Cardiotoxicity of adrenochrome in isolated rabbit hearts assessed by epicardial NADH fluorescence

Noradrenaline in a micromolar concentration has recently been shown to contribute to ischemic tissue injury by direct cardiotoxic effects independent of functional alterations. Oxygen free radicals, generated during the auto-oxidation of catecholamines, are important mediators of catecholamine cardiotoxicity. However, the role of the oxidative products (aminochromes) is still unclear. We examined the effects of adrenochrome on functional parameters and on regional myocardial ischemia (MI) in isolated electrically-driven rabbit hearts with depleted catecholamine stores (reserpine 7.0 mg/kg i.p. 16 – 24 h before preparation, Langendorff, constant pressure: 70 cm H₂O, Tyrode solution, Ca⁺⁺ 1.8 mmol/l, 37°C). Repetitive MI, separated by a reperfusion period of 50 min, was induced by coronary artery branch ligature, and MI was quantitated from epicardial NADH fluorescence photography. Adrenochrome-treatment (10^– 6 M or 10^– 4 M) was started after a reperfusion period of 20 min. The left ventricular pressure (LVP) was significantly enhanced by adrenochrome (p <0.05), but it fell thereafter to below its initial value in hearts treated with adrenochrome 10^– 4 M. The global coronary flow (CF) was not affected by adrenochrome 10^– 6 M (P >0.05), but it was significantly decreased by adrenochrome 10^– 4 M (P <0.05). The relative CF (= CF/LVP × heart-rate) was numerically decreased by adrenochrome 10^– 6 M (p >0.05) and more markedly by adrenochrome 10^– 4 M (p <0.05). Whereas epicardial NADH fluorescence was similar after repetitive coronary artery occlusions in controls and in hearts treated with adrenochrome 10^– 6 M (p >0.05), it was significantly enhanced by adrenochrome 10^– 4 M (p <0.05). In isolated rabbit hearts, adrenochrome possesses deleterious effects on MI only at a very high concentration but not in a micromolar concentration. Therefore, it seems that aminochromes may be less cardiotoxic than catecholamines. Received: 28 February 1994 / Accepted: 2 May 1994     

Aluminum effects on blood chemistry and long bone development in the chick embryo

Body growth, blood chemistry, and long bone development of 10- to 16-day chick embryos (Gallus gallus) treated with aluminum (Al) citrate, sodium (Na) citrate, or sodium chloride (NaCl) were investigated. Two administration protocols were used. Acutely-treated embryos received 6.0 μmol Al citrate or Na citrate on day 8 of incubation. Chronically-treated embryos received a daily dose of 1.5 μmol Al citrate or Na citrate beginning on day 8 of incubation. For both protocols, Al citrate and Na citrate had no significant influence on viability or body weight. Al citrate-treated embryos had: (a) significantly shorter mean tibia lengths by day 16 of incubation, (b) a consistently lower ratio of tibia length: body weight on all days investigated, and (c) a persistent mid-diaphyseal malformation (angulation) of the femur and tibia. Spatially correlated with the malformation was a calcification defect detected by alizarin red S staining of intact tibias and the accumulation of aluminum as demonstrated by acid solochrome azurine staining of histological sections. Aluminum was localized at the mineralization front of the osteogenic collar surrounding the cartilage core of the tibia. Aluminum citrate or Na citrate had no significant effect on serum total calcium, inorganic phosphorus, total alkaline phosphatase activity, or creatinine, except for a transitory hypercalcemia (day 10) and phosphatemia (days 10 and 12) in Al citrate-treated embryos. The concomitant localization of Al and the early calcification defect in the region of tibial malformation implicate aluminum in the pathogenesis of the skeletal abnormality. Received: 8 March 1993/Accepted: 19 April 1994     

Mercury toxicokinetics in Wistar rats exposed to elemental mercury vapour: modeling and computer simulation

