Forms and prevalence of intersexuality and effects of environmental contaminants on sexuality in cricket frogs (Acris crepitans).

Cricket frogs (Acris crepitans) from several different sites in Illinois were collected to assess the effects of environmental contamination on the prevalence of intersex gonads. Of 341 frogs collected in 1993, 1994, and 1995, 2.7% were intersex individuals. There was no statistically significant relationship between the chemical compounds detected and cricket frog intersexuality. However, there was an association approaching significance (p = 0.07) between the detection of atrazine and intersex individuals. A comparison of reference sites with sites that had point polychlorinated biphenyl (PCB) and polychlorinated dibenzofuran (PCDF) contamination revealed a significant relationship between sex-ratio reversal and contamination with PCBs and PCDFs. The sex ratio of juvenile frogs studied from three sites with PCB and PCDF point contamination favored males over females, which was the opposite of the sex ratio in control ponds (p = 0.0007). The statistically significant correlation between organochlorine contamination and sex-ratio reversal suggests PCBs and PCDFs can influence cricket frog sexual differentiation. The current study suggests that in cricket frogs, sex ratios and the prevalence of intersex gonads are altered by environmental contamination.

Electromyographic assessment of the neuromuscular blockade produced in vivo by anatoxin-a in the rat.

The indirectly evoked compound action potentials (ECAP) of the plantar muscles of the rat were used to investigate the pharmacodynamics in vivo of the neuromuscular blockade produced by anatoxin-a. Onset time to maximum depression and the magnitude of maximum depression in amplitude of the ECAP were dose-dependent. The mean maximum percent depression (+/- S.D.) of the ECAP induced by single, supramaximal stimulations of the posterior tibial nerve after i.v. doses of (+)anatoxin-a hydrochloride at 0, 50, 100, 200 and 800 micrograms/kg were 3 (4), 53 (15), 82 (7), 95 (2), and 100 (1), respectively. The ED50 (95% confidence limits) for depression of the ECAP was 47 mg/kg (39-57 micrograms/kg). Rats administered 200 micrograms/kg or less of (+)anatoxin-a hydrochloride had 75% return of the pretoxin amplitude of the ECAP within 93 min. Animals dosed at 800 micrograms/kg did not have return of neuromuscular function and died despite mechanical ventilation, suggesting a lethal mechanism(s) of action in addition to respiratory paralysis. Percent decrements (+/- S.D.) in the amplitude of the fourth ECAP following repetitive stimulation at 10 Hz were 6 (5), 13 (22), 46 (18) and 59 (8) from (+)anatoxin-a hydrochloride given i.v. at 0, 50, 100 and 200 micrograms/kg, respectively. The decrement observed following repetitive stimulation was attributed to a presynaptic site of action. No change in maximal motor nerve conduction velocity or latency of the ECAP was observed after i.v. administration of (+)anatoxin-a hydrochloride at 100 micrograms/kg. LD50 values (95% confidence limits) for anatoxin-a administered i.v. to mice were 386 micrograms/kg (365-408 micrograms/kg, for (+)anatoxin-a hydrochloride and 913 micrograms/kg (846-985 micrograms/kg) for racemic anatoxin-a hydrochloride. No deaths were observed in mice after i.p. administration of (-)anatoxin-a hydrochloride at doses up to 73 mg/kg.

Assessment of potential therapies for acute T-2 toxicosis in the rat.

The efficacy of a variety of approaches for the treatment of animals with acute T-2 toxicosis was assessed utilizing young female rats. A single large dose of the water soluble salt of methylprednisolone significantly prolonged survival times in T-2 toxin treated animals. The use of diltiazem hydrochloride, dazemgrel, N-acetylcysteine, dimethyl sulfoxide, adenosine triphosphate (ATP), ATP combined with magnesium chloride, ascorbic acid, and aprotinin did not prolong survival times at the dosages administered. Trichodermin, a trichothecene similar in structure and biochemical activity to T-2 toxin but much less acutely toxic, had a detrimental effect on survival times whether given 1 hr prior to or after T-2 toxin.

Improved method for purification of toxic peptides produced by cyanobacteria.

