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Archives of Toxicology (v.71, #3)
Intranephron distribution of cysteine S -conjugate β-lyase activity and its implication for hexachloro-1,3-butadiene-induced nephrotoxicity in rats by H. S. Kim; S. H. Cha; D. G. Abraham; Arthur J. L. Cooper; H. Endou (pp. 131-141).
The intranephron distribution of two major cysteine S-conjugate β-lyases was determined in order to clarify the role of these enzymes in promoting the nephrotoxicity associated with certain halogenated xenobiotics. Various nephron segments [i.e., glomerulus, early, middle, and terminal portions of the proximal tubule (S1, S2, and S3 respectively), the thick ascending limb, the distal tubule, and the collecting tubule] were isolated by microdissection from collagenase-treated rat kidneys. Each segment was dissected in Hanks' solution, solubilized with Triton X-100, and applied to a micropolyacrylamide gel constructed with a continuous gradient. The gels were subjected to electrophoresis and then incubated in the dark in a solution containing S-(1,2 dichlorovinyl)-l-cysteine (DCVC), sodium α-keto-γ-methiolbutyrate, phenazine methosulfate, and nitroblue tetrazolium. The position of cysteine S-conjugate β-lyase- and l-amino acid oxidase activities in the gels was revealed by the presence of blue formazen dye bands. The relative intensities of the bands were determined by optical scanning with a microdensitometer. Three bands were detected: band I (Mr ˜ 330 000) corresponds to a recently described high Mr cysteine S-conjugate β-lyase whereas band III (Mr ˜ 90 000) corresponds to a lower Mr cysteine S-conjugate β-lyase (identical to cytosolic glutamine transaminase K). Band II (Mr ˜ 240 000) corresponds to l-amino acid oxidase (a unique activity of the B isoform of rat kidney l-hydroxy acid oxidase). β-Lyase activity with DCVC as substrate was detected in the S1, S2, and S3 segments of the nephron but not in other regions of the kidney. The activity was in the order: S2 = S3 > S1. In another series of experiments, rats were killed 24 h after treatment with hexachloro- 1,3-butadiene (HCBD). In whole kidney homogenates the relative intensity of band III (per 22.2 μg tissue wet weight) after a 30 min incubation was induced significantly (by 50%), but the relative intensities of the other two bands were unchanged. On the other hand, in proximal tubules isolated from HCBD-treated rats the relative intensities (per 5 mm of nephron) of peak I of S2, peak II of S3, and peak III of S3 were significantly reduced by 28, 33, and 72%, respectively. These findings suggest that the low Mrβ-lyase is induced by HCBD and that impaired cell function in the segments (especially S3) results in proteins leaking out of the target cells. To examine the relationship between the nephrotoxic effect of HCBD and cysteine S-conjugate β-lyase activity, the intracellular ATP:protein ratio was quantitated in each nephron segment and in whole kidney homogenates. In HCBD-treated rats the ATP:protein ratio of the S1, S2, and S3 segments was unchanged, decreased by ˜50%, and increased by ˜30%, respectively. In the kidney homogenates of HCBD-treated rats the ATP content was decreased by 32%. However, the loss of ATP was significantly less when the rats were pretreated with aminooxyacetate (a general inhibitor of pyridoxal 5′-phosphate-dependent enzymes, including β-lyase) 1 h before HCBD administration. The results strongly suggest that HCBD is converted to toxic metabolites within the kidney and that this process leads to metabolic derangement and reduction of ATP in susceptible kidney cells.
Keywords: Key words Cysteine S-conjugates ; Damage to the S3 region of proximal tubules by halogenated xenobiotics ; Intranephron distribution of cysteine S-conjugate β-lyase ; β-Lyase activity staining with S-(1; 2-dichlorovinyl)-l-cysteine ; Nephrotoxicity of hexachloro-1; 3-butadiene
Effects of all-trans-retinoyl-β-d-glucuronide and all-trans-retinoic acid on chondrogenesis and retinoid metabolism in mouse limb bud mesenchymal cells in vitro by J. O. Sass; Bernd Zimmermann; Ralph Rühl; Heinz Nau (pp. 142-150).
Retinoids, derivatives of vitamin A, are essential for many vertebrate functions. Furthermore, several drugs of this class of compounds are valuable in the treatment of certain forms of skin disorders and cancer. However, the therapeutic application of retinoids is limited by their teratogenic potency. The limbs are important sites of retinoid-induced malformations in rodents. Therefore, organoid cultures of limb bud mesenchymal cells have been established for screening of the teratogenic potency of retinoids. We have now applied this system to compare the effects of all-trans-retinoyl-β-d-glucuronide (all-trans-RAG) with those of all-trans-retinoic acid (all-trans-RA) on chondrogenesis, as assessed by the Alcian blue binding assay and by electron microscopic evaluation including quantitative morphometric analysis. First data of retinoid toxicokinetics in the culture media as well as retinoid concentrations in the cultured mesenchymal limb bud cells were established. While all-trans-RA inhibited chondrogenesis at 10−7 M by ca. 50%, tenfold higher concentrations of all-trans-RAG were necessary to obtain the same effect. This difference reflects the ratio of RA isomers which were found in the medium after incubation with either all-trans-RAG or all-trans-RA. A pulse experiment (10−5 M all-trans-RAG or all-trans-RA for the first 2 h of a 6-day incubation period) demonstrated inhibition of chondrogenesis with all-trans-RA, but not with all-trans-RAG. The data indicate that RAG inhibits chondrogenesis upon hydrolysis to RA. Surprisingly, the rather polar RAG isoforms were extensively accumulated in the limb bud mesenchymal cells when compared to the medium. Both all-trans-RAG and all-trans-RA also induced a large increase of retinyl ester concentrations in the chondrocytes compared to vehicle-treated cells. This finding further supports a recent suggestion that RA regulates retinol metabolism via feedback inhibition of retinol oxidation and stimulation of the esterification of retinol.
Keywords: Key words Chondrogenesis; Limb bud; Retinoic acid; Retinoyl glucuronide; Retinol metabolism
Cooperative inhibition of acetylcholinesterase activities by hexachlorophene in human erythrocytes by Hirohiko Matsumura; Masato Matsuoka; H. Igisu; Masato Ikeda (pp. 151-156).
Hexachlorophene (HCP), pentachlorophenol (PCP), 2,4,5-trichlorophenol (2,4,5-TCP), and 2,4,6-trichlorophenol (2,4,6-TCP) all hemolyzed washed human erythrocytes and inhibited acetylcholinesterase (AchE) activities in erythrocyte membrane. HCP was much more potent in either effect than any other compound examined. The inhibition of AchE activities by HCP was reversed on adding albumin. The dose-response curves by HCP and PCP were sigmoidal, indicating cooperative