The kinetics of total mercury (Hg) absorption, distribution and elimination in Wistar rats exposed for long periods to elemental mercury vapour (Hg°) in the Idrija mercury mine were studied. From the experimental data base a compartmental model was built as a framework for experimental data interpretation and prediction of organ mercury levels under different conditions. Using the model the exposures of rats under conditions comparable to those of professionally exposed workers (mercury miners, workers in the chloralkali industry) and individuals with amalgam fillings were simulated. Received: 18 October 1993/Accepted: 7 March 1994     

Genotoxic risk for humans due to work place exposure to ethylene oxide: remarkable individual differences in susceptibility

Single strand breaks of DNA of peripheral mononuclear blood cells from 97 male and female workers occupationally exposed to ethylene oxide were analysed by the alkaline elution method. These individuals were occupied with the sterilization of medical devices in hospitals and in commercial plants. Ethylene oxide in the air of the working areas was detected up to a maximal concentration of 16.5 mg/m³ calculated as 4-h time-weighted average (4h TWA). Mean value was 1.47±0.52 mg/m³ (1 mg/m³ = 0.55 ppm). Compared to the mean elution rate of the DNA from non-smoking workers exposed to air concentrations of ethylene oxide below the detection limit of 0.1 mg/m³ (4h TWA) the non-smokers working in rooms with a concentration of ethylene oxide between 0.5 mg/m³ and 2 mg/m³ showed a statistically significant (P <0.05) 119% higher mean elution rate and even for the non-smokers exposed to 0.1 – 0.5 mg/m³ of ethylene oxide a statistically significant (P <0.05) 53% higher mean elution rate was observed. For smokers a similar tendency was found but the increase in elution rates in response to the external exposure was smaller than in non-smokers and no statistical significance was obtained. According to their sensitivity to ethylene oxide the non-smoking workers could be classified into two subpopulations. In the majority of the non-smokers (67%) approximately 5-fold more DNA strand breaks were induced by ethylene oxide than in the other non-smokers. A lowest detectable effect level could only be specified for non-smokers. For the “higher sensitive” group the lowest detectable effect level in an examination of a single individual was calculated to be 0.6 mg/m³ ethylene oxide in the air (4h TWA). For the “lower sensitive” group a lowest detectable effect level was calculated to be 3.5 mg/m³. Received: 18 October 1993/Accepted: 16 February 1994     

Influence of inducers and inhibitors of cytochrome P450 on the hepatotoxicity of hydrazine in vivo

Hydrazine hepatotoxicity in vivo, as manifested by triglyceride accumulation, depletion of ATP and reduced glutathione (GSH) was shown to be dose related. The effect of pretreatment of rats with various inhibitors and inducers of cytochrome P450 on these dose-response relationships was investigated. Pretreatment with the inhibitor piperonyl butoxide increased triglyceride accumulation whereas pretreatment with the inducers phenobarbital and β-naphthoflavone (BNF) resulted in reduced triglyceride accumulation. Pretreatment with the inducers acetone and isoniazid also enhanced triglyceride accumulation. Only phenobarbital pretreatment also significantly reduced GSH and ATP depletion. A linear correlation was found between hepatic glutathione and ATP levels in non-pretreated animals given various doses of hydrazine. However, exponential relationships were found between hepatic triglycerides and both hepatic ATP and glutathione. The results suggest that i) the hepatotoxicity of hydrazine can be modulated by inducing or inhibiting particular isoenzymes of cytochrome P450, ii) ATP and GSH depletion may not be directly involved in the development of fatty liver. Received: 29 November 1993/Accepted: 16 February 1994     

Pharmacokinetics of the oximes HI 6 and HLö 7 in dogs after i.m. injection with newly developed dry/wet autoinjectors

