Preclinical Characterization of an Antibody-Drug Conjugate Targeting CS-1 and the Identification of Uncharacterized Populations of CS-1-Positive Cells.

Multiple myeloma is a hematologic cancer that disrupts normal bone marrow function and has multiple lines of therapeutic options, but is incurable as patients ultimately relapse. We developed a novel antibody-drug conjugate (ADC) targeting CS-1, a protein that is highly expressed on multiple myeloma tumor cells. The anti-CS-1 mAb specifically bound to cells expressing CS-1 and, when conjugated to a cytotoxic pyrrolobenzodiazepine payload, reduced the viability of multiple myeloma cell lines in vitro In mouse models of multiple myeloma, a single administration of the CS-1 ADC caused durable regressions in disseminated models and complete regression in a subcutaneous model. In an exploratory study in cynomolgus monkeys, the CS-1 ADC demonstrated a half-life of 3 to 6 days; however, no highest nonseverely toxic dose was achieved, as bone marrow toxicity was dose limiting. Bone marrow from dosed monkeys showed reductions in progenitor cells as compared with normal marrow. In vitro cell killing assays demonstrated that the CS-1 ADC substantially reduced the number of progenitor cells in healthy bone marrow, leading us to identify previously unreported CS-1 expression on a small population of progenitor cells in the myeloid-erythroid lineage. This finding suggests that bone marrow toxicity is the result of both on-target and off-target killing by the ADC.

The enhanced immunopharmacology of VIB4920, a novel Tn3 fusion protein and CD40L antagonist, and assessment of its safety profile in cynomolgus monkeys.

BACKGROUND AND PURPOSE: Inhibition of the T- and B-cell interaction through the CD40/CD40 ligand (L) axis is a favourable approach for inflammatory disease treatment. Clinical studies of anti-CD40L molecules in autoimmune diseases have met challenges because of thromboembolic events and adverse haemostasis. VIB4920 (formerly MEDI4920) is a novel CD40L antagonist and Tn3 fusion protein designed to prevent adverse haemostasis and immunopharmacology. We evaluated the pharmacokinetics, activity and toxicity of VIB4920 in monkeys. EXPERIMENTAL APPROACH: Cynomolgus monkeys received i.v. or s.c. 5-300 mg·kg-1 VIB4920 or vehicle, once weekly for 1 month (Studies 1 and 2) or 28 weeks (Study 3). VIB4920 exposure and bioavailability were determined using pharmacokinetic analyses, and immune cell population changes via flow cytometry. Pharmacological activity was evaluated by measuring the animals' capacity to elicit an immune response to keyhole limpet haemocyanin (KLH) and tetanus toxoid (TT). KEY RESULTS: VIB4920 demonstrated linear pharmacokinetics at multiple doses. Lymphocyte, monocyte, cytotoxic T-cell and NK cell counts were not significantly different between treatment groups. B-cell counts reduced dose-dependently and the T-cell dependent antibody response to KLH was suppressed by VIB4920 dose-dependently. The recall response to TT was similar across treatment groups. No thromboembolic events or symptoms of immune system dysfunctionality were observed. CONCLUSIONS AND IMPLICATIONS: VIB4920 demonstrated an acceptable safety profile in monkeys. VIB4920 showed favourable pharmacokinetics, dose-dependent inhibition of a neoantigen-specific immune response and no adverse effects on immune function following long-term use. Our data support the use of VIB4920 in clinical trials.

Talaromycosis (Penicilliosis) in a Cynomolgus Macaque.

A sexually mature Chinese-origin female Macaca fascicularis assigned to the high-dose group in a 26-week toxicology study with an experimental immunomodulatory therapeutic antibody (a CD40 L antagonist fusion protein) was euthanized at the scheduled terminal sacrifice on study day 192. The animal was healthy at study initiation and remained clinically normal throughout the study. On study day 141, abnormal clinical pathology changes were found during a scheduled evaluation; splenomegaly was detected on study day 149 and supported by ultrasound examination. At the scheduled necropsy, there was marked splenomegaly with a nodular and discolored appearance. Cytologic examination of a splenic impression smear revealed yeast-like organisms within macrophages. Histologically, there was disseminated systemic granulomatous inflammation with 2- to 3-µm oval, intracytoplasmic yeast-like organisms in multiple organs identified as Talaromyces (Penicillium) marneffei. This organism, not previously reported as a pathogen in macaques, causes an important opportunistic infection in immunosuppressed humans in specific global geographic locations.

