Clinical Signs Of Neurotoxicity Research Articles

CD19-targeted chimeric antigen receptor T cell therapy in two patients with multiple sclerosis

Progressive multiple sclerosis (MS) is characterized by compartmentalized smoldering neuroinflammation caused by the proliferation of immune cells residing in the central nervous system (CNS),including B cells. Although inflammatory activity can be prevented by immunomodulatory therapies during early disease, such therapies typically fail to halt disease progression. CD19 chimeric antigen receptor (CAR)-T cell therapies have revolutionized the field of hematologic malignancies. Although generally considered efficacious, serious adverse events associated with CAR-T cell therapies such as immune effector cell-associated neurotoxicity syndrome (ICANS) have been observed. Successful use of CD19 CAR-T cells in rheumatic diseases like systemic lupus erythematosus and neuroimmunological diseases like myasthenia gravis have recently been observed, suggesting possible application in other autoimmune diseases. Here, we report the first individual treatment with a fully human CD19 CAR-T cell therapy (KYV-101) in two patients with progressive MS. CD19 CAR-T cell administration resulted in acceptable safety profiles for both patients. No ICANS was observed despite detection ofCD19 CAR-T cells in the cerebrospinal fluid. In case 1, intrathecal antibody production in the cerebrospinal fluid decreased notably after CAR-T cell infusion and was sustained through day 64. CD19 CAR-T cell administration in progressive MS resulted in an acceptable safety profile. CAR-T cell presence and expansion were observed in the cerebrospinal fluid without clinical signs of neurotoxicity, which, along with intrathecal antibody reduction, indicates expansion-dependent effects of CAR-T cells on CD19+ target cells in the CNS. Larger clinical studies assessing CD19 CAR-T cells in MS are warranted. Both individual treatments as well the generated data were not based on external funding.

Bilirubin nomogram: A prediction tool or natural history profile?

Bilirubin nomograms have dual functions: (a) to track an individual baby using an evidencebased predictive risk assessment tool; and, (b) to inform the public health perspective on adverse outcome reduction by altering the natural course of the disease through early intervention. The original report that demonstrated the predictive value of a routine predischarge total serum bilirubin (TSB), measured as a universal screen, was simplified for immediate applicability to plot on an hour-specific bilirubin nomogram described for a population of racially diverse, healthy, term and near-term newborns during the first postnatal week in a Philadelphia hospital [1]. Subsequent studies have validated this predictive ability and the usefulness of this nomogram in several regional and multi-national populations [2]. When used with combination of TSB and transcutaneous testing, other centers have effectively promoted universal bilirubin screening [3, 4]. The outcome measure is the likelihood (LR) of TSB >95th percentile for age in hours. Based on the prevalence of “disease”: TSB in the high-risk zone, >95th percentile post-discharge for any population is (present / (present + absent)). One out of 23 infants in this study evidenced “disease”. The LR may calculate a revised or posttest probability of disease: posttest odds = pretest odds × LR where odds = probability ÷ (1 – probability). Because LR is prevalence independent, it can be used to calculate the posttest probability at any site in the world. Alternatively, the revised probability may be obtained for any site by dividing the absent component of the present : absent ratio for the total population by the LR to calculate the revised present : absent ratio for each risk zone (legend for Table 3 in reference 1). The clinical goal for any predictive test is defined by its outcome. We selected an identification of a higher-risk population to ensure a safety margin because of known imprecision of clinical diagnostic tests. Biological risk factors, accuracy as well as precision of bilirubin, pattern of clinical practice and access to healthcare markedly influence the predictive performance of the bilirubin tests. Gestational age, race and ethnicity are also singularly important [4, 5]. Subsequent consensus for TSB thresholds for use phototherapy governed by prematurity, hemolysis and clinical signs of neurotoxicity [2,4] have informed clinical practices similar to those in North America and Europe. However, geography and nationality are poor surrogates for biology, race, ethnicity and prematurity and have yet to be recognized as specific risk factors. Performance of a predictive test is also largely defined, in a research setting, with meticulous attention to study design, definitions, minimization of bias, subsequent validation in diverse cohorts and an anticipation of variable implementation in actual clinical practice. In this issue of Indian Pediatrics, Pathak, et al., [6] have attempted to replicate a predictive study without addressing a precise hypothesis and have expended immense effort without studying the vulnerability (if any) for their specific population cared for at their institution. Their data could be influenced by prevailing clinical practice pattern, race or ethnicity but not geography. Assessment of proven predictive tools for local institutional quality improvement should be benchmarked to best practice models and encouraged. On the other hand, novel questions impact nations with additional clinical or public health burden for bilirubin neurotoxicity that includes sepsis, small for dates, postnatal hypoalbuminemia, unrecognized hemolysis, insufficient breast milk intake or access to healthcare. Here, the risk of neurotoxicity can be altered by natural history of the disease itself. A national endeavor to define thresholds for interventions relevant to the diverse population is an achievable newborn health priority to reduce the prevalence of infants with TSB >95th percentile, use of exchange transfusion or possibly prevent kernicterus in India.

