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Archives of Toxicology (v.69, #6)
Inhibitory effect of methylmercury on migration and tube formation by cultured human vascular endothelial cells by Takuji Kishimoto; Tetsuhisa Oguri; Miyoko Abe; Hiroko Kajitani; Manabu Tada (pp. 357-361).
The effect of methylmercury chloride (MeHg) on migration and tube formation by cultured human umbilical vein endothelial cells (HUVECs) was quantitatively analyzed. The distance of endothelial cell outgrowth from the scraped edge of a monolayer was measured. HUVEC outgrowth was inhibited by MeHg (1.0–5.0 μM) treatment in a dose-dependent manner. Tube formation was studied by culturing the cells on gelled basement membrane matrix (Matrigel). Treatment of HUVECs with 0.1–5.0 μM MeHg for 24 h inhibited tube formation dose-dependently. These results suggest that migration and tube formation by HUVECs are susceptible to MeHg cytotoxicity, and that MeHg could be injurious to endothelial cell function, which may be involved in the pathogenesis of arteriosclerosis.
Keywords: Endothelial cell; Methylmercury; Migration; Tube formation; Arteriosclerosis
Formation of epoxide and quinone protein adducts in B6C3F1 mice treated with naphthalene, sulfate conjugate of 1,4-dihydroxynaphthalene and 1,4-naphthoquinone by Laurie S. Tsuruda; Michael W. Lamé; A. Daniel Jones (pp. 362-367).
Naphthalene (NA) is metabolically activated to the reactive intermediates, naphthalene oxide (NO) and naphthoquinones. To investigate the role of circulating reactive metabolites in NA toxicity, the half-life of NO was examined. The in vitro half-life of NO in both whole blood and plasma was 10 min. Detectable levels of NO were seen in perfusate leaving the isolated perfused liver of B6C3F1 mice infused with 10 μmol/h NA. Identification of protein sulfhydryl adducts in NA-exposed mice (50 and 100 mg/kg, IP, 24h) revealed a predominance of quinone adducts in liver, lung, kidney, red blood cells and brain. The epoxide adduct predominated in plasma protein. Administration of the sulfate conjugate of 1,4-dihydroxynaphthalene (NHQS) (100 mg/kg) resulted in formation of naphthoquinone protein sulfhydryl adducts in lung, liver and kidney. Administration of 1,4-naphthoquinone (NQ) (5mg/kg) produced NQ adducts in liver, lung, kidney, plasma and brain.
Keywords: Naphthalene; Naphthalene oxide in vitro half-life; 1,4-Naphthoquinone; Sulfate ester of 1,4-dihydroxynaphthalene; Protein binding
Interactive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and retinoids on proliferation and differentiation in cultured human keratinocytes: quantification of cross-linked envelope formation by Jos A. M. Berkers; Ine Hassing; Bert Spenkelink; Abraham Brouwer; Bas J. Blaauboer (pp. 368-378).
Dioxins are potent inducers of chloracne in humans. This skin aberration can be interpreted as an altered differentiation pattern of acinar sebaceous base cells and a change in the rate of terminal differentiation of the keratinocytes. We measured this rate induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in primary cultures of human keratinocytes. As parameters for differentiation, we quantified the 35S-methionine incorporation into cross-linked envelopes (revealing the total CLE biomass), as well as the number of microscopically visible CLEs. It was shown that TCDD is a very potent inducer of both CLE biomass and number with a half-maximal effect concentration (EC50) of 1.4 nM. CLE biomass was maximally increased 10-fold and the number of cells in culture producing a CLE was increased from 15% in control cultures to maximally 75% of the cells in TCDD-treated cultures. Both effects were Ca2+-dependent and increased with elevated cell density, being optimal in post-confluent cultures. Retinoic acid dose-dependently decreased the effect of 10−8 M TCDD, 10−6 M having a nearly complete antagonistic action. This interaction of retionic acid with TCDD-induced differentiation was non-competitive. Retinol was equally potent as an antagonist of the TCDD-induced elevation of CLE formation as compared with retinoic acid. Retinyl palmitate and etretinate were not very effective as TCDD antagonists. Supplementation of hydrocortisone suppressed the TCDD-induced keratinocyte differentiation. It was concluded that CLE biomass quantification provides a reliable and sensitive parameter for keratinocyte differentiation. In this in vitro system it is shown that TCDD strongly induces a switch from proliferation to terminal differentiation and that this effect can be antagonized effectively by retinoic acid and retinol.
Keywords: Cell culture; Differentiation; Dioxion; Keratinocytes; Retinoids
Acute toxicity of cyclohexylmethylphosphonofluoridate (CMPF) in rhesus monkeys: serum biochemical and hematologic changes by G. David Young; Irwin Koplovitz (pp. 379-383).