Pharmacological enhancement of endocannabinoid signaling reduces the cholinergic toxicity of diisopropylfluorophosphate.

Diisopropylfluorophosphate (DFP) elicits cholinergic toxicity by inhibiting acetylcholinesterase, leading to accumulation of the neurotransmitter acetylcholine and excessive stimulation of cholinergic receptors throughout the body. Endocannabinoids inhibit the release of neurotransmitters including acetylcholine via a widely distributed retrograde signaling pathway. Endocannabinoid signaling is therefore a potential therapeutic target for the management of OP poisoning. We first evaluated the relative in vitro and in vivo (2.5mg/kg, sc) effects of DFP on cholinesterase, fatty acid amide hydrolase (FAAH, an endocannabinoid degrading enzyme), monoacylglycerol lipase (MAGL, another endocannabinoid degrading enzyme) and cannabinoid receptor (CB1) binding in rat hippocampus. The effects of WIN 55212-2 (cannabinoid receptor agonist, 1.5mg/kg), URB597 (FAAH inhibitor, 3mg/kg), URB602 (MAGL inhibitor, 10mg/kg) or AM404 (endocannabinoid uptake inhibitor, 10mg/kg) on DFP toxicity were then examined. Adult male rats were given either peanut oil or DFP followed immediately by vehicle or one of the four cannabinomimetic drugs. Functional signs of toxicity were evaluated for 24h and then rats were sacrificed for neurochemical measurements. DFP inhibited cholinesterase, FAAH, MAGL and CB1 receptor binding in vitro in a concentration-dependent manner, with highest and lowest potency against cholinesterase and FAAH, respectively. In vivo, DFP inhibited hippocampal cholinesterase (89%) and FAAH (42%), but had no significant effect on MAGL or CB1 binding. Rats treated with DFP alone showed typical signs of cholinergic toxicity including involuntary movements and excessive secretions (SLUD signs). WIN 55212-2, URB597, URB602 and AM404 all significantly reduced involuntary movements following DFP exposure in a time-dependent manner, and most (URB597, URB602 and AM404) also significantly reduced DFP-induced SLUD signs. These results suggest that enhancing endocannabinoid signaling can attenuate the acute toxicity of DFP and provide rationale for further investigations on the role of endocannabinoids in cholinergic toxicity.

Selective inhibition of butyrylcholinesterase in vivo in horses by the feed-through larvacide Equitrol.

The organophosphate insecticide tetrachlorvinphos (TCVP, Rabon) is the active ingredient in "feed-through" larvacides (e.g., Equitrol) for fly control around horse stables. As with other organophosphates, TCVP elicits toxicity by inhibiting acetylcholinesterase, leading to accumulation of the neurotransmitter acetylcholine and cholinergic signs. Relatively little is known, however, on the effects of TCVP-containing larvacides on acetylcholinesterase or other esterases in horses. Previous in vitro studies indicated that horse plasma cholinesterase activity was substantially (>10,000-fold) more sensitive than erythrocyte cholinesterase activity to inhibition by TCVP. In the current study, we examined the relative proportion of acetylcholinesterase and butyrylcholinesterase activities in horse plasma and muscle, and evaluated the in vivo effects of Equitrol on target and non-target esterases following oral feeding in horses. In vitro inhibition studies suggested that essentially all cholinesterase activity in horse plasma was butyrylcholinesterase, while muscle contained >90% acetylcholinesterase activity. For in vivo studies, adult, male horses (364-590kg; n=3/treatment group) were given either sweet feed alone or sweet feed supplemented with Equitrol daily for 21 consecutive days at the recommended rate. Clinical signs (vital signs, abdominal auscultation, ophthalmic exam, body temperature) were recorded on a daily basis. Heparinized blood samples were taken at days -1, 1, 3, 7, 21, 28, and 42 while muscle (semimembranosus) biopsies were taken under aseptic conditions on days -1 and 21. No signs of overt toxicity were noted at any time during the study. Plasma cholinesterase activity was significantly inhibited (33%) in larvacide-treated horses as early as one day after treatment and peak inhibition (69-71%) was noted at days 7 and 21. Following cessation of dosing, plasma cholinesterase activity recovered (46% and 83% of control on days 28 and 42, respectively). Neither erythrocyte cholinesterase activity nor plasma carboxylesterase activity was affected by larvacide treatment in vivo. Muscle cholinesterase activity was highly variable among individual horses (pre-treatment range: 0.50-4.92nmole/min/mg protein), but there was no suggestion of a treatment-related reduction in muscle cholinesterase activity. These in vivo results confirm our previous in vitro studies indicating marked differential sensitivity of horse plasma and erythrocyte cholinesterase to inhibition by TCVP. Furthermore, the results suggest that recommended dosing levels of the TCVP-containing larvacide in horses are unlikely to affect acetylcholinesterase activities or disrupt cholinergic neurotransmission in target tissues.

