ergot alkaloids, ergovaline and ergotamine, cause dysregulation of physiological functions, characterised by vasoconstriction as well as thermoregulatory and cardiovascular effects in grazing livestock. To assess the effect of the mycotoxins, blood pressure and heart rate of male mice were measured, and metabolite profiling undertaken to determine relative abundances of both ergotamine and its metabolic products in body and brain tissue. Ergotamine showed similar cardiovascular effects to ergovaline, causing elevations in blood pressure and reduced heart rate. Bradycardia was preserved at low-levels of ergovaline despite no changes in blood pressure. Ergotamine was identified in kidney, liver and brainstem but not in other regions of the brain, which indicates region-specific effects of the toxin. The structural configuration of two biotransformation products of ergotamine were determined and identified in the liver and kidney, but not the brain. Thus, the dysregulation in respiratory, thermoregulatory, cardiac and vasomotor function, evoked by ergot alkaloids in animals observed in various studies, could be partially explained by dysfunction in the autonomic nervous system, located in the brainstem.

Effects and mechanisms of action of the ergopeptides ergotamine and ergovaline and the effects of peramine on reticulum motility of sheep.

OBJECTIVE: To investigate effects and mechanisms of ergotamine and ergovaline and effects of peramine on reticulum motility of sheep. SAMPLE POPULATION: 3 sheep with indwelling electrodes in the reticulum and samples of reticulum collected from 126 sheep at an abattoir. PROCEDURES: In conscious sheep, motility was recorded as integrated electromyograms from the reticulum. Ergotamine was administered IV alone or in combination with the cholinergic muscarinic receptor antagonist atropine to sheep, and motility of the reticulum was assessed. In vitro, whole wall strips of the reticulum, cut in a direction to record longitudinal muscle activity via force transducers, were placed in 10-mL organ baths and superfused with Tyrode Ringer's solution at 37 degrees C and oxygenated with 95% oxygen and 5% carbon dioxide. Testing involved incubation of reticulum strips with ergotamine, ergovaline, and peramine and measurement of motility of the reticulum tissues. RESULTS: Administration of ergotamine to sheep reduced the frequency of reticulum contractions and increased baseline electromyographic activity (tonus). Frequency was unaffected by atropine, whereas tonus was significantly reduced. In vitro, ergotamine and ergovaline increased tonic contractions and stimulated phasic contractions of reticulum tissues and potentiated electrically stimulated contractions. Atropine and tetrodotoxin reduced tonic contractions, but stimulation of large-amplitude phasic contractions remained. Peramine had no effect on motility of reticulum tissues. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the study indicated that peripheral excitatory effects of the ergopeptides on motility of the reticulum appear to be mediated partly through myenteric neurons and muscarinic receptors and also through direct effects on the muscles.

Effects of endophyte-infected tall fescue seed and protein supplementation on stocker steers: II. Adaptive and innate immune function.

Fescue toxicosis is a multifaceted syndrome common in cattle grazing endophyte-infected tall fescue that affects performance; however, little information is available pertaining to its effects on immunity. Recently, it has been shown that supplemental CP can improve performance in weaned steers postvaccination. Thus, the objective of this study was to evaluate the effect of supplemental CP on innate and adaptive immune responses in stocker steers chronically exposed to ergovaline. Angus steers (n = 12 pens; 3 steers/pen) were stratified by weight and assigned to a 2 × 2 factorial arrangement to examine crude protein levels of supplement (14% or 18%) and ergovaline exposure (0 or 185 µg ergovaline/kg BW/d via ground endophyte-free (EF) or endophyte-infected (EI) tall fescue seed, respectively) on immune response. Consumption of low to moderate concentration of ergovaline from EI tall fescue seed was sufficient to induce mild symptoms associated with fescue toxicosis. Blood samples were collected at day 0, 42, and 56 to evaluate infectious bovine rhinotracheitis (IBR) and bovine viral diarrhea virus (BVDV) type 1b titers following vaccine challenge. Additionally, serum cytokine concentrations were evaluated using Quantibody Bovine Cytokine Arrays on day 0, 28, and 42. Data were analyzed using PROC MIXED of SAS with repeated measures. Regardless of treatment, no differences were observed in IBR and BVDV-1b seroconversion following vaccine challenge (P > 0.05). Regardless of crude protein concentration, EI steers had greater concentrations of proinflammatory cytokines (TNF-α, IFN-γ, IL-1α), chemokines (CCL2, CCL4, MIG), anti-inflammatory cytokines (IL-2, -13, -15, -21), and various growth factors (FGF-1, IGF-1, VEGF-A) when compared to EF steers (P < 0.05). Furthermore, VEGF-A and IGF-1 concentrations were greater in EI-14 steers on day 28 compared to EI-18, EF-14, and EF-18 steers (P < 0.05), however, this difference was not observed on day 0 or 42 (P > 0.05). Based on these data, steers exposed to ergovaline have an increase in pro- and anti-inflammatory cytokines and supplemental CP had minimal impact to mitigate this response. However, in the current study, exposure to ergovaline had little to no effect on adaptive immunity and response to vaccination. Together, chronic exposure to ergovaline results in a hyperactive innate immune response, which may lead to an immuno-compromised animal.