The improved method consists of ODS-silica gel extraction, and separation on silica gel and HPLC with UV (238 nm) detector. The method has been successfully applied to the isolation of toxic peptides from the Monroe and M-228 strains of Microcystis aeruginosa. This method reduces toxin extraction and separation time, and so enables a rapid isolation of peptide toxins from cyanobacteria.

Isolation and characterization of the minor components associated with microcystins LR and RR in the cyanobacterium (blue-green algae).

Two structurally similar analogues of microcystins LR and RR, cyclic peptide hepatotoxins from Microcystis, were isolated by chromatographic methods. Although they have the same mol. wt and amino acid compositions as those of the parent toxins, they do not possess similar toxicities. Ultraviolet and 1H-NMR spectral data for both components demonstrate clear structural difference of these cyclic peptides from the parent toxins, which are probably responsible for the marked decreases in their observed toxicities.

Uptake and subcellular localization of tritiated dihydro-microcystin-LR in rat liver.

Microcystin-LR (MC-LR), a cyclic heptapeptide hepatotoxin (mol. wt = 994) produced by the blue-green alga (cyanobacterium), Microcystis aeruginosa, was reduced with tritium labeled sodium borohydride, converted to [3H]-dihydro-microcystin-LR ( [3H]-2HMC-LR), and purified to greater than 99% purity by C-18 reverse-phase high-performance liquid chromatography. The uptake and subcellular distribution of [3H]-2HMC-LR were determined in suspensions of hepatocytes at 0 degrees C and 37 degrees C, or following rifampicin pretreatment, and in perfused rat liver. The remaining cells were homogenized and subfractionated using sucrose gradient centrifugation. Suspensions of 7.5 x 10(6) hepatocytes also were incubated with 10 micrograms/ml of toxin, solubilized in Triton X-100, and ultracentrifuged to pellet the detergent insoluble fraction (containing actin). Isolated rat livers were perfused with media containing [3H]-2HMC-LR and the uptake of radiolabel was determined. Sequential biopsy samples were collected for histologic examination. The remaining liver was homogenized and subcellular fractions prepared. Uptake of radiolabel was rapid in both cell suspension at 37 degrees C and perfused liver; however, uptake in cell suspensions was reduced by about 50% at 0 degrees C and by rifampicin (50 micrograms/ml) pretreatment. Hepatocyte necrosis was observed in isolated perfused livers 45 min after initiation of perfusion with [3H]-2HMC-LR. In both hepatocyte suspensions and perfused livers 65 to 77% of the radiolabel was in the cytosolic fraction. In the hepatocyte suspensions, 13 to 18% of the radiolabel was present in the plasma membrane/nuclear fraction with lesser amounts in the other fractions. Trichloroacetic acid treatment of cytosolic fractions indicated that in hepatocyte suspensions, 50-60% of the radiolabel was bound to cytosolic protein. Studies using the perfused liver confirmed that the majority of the radiolabeled MCLR (78-88%) was bound to cytosolic protein. These data suggest that the uptake of [3H]-2HMC-LR occurs primarily by an energy-dependent transport process involving the rifampicin-sensitive hepatic bile acid carrier and that once inside the hepatocyte, the toxin binds to a cytosolic protein(s).

Distribution of tritiated dihydromicrocystin in swine.