Comparative in vivo effects of parathion on striatal acetylcholine accumulation in adult and aged rats.

Aged rats are more sensitive to the acute toxicity of the prototype organophosphate insecticide, parathion. We compared the acute effects of parathion on diaphragm and brain regional cholinesterase activity, muscarinic receptor binding and striatal acetylcholine levels in 3- and 18-month-old male Sprague-Dawley rats. Adult and aged rats were surgically implanted with a microdialysis cannula into the right striatum 5-7 days prior to parathion treatment. Rats were given either vehicle (peanut oil, 2 ml/kg) or one of a range of dosages of parathion (adult: 1.8, 3.4, 6.0, 9.0, 18 and 27 mg/kg, s.c.; aged: 1.8, 3.4, 6 and 9 mg/kg, s.c.) and body weight, functional signs of toxicity, and nocturnal motor activity were recorded for seven days. Three and seven days after parathion treatment, microdialysis samples were collected and rats were subsequently sacrificed for biochemical measurements. Higher dosages of parathion led to significant time-dependent reductions in body weight in both age groups. Rats in both age groups treated with lower dosages showed few overt signs of cholinergic toxicity while equitoxic high dosages (adult, 27 mg/kg; aged, 9 mg/kg) elicited marked signs of cholinergic toxicity (involuntary movements and SLUD [i.e., acronym for Salivation, Lacrimation, Urination and Defecation] signs) with peak effects being noted 3-4 days after treatment. Nocturnal activity (ambulation and rearing) was reduced in both age groups following parathion dosing, with more prominent effects in adults and rearing being more consistently affected. Dose- and time-dependent inhibition of cholinesterase activity was noted in both diaphragm and striatum. Total muscarinic receptor ([(3)H]quinuclidinyl benzilate, QNB) binding was significantly lower in aged rats, and both total binding and muscarinic agonist ([(3)H]oxotremorine methiodide] binding was significantly reduced in both age-groups treated with the highest dosages of parathion (adult, 27 mg/kg; aged, 9 mg/kg). In contrast to relatively similar levels of cholinesterase inhibition, striatal extracellular acetylcholine levels were significantly lower (2.2- to 2.9-fold) in aged rats at both 3 and 7 day time-points compared to adult rats treated with equitoxic dosages (i.e., 9 and 27 mg/kg, respectively). No age-related differences in in vitro striatal acetylcholine synthesis or in vivo acetylcholine accumulation following direct infusion of the cholinesterase inhibitor neostigmine (1 microM) were noted. While aged rats are more sensitive than adults to the acute toxicity of parathion, lesser acetylcholine accumulation was noted in the striatum of aged rats exhibiting similar levels of cholinesterase inhibition. These findings suggest that lesser acetylcholine accumulation may be required to elicit cholinergic signs in the aged rat, possibly based on aging-associated changes in muscarinic receptor density.

Comparative effects of oral chlorpyrifos exposure on cholinesterase activity and muscarinic receptor binding in neonatal and adult rat heart.