Effects of ergotamine and ergovaline on the electromyographic activity of smooth muscle of the reticulum and rumen of sheep.

OBJECTIVE: To investigate the effects of IV administration of ergotamine and ergovaline and intraruminal administration of ergotamine on electromyographic (EMG) activity of reticuloruminal smooth muscle in conscious sheep. ANIMALS: 3 sheep with indwelling electrodes in the musculature of the reticulum and rumen. PROCEDURE: In a crossover design study, reticuloruminal motility before and after IV administration of ergotamine (5, 10, 20, and 40 nmol/kg) or ergovaline (2.5, 5, and 10 nmol/kg) was evaluated; EMG effects were compared with those of corresponding control treatments (IV administration of saline [0.9% NaCl] solution or acetone, respectively) in sheep. Ergotamine (800 nmol/kg) or water was also administered intraruminally and their effects compared. RESULTS: After IV administration of ergopeptides, vagally dependent cyclical A and B sequences of contraction of the reticulorumen were immediately inhibited, preceding increases in baseline EMG activity (tonus). The return of cyclical contractions was associated with an increase in contraction amplitude. The effects were dose dependent; administration of 40 nmol of ergotamine/kg resulted in responses that continued for 3 to 4 hours. The effects of intraruminal administration of ergotamine were variable; after 8 hours, EMG activity was increased from baseline for < 2 hours in 1 sheep, 10 hours in another, and > 15 hours in the third. CONCLUSIONS AND CLINICAL RELEVANCE: In sheep, the effects of ergotamine and ergovaline on reticuloruminal motility after IV administration and the duration of responses following intraruminal administration suggest that disruption of digestion may occur in animals grazing endophyte-infected pasture that has a high ergopeptide content.

Effects of the potential antidepressant dihydroergosine in rats forced to swim: influence on plasma corticosterone.

The potential antidepressant properties of the ergot alkaloid dihydroergosine (DHESN) were studied in rats forced to swim in a restricted space. DHESN (50 mg/kg, intraperitoneally) reduced the duration of immobility in rats forced to swim 1 hr after administration. This effect was still present after 48 hr, but 7 hr. In non-swimming rats, the same dose of DHESN increased the plasma corticosterone concentration when administered 1 hr prior to sacrifice, but was without effect 48 hr after administration. In rats forced to swim, DHESN elicited two opposite effects. One hr following a single administration, it increased plasma secretion of corticosterone, whereas administered 48 hr prior to forced swimming, it decreased plasma corticosterone. These results, along with our previous data, give evidence that DHESN might possess antidepressant properties.

Effect of dihydroergosine (DHESN) on the serotoninergic system and behaviour: is DHESN a new antidepressive agent?

Acute (50.0 mg/kg) and repeated (0.1-10.0 mg/kg) administration of dihydroergosine (DHESN) to rats over 5 days lowered the concentration of 5-HIAA in the brain. DHESN given acutely increased the brain 5-HT in p-CPA-treated animals and diminished the probenecid-induced increase in brain 5-HIAA. In pargyline-treated rats DHESN enhanced the 5-HT/5-HIAA ratio. DHESN administered to rats repeatedly over 5 days decreased the level of 5-HT in blood platelets, and in vitro at concentrations of 10(-4) M and 10(-3) M inhibited the uptake of [14C]-5-HT in platelets. DHESN (10.0-100.0 mg/kg) potentiated the 5-HT syndrome produced in rats by pargyline and 5-HTP. This potentiation was blocked with cyproheptadine but not with haloperidol. DHESN (1.0 and 10.0 mg/kg) lowered the locomotor activity of rats and 10.0 mg/kg DHESN also reduced the duration of immobility in rats forced to swim in a restricted space. The results indicate that DHESN, like antidepressants, decreases the turnover of serotonin in the brain and potentiates the 5-HT-mediated behaviour. This might suggest that the drug should be further investigated for its potential antidepressive properties.

Serotoninergic neurons: long-lasting activation by a possible antidepressant dihydroergosine.