Absence of neurotoxic effects in leopard sharks, Triakis semifasciata, following domoic acid exposure

Domoic acid (DA), a potent neurotoxin produced by select species of algae and diatoms, kills neurons bearing kainic acid-type glutamate receptors. Studies have shown that DA bioaccumulates in invertebrates and fish that consume the diatoms. In every vertebrate species tested or observed in the wild, dietary or systemic DA causes neuronal damage or clinical signs of neurotoxicity. Sharks, like marine birds and mammals, are exposed to DA through their diet; however, no research has demonstrated the effect of DA on shark behavior or physiology. In this study, juvenile leopard sharks ( Triakis semifasciata) were given DA by intracoelomic injection at doses of 0, 1, 3, 9, and 27 mg/kg and observed for 7 days. The sharks failed to demonstrate behavioral or histological changes in response to the toxin. We identified putative brain glutamate receptors by probing western blots with an antibody specific for kainic acid-type glutamate receptors and demonstrated receptor localization in the cerebellum with immunohistochemistry. Blood levels of DA in three sharks dosed at 9 mg/kg fell rapidly within 1.5 h of injection. We show that leopard sharks possess the molecular target for DA but are resistant to doses of DA known to be toxic to other vertebrates.

Neuropathologic toxicity of artemisinin derivatives in a mouse model.

Intramuscular administration of high doses of artemether and arteether to experimental mammals produces selective damage to brain stem centers involved predominantly in auditory processing and vestibular reflexes. The relationship between clinical signs of neurotoxicity and neuropathologic toxicity was studied in the mouse. Intramuscular artemether (50-100 mg/kg/day for 28 days) caused dose-dependent neuropathologic damage to the brain stem. There was no pathologic evidence of neuronal death in mice receiving either oral artemether, or oral or intramuscular artesunate, in doses up to 300 mg/kg/day. The neurons in the lower brain stem trapezoid nucleus, the gigantocellular reticular nucleus, and the inferior cerebellar peduncle were the most sensitive to the toxic effects of artemether. All mice with neuropathologic changes also showed behavioral changes, whereas in some mice with gait disturbance, no corresponding histopathologic damage could be detected. Thus clinical assessment was a sensitive measure of neurotoxicity. There may be a reversible component to artemether neurotoxicity.