Organophosphorus (OP) pesticides elicit acute toxicity by inhibiting acetylcholinesterase (AChE), the enzyme responsible for inactivating acetylcholine (ACh) at cholinergic synapses. A number of OP toxicants have also been reported to interact directly with muscarinic receptors, in particular the M(2) muscarinic subtype. Parasympathetic innervation to the heart primarily regulates cardiac function by activating M(2) receptors in the sinus node, atrial-ventricular node and conducting tissues. Thus, OP insecticides can potentially influence cardiac function in a receptor-mediated manner indirectly by inhibiting acetylcholinesterase and directly by binding to muscarinic M(2) receptors. Young animals are generally more sensitive than adults to the acute toxicity of OP insecticides and age-related differences in potency of direct binding to muscarinic receptors by some OP toxicants have been reported. We thus compared the effects of the common OP insecticide chlorpyrifos (CPF) on functional signs of toxicity and cardiac cholinesterase (ChE) activity and muscarinic receptor binding in neonatal and adult rats. Dosages were based on acute lethality (i.e., 0.5 and 1x LD(10): neonates, 7.5 and 15 mg/kg; adults, 68 and 136 mg/kg). Dose- and time-related changes in body weight and cholinergic signs of toxicity (involuntary movements) were noted in both age groups. With 1x LD(10), relatively similar maximal reductions in ChE activity (95%) and muscarinic receptor binding (approximately 30%) were noted, but receptor binding reductions appeared earlier in adults and were more prolonged in neonates. In vitro inhibition studies indicated that ChE in neonatal tissues was markedly more sensitive to inhibition by the active metabolite of chlorpyrifos (i.e., chlorpyrifos oxon, CPO) than enzyme in adult tissues (IC(50) values: neonates, 17 nM; adults, 200 nM). Chelation of free calcium with EDTA had relatively little effect on in vitro cholinesterase inhibition, suggesting that differential A-esterase activity was not responsible for the age-related difference in cholinesterase sensitivity between age groups. Pre-incubation of neonatal and adult tissues with selective inhibitors of AChE and butyrylcholinesterase (BChE) indicated that a majority (82-90%) of ChE activity in the heart of both neonates and adults was BChE. The rapid onset (by 4h after dosing) of changes in muscarinic receptor binding in adult heart may be a reflection of the more potent direct binding to muscarinic receptors by chlorpyrifos oxon previously reported in adult tissues. The results suggest that ChE activity (primarily BChE) in neonatal heart may be inherently more sensitive to inhibition by some anticholinesterases and that toxicologically significant binding to muscarinic receptors may be possible with acute chlorpyrifos intoxication, potentially contributing to age-related differences in sensitivity.

Modulation of parathion toxicity by glucose feeding: Is nitric oxide involved?

Glucose feeding can markedly exacerbate the toxicity of the anticholinesterase insecticide, parathion. We determined the effects of parathion on brain nitric oxide and its possible role in potentiation of toxicity by glucose feeding. Adult rats were given water or 15% glucose in water for 3 days and challenged with vehicle or parathion (18 mg/kg, s.c.) on day 4. Functional signs, plasma glucose and brain cholinesterase, citrulline (an indicator of nitric oxide production) and high-energy phosphates (HEPs) were measured 1-3 days after parathion. Glucose feeding exacerbated cholinergic toxicity. Parathion increased plasma glucose (15-33%) and decreased cortical cholinesterase activity (81-90%), with no significant differences between water and glucose treatment groups. In contrast, parathion increased brain regional citrulline (40-47%) and decreased HEPs (18-40%) in rats drinking water, with significantly greater changes in glucose-fed rats (248-363% increase and 31-61% decrease, respectively). We then studied the effects of inhibiting neuronal nitric oxide synthase (nNOS) by 7-nitroindazole (7NI, 30 mg/kg, i.p. x4) on parathion toxicity and its modulation by glucose feeding. Co-exposure to parathion and 7NI led to a marked increase in cholinergic signs of toxicity and lethality, regardless of glucose intake. Thus, glucose feeding enhanced the accumulation of brain nitric oxide following parathion exposure, but inhibition of nitric oxide synthesis was ineffective at counteracting increased parathion toxicity associated with glucose feeding. Evidence is therefore presented to suggest that nitric oxide may play both toxic and protective roles in cholinergic toxicity, and its precise contribution to modulation by glucose feeding requires further investigation.

Modulation of paraoxon toxicity by the cannabinoid receptor agonist WIN 55,212-2.