Changes in serum biochemical and hematological parameters were studied in 20 male rhesus monkeys following acute poisoning by the organophosphate nerve agent cyclohexylmethylphosphonofluoridate (CMPF or GF). Animals were challenged with 5 × LD50 GF (233 μg/kg, IM) following pretreatment with pyridostigmine (0.3–0.7 mg/kg per 24 h) and treated with atropine (0.4 mg/kg, IM) and either 2-PAM (25.7 mg/kg, IM) or HI6 (37.8 mg/kg, IM) at the onset of clinical signs or at 1 min after exposure. Muscle fasciculations, tremors, or convulsions occurred in 19 of 20 animals. Serum biochemical and hematologic parameters were analyzed 2 days and 7 days after exposure and compared to pre-exposure baseline values. Significant increases in creatine kinase (CK), lactate dehydrogenase (LD), aspartate transaminase (AST), alanine transaminase (ALT) and potassium ion (K+), associated with damage to striated muscle and metabolic acidosis, occurred in both oxime-treated groups 2 days after exposure. Total protein, albumin, red blood cell (RBC) count, hemoglobin concentration (Hb) and hematocrit (Hct), were decreased in both oxime-treated groups at 7 days. The results demonstrate that animals exposed to a single high dose of GF and treated with standard therapy exhibit changes in serum biochemical and hematological indices directly and indirectly associated with their clinical presentations.
Keywords: Cyclohexylmethylphosphonofluoridate; Rhesus monkey; Serum; Biochemistry; Hemotology
Assessment of primary neuronal culture as a model for soman-induced neurotoxicity and effectiveness of memantine as a neuroprotective drug by Sharad S. Deshpande; Catherine D. Smith; Margaret G. Filbert (pp. 384-390).
An in vitro mammalian model neuronal system to evaluate the intrinsic toxicity of soman and other neurotoxicants as well as the efficacy of potential countermeasures was investigated. The link between soman toxicity, glutamate hyperactivity and neuronal death in the central nervous system was investigated in primary dissociated cell cultures from rat hippocampus and cerebral neocortex. Exposure of cortical or hippocampal neurons to glutamate for 30 min produced neuronal death in almost 80% of the cells examined at 24 h. Hippocampal neurons exposed to soman for 15–120 min at 0.1 μM concentration caused almost complete inhibition (⋝90%) of acetylcholinesterase but failed to show any evidence of effects on cell viability, indicating a lack of direct cytotoxicity by this agent. Acetylcholine (ACh, 0.1 mM), alone or in combination with soman, did not potentiate glutamate toxicity in hippocampal neurons. Memantine, a drug used for the therapy of Parkinson’s disease, spasticity and other brain disorders, significantly protected hippocampal and cortical neurons in culture against glutamate and N-methyl-D-aspartate (NMDA) excitotoxicity. In rats a single dose of memantine (18 mg/kg) administered 1 h prior to a s.c. injection of a 0.9 LD50 dose of soman reduced the severity of convulsions and increased survival. Survival, however, was accompanied by neuronal loss in the frontal cortex, piriform cortex and hippocampus.
Keywords: Memantine pretreatment; Soman neurotoxicity; Glutamate excitotoxicity; Hippocampal neurons; Neuronal loss
Lipidosis of the dorsal root ganglia in rats treated with an almitrine metabolite by Yoshihiro Yamanaka; Emiko Sakamoto; Yasuji Sakuma; Hiroshi Uno; Tamotsu Koyama; Yoshihiro Izawa; Kosaku Fujiwara (pp. 391-396).
Toxic effects of a detriazinyl metabolite of almitrine (DTMA) were evaluated in rats and on cultured rat macrophages. In rats daily treated with DTMA for 16 weeks, spastic gaits with heel-lifting appeared, and lamellated and/or crystalloid bodies formed in sensory neurons, satellite cells, Schwann cells, and vascular endothelial cells of the dorsal root ganglia. The lysosomal lamellated bodies, which were not induced by almitrine, were produced also in cultured rat macrophages exposed to over 1 ξ 10−5 M DTMA.
Keywords: Almitrine bismesylate; Detriazinyl metabolite of almitrine; Cationic amphiphilic drugs; Drug-induced lipidosis; Neuropathy
Acute and subacute inhalation toxicity of diborane in male ICR mice by Takamoto Uemura; Kazuyuki Omae; Hiroshi Nakashima; Haruhiko Sakurai; Kazuto Yamazaki; Toshikatsu Shibata; Koji Mori; Mitsuhiro Kudo; Hirokazu Kanoh; Masatomo Tati (pp. 397-404).