GLUCOSE FEEDING EXACERBATES PARATHION-INDUCED NEUROTOXICITY

Excessive dietary intake of sugars could alter various biotransformation processes and the pharmacological and toxicological properties of numerous xenobiotics. In the present study, the effects of glucose supplementation were examined on the neurotoxicity of the organophosphorus (OP) pesticide parathion (PS) and its active metabolite, paraoxon (PO), a potent inhibitor of acetylcholinesterase (AChE). Rats (n = 6-12/treatment group) were given free access to tap water or 15% glucose (w/v) in tap water beginning 7 d prior to OP toxicant exposure. Food, caloric intake, and body weight were measured daily. Animals were challenged with either PS (4.5, 9, or 18 mg/kg, sc) or PO (0.3, 0.5, or 0.7 mg/kg, sc) and clinical signs of neurotoxicity (i.e., autonomic dysfunction, involuntary movements) were recorded daily for the following 13 d. Glucose feeding was associated with a dramatic drop (~50%) in feed intake and an increase (~20%) in total caloric consumption over the 7 d prior to OP exposure. Functional toxicity associated with PS exposure was increased in glucose-fed (GF) rats, but the glucose diet had no apparent effect on clinical signs of toxicity following PO treatment. Glucose feeding increased the magnitude of AChE inhibition in the frontal cortex and plasma at lower dosages (i.e., 4.5 and 9 mg/kg) 3 d following PS treatment. Time-course studies (3, 7, and 11 d after PS exposure, 18 mg/kg, sc) indicated significantly greater brain and plasma AChE inhibition in glucosefed animals at later time points. In contrast, glucose feeding had no effect on the degree of AChE inhibition following PO exposure. Neither liver microsomal oxidative desulfuration of PS, nor liver or plasma paraoxonase, nor liver or plasma carboxylesterase activities were measurably affected by glucose feeding. Downregulation of muscarinic receptors 7 d after PS exposure (18 mg/kg, sc) was more extensive in GF rats. It is postulated that excessive glucose consumption decreases the intake of other dietary components, in particular amino acids, limiting the de novo synthesis of AChE and consequent recovery of synaptic transmission. Due to the shorter duration of inhibition following PO exposure, spontaneous reactivation of AChE may be more important than de novo protein synthesis in recovery of function, and thus with the effects of glucose feeding on its toxicity. Individuals that derive a large proportion of their calories from sugars may be at higher risk of acute toxicity from organophosphorus pesticides such as PS.

The effect of postnatal age on L -2-chloropropionic acid-induced cerebellar granule cell necrosis in the rat

L-2-Chloropropionic acid (L-2-CPA) selectively damages the cerebellum in adult rats. The rat cerebellum continues to develop postnatally during the first 4 weeks of life. In this study we examined the neurotoxic effect on rats of increasing postnatal age. Daily oral dosing of rats aged 56 days with 250 mg/kg per day of L-2-CPA for 3 days produced necrosis to neurons in the cerebellar granule cell layer and to neurons in the medial/ventral region of the habenular nucleus. Rats aged 22 days were resistant to the cerebellar toxicity while rats aged 32 days and older were sensitive. A single large oral dose of 500 or 750 mg/kg L-2-CPA produced no clinical signs of neurotoxicity or lesions in the cerebellum 48 h after dosing in 22-day-old rats. Daily dosing of 22-day-old rats at 250 mg/kg per day L-2-CPA for 10 days also produced no signs of neurotoxicity or reduction in body weight gain, although histological examination of the brain revealed slight neuronal cell necrosis in the granule cell layer of the cerebellum with a minimal effect in the medial/ventral region of the habenular nucleus. In contrast, daily dosing of rats aged 32, 38, 48 and 58 days with 250 mg/kg per day of L-2-CPA for 3 days produced clear signs of neurotoxicity which were associated with reduced body weight gain and loss of hindlimb function. In these rats there was clear evidence of neuronal cell loss in the cerebellar granule cell layer and medial/ventral region of the habenular nucleus. This study showed that the postnatal developing cerebellum is resistant to L-CPA-induced injury in rats up to 25 days of age, but becomes vulnerable to the toxicity by 32 days of age. The basis for the resistance of the developing cerebellum to L-CPA is discussed.

Effects of racecadotril and loperamide on bacterial proliferation and on the central nervous system of the newborn gnotobiotic piglet.