The rapid onset of cholinergic crisis after intoxication with highly toxic organophosphorus compounds calls for pre-clinical administration of effective antidotes as early as possible. For this purpose, i.m. administration of the antidotes by autoinjectors is desired to allow early treatment also in the absence of a physician. Besides atropine, oximes with broad antidotal spectrum are considered valuable adjuncts that should be included in antidotal mixtures. To circumvent the problem of limited stability of the new-generation oximes, dry/wet autoinjectors were developed in which the unstable solid is dissolved by a diluent in an adjacent chamber upon activation of the device. In this study the tolerance, bioavailability and pharmacokinetics of 500 mg HI 6 [1-(((4-(aminocarbonyl) pyridinio)methoxy) methyl)-2-((hydroxyimino)methyl) pyridinium dichloride monohydrate] or 200 mg HLö 7 [1-(((4-(aminocarbonyl) pyridinio)methoxy)methyl)-2,4-bis ((hydroxyimino)methyl)pyridinium dimethanesulfonate] in combination with 2 mg atropine sulfate versus atropine alone, delivered by two dry/wet autoinjector types, were investigated in eight male beagle dogs (16 kg) in a complete cross-over design. The dogs tolerated the six injections with 3-week intervals without any symptoms of discomfort. Nonetheless, CPK activity increased, peaking at 6 h after injection. In contrast to atropine which merely led to a marginal increase, HI 6 plus atropine increased the baseline CPK activity about 10-fold, and HLö 7 plus atropine about 20-fold, regardless of the injector type. The HI 6 autoinjectors from Astra Tech were from an irregular production batch which did not deliver the declared HI 6 dose. The HLö 7 autoinjectors from Astra Tech and both Binjaect autoinjectors from STI functioned well: the bioavailability was complete with t_max values of about 25 min as observed after conventional i.m. injection. The absorption half-time was about 8 min, elimination t_1/2 about 50 min, and V_app 0.26 l/kg. The urinary recovery of unchanged oximes was 70–80%, the renal clearance being the same as for inulin. Unexpectedly, hematocrit and hemoglobin content of blood decreased by about 15% within 2 h and reached pre-treatment values after 6–24 h. This decrease was observed with all three drug treatments and could not be accounted for by blood loss (<4%), thus pointing to an atropine effect. In conclusion, the newly developed dry/wet autoinjectors appear suitable for the administration of atropine and an oxime stored in solid form.     

Erythroid progenitor cells that survive benzene exposure exhibit greater resistance to the toxic benzene metabolites benzoquinone and hydroquinone

Benzene is a well known hematotoxicant which induces hematopoietic dyscrasias of varying intensities in different individuals and even in different strains of the same experimental animal species. Although there is ample evidence that diverse responses to benzene are related to differences in benzene metabolism, we have recently provided evidence implicating differences in host target cell susceptibility to these diverse responses to benzene. The present study extends our previous work and concerns strain-specific differences in marrow progenitor cells that survive benzene exposure. Two mouse strains (Swiss-Webster and C57Bl/6J) which respond to benzene exposure with different intensities of bone marrow cytotoxicity were used. Bone marrow cells from benzene-exposed and untreated mice were cultured with one of five benzene metabolites: 1,4-benzoquinone (BQ), catechol (C), hydroquinone (HQ), muconic acid (MA) or phenol (P) and the abilities of these cells to produce erythroid (CFU-e) or granulocyte/macrophage colonies (GM-CFU-c) were assessed. In both strains, marrow cells isolated from benzene-exposed mice showed a higher percentage of plated CFU-e surviving culture with BQ, HQ or MA than marrow cells isolated from control mice. In contrast, both strains of benzene-exposed mice displayed decreased percentages of plated CFU-e surviving culture with catechol than cells isolated from control mice. Only one condition (the culturing of cells with HQ under GM-CFU-c forming conditions) showed any strain-specific difference in plating efficiency. In all, 20 possible combinations of benzene metabolites and cell types were examined (5 metabolites × 2 progenitor cell types × 2 strains). With seven of these combinations, the colony-forming efficiencies were higher for plated cells isolated from benzene-exposed mice than from untreated mice. With three combinations, the colony-forming efficiencies were lower for cells from benzene-exposed mice, and for ten combinations, there were no changes in plating efficiencies. Possible mechanisms for an acquired resistance to the toxicities of benzene metabolites were explored by measuring the concentrations of hepatic and bone marrow sulfhydryl (SH) groups in cells isolated from benzene-exposed and untreated mice. In both strains, benzene exposure induced no changes in hepatic SH concentrations, but the SH content of bone marrow was more than doubled after benzene exposure in both strains. These results suggest that a fraction of hematopoietic progenitor cells are able to survive severe benzene exposure and produce progeny because of a marked increase in marrow SH groups which react with electrophilic benzene metabolites. Moreover, this protective mechanism occurs in two mouse strains with differing susceptibilities to benzene. Received: 23 November 1993/Accepted: 26 April 1994     