The distribution of tritiated dihydromicrocystin [3H]2H-MCLR was studied in anesthetized specific-pathogen-free pigs. Two doses were administered i.m. and one dose was given via an isolated ileal loop. At 4 hr after i.v. administration of the toxin at 25 micrograms/kg, 64.6% of the total dose (%TD) was located in the liver, with smaller amounts distributed to the kidneys (1.2% TD), lungs (1.75% TD), heart (0.22% TD), ileum (0.13% TD) and spleen (0.04% TD). A similar distribution was found at 4 hr postdosing in pigs given 75 micrograms/kg, although the liver contained a lower fraction of the total dose, at 46.99% TD, and the kidneys had somewhat more, at 2.19% TD, than the low dose. At the high dose, the fractions of the amount given accounted for by the lungs (0.55% TD), heart (0.23% TD), ileum (0.20% TD) and spleen (0.07% TD) were similar to those at the low dose. The livers of the pigs given 75 micrograms/kg via the ileal loop, at 5 hr postdosing, contained 49.5% TD and the ileum had 33.94% TD. Smaller amounts were distributed to kidneys (1.04% TD), lungs (0.65% TD), heart (0.81% TD) and spleen (0.16% TD). The livers of both groups dosed at 75 micrograms/kg contained higher concentrations of toxin, but lower percentages of the total dose, than the livers of pigs dosed at 25 micrograms/kg. Larger increases in serum arginase in the two 75 micrograms/kg groups were associated with histological evidence of more severe liver damage than at the 25 micrograms/kg dose. Analysis of radiolabeled compounds from hepatic tissue using fast atom bombardment mass spectrometry determined that the primary constituent was [3H]2H-MCLR, but two minor radioactive components were also isolated. These findings indicate that [3H]2H-MCLR is rapidly concentrated in the liver of swine, whether given i.v. or via an isolated ileal loop, that at extremely toxic doses uptake is slowed, and that it is as toxicologically active as the parent compound.

Toxicokinetics of tritiated dihydromicrocystin-LR in swine.

The toxicokinetics of tritiated dihydromicrocystin-LR ([3H]2H-MCLR) were studied in anesthetized, specific-pathogen-free pigs. Pigs were dosed with radiolabeled plus non-labeled 2H-MCLR at 25 or 75 micrograms/kg i.v., or via an isolated ileal loop at 75 micrograms/kg. The i.v. doses were rapidly removed from the blood. At either i.v. dose, more than half the radiolabel from [3H]2H-MCLR present in the blood at 1 min postdosing was cleared by 6 min. The blood clearance at the 75 micrograms/kg dose was slower than at the 25 micrograms/kg dose. Accordingly, at the high dose, the concentrations of the toxin in blood were disproportionately higher from 10 min after dosing until the study ended 4 hr later. The decreased clearance is presumably due to decreased elimination from the blood as a consequence of the hepatic injury that was observed histologically. Following administration of [3H]2H-MCLR at 75 micrograms/kg via the ileum, the maximal toxin concentration in blood was achieved at 90 min after dosing. At that time the [3H]2H-MCLR concentration in portal venous blood was 3.6 times higher than in peripheral venous blood. Although bile production varied, following i.v. dosing radioactivity was detected in bile as early as 12 min postdosing in one animal. This study demonstrated that [3H]2H-MCLR is rapidly removed from the blood of anesthetized swine and that excretion of the radiolabel into bile may begin within 30 min of dosing.

Comparison of effects of anatoxin-a(s) and paraoxon, physostigmine and pyridostigmine on mouse brain cholinesterase activity.

Anatoxin-a(s), an alkaloid neurotoxin from the freshwater cyanobacterium, Anabaena flos-aquae NRC-525-17, was compared to paraoxon, physostigmine and pyridostigmine for effects on brain cholinesterase after i.p. injection into Balb/c mice. The duration of clinical signs in mice injected with anatoxin-a(s) persisted longer than in mice given the carbamates and was comparable with that of paraoxon. Anatoxin-a(s) did not inhibit brain cholinesterase activity suggesting that this toxin is unable to cross the blood-brain barrier.

A new procedure for the analysis and purification of naturally occurring anatoxin-a from the blue-green alga Anabaena flos-aquae.

Anabaena flos-aquae produces at least two neurotoxins termed anatoxin-a (ANTX-a) and -a(s) [ANTX-a(s)]. ANTX-a is a potent postsynaptic depolarizing neuromuscular blocking agent. Its structure is the secondary amine, 2-acetyl-9-azabicyclo[4.2.1]non-2-ene. Known isolation and analysis methods have not always given satisfactory results. Therefore, an improved procedure was developed for both isolation and analysis of ANTX-a which involves four steps; extraction, clean-up, separation and determination. Extraction was efficiently done with 0.05 M acetic acid, and a reversed phase. ODS (octadecylsilanized) and a cation exchanger (COOH) organosilans bonded to silica gels were applied to a clean-up step. Separation and determination were then performed using two TLC and HPLC chromatographies.

Structural modifications imparting reduced toxicity in microcystins from Microcystis spp.