The duration of the stimulating effect of one single i.p. injection of a possible antidepressant dihydroergosine (50 mg/kg) on the 5-HT syndrome was studied in rats. Dihydroergosine produced a very pronounced stimulation of the 5-HT syndrome which was still present after 1-144 h (6 days). The results suggest that dihydroergosine produces a long-lasting stimulation of serotoninergic neurons.

Dihydroergosine: anticonflict effect in rats and enhancing effects on [3H]muscimol binding in the human brain post mortem.

The anticonflict activity of the ergot alkaloid, dihydroergosine, a drug which binds to 5-hydroxytryptamine1 (5-HT1) receptors and to gamma-aminobutyric acidA (GABAA) receptor-associated Cl- ionophore, was studied in water-deprived rats. In vitro effects of this drug on [3H]muscimol and [3H]flunitrazepam binding to the crude synaptosomal pellet of the human frontal cortex post-mortem were also investigated. Dihydroergosine, given 2 h prior to testing, enhanced drinking under punished (0.8 mA) conditions, and diminished it under unpunished conditions. The mechanism of this effect was (-)-propranolol- and pindolol-insensitive and picrotoxin-sensitive. Flumazenil either failed to affect, or at a higher dose (10 mg/kg), counteracted the dihydroergosine-induced enhancement of punished drinking. This dose of flumazenil was itself anxiogenic. Dihydroergosine had mild sedative and analgesic properties. Low concentrations of dihydroergosine (10 nM to 100 microM) enhanced the binding of [3H]muscimol but not of [3H]flunitrazepam. The results suggest that dihydroergosine may possess anxiolytic properties presumably mediated by its specific action at the GABA/benzodiazepine/chloride channel complex.

Dihydrogenated ergot compounds bind with high affinity to GABAA receptor-associated Cl- ionophore.

The binding of t-[3H]butylbicycloorthobenzoate ([3H]TBOB) to crude synaptosomal membranes of the mouse brain (cerebrum minus cortex) in the presence of dihydroergotoxine, dihydroergosine, dihydroergotamine and gamma-aminobutyric acid (GABA) was studied in vitro. [3H]TBOB binding was inhibited by all drugs used. The rank order of potency was dihydroergotoxine greater than GABA greater than dihydroergosine greater than dihydroergotamine. This suggests that dihydrogenated ergot compounds, especially dihydroergotoxine, possess appreciable binding activity (comparable to that of benzodiazepines and barbiturates) at the GABAA receptor-associated C1- ionophore.

Intima-related vasodilatation of the perfused rat caudal artery.

We examined the endothelial dependence of responses to ACh and some vasodilator drugs by using the central tail artery of the rat perfused with Krebs buffer. Perfusion with ACh (100 nM-100 microM) produced dose-dependent vasodilatation of arteries preconstricted with norepinephrine and antagonized pressor responses to periarterial electrical stimulation. Endothelium was removed by introducing a fine catheter through the lumen or a stream of gas (O2 95%-CO2 5%) intraluminally. Both procedures prevented the vasodilator effect of ACh. Gassing also abolished the vasodilatation in response to hydralazine 334 nM but not to equidilator amounts of papaverine 13 microM, or nitroglycerin 50 nM. These results indicating endothelial dependence of hydralazine and ACh responses are in accord with our previous studies on vascular rings.

Evidence that ergovaline acts on serotonin receptors.

Ergovaline is a potent vasoconstrictor of isolated bovine uterine and umbilical arteries. Contractions to ergovaline were slow to develop and required 120 min or more to reach their maximum effect. Tissues contracted to ergovaline did not relax over a 3 hr period despite repeated changing of the bath fluid with fresh Krebs solution. This indicates a high affinity of ergovaline for its tissue receptor. Contractions to ergovaline were antagonized by ketanserin and phenoxybenzamine but not by prazosin or phentolamine. This indicates that 5-HT2 receptors but not adrenergic receptors are involved in the vasoconstriction. The results suggest that drugs effective in blocking 5-HT2 receptors may be useful in preventing or reducing the toxic symptoms due to ergovaline in animals consuming Acremonium coenophialum infected tall fescue.

Do imipramine and dihydroergosine possess two components--one stimulating 5-HT1 and the other inhibiting 5-HT2 receptors?