Toxicity of β-blockers in a rat whole embryo culture: concentration-response relationships and tissue concentrations

Beta-adrenoceptor blockers are widely used drugs for the treatment of cardiovascular diseases. Since β-blockers cross the placenta, it is essential to consider possible adverse effects on the embryo. Six β-adrenoceptor blockers were tested at various concentrations (10 – 5000 μM) in a rat whole embryo culture. Although inducing a very similar pattern of dysmorphogenetic effects (incomplete flexure, disturbed development of the neural tube, the head, the heart and the tail bud), the compounds exhibited a wide range of embryotoxic potency. Estimation of the EC₅₀ (median-concentration producing dysmorphogenesis in 50% of the embryos) for the six compounds revealed differences of more than two orders of magnitude: propranolol 25 μM, alprenolol 30 μM, metoprolol 100 μM, pindolol 150 μM, acebutolol 500 μM, atenolol 4000 μM. Measurements of the concentrations of the various drugs in the cultured embryos at corresponding EC₅₀ levels showed differing values: metoprolol 4.5 μM, propranolol 5.2 μM, alprenolol 8.4 μM, pindolol 9.0 μM, acebutolol 12.5 μM and atenolol 77.0 μM. With regard to the EC₅₀ and the degree of substance transfer to the embryo it can be stated that propranolol and metoprolol show a much higher intrinsic potency to interfere with normal in vitro embryonic development than, e.g. atenolol. Received: 1 September 1993 / Accepted: 16 February 1994     

Toxic effects and excretion in urine of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in the rat after a single oral dose

Toxic effects and excretion in urine of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), the potent mutagenic compound in chlorinated drinking water, was evaluated in male Wistar rats by the up-and-down method. MX was dosed by gavage in deionized water at doses between 200 mg/kg and 600 mg/kg, for one animal at a time, and effects were observed for 14 days. Urine was collected in metabolism cages up to 72 h after dosing for chemical analysis of MX in urine. The animals receiving 200 mg/kg did not display clear clinical signs but at higher doses the signs of ill effects included dyspnea, laborious, wheezing and gasping breathing, decreased spontaneous motor activity, ataxia, nostril discharges, catalepsia and cyanosis. In necropsy bronchi contained foamy liquid and the lungs appeared edematous and spongy. The stomach cavity was expanded due to accumulation of fluid and gas and the gastrointestinal tract from stomach to caecum was reddish. Microscopically, the main target organ of toxicity was the gastrointestinal tract (diffuse congestion and necrosis in the mucosa). Signs of toxicity were recorded also in lungs (slight edema) and kidneys (dilated tubules, thin tubular epithelium, brownish tubular and interstitial concretion). The LD₅₀ in 48 h was 230 mg/kg. Only 0.03 – 0.07% of the dose (200 mg/kg or 300 mg/kg) was excreted in urine as intact MX. The results indicate that at high doses MX has a strong local irritating effect in the gastrointestinal tract and it probably increases liquid permeability in lungs. MX may also cause tubular damage in kidneys. Data also indicate that after an oral dose only traces of MX are excreted in urine as intact compound, suggesting that MX is subjected to intense metabolism before excretion, even at lethal doses. Received: 14 December 1993/Accepted: 28 February 1994     