Cannabinoids can reduce the pre-synaptic release of acetylcholine and other neurotransmitters in the mammalian brain through a retrograde signaling pathway. Organophosphorus insecticides elicit toxicity by inhibiting acetylcholinesterase and thereby increasing synaptic acetylcholine levels. Several studies suggest that some organophosphorus toxicants can potentially modify cannabinergic signaling by direct binding to cannabinoid receptors and inhibition of enzymes responsible for cannabinoid degradation (i.e., fatty acid amide hydrolase and monoacylglycerol lipase). We hypothesized that exposure to the cannabinoid receptor agonist WIN 55,212-2 (WIN) could alter the acute toxicity of the prototype anticholinesterase, paraoxon. In vitro, paraoxon inhibited hippocampal cholinesterase and fatty acid amide hydrolase activities, and displaced specific binding to the cannabinoid receptor ligand ([(3)H]CP 55,940) in a concentration-dependent manner. WIN (0.5, 1.5 or 5mg/kg/day) had a complex dose-related effect on locomotor activity when evaluated for 2h after either the first or last of seven daily exposures, and significantly decreased hippocampal CB1 binding following repeated dosing. Four hours after dosing, paraoxon (0.4 mg/kg, sc) elicited classical signs of cholinergic toxicity and significantly reduced hippocampal cholinesterase and fatty acid amide hydrolase activities as well as [(3)H]CP 55,940 binding. A single exposure to WIN (1.5 mg/kg) significantly reduced involuntary movements and SLUD signs following acute paraoxon exposure (0.4 and 0.6 mg/kg, sc). In contrast, when rats were challenged with paraoxon (0.4 mg/kg) after the seventh daily exposure to WIN (1.5mg/kg/day), involuntary movements were significantly increased at later timepoints, while SLUD signs were unaffected. These results suggest that acute and repeated exposure to cannabinoid agonists may differentially modify acute cholinergic toxicity, possibly through modulation of acetylcholine release and adaptation in cannabinergic signaling associated with repeated cannabinoid exposures.

Effects of acute chlorpyrifos exposure on in vivo acetylcholine accumulation in rat striatum.

This study examined the acute effects of chlorpyrifos (CPF) on cholinesterase inhibition and acetylcholine levels in the striatum of freely moving rats using in vivo microdialysis. Adult, male Sprague-Dawley rats were treated with vehicle (peanut oil, 2 ml/kg) or CPF (84, 156 or 279 mg/kg, sc) and functional signs of toxicity, body weight and motor activity recorded. Microdialysis was conducted at 1, 4 and 7 days after CPF exposure for measurement of acetylcholine levels in striatum. Rats were then sacrificed and the contralateral striatum and diaphragm were collected for biochemical measurements. Few overt signs of cholinergic toxicity were noted in any rats. Body weight gain was significantly affected in the high-dose (279 mg/kg) group only, while motor activity (nocturnal rearing) was significantly reduced in all CPF-treated groups at one day (84 mg/kg) or from 1-4 days (156 and 279 mg/kg) after dosing. Cholinesterase activities in both diaphragm and striatum were markedly inhibited (50-92%) in a time-dependent manner, but there were relatively minimal dose-related changes. In contrast, time- and dose-dependent changes in striatal acetylcholine levels were noted, with significantly higher levels noted in the high-dose group compared to other groups. Maximal increases in striatal acetylcholine levels were observed at 4-7 days after dosing (84 mg/kg, 7-9-fold; 156 mg/kg, 10-13-fold; 279 mg/kg, 35-57-fold). Substantially higher acetylcholine levels were noted when an exogenous cholinesterase inhibitor was included in the perfusion buffer, but CPF treatment-related differences were substantially lower in magnitude under those conditions. The results suggest that marked differences in acetylcholine accumulation can occur with dosages of CPF eliciting relatively similar degrees of cholinesterase inhibition. Furthermore, the minimal expression of classic signs of cholinergic toxicity in the presence of extensive brain acetylcholine accumulation suggests that some compensatory process(es) downstream from synaptic neurotransmitter accumulation limits the expression of toxicity following acute CPF exposure.

Comparative carboxylesterase activities in infant and adult liver and their in vitro sensitivity to chlorpyrifos oxon.

Maturational expression of carboxylesterase activity in laboratory animals has been correlated with age-related differences in sensitivity to many organophosphorus insecticides including chlorpyrifos. Little information is available, however, on the maturational expression of liver carboxylesterases in humans. Human liver carboxylesterase activity was compared in tissues from infants (2-24 months) and adults (20-36 years). There was no significant difference between mean infant and adult carboxylesterase activities. The carboxylesterase activity rank order was: 2 months<3 months<20 years<24 months<4 months<36 years<21 years<8 months<34 years<35 years. Proteins (3 microg) were separated and blotted using antibodies against rat hydrolase S (HS), human carboxylesterase (HCE) types 1 and 2, and CYP3A4. Again, there were no significant differences in staining density between infant and adult tissues with any isozyme. Aliquots of each sample were pre-incubated (30 min, 37 degrees C) with chlorpyrifos oxon to evaluate in vitro sensitivity. Based on 95% confidence intervals, no significant differences in IC50 values were obtained in 3-month to 36-year samples (range: 1.42-2.12 nM), while the IC50 was significantly lower in the 2-month sample (0.45 nM). Carboxylesterase activity across samples was correlated with cytochrome b5 content and HS immunosignal but not with other microsomal activities (total cyt P450 content, testosterone hydroxylation, coumarin hydroxylation, and EROD). The results suggest that, in contrast to rodents, human liver carboxylesterase expression changes relatively little during postnatal maturation.