The effects of 4 days of oral administration of different doses of two drugs, an enkephalinase inhibitor (the antisecretory agent, racecadotril) and a mu-receptor agonist (loperamide), on intestinal growth of a bacterial nonpathogenic strain (Escherichia coli E 404) and on the central nervous system (CNS) were compared in newborn gnotobiotic piglets. The E. coli content of the proximal jejunum (segment S1) and the E. coli ratio of stomach:segment S1 were similar in the racecadotril (20 mg/kg b.d., n = 5) and control groups. In contrast, in the loperamide group (1 mg/kg b.d., n = 4), the E. coli content of segment S1 and the E. coli ratio stomach:S1 were both significantly higher than with racecadotril or control (P = 0.04 and 0.005, respectively, for E. coli content; P = 0.05 and 0.03, respectively, for stomach:S1). There were no clinical signs of neurotoxicity and no deaths with racecadotril given orally at a high dose of 130 mg/kg b.d. (n = 5)--nearly 60 times the paediatric dosage. In contrast, an equivalent high dose of loperamide (5 mg/kg b.d.) resulted in death in three out of four piglets. In contrast to loperamide, racecadotril did not induce bacterial overgrowth and did not produce central neurotoxicity.

Differential effects of orally versus parenterally administered qinghaosu derivative artemether in dogs

SummaryArtemether (AM) is an antimalarial drug derived from artemisinin (Qinghaosu), an extract of the herb Artemisia annua L., sweet wormwood. Its antiparasitic effect is that of a schizontocide and is explained by rapid uptake by parasitized erythrocytes and interaction with a component of hemoglobin degradation resulting in formation of free radicals. It has been shown to exhibit a high clinical cure rate. Previous animal safety studies with Qinghaosu derivatives revealed dose-dependent neurotoxicity with movement disturbances and neuropathic changes in the hindbrain of intramuscularly treated dogs, rats and monkeys. Such effects have not been seen in man. The objective of our present studies was to compare the effects of high levels of AM administered to dogs p.o. versus i.m. In a pilot study 20 mg/kg/day of AM was given i.m. to groups of 3 male Beagle dogs for 5 and 30 days, respectively. Clinical signs of neurotoxicity were noted in some individual dogs from test day 23 on. One dog had to be sacrificed pre-term. Hematologic findings indicated a hypochromic, microcytic anemia. Microscopic examination demonstrated neuropathic changes only at 30 days, but not at 5 days. The animals had neuronal and secondary axonal damage, most prominent in the cerebellar roof, pontine and vestibular nuclei, and in the raphe/paralemniscal region. The affected neurons showed loss of Nissl substance, cytoplasmic eosinophilia, shrinkage of the nucleus and in advanced stages scavenging by microglia. In a subsequent experiment, AM was administered to groups of 4 male and 4 female dogs, respectively, at 8 daily doses of 0, 20, 40 and 80 mg/kg i.m., or 0, 50, 150 and 600 mg/kg p.o. Neurologic signs were seen at high i.m. doses only. In most animals they were inconspicuous and consisted of reduced activity with convulsions seen in single dogs shortly before death. Neuronal damage occurred in all animals at 40 and 80 mg/kg following i.m. treatment. At 20 mg/kg minimal effects occurred in 5/8 dogs only, indicating that this level was close to tolerated exposure. No comparable lesions were observed after oral administration. Both i.m. and p.o. exposure at high dose levels was associated with a prolongation of mean QT interval of ECG, suggesting slowing of repolarization of the myocardium. Individual data indicated that in 1 of 4 females at 80 mg/kg i.m. this prolongation was above the 25 % level considered as threshold for concern. After intramuscular administration pharmacokinetics indicated peak plasma levels of AM at 2 to 4 hours post-dose, slow elimination and a tendency to accumulate after repeated administration. Only low levels of the major metabolite, dihydroartemisinin (DHA), were found. AM levels in the cerebrospinal fluid (CSF) were < 10 % of plasma levels. After oral administration AM concentrations were considerably lower than after i.m. administration. The concentration of DHA was high on day 1 but almost nil on day 7 indicating its fast inactivation in dogs. Two hours after the 8th oral administration neither AM nor DHA was detected in CSF which may explain the absence of neurotoxicity in dogs after oral administration of AM.