Inhalation toxicokinetics of butoxyethanol and its metabolite butoxyacetic acid in the male Sprague-Dawley rat

A total of 16 male Sprague-Dawley rats were continuously exposed to 20 ppm or 100 ppm butoxyethanol (BE) vapor for 1, 2, 3, 4, 6, 8, 10, or 12 days. Urine was collected in 24-h intervals and stored at –70°C. At the end of the exposure the animals were euthanized by decapitation and tissue samples of blood, muscle, liver and were rapidly collected and frozen to –70°C. The samples were later derivatized and analyzed for BE and its major metabolite butoxyacetic acid (BAA) by electron capture gas chromatography. BE and BAA were rapidly distributed to the tissues examined. The concentration of BE in blood was slightly higher, and that of BAA markedly higher than in other tissues, indicating weak (BE) and pronounced (BAA) blood protein binding, respectively. BE was efficiently metabolized and the blood clearance averaged 2.6 l/h per kg, corresponding to a hepatic extraction ratio of about 0.75. The renal clearance of BAA (average 0.53 l/h per kg) corresponded to approximately 15% of the renal blood flow. The kinetics of BE and BAA were linear up to 100 ppm. There were no clear indications of changes in the toxicokinetics, such as metabolic induction or inhibition of metabolism or excretion, during the course of the exposure. The recovery of BAA in urine was 64% of the calculated inhaled amount of BE, on an equimolar basis. Received: 15 February 1994/Accepted: 3 May 1994     

Computer assisted recognition and quantitation of the effects of airborne chemicals acting at different areas of the respiratory tract in mice

The pattern and timing of a normal breath in unanesthetized mice was analyzed from measurement of inspiratory and expiratory airflows (V^.I and V^.E). Airflow was measured via a differential pressure transducer, attached to a pneumotachograph, which itself was attached to a body plethysmograph into which a mouse was placed. The analog voltage from the differential pressure transducer was digitized and stored for analysis on a microcomputer. Criteria were developed to classify each breath as normal (N) or belonging into one of seven abnormal categories. The abnormal categories were arrived at by computer analysis, recognizing specific modifications of the normal pattern into patterns of: sensory irritation of the upper respiratory tract (S), airflow limitation within the conducting airways of the lungs (A) or pulmonary irritation at the alveolar level (P). Combinations of these effects, i.e., S+A, P+A, P+S and P+S+A were also recognized. Computer analysis of each breath also permitted quantitative evaluation of the degree of S, A or P abnormalities. To induce each type of effect we used inhalation exposures to 2-chlorobenzylchloride, carbamylcholine or propranolol. We propose that this approach will permit rapid evaluation of the possible effects of airborne chemicals at three levels of the respiratory tract, with the classification of the type of effect easily obtained in an objective way using well defined criteria, followed by quantitation of the degree of each effect. Received: 16 December 1993 / Accepted: 11 April 1994     

Acute effects of methylmercury on hepatic and renal glutathione metabolisms in mice

Because of its high affinity to the sulfhydryl group, the in vivo fate of methylmercury (MeHg) is closely related to the glutathione (GSH) metabolism. Here, to examine the possible effects of MeHg on the GSH metabolism, C57BL female mice were challenged by this heavy metal at a marginal dose level to induce slight renal dysfunction. Liver and blood GSH levels decreased by 16% and 20%, respectively, 24 h after MeHg (160 μmol/kg) administration, whereas kidney and plasma levels drastically increased. The GSH half-lives obtained using L-buthionine-(S,R)-sulfoximine were shortened by 17% in the liver, but lengthened by 28% in the kidney. The accelerated secretion of GSH from the liver and/or blood cells might have caused increased plasma levels of the tripeptide, which in turn could increase the supply of the constituent amino acids for GSH synthesis to the kidney. Furthermore, renal γ-glutamylcysteine synthetase activity, a rate-determining enzyme in GSH biosynthesis, was found to be enhanced in the MeHg-treated group. The marked increase in the renal GSH levels induced by MeHg could be due to the increased synthesis and the decreased efflux of the tripeptide in this tissue. The MeHg-induced alterations of GSH metabolism described here might reflect one of the defense mechanisms of bioorganisms against the challenge by MeHg. Received: 29 November 1993 / Accepted: 25 April 1994     