To clarify the toxicity of diborane, we conducted acute (15 ppm for 1, 2, 4 or 8h) and subacute (5 ppm for 2 or 4 weeks) inhalation studies on ICR mice. The concentration resulting in a 50% kill after 4 h exposure was 31.5 ppm. body weight gain was suppressed and the lung weight was increased in diborane-exposed mice in both acute and subacute studies. In the acute study, diffuse pan bronchiolitis-like lesions developed in the lung in various degrees depending on exposure time, which can be pathologically characterized as infiltration of inflammatory cells into the terminal bronchioles and surrounding alveoli, pulmonary congestion and bleeding and/or edema. In the subacute study, we observed lymphoid hyperplasia in the perivascular and peribronchial areas, and infiltration of macrophage and plasma cells into the alveoli. In the mice exposed for 4 weeks, the lesions were more severe than in those exposed for 2 weeks, consisting of hyperplasia and desquamation of Clara cells. In the nasal cavity, we saw mucous exudate and inflammatory cells, suggesting irritation caused by diborane. The histopathological findings, except for the respiratory organs, did not reveal any exposure-related changes. No significant changes were seen in hematological and serum biochemical examinations either. In conclusion, the target organ of diborane inhalation is the respiratory organs, particularly the lung. Further inhalation experiments are essential to investigate the safety exposure levels of diborane.
Keywords: Diborane; Mice; Toxicity; Inhalation
Toxicity of 3-methylindole, 1-nitronaphthalene and paraquat in precision-cut rat lung slices by Roger J. Price; Anthony B. Renwick; Paula T. Wield; Jenny A. Beamand; Brian G. Lake (pp. 405-409).
The toxicity of 3-methylindole, 1-nitronaphthalene and paraquat has been studied in precision-cut rat lung slice cultures. Lung slices were prepared from male Sprague-Dawley rats using an agarose gel instilling technique with a Krumdieck tissue slicer and cultured for 24 h in a dynamic organ culture system. Treatment of rat lung slices with 3-methylindole, 1-nitronaphthalene or paraquat produced concentration dependent decreases in lung slice protein synthesis and potassium content. EC50 values (concentration to produce a 50% inhibition) for protein synthesis were 0.024, 0.27 and 0.57 mM for paraquat, 1-nitronaphthalene and 3-methylindole, respectively. These results demonstrate that precision-cut lung slices are a useful in vitro model system for studying the pulmonary toxicity of xenobiotics. Lung slices offer the potential as a rapid in vitro screen for identifying pulmonary toxicants and to evaluate species differences in response.
Keywords: Rat lung slices; In vitro toxicity; 3-Methylindole; 1-Nitronaphthalene; Paraquat
Changes in markers of oxidative status in brain, liver and kidney of young and aged rats following exposure to aromatic white spirit by S. C. Bondy; H. R. Lam; G. Østergaard; S. X. Guo; Ø. Ladefoged (pp. 410-414).
Levels of glutathione and activity of glutamine synthetase were assayed in organs of rats following inhalation of a heterogeneous solvent mixture containing both aliphatic and aromatic hydrocarbons. This mixture was administered for 3 weeks (6 h daily) at two levels in the inhaled air (400 and 800 ppm) to young adult (5-month-old) and aged (14-month-old) rats. Depression of levels of glutamine synthetase in the P2 fraction of kidney was observed, which was more severe in aged than young adult rats. Glutamine synthetase is a cytosolic enzyme especially susceptible to oxidative damage. A parallel depression of this enzyme was also seen in the corresponding hepatic fractions. However, levels of glutamine synthetase in the hippocampus were elevated by this exposure. Glutathione levels were depressed in P2 fractions of livers of exposed rats, and also in the corresponding renal fraction. Glutathione concentration was unchanged in cerebral fractions. Overall results were interpreted to imply that pro-oxidant events were elevated in kidney and liver following prolonged inhalation of the solvent mixture. The changes found in brain tissue did not reveal evidence of oxidative stress but, however, suggested that glial activation was taking place.
Keywords: Solvents; Aromatics; Free radicals; Reactive oxygen; Oxidative stress; Synaptosomes
Inhibition of intercellular communication by condensates of high and low tar cigarettes by Ole Vang; Håkan Wallin; Herman Autrup (pp. 415-420).
Inhibition of gap junctional intercellular communication (GJIC) is a predictive short term test for tumor promoting activity. A new metabolic cooperation assay has been developed, which takes the cytochrome P-450 metabolism into account. In this assay the inhibitory activity of tobacco smoke condensates (CSC) and CSC fractions from high and low tar cigarettes was tested. CSC of both high and low tar cigarettes and fractions thereof contained tumor promoting activity. The tar yield of the cigarettes did not closely reflect the effects in the GJIC assay and the major constituent nicotine had no effect. The effect was only marginally greater in cells expressing different cytochrome P-450 enzymes, indicating that the active substances are not metabolized by these enzymes. The activities of CSC fractions were considerably lower than the activities in the unfractionated CSC. This may indicate that compounds in the CSC act strongly synergistically. Furthermore, CSC and CSC fractions synergistically inhibit GJIC with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, indicating different mechanisms of action.