Dietary modulation of parathion-induced neurotoxicity in adult and juvenile rats.

Previous studies indicated that dietary glucose (15% in drinking water) could markedly exacerbate the toxicity of parathion in adult rats. The present study evaluated the effect of consumption of the commonly used sweetener, high fructose corn syrup (HFCS), on parathion toxicity in adult and juvenile rats. Animals were given free access to either water or 15% HFCS in drinking water for a total of 10 days and challenged with parathion (6 or 18 mg/kg, s.c., for juveniles or adults, respectively) on the 4th day. Signs of cholinergic toxicity, body weight and chow/fluid intake were recorded daily. Acetylcholinesterase (AChE) activity and immunoreactivity (AChE-IR) in frontal cortex and diaphragm were measured at 2, 4, and 7 days after parathion. As HFCS was associated with significant reduction in chow intake, adult rats were also pair-fed to evaluate the effect of similar reduced chow intake alone on parathion toxicity. The results indicated that the cholinergic toxicity of parathion was significantly increased by HFCS feeding in both age groups. The excess sugar consumption, however, did not significantly affect parathion-induced AChE inhibition in either tissue or either age group. Enzyme immunoreactivity in frontal cortex was generally not affected in either age group while diaphragm AChE-IR was significantly reduced by parathion and HFCS alone in adult animals at 2 and 4 days timepoints, and more so by the combination of sugar feeding and parathion exposure in both age groups. Food restriction alone did not exacerbate parathion toxicity. While the mechanism(s) remains unclear, we conclude that voluntary consumption of the common sweetener HFCS can markedly amplify parathion acute toxicity in both juvenile and adult rats.

Pharmacology and toxicology of cholinesterase inhibitors: uses and misuses of a common mechanism of action.

Cholinesterase inhibitors have been used in the treatment of human diseases, the control of insect pests, and more notoriously as chemical warfare agents and weapons of terrorism. Most uses of cholinesterase inhibitors are based on a common mechanism of action initiated by inhibition of acetylcholinesterase (AChE). Extensive inhibition of this enzyme leads to accumulation of the neurotransmitter acetylcholine and enhanced stimulation of postsynaptic cholinergic receptors. This action is beneficial in cases where a reduction in cholinergic transmission contributes to clinical symptoms, e.g., low muscle tone in the autoimmune disorder myasthenia gravis due to loss of nicotinic receptors. Under normal conditions, however, extensive inhibition of AChE leads to excess synaptic acetylcholine levels, over-stimulation of cholinergic receptors, alteration of postsynaptic cell function and consequent signs of cholinergic toxicity. This biochemical cascade forms the basis for the use of anticholinesterase insecticides in pest control as well as for nerve agents in chemical warfare. Paradoxically, the short-acting cholinesterase inhibitor pyridostigmine, an important therapeutic agent in the treatment of myasthenia gravis, was used during the Persian Gulf War to prevent the long-term clinical consequences of possible organophosphate nerve agent exposure. As shown in the attacks in Matsumoto and Tokyo, these same nerve agents can be effectively used to inflict urban terror. Cholinesterase inhibitors thus share a common mechanism of pharmacological or toxicological action, ultimately modifying cholinergic signaling through disruption of acetylcholine degradation. While the use of cholinesterase inhibitors relies on their interaction with AChE, a variety of reports indicate that a number of cholinesterase inhibitors have additional sites of action that may have pharmacologic or toxicologic relevance. A variety of esterase and non-esterase enzymes, neurotransmitter receptors and elements of cell signaling pathways are targeted by some anticholinesterases. In some cases, these actions may occur at concentrations/dosages below those affecting cholinergic transmission. Studies of interactive toxicity of binary mixtures of common organophosphorus insecticides indicate that non-cholinesterase targets may be important in cumulative toxicity. Exposure to multiple anticholinesterases having selective effects on other macromolecules could confound the assumption of additivity in cumulative risk assessment. Knowledge of such selective additional targets may aid, however, in the optimization of strategies for poisoning therapy and in the further elucidation of mechanisms of toxicity for this class of compounds.