A cyanobacterial (blue-green algal) bloom containing Microcystis aeruginosa (dominant), M. viridis, and M. wesenbergii, was collected from Homer Lake (Illinois, U.S.A.) in the summer of 1988 and microcystins were isolated. One microcystin of substantially reduced toxicity was isolated, together with ten hepatotoxic microcystins. The compound with reduced toxicity was nonlethal at 1 mg/kg (i.p. mouse) and was determined to have a (C3H7O2) mono-ester of the alpha-carboxyl on the Glu unit of microcystin-LR. The other nine microcystins apart from MCLR had approximate LD50S ranging from 97 micrograms/kg to 750 micrograms/kg.

Pharmacokinetics of the trichothecene mycotoxin, T-2 toxin, in swine and cattle.

The pharmacokinetics of the trichothecene mycotoxin, T-2 toxin, were determined in growing gilts and heifers. Following intra-aortal administration in swine and intravenous administration in calves, the disappearance of the parent T-2 toxin followed a 2-compartment open model. Mean elimination phase half-lives were 13.8 and 17.4 min and mean apparent specific volumes of distribution were 0.366 and 0.376 l/kg in swine and calves, respectively. The fraction of T-2 toxin eliminated as parent compound in the urine was negligible. In spite of administration of a lethal oral dose in swine (2.4 mg/kg) and toxic oral doses (up to 3.6 mg/kg) in calves, no parent T-2 toxin was detected in plasma or urine. After intra-aortal administration in swine, tissue concentrations of T-2 toxin were consistently highest in lymphoid organs. Tissue residues of T-2 toxin were rapidly depleted such that, in spite of administration of a potentially lethal intra-aortal dose, no quantifiable T-2 toxin was present in any of the tissues collected at 4 hr after dosing. No T-2 toxin could be detected in liver, even at 1 hr after dosing.

Clinical, biochemical, electrophysiologic, and histologic assessment of chlorpyrifos induced delayed neuropathy in the cat.

The ability of a supralethal dose of chlorpyrifos to produce delayed neuropathy was examined using assessments of clinical signs, electromyography (EMG), motor nerve conduction velocity (MNCV), lymphocyte neuropathy target esterase activity (LNTE), and histologic changes in nervous system tissues. Cats were exposed to a single, im injection of corn oil (vehicle control), DFP (positive control) at 5.0 mg/kg, or chlorpyrifos at 300 mg/kg and observed for 60 days. Atropine and 2-PAM were administered to chlorpyrifos exposed cats one to two times a day for 14 to 24 days in response to the appearance of cholinergic signs. Anorectic cats during the acute toxicosis were force fed by hand and hydration was maintained by administering fluids sc. Onset of ataxia (mean +/- SD) for the positive control and chlorpyrifos exposed cats were 16.2 +/- 1.8 days (range of 14-19 days) and 19.0 +/- 1.4 days (range of 17-21 days), respectively. Functional deficits for both groups were confined to the hindlimbs and characterized by a crouched-waddling gait, hypermetria, and proprioceptive deficits. Maximal inhibition of LNTE activity was 96% at 24 hr postdosing in the positive control group and 46% at 7 days postdosing in the chlorpyrifos group. No EMG or MNCV abnormalities were detected in any of the treatment groups. Axonal degeneration was similar for the positive control and chlorpyrifos exposed cats. Ascending tracts of the cervical spinal cord and descending tracts of the thoracic and lumbar spinal cord were most severely affected and peripheral nerves were only mildly affected. The clinical and histologic effects produced indicate that chlorpyrifos can cause delayed neuropathy in the domestic cat. The moderate but prolonged inhibition of LNTE produced by chlorpyrifos is atypical of classic organophosphorus delayed neurotoxicants.

Regional brain cholinesterase activity in rats injected intraperitoneally with anatoxin-a(s) or paraoxon.