The Neurotoxicity of Drugs Given Intrathecally (Spinal)

A growing understanding of the neuropharmacology of spinal cord processing of nociceptive input has led to intense interest in the use of spinal drugs in anesthesia and pain management. The direct application of receptor-specific therapeutics at the spinal cord can potentially interrupt specific pain pathways and limit systemic side effects, but this practice also carries the inherent risk of injury to the central nervous system. Thus, the neurotoxicity of spinal drugs is a central safety issue. Spinal cord or nerve root toxicity may manifest itself as histologic, physiologic, or behavioral/clinical derangements after exposure to a spinal drug. Neurohistopathology is broadly classified as neural injury, gliosis, or damage to the myelin sheath, and it also describes inflammatory changes and involvement of the arachnoid cell layers. Physiologic neurotoxicity of spinal drugs includes changes in spinal cord blood flow, disruption of the blood-brain barrier, and changes in the electrophysiology of impulse conduction. Behavioral and clinical signs of neurotoxicity include pain, motor and sensory deficits, and bowel and bladder dysfunction. Ideally, a complete roster of histological, physiologic, and behavioral testing would be performed on spinal drugs in several animal species, followed by safety trials in humans before widespread use. In practice, drugs have taken a variety of roads from conception to application, and often without safety data. In this article, we review available neurotoxicity data on drugs that have a clinical application, classified as spinal local anesthetics, spinal analgesics, or spinal adjuvants.

Toxicity of the novel animal-derived anticancer agent, VRCTC-310: acute and subchronic studies in beagle dogs

Acute and subchronic toxicities of VRCTC-310, a combination product of crotoxin (CT) and cardiotoxin (CD), which has shown antitumor activity in vivo, have been studied in Beagle dogs. Single i.m. doses of 0.25, 0.5 and 1.0 mg/kg resulted in dose-dependent local muscular toxicity consisting of myofiber atrophy, interstitial edema and macrophage infiltration. Also, AST, ALT and LDH levels increased on day 2, returning to normal values on days 6–8. Local lesions were absent after recovery on day 45. At 2.0 mg/kg, signs of neurotoxicity (ataxia) appeared, in addition to vomitus, salivation, hematuria and myotoxicity in tongue and diaphragm on day 8. Local lesions healed with fibrosis at the site of injection on day 45. Administration of fixed (0.025 and 0.05 mg/kg) or escalating (0.025–0.1 mg/kg) daily doses for 30 days also produced local muscular damage, which was absent at day 75. The increases in AST, ALT and LDH serum activities on days 2–4 were independent of dosing schedule and sharply decreased on day 8, despite continuation of treatment. An escalating dose schedule of 0.025-2.0 mg/kg showed local muscle damage at the site of injection on day 31, however, there were no lesions of myotoxicity in the tongue or diaphragm and no clinical signs of neurotoxicity were observed. Animals tolerated the subchronic treatment better than the acute. The resolution of serum enzymes to normal values during treatment may be attributed to a decrease of sensitivity to VRCTC-310-mediated myotoxic effects.

Role of arteether in the treatment of malaria and plans for further development

Arteether has potent antimalarial activity in vitro against drug resistant Plasmodium falciparum. Preclinical studies of arteether injection have been completed, and phase I safety, tolerance and pharmacokinetic studies are in progress in The Netherlands. No intolerance has yet been observed. Production has been established in a pilot scale in The Netherlands by A.C.F. Beheer BV. Toxicity studies have been conducted in rats and dogs: 3 mg/kg/d for 28 d had no effect. At higher dosages, toxic effects on heart, brain, bone marrow, kidney and liver were observed. Cardiotoxicity is characterized in the dog by a dose-related prolongation of the QTc interval and inversion of the T-wave in some animals. Arrhythmias have not been observed. Electrocardiographs changes returned to baseline values after cessation of daily drug administration. Neuronal toxicity was observed in dogs given daily doses of 6·75 or 15 mg/kg/d intramuscularly for 28 d. Signs of lethargy, incoordination, and abnormal responses appeared in the fourth week. Electroencephalograms exhibited no abnormality. Neuronal degeneration and subsequent myelin degeneration, particularly affecting the cerebellum and other portions of the mid- and hind-brain, were observed. Clinical signs of neurotoxicity did not resolve completely within 30 d after cessation of dosing, and histopathological damage in the brain was still evident. Behavioural and histological evidence of neurotoxicity was also observed in rats. The neurotoxic effects of arteether and artemether in rats and dogs were similar.