Stress regulation of sulfotransferases in male rat liver.

Sulfotransferase (SULT) catalyzed sulfation is responsible for hormone regulation and xenobiotic detoxification. Induction of SULTs by various hormones has been reported. Stress regulation of SULTs has not been reported, however. Here we report that rat liver SULTs can be regulated by physical stress (forced running, EX) and chemical stress (the organophosphorus pesticide parathion, PS). Both EX and PS increased rat liver phenol-sulfating SULT1A1 and hydroxysteroid-sulfating SULT2A1 activities. The increase in SULT1A1 activity did not correlate with protein (Western blot) or mRNA (RT-PCR) results but correlated well with increased non-protein soluble thiols. This suggests a possible Cys modification mechanism for stress regulation of SULT1A1. In vitro studies on GSH/GSSG effects on SULT1A1 activity support this conclusion. In contrast, SULT2A1 activity following physical or chemical stress treatments correlated well with protein and mRNA levels. This suggests a stress regulation mechanism of SULT2A1 at the gene transcription level, possibly occurring via hormones.

Interactive toxicity of the organophosphorus insecticides chlorpyrifos and methyl parathion in adult rats.

The acute interactive toxicity following exposure to two common organophosphorus (OP) insecticides, chlorpyrifos (CPF) and methyl parathion (MPS), was investigated in adult male rats. Oral LD1 values were estimated by dose-response studies (CPF = 80 mg/kg; MPS = 4 mg/kg, in peanut oil, 1 ml/kg). Rats were treated with both toxicants (0.5 or 1 x LD1) either concurrently or sequentially, with 4-h intervals between dosing. Functional signs of toxicity (1-96 h) and cumulative lethality (96 h) were recorded. Rats treated with CPF (1 x LD1) did not show any signs of toxicity although MPS (1 x LD1) elicited slight to moderate signs (involuntary movements) within 1-2 h. Concurrent exposure (LD1 dosages of both CPF and MPS) caused slight signs of toxicity only apparent between 24 and 48 h after dosing. When rats were treated sequentially with MPS first followed by CPF 4 h later, slight signs of toxicity were noted between 6 and 24 h, whereas reversing the sequence resulted in 100% lethality within 1 h of the second dosage. Following exposure to lower dosages (0.5 x LD1), the CPF first group showed higher signs of cholinergic toxicity compared with MPS first or concurrent groups. Cholinesterase inhibition in plasma, diaphragm, and frontal cortex was generally higher in rats treated sequentially with CPF first than in those treated initially with MPS from 4 to 24 h after dosing. Plasma and liver carboxylesterase inhibition at 4 h was also significantly higher in the CPF first (62-90%) compared with MPS first (22-43%) group, while at 8 and 24 h, there was no significant difference between any of the treatment groups. ChE inhibition assays to evaluate in vitro hepatic detoxification of oxons indicated that carboxylesterase (CE)- and A-esterase-mediated pathways are markedly less important for methyl paraoxon (MPO) than chlorpyrifos oxon (CPO) detoxification. CPF pretreatment blocked hepatic detoxification of methyl paraoxon while MPS pretreatment had minimal effect on hepatic CPO detoxification ex vivo. These findings suggest that the sequence of exposure to two insecticides that elicit toxicity through a common mechanism can markedly influence the cumulative action at the target site (acetylcholinesterase, AChE) and consequent functional toxicity.

In vitro inhibition of blood cholinesterase activities from horse, cow, and rat by tetrachlorvinphos.