Adult male Long-Evans rats were injected intraperitoneally with 1.5, 3.0 or 9.0 micrograms/kg of anatoxin-a(s) that had been extracted from laboratory-grown Anabaena flos-aquae NRC-525-17, 800 micrograms/kg of paraoxon, or a control solution. Blood, anterior spinal cord, and brain cerebellar, cortical, medullary, midbrain, hippocampal, hypothalamic, olfactory and striatal cholinesterase activity was determined in rats that died prior to 2 hours or were anesthetized and killed at 2 hours. Unlike paraoxon, anatoxin-a(s) did not cause detectable inhibition of cholinesterase in the central nervous system, but did cause inhibition of cholinesterase in blood, suggesting that anatoxin-a(s) is strictly a peripheral cholinesterase inhibitor.

Effect of anatoxin-a(s) from Anabaena flos-aquae NRC-525-17 on blood pressure, heart rate, respiratory rate, tidal volume, minute volume, and phrenic nerve activity in rats.

The effects of anatoxin-a(s) [antx-a(s)] from the cyanobacterium Anabaena flos-aquae NRC-525-17 on mean arterial blood pressure, heart rate, respiratory rate, tidal volume, minute volume, and phrenic nerve activity were evaluated in anesthetized Sprague-Dawley rats. Anatoxin-a(s) was administered by continuous intravenous infusion. The initial effect of the toxin was to slow the heart rate and reduce arterial blood pressure, followed by much more pronounced reductions in these parameters. The marked decline in heart rate and blood pressure frequently occurred before there was a large decrease in respiratory minute volume [reduced by only 15.4 +/- 3% (mean +/- S.E.) compared to the predose period], suggesting that antx-a(s) has an important muscarinic action on the cardiovascular system in vivo. Phrenic nerve amplitude increased, but, nevertheless, tidal and minute volumes decreased progressively, indicating that antx-a(s), unlike most low-molecular-weight organophosphorus cholinesterase inhibitors, does not have any remarkable inhibitory action on central mediation of respiration.

Toxicity of intraperitoneal doses of microcystin-LR in two strains of male mice.

Male Balb/C and Swiss Webster (SW) mice were administered various i.p. doses of microcystin-LR (MCLR) to establish dose-response curves and to determine if a sublethal dose of MCLR would protect against an approximate LD100 min given 2 or 3 days later. Micocystin-LR has an extremely steep dose-lethal response curve in BC mice--LD50 = 32.5 micrograms (micrograms)/kg, approximate LD0 max = 25 micrograms/kg and approximate LD100 min = 40 micrograms/kg. Liver weights increased 64% (BC) and 51% (SW) and kidney weights increased 32% (BC) and 20% (SW) within 200 minutes following administration of an approximate LD100 min of MCLR in naive mice. Grossly and histologically the marked increase in liver weight appeared to be caused primarily from intrahepatic hemorrhage and death is probably a result of hemorrhagic shock. Twenty-four hours following administration of a sublethal dose of MCLR to naive BC mice, liver weights were increased significantly (8.7%), but no clinical signs or histologic lesions were observed. In SW mice, administration of a LD23 of MCLR resulted in significantly increased survivability and survival times when an approximate LD100 min of MCLR was given 3 days later. Survivors of the LD23/LD100 min regimen had 96 hour postdosing liver weights not significantly different from those of mice which died acutely after the same hepatotoxin treatments. These survivors showed weakness, recumbency, anorexia, and icterus, and had marked gross liver lesions. Histologically these lesions were undergoing rapid reparative processes.

Microcystin-LR decreases hepatic and renal perfusion, and causes circulatory shock, severe hypoglycemia, and terminal hyperkalemia in intravascularly dosed swine.