Oxidation pathways for the intracellular probe 2′,7′-dichlorofluorescin

The oxidation of 2’,7’-dichlorofluorescin (DCFH) to a fluorescent product is currently used to evaluate oxidant stress in cells. However, there is considerable uncertainty as to the enzymatic and nonenzymatic pathways that may result in DCFH oxidation. Iron/hydrogen peroxide-induced DCFH oxidation was inhibited by catalase or by the hydroxyl radical scavenger dimethylsulfoxide; however, superoxide dismutase (SOD) had no effect on DCFH oxidation. The formation of hydroxyl radical (indicated by the oxidation of salicylic acid to 2,3-dihydroxybenzoic acid) was proportional to DCFH oxidation, suggesting that the hydroxyl radical is responsible for the iron/peroxide-mediated oxidation of DCFH. Utilizing a superoxide generating system consisting of hypoxanthine/xanthine oxidase, oxidation of DCFH was unaffected by SOD, catalase or desferoxamine, and stimulated by removing hypoxanthine from the reaction mixture. In contrast, SOD or elimination of hypoxanthine abolished superoxide formation. In addition, potassium superoxide did not support the oxidation of DCFH. Thus, superoxide is not involved in DCFH oxidation. Boiling xanthine oxidase eliminated its concentration-dependent oxidation of 1 μM DCFH, indicating that xanthine oxidase can enzymatically utilize DCFH as a high affinity substrate. Kinetic studies of the oxidation of DCFH by xanthine oxidase indicated a K _{m (app)} of 0.62 μM. Hypoxanthine competed with DCFH with a K _{i (app)} of 1.03 mM. These studies suggest that DCFH oxidation may be a useful indicator of oxidative stress. However, other types of cellular damage may produce DCFH oxidation. For example, conditions or chemicals that damage intracellular membranes may release to the cytoplasm oxidases or peroxidases that might use DCFH as a substrate, similar to xanthine oxidase Received: 25 October 1993 / Accepted: 14 March 1994     

Metabolism of dichloromethane (methylene chloride) to formaldehyde in human erythrocytes: influence of polymorphism of glutathione transferase Theta (GST T1-1)

Human hemolysate was incubated in vitro with different concentrations of dichloromethane (methylene chloride). The resulting enzymatically mediated production of formaldehyde was determined by two independent analytical methods (Nash-reaction/colorimetry or HPLC). The formation of formaldehyde from dichloromethane is influenced by the polymorphism of glutathione-S-transferase (GST) Theta, in the same way as the metabolism of methyl bromide, methyl chloride, methyl iodide and ethylene oxide. Three quarters of the population (“conjugators”) possess, whereas one quarter (“non-conjugators”) lack this enzyme activity in human erythrocytes. The metabolism of dichloromethane in hemolysate in vitro can be described by Michaelis-Menten kinetics; for an individual with high GST T1-1 enzyme activity, the maximum velocity of formaldehyde production was calculated to be approximately 180 pmol/min per mg Hb, the k_M being approximately 60 mM dichloromethane. Carcinogenicity of dichloromethane in long-term inhalation exposure of rodents has been attributed to metabolism of the compound via the GST-dependent pathway. Extrapolation of the results to humans for risk assessment should consider the newly discovered polymorphic enzyme activity of GST Theta. Furthermore, the possible existence of a “high-risk” population among humans should be considered in epidemiological research. Received: 1 February 1994/Accepted: 30 March 1994     

Effect of sodium pyridinethione on the uptake and distribution of nickel in rats,ferrets and guinea-pigs