Keywords: Intercellular communication; Cytochrome P450; Cigarette smoke condensate; High tar; Low tar; Nicotine
Influence of glucose on the toxicity of oxophenylarsine in MDCK cells by B. Liebl; H. Mückter; E. Doklea; F. X. Reichl; B. Fichtl; W. Forth (pp. 421-424).
Trivalent arsenicals like oxophenylarsine (PhAsO) inhibit cellular pyruvate dehydrogense, thus leading to a drop of acetylCoA formation and a slowdown of the citric acid cycle. Glucose may protect cells from arsenic toxicity, because increased glycolysis may prevent fatal shortage of ATP. On the other hand, PhAsO has been shown to inhibit glucose uptake in Madin-Darby canine kidney (MDCK) cells. We have investigated the effect of PhAsO on viability, ATP levels and glucose uptake of MDCK cells in the presence of normal (5 mmol/1) and low (0.01 mmol/1) glucose concentrations. At normal as well as at low glucose concentration was not affected by PhAsO concentrations formationswas not affected by PhAsO concentrations up to 2 μmol/1 within 3 h of observation. At higher PhAsO concentrations viability was diminished earlier and more pronounced in the presence of low glucose concentrations. 10μmol/1 PhAsO induced a drastic drop of ATP within 30 min which was followed by an almost complete loss of viable cells after 180 min in the presence of low glucose concentrations, while at normal glucose levels no influence on ATP contents or on cell viability was detected within 60 min of incubation. On the other hand, glucose uptake, determined as 14C accumulation by cells incubated for 10min with D-[6-14C]-glucose, was inhibited by PhAsO at low as well as at normal glucose concentrations in a dose dependent manner. At normal glucose concentrations, however, basal uptake was higher (57 nmol/mg protein) and half-maximum inhibition (IC50) was shifted to higher PhAsO concentrations (2×10−4mol/1) than at low glucose levels (basal uptake: 1.6nmol/mg protein; IC50: 5×10−5mol/1). It is concluded that 1) in PhAsO-exposed MDCK cells different uptake mechanisms operate in high and low glucose states and 2) that glucose exerts a beneficial effect on the toxicity of PhAsO in these cells.
Keywords: Oxophenylarsine; Glucose; MDCK cells
Interaction of phosphamidon with neuropathy target esterase and acetylcholinesterase of hen brain by Milan Jokanović; Matej Maksimović; Radica M. Stepanovic (pp. 425-428).
Phosphamidon (PSM) is an organophosphorus insecticide widely used in agriculture. This study was undertaken to examine the interaction of PSM with acetylcholinesterase (AChE) and neuropathy target esterase (NTE) of hen brain in vitro and in vivo. PSM was a potent inhibitor of AChE, with an I50 of 2.9μM and second-order rate constant (ka) of 1.2×104 M−1 min−1 at 37°C. PSM-inhibited AChE aged rapidly (t1/2=1.9h). Pyridinium oximes pralidoxime, trimedoxime, obidoxime and HI-6 were effective reactivators of PSM-inhibited AChE, providing up to 75% reactivation. PSM was one of the weakest inhibitors of NTE among organophosphorus compounds, with an I50 of 19 mM and ka of 1.8 M−1 min−1 at 37°C. Inhibited NTE did not reactivate spontaneously and KF-induced reactivation was not obtained even at the earliest tested moments, so it was not clear whether aging of PSM-inhibited NTE occurred very quickly or the KF molecule could not affect the stability of phosphoryl-NTE bond. From the ratio of kas for NTE and AChE (0.00015) it was predicted that delayed neuropathic effects of PSM in vivo would appear only at doses far above the acute LD50. The LD50 value of PSM p.o. for hens was 9 mg/kg. Hens were treated with a single oral dose of PSM, combined with standard antidotal treatment which included atropine, physostigmine, pralidoxime and anticonvulsant midazolam. Doses of 90 and 250 mg/kg caused up to 27% and 45% NTE inhibition 48h after poisoning, respectively. Clinical signs of neuropathy were not seen up to 25 days after treatment, which could be expected, since the proposed level (>70%) of NTE inhibition necessary for the occurrence of delayed neuropathy was not achieved. The results suggest that PSM, at doses tested, has no ability to cause delayed neuropathy in hens without showing signs of severe cholinergic intoxication.
Keywords: Phosphamidon; Organophosphorus compounds; Acetylcholinesterase; Neuropathy target esterase