The mechanisms by which imipramine and dihydroergosine stimulate the 5-HT syndrome in rats and inhibit the head-twitch response in rats and mice were studied. Imipramine- and dihydroergosine-induced stimulation of the 5-HT syndrome was inhibited stereoselectively by propranolol, a high affinity ligand for 5-HT1 receptor sites, but not by ritanserin, a specific 5-HT2 receptor antagonist. (-)-Propranolol potentiated the inhibitory effect of imipramine, but not of dihydroergosine on the head-twitch response, while ritanserin was without effect. Neither imipramine nor dihydroergosine were able to stimulate the 5-HT syndrome in the animals pretreated with p-chlorophenylalanine. As expected, 8-OH-DPAT, a selective 5-HT1A receptor agonist, stimulated, and 5-HT1B agonists CGS 12066B and 1-(trifluoromethylphenyl)piperazine (TFMPP) failed to stimulate the 5-HT syndrome induced in rats by pargyline and 5-HTP administration. A higher dose of ritanserin inhibited the syndrome. While 8-OH-DPAT alone produced all behavioral components of the 5-HT syndrome, dihydroergosine or imipramine alone even at very high doses never produced tremor or a more intensive forepaw padding as seen when these drugs were given in combination with pargyline and 5-HTP. A single administration of (-)-propranolol also inhibited the head-twitch response. This effect lasted in mice longer than after ritanserin administration. In in vitro experiments dihydroergosine expressed approximately twenty-fold higher affinity for 3H-ketanserin binding sites than imipramine. The results suggest that imipramine and dihydroergosine possess two components--one stimulating the 5-HT syndrome in rats by a presynaptic, presumably 5-HT1A-mediated mechanism, and the other inhibiting 5-HT2 binding sites.

Behavioural evidence for simultaneous dual changes of 5-HT receptor subtypes: mode of antidepressant action?

Effects of the classic antidepressant imipramine and of an imipramine-like potential antidepressant dihydroergosine were studied in mice, rats and guinea pigs using behavioural models associated with the activation of 5-HT2 and 5-HT1 receptors respectively. Both drugs given in a single dose inhibited the 5-HT2 mediated behaviour for 24 and 48 h respectively and simultaneously stimulated 5-HT1 mediated behaviour for 6 days. Blockade of 5-HT2 receptors could have reduced their inhibitory influence on 5-HT1 receptors. We propose that the interplay between the two receptor subtypes controls the serotoninergic transmission. This idea throws a new light on the mode of action of antidepressants.

Species dependent dual modulation of the benzodiazepine/GABA receptor chloride channel by dihydroergosine.

Dihydroergosine (50 and 100 mg/kg) enhanced the incidence of bicuculline (3 mg/kg)-induced convulsions in female rats, while 100 mg/kg of dihydroergosine given to female mice made 45% convulsive dose of bicuculline (2.5 mg/kg) to be subconvulsive. The same dose of dihydroergosine enhanced in mice the latency of bicuculline (4 mg/kg)-induced convulsions. Although, in in vitro experiments dihydroergosine showed very weak ability to prevent the binding of 3H-muscimol, the drug was able to diminish and to augment the IC50 of bicuculline and GABA when added to crude synaptosomal pellet of the rat and mouse brain respectively. Lower concentrations of dihydroergosine stimulated and higher inhibited 3H-TBOB binding to the crude synaptosomal pellet of the rat brain. In the preparation of mouse brain dihydroergosine produced only inhibition of 3H-TBOB binding. Only slight quantitative differences were observed in bicuculline-induced stimulation and in GABA- and diazepam-induced inhibition of 3H-TBOB binding between the two species. The results suggest that the opposite species-dependent effects of dihydroergosine on bicuculline-induced convulsions are due to the ability of this drug to modulate species-dependently the benzodiazepine/GABA receptor chloride channel complex.

Monoamine sensitivity of smooth muscle in vivo in nociception disorders.

A significant degree of supersensitivity to 5-HT and DA was detected when carrying out the computerized venotest on migraine patients during an attack. A similar supersensitivity was observed during morphine abstinence and naloxone-precipitated withdrawal in addicts. Mild abstinence after slight and short morphine treatment provoked monoamine supersensitivity in volunteers. In these conditions, the administration of morphine inhibited the 5-HT and DA supersensitivity. In spontaneous central panalgesia, monoamine supersensitivity is detectable, as well as in panalgesia induced in headache sufferers by means of PCPA 5-HT deprivation. By means of the venotest, the ergot derivatives were confirmed as being partial 5-HT agonists. These drugs can also carry out their therapeutic activity by potentiating 5-HT at a central level in 5-HT-deficient neurons. The presence of opiate receptors in the human vein is stressed. The high supersensitivity of the venous smooth muscle to 5-HT and DA both in headache and systemic pain sufferers and during morphine withdrawal suggests a pathophysiological analogy between these conditions.

Inhibitory influence of dihydroergosine on the aggressiveness of rats and mice.