Cross-bred, anesthetized female swine were given intravascularly a lethal (72 microg/kg; n = 6) or toxic-sublethal (25 microg/kg; n = 6) dose of microcystin-LR (MCLR), from Microcystis aeruginosa, or the vehicle (n = 4). At the high dose, from 12 to 18 min after administration, central venous pressure and hepatic perfusion were significantly lower, and shortly thereafter, portal venous pressure was significantly higher and aortic mean pressure was significantly lower than controls. By 45 min postdosing, serum bile acids, lactate, potassium, and total bilirubin, as well as blood pO2, were significantly higher, while hematocrit, platelet count, and blood bicarbonate, pCO2, and base excess were significantly lower than controls. By 90 min, serum arginase, urea nitrogen, inorganic phosphorus, and creatinine were significantly higher, while glucose and blood pH were significantly lower than in controls. By 150 min, serum alanine and aspartate aminotransferases, alkaline phosphatase, lactate dehydrogenase, and creatinine phosphokinase activities were significantly higher than controls. At the low dose, significant differences from controls occurred in hemodynamic, organ perfusion, and serum chemistry parameters, but such changes generally took longer to occur and were of a lesser magnitude than at the high dose. Livers of the high-dose swine were swollen and dark red-purple, and exuded excessive blood on the cut surface. Based on increases in liver weight and liver hemoglobin, 38% of the total blood volume was lost into the liver. Terminally, all high-dose swine experienced hyperkalemia, and most had severe hypoglycemia. Death due to acute MCLR toxicosis in intravascularly dosed swine appears to result from severe intrahepatic hemorrhage, partial obstruction of blood flow through the liver, circulatory shock, severe hypoglycemia, and/or terminal hyperkalemia.

Chemical study of the hepatotoxins from Microcystis aeruginosa collected in California.

Four cyclic peptide toxins were purified and quantified from the aqueous extract of algal cell material utilizing high performance liquid chromatography, thin layer chromatography, and fast atom bombardment mass spectrometry. The cyclic peptide toxins appear to be similar structurally to hepatotoxins from previously identified blooms of the blue-green alga Microcystis aeruginosa.

Neutral red assay modification to prevent cytotoxicity and improve reproducibility using E-63 rat skeletal muscle cells.

Cellular uptake of neutral red dye (NR) is currently used as an indirect measure of viable cells in cultures. We used E-63 rat skeletal muscle cells to identify causes of NR assay variability and to develop modifications that substantially reduce it. Three methods of NR preparation and/or addition to cells were used. When NR medium was prepared, incubated overnight, and filtered to remove precipitates, the amount of dye precipitated varied greatly. Coefficients of variation (CVs) in NR uptake were greater than 25% between assays. Higher NR concentrations, longer incubation times, increased pH, and decreased temperature promoted NR precipitation in media. NR media prepared and filtered just prior to use or direct addition of prefiltered NR stock solution to cell cultures resulted in much smaller CVs between assays. NR was cytotoxic to E-63 rat muscle and primary quail myoblasts in a time- and concentration-dependent manner. NR exposure to E-63 cells for greater than 1.25 and 2 hr at 157 or 127 microg/ml, respectively, was associated with swelling and rupture of lysosomes. By contrast, there was no evidence of cytotoxicity when E-63 cells were exposed to NR for 1 hr at either 127 or 157 microg/ml. Primary quail myoblasts developed lysosomal swelling and ruptured more rapidly than E-63 cells when exposed to NR at either 127 or 157 microg/ml. For confluent 10-day cultures of E-63 cells exposed to NR at 127 microg/ml for 1 hr, the CVs within assay and between assays were 3.3-3.9% and 5.1%, respectively. For similarly exposed, actively replicating 3-day cultures of E-63 cells, the CVs within and between assays were 6.2-9.6% and 2.4%, respectively. NR uptake by the E-63 cells was linear with respect to viable cell number.

Black walnut (Juglans nigra) toxicosis: a model for equine laminitis.

Twelve light horse geldings developed laminitis within 8 to 12 h of being dosed by nasogastric tube with an aqueous extract of black walnut (Juglans nigra). Four of the 12 horses developed the severe signs of grade 3 laminitis (lame at a walk, refused to lift feet). Laminitis was accompanied by mild depression and limb oedema. There was no evidence of shock or colic. The horses developed neutropenia by 4 h after dosing with the extract, which shifted to a relative neutrophilia by 8 to 12 h. Minimal increases in plasma epinephrine and cortisol concentrations were suggested in severely affected horses. Severe laminitis was characterized by necrosis of dermal tips of dorsal primary epidermal laminae. A proliferative epithelial response in these laminae was distinguished by numerous mitotic figures and clusters of epithelial cells. This evidence suggests that black walnut toxicosis is not only a consistent clinical model, but is also a reliable clinico-pathological and pathological model for study of the pathogenesis and treatment of laminitis.