The organophosphorus insecticide tetrachlorvinphos (TCVP) is commonly used as a feed-through larvicide in many livestock species, including cattle and horses. Cholinesterase (ChE) activity in blood (generally plasma or whole blood) is often employed to assess organophosphorus insecticide intoxication in animals as well as humans. In many species, including horse and man, plasma contains predominantly butyrylcholinesterase whereas red blood cells in all species express exclusively acetylcholinesterase. To evalulate the comparative interaction of TCVP with blood ChEs in different species, we compared the in vitro sensitivity of ChE activity in plasma and erythrocytes from horse, cow, and rat. Horse plasma ChE was most sensitive (IC(50), 30 minutes, 30 degrees C = 97 nM), whereas horse erythrocyte ChE activity was least sensitive (IC(50) > 1 mM). In contrast, cow plasma ChE showed lower sensitivity (IC(50) = 784 microM) to inhibition by TCVP than erythrocyte ChE (IC(50) = 216 microM). Rat plasma and erythrocyte ChE activities had relatively similar sensitivity to TCVP (IC(50) = 54 microM and 78 microM, respectively). The results suggest that plasma and erythrocyte ChE from horse, cow, and rat show marked species- and blood fraction-dependent differences in sensitivity to TCVP. Knowledge of such differences in sensitivity of blood ChE activities to TCVP may be important in the clinical interpretation of intoxication with this pesticide across species.

Effects of daily stress or repeated paraoxon exposures on subacute pyridostigmine toxicity in rats.

Pyridostigmine (PYR) is a carbamate cholinesterase (ChE) inhibitor used during the Persian Gulf War as a pretreatment against possible chemical nerve agent attack. Because of its quaternary structure, PYR entry into the central nervous system is limited by the blood-brain barrier (BBB). Following reports of unexplained illnesses among Gulf War veterans, however, central nervous system effects of PYR have been postulated through either stress-induced alteration of BBB permeability or via interactions with other neurotoxic agents. We evaluated the effects of daily physical (treadmill running) stress or daily exposure to a subclinical dosage of the organophosphate ChE inhibitor paraoxon (PO) on ChE inhibition in blood, diaphragm and selected brain regions in young adult male Sprague-Dawley rats following subacute PYR exposures. In physical stress studies, rats were placed on a treadmill for 90 min each day for 14 days just prior to PYR (0, 3, or 10 mg/kg per day) administration. In PO-PYR interaction studies, rats were treated with PO (0, 0.05, or 0.1 mg/kg per day) 1 h prior to daily PYR (0 or 3 mg/kg per day) administration for 14 consecutive days. Rats were evaluated daily for signs of cholinergic toxicity and were killed 1 h after the final PYR treatment. Forced running increased plasma corticosterone levels throughout the experiment (on days 1, 3, 7 and 14) when measured immediately after termination of stress. PYR-treated rats in the high dosage (10 mg/kg per day) group exhibited slight signs of toxicity (involuntary movements) for the first 6 days, after which tolerance developed. Interestingly, signs of cholinergic toxicity following PYR were slightly but significantly increased in rats forced to run on the treadmill prior to dosing. ChE activities in whole blood and diaphragm were significantly reduced 1 h after the final PYR challenge, and ChE inhibition in diaphragm was significantly greater in stressed rats than in non-stressed controls following high dose PYR (10 mg/kg per day). No significant effects of treadmill running on PYR-induced ChE inhibition in brain regions were noted, however. Repeated subclinical PO exposure had no apparent effect on functional signs of PYR toxicity. As with repeated treadmill running, whole blood and diaphragm ChE activities were significantly reduced 1 h after the final PYR administration, and ChE inhibition was significantly greater with combined PO and PYR exposures. Brain regional ChE activity was significantly inhibited after daily PO exposure, but no increased inhibition was noted following combined PO and PYR dosing. We conclude that, while some stressors may under some conditions affect functional signs of toxicity following repeated pyridostigmine exposures, these changes are likely to occur via alteration of peripheral cholinergic mechanisms and not through enhanced entry of pyridostigmine into the brain.

Age-related effects of chlorpyrifos and parathion on acetylcholine synthesis in rat striatum.

We compared the in vivo effects of two organophosphorus (OP) insecticides, chlorpyrifos (CPF) and parathion (PS) on acetylcholine (ACh) synthesis in neonatal, juvenile and adult rats. Basal levels of ACh synthesis were highest in adult rats, intermediate in juveniles and lowest in neonates. Following high (maximum tolerated dosage) subcutaneous exposure to either insecticide, relatively similar degrees of cholinesterase inhibition were noted, but the time to peak reduction varied among the age groups. CPF had no effect on ACh synthesis in neonates, increased synthesis in juveniles and decreased synthesis in adults, but only in the low dose group. PS had more consistent effects on ACh synthesis, decreasing transmitter synthesis in neonates (24 h after dosing) but increasing synthesis in juveniles and adults at both 4 and 24 h after exposure. Selective changes in neurotransmitter synthesis may contribute to differential age-related toxicity of these agents.