Fifty mg/kg of ergot alkaloid dihydroergosine (DHESN) inhibited the mouse-killing behavior of isolated male rats, while 10 mg/kg did not. This effect of DHESN (50 mg/kg) lasted for 24 hr. When an additional injection of DHESN (50 mg/kg) was given to mouse-killer rats 6 days following the first, the mouse-killing behavior was again inhibited. The effect of the second drug injection also persisted for 24 hr and was accompanied by an increased concentration of 5-HT in the raphe nuclei and hypothalamus and by a decreased concentration of GABA in the olfactory bulbs. DHESN also inhibited aggressiveness in isolated mice. Two hr following the administration of 10 mg/kg DHESN the fighting was inhibited in 46% of pairs tested, while 50 mg/kg abolished it completely. The effect of 50 mg/kg lasted 24 hr. These results, showing the antiaggressive effects of DHESN, support our previous suggestion that DHESN might presumably be a new antidepressant, and suggest that besides the serotoninergic, the GABA-ergic system might also be involved in the modification of behavior induced by this drug.

Antagonistic effects of simultaneous exposure of ergot alkaloids on kidney adenosine triphosphatase system.

Much of the research on fescue toxicosis has concentrated on evaluating animal response to grazing endophyte-infected (E+) versus endophyte-free tall fescue or the effects of single toxins such as ergonovine (EN), ergovaline (EV), or ergotamine (ET) on animal performance. Such approaches have eliminated the opportunity to test the possible additive, synergistic, or antagonistic interactions of one or more ergot alkaloids with the other ergot alkaloids found in E+ tall fescue. This study was conducted to determine the effects of simultaneous exposure of pairs of EN, EV, and ET on the kidney adenosine triphosphatase (ATPase) system in vitro. Tests were performed using three separate rat kidney homogenates and were repeated four times at concentrations of 0, 75, and 200 microM. Individually, EN, EV, and ET induced dose-dependent inhibitions of kidney Na(+)/K(+) ATPase, with EN being most potent, followed by purified EV, and then by ET. The ergot alkaloids inhibited Mg(2+) ATPase to a lesser degree than Na(+)/K(+) ATPase, with EN again being the most potent toxin. Simultaneous exposure to any combination of the ergot alkaloid pairs tested (EV + ET, EV + EN, and ET + EN) resulted in significant interactions (P < 0.05), indicating antagonistic effects on the inhibition of Na(+)/K(+) ATPase and Mg(2+) ATPase for most concentration combinations. These interactions suggest that in studies of the effects of any ergot alkaloid on animal performance, effects of other ergot alkaloids may also be present. Effects may not be additive, as was the case in this study, and the presence of one toxin may enhance or hinder the effectiveness of others.

Ergovaline toxicity on Caco-2 cells as assessed by MTT, alamarBlue, and DNA assays.

The exact mechanisms of fescue toxicity in animals have yet to be established, but it has been associated with an inability to thrive. Ergovaline is the major ergopeptine alkaloid associated with fungal infections of tall fescue. Gastrointestinal (GI) toxicity of ergovaline (10(-11) to 10(-4) M) was evaluated in Caco-2 cells (mimicking the GI epithelium) beginning on days 1, 8, and 18 of culture. Acute and chronic toxicity was assessed after 24 and 72 h of exposure. Treatment periods were chosen to study undifferentiated, semidifferentiated, and completely differentiated cells. Cell loss and metabolic activity were assessed by thiazolyl blue reduction (3-(4,5-dimethylthiozole-2-yl)-2,5,-biphenyl tetrazolium bromide [MTT], mitochondrial succinate dehyrdogenase activity), alamarBlue assay (cytochrome oxidase activity), and deoxyribonucleic acid (DNA) quantitation. Undifferentiated cells were sensitive to 1 x 10(-4) M ergovaline after acute exposure (from 52 to 74% of control values depending on assay). After 72 h of exposure to 1 x 10(-4) M ergovaline, in all three assays, treatment means were reduced to approximately 10% of the control means. By day 11 in culture, ergovaline toxicity to cells had decreased. With 24 h exposure, an apparent paradoxical increase in MTT was seen at some concentrations. This increase in MTT was also found in fully differentiated cells (day 21), whereas alamarBlue activity decreased. No change in DNA was found until 72 h of exposure, when DNA was reduced approximately 12% over most concentrations. These findings indicate differentiation state-dependent sensitivity of Caco-2 cells to ergovaline, potential problems of the MTT assay as an indicator of cellular toxicity, and usefulness of alamarBlue assay over DNA assay for toxicity assessment.