Oral administration of sodium pyridinethione together with Ni²⁺ (using ⁶³Ni²⁺ as a tracer) to rats, ferrets and guinea-pigs produced highly increased tissue levels of the metal in several tissues in comparison with animals given the Ni²⁺ alone. Ni²⁺ forms a lipophilic complex with pyridinethione and it can be assumed that a facilitated passage of the Ni²⁺ across the cellular membranes of various tissues is important for the observed effects. Pigmented tissues (e. g. the eye melanin), the pancreatic islets, the nervous system and striated muscles showed high levels of Ni²⁺ in animals given sodium pyridinethione. However, in some instances marked species differences were observed. Thus, microautoradiography indicated an uptake of Ni²⁺ both in the β- and α-cells in the pancreatic islets in the rat, whereas in the guinea-pig only some cells (probably the α-cells) accumulated high levels of Ni²⁺. In the ferret sodium pyridinethione induced a high uptake of Ni²⁺ in the heart muscle, which was not seen in the other species. The Ni²⁺ is probably taken up in the various tissues complexed to pyridinethione. Within the tissues the complex may dissociate and the Ni²⁺ may bind to some endogeneous tissue components. The affinity of the Ni²⁺ for the endogeneous ligands in relation to the affinity for the pyridinethione may be of importance for the effects on the disposition of the Ni²⁺. The species variations may be related to differences in the structural conformations of the endogeneous Ni²⁺-binding ligands. Received: 25 October 1993/Accepted: 25 January 1994     

Electrophysiological and biochemical effects following single doses of organophosphates in the mouse

Single doses of organophosphates (mipafox or ecothiopate) were given subcutaneously to mice. At intervals up to 77 days after dosing animals were killed and muscle action potentials and endplate potentials were recorded intracellularly in mouse phrenic-nerve/hemidiaphragm preparations. Activities of acetylcholinesterase and neuropathy target esterase in brain and acetylcholinesterase in diaphragm were also measured. Mipafox (0.11 mmol/kg), a neurotoxic organophosphate, produced an increase in prejunctional jitter (i. e. the variabilities of the latencies) of endplate potentials. This increase began 14 – 21 days after administration and lasted more than 23 days. No clinical signs of neuropathy were observed during this study. Mipafox also produced an increase in postjunctional (muscle action potential) jitter. Mipafox inhibited brain and diaphragm acetylcholinesterase and brain neuropathy target esterase. By comparison, a non-neurotoxic organophosphate, ecothiopate (0.5 μmol/kg), was a potent inhibitor of diaphragm acetylcholinesterase and produced a large increase in postjunctional jitter but ecothiopate did not inhibit brain neuropathy target esterase and had no effect on prejunctional jitter. Doses were chosen so that the inhibition of diaphragm acetylcholinesterase by each of the two organophosphates was similar. It is concluded that the neurotoxic organophosphate, mipafox, produced measurable changes in nerve function. These long-term changes may represent a new phenomenon, unrelated to the classical organophosphate induced delayed neuropathy. Alternatively, they may represent a neuropathic process which precedes or is below the threshold for clinical signs. Received: 4 November 1993/Accepted: 2 February 1994     

Piperonyl butoxide induces hepatocellular carcinoma in male CD-1 mice

Male CD-1 mice in groups of 52, 53 or 100 were administered piperonyl butoxide (α-[2-(2-butoxyethoxy)ethoxy-4,5-methylenedioxy-2-propyltoluene) in the diet at levels of 0 (control), 0.6 and 1.2% for 12 months. Hepatocellular carcinoma was induced in treated groups in a dose-dependent manner but not in the control group. The incidences of hepatocellular carcinoma were 11.3 and 52.0% in mice given 0.6 and 1.2% piperonyl butoxide, indicating that piperonyl butoxide can cause hepatocellular carcinoma in mice as it is known to do in rats. Received: 18 January 1994/Accepted: 8 March 1994