Induction of cytochrome P450 isozymes by simultaneous inhalation exposure of hens to n-hexane and methyl iso-butyl ketone (M iBK)

Chickens were exposed simultaneously to the industrial hexacarbon solvents n-hexane and methyl iso-butyl ketone (M iBK). n-Hexane has been shown to be neurotoxic in both humans and other vertebrates. While M iBK is not neurotoxic, it has been shown to greatly synergize the clinical appearance of neurotoxicity in animals exposed to both of these solvents. Groups of hens were exposed for 29 days in inhalation chambers to 1000 ppm n-hexane in combination with 10, 100, 250, 500, or 1000 ppm M iBK. Other groups received either 1000 ppm n-hexane, 1000 ppm M iBK, or ambient air and served as controls. A dose-dependent decrease in body weight and an increase in clinical effects were noted for the highest exposure groups (1000 ppm n-hexane combined with 1000, 500 or 250 ppm M iBK). There was an M iBK dose-dependent increase in cytochrome P450 content and benzphetamine N-demethylase activity, but there was no distinct pattern for ethoxyresorufin O-deethylase or cytochrome c reductase activities. Mixed-function oxidase levels and activities (cytochrome P450 content and benzphetamine N-demethylase) were elevated significantly (P < 0.05) over controls even in the lowest M iBK group (10 ppm), although there were no clinical signs of neurotoxicity. Four different isozymes of cytochrome P450 were measured immunologically. There was a dose-dependent increase in three of the isozymes, two of which were phenobarbital inducible and one of which was induced by β-napthoflavone. Quantitatively, the largest increase was in the PB-A isozyme, a phenobarbital-inducible isozyme which accounted for approximately 70% of the cytochrome P450 present in animals treated with M iBK. The results suggest that M iBK selectively induces cytochrome P450 isozymes leading to the metabolic activation of the weak neurotoxicant n-hexane to the potent neurotoxicant 2,5-hexanedione (2,5-HD).

Comparison of neuropsychologic functioning and clinical indicators of neurotoxicity in long-term survivors of childhood leukemia given cranial radiation or parenteral methotrexate: a prospective study.

We prospectively compared neuropsychologic functioning and clinical indicators of neurotoxicity in 49 consecutive childhood leukemia patients in long-term continuous complete remission (CR) who had received two different regimens of CNS prophylaxis by random assignment. Twenty-three patients were treated with 1,800 cGy cranial radiation and intrathecal methotrexate (RT group) and 26 with parenteral methotrexate only (MTX group). Over half of the RT group had somnolence syndrome, and four developed cerebral calcifications late in their clinical course. Abnormal electroencephalograms (EEGs) were seen in 15 patients in the MTX group, and six had early, transient white-matter hypodensities apparent on computed tomographic (CT) scans. Mean scores on standard tests of intelligence and academic achievement, administered after remission induction and again at a median of 6 years after treatment cessation, did not differ significantly between the two groups. However, statistically significant decreases in overall and verbal intelligence quotients (IQs) and in arithmetic achievement were found within both treatment groups. Sixteen of 26 in the MTX group and 14 of the 23 in the RT group had clinically important decreases (greater than or equal to 15 points) on one or more neuropsychologic measures. These changes did not correlate with findings on CT scans, EEGs, or other clinical signs of neurotoxicity. We conclude that 1,800 cGy cranial radiation and parenteral methotrexate, as used in this study, are associated with comparable decreases in neuropsychologic function.

Acute Parenteral Neurotoxicity of Domoic Acid in Cynomolgus Monkeys (M. fascicularis)

To study the CNS effects of domoic acid (D.A.), 6 adult Cynomolgus monkeys (M. fascicularis) were dosed intraperitoneally (4 mg/kg) or intravenously (0.025-0.5 mg/kg) with D.A. obtained from cultured mussels contaminated with this neurotoxin. Clinical signs of neurotoxicity were preceded by a short presymptomatic period (2-3 min) and an even shorter prodromal period (0.5-1 min). The symptomatic period proper was characterized by persistent chewing with frothing, varying degrees of gagging, and vomit. Monkeys in the higher dose regimen exhibited additional signs including abnormal head and body positions, rigidity of movements and loss of balance, and tremors. The duration of the symptomatic period was dose dependent. Excitotoxic lesions consisting of vacuolation of the neuropil, astrocytic swelling, and neuronal shrinkage and hyperchromasia were detected in the area postrema, the hypothalamus, the hippocampus, and the inner layers of the retina in monkeys given D.A. at 0.5 mg/kg intravenously and 4 mg/kg intraperitoneally. It was concluded that D.A., administered intravenously, is neuroexcitatory and a powerful emetic at doses of 0.025 to 0.2 mg/kg. At higher doses (0.5 mg/kg intravenously and 4 mg/kg intraperitoneally), D.A. is strongly excitotoxic.

Pattern of neurotoxicity of n-hexane, methyl n-butyl ketone, 2,5-hexanediol, and 2,5-hexanedione alone and in combination with O-ethyl O-4-nitrophenyl phenylphosphonothioate in hens.

This investigation was designed to study the neurotoxicity produced in hens by the aliphatic hexacarbons n-hexane, methyl n-butyl ketone (MnBK), 2,5-hexanediol (2,5-HDOH), and 2,5-hexanedione (2,5-HD) following daily dermal application of each chemical alone and in combination with O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN). Dermal application was carried out on the unprotected back of the neck. To assess whether the joint neurotoxic action of various chemicals is caused by the enhancement of absorption through the skin or by interaction at the molecular level, two additional experiments were performed. In the first experiment, EPN was dissolved in each of the aliphatic hydrocarbons prior to their topical application. In the second experiment, EPN was dissolved in acetone and applied at a different location from that of the aliphatic hexacarbons. Dermal application was carried out for 90 d followed by a 30-d observation period. The results show that hens treated with EPN developed severe ataxia followed by improvement during the observation period; n-hexane produced leg weakness with subsequent recovery, whereas the same dose of MnBK, 2,5-HDOH, or 2,5-HD produced clinical signs of neurotoxicity characterized by gross ataxia; concurrent dermal application of EPN with n-hexane or 2,5-HDOH at the same site or at different sites produced an additive neurotoxic action; simultaneous dermal application of EPN and MnBK at different sites resulted in an additive effect, whereas it caused potentiation when applied at the same site; and concurrent topical application of EPN and 2,5-HD produced a potentiating neurotoxic effect. While no histopathologic lesion was produced at the end of the observation period when any test chemical was applied alone, binary treatments of EPN and aliphatic hexacarbons resulted in histopathologic changes in some hens, with morphology and distribution characteristic of EPN neurotoxicity. The joint potentiating or additive action of aliphatic hexacarbons on EPN neurotoxicity was: 2,5-HD greater than MnBK greater than 2,5-HDOH greater than n-hexane. The mechanism of this joint action seems to be related both to enhancing skin absorption of EPN and/or its metabolic activation by n-hexane and its related chemicals.

Studies on the delayed neurotoxicity of organophosphorus compounds- (I).

Delayed neurotoxicity experiments of an organophosphorus compound, TOCP, on hens and quails were carried out. The animals were orally exposed with TOCP in dose of 400 mg/kg, and maintained for 25 and 50 days under observation, respectively. They were sacrificed and dissected at different periods after the exposure, and the histopathological examinations were made on those animals. During the periods of 50 days, no abnormal symptoms except for acute poisoning were noted in the quails. As to the hens, neurological disorders were observed from around 12 days of the experiment. In hens, the morphological alteration in the earlier stage of the experiment was perivascular cuffing of small round cells seen in the cerebrum, cerebellum and spinal cord. The major alteration was degeneration of axons and myelin in the white matter of the spinal cord. A minimal degree of these changes was found even in the hens in which the clinical signs of neurotoxicity had not yet been observed. Later the process expanded to the whole spinal cord as well as the sciatic nerve. The electron microscopy performed after 15 days revealed moderate change of myelin and axons. From these results, it was concluded that the clinical signs as well as the morphological changes were closely related to the exposure of the compound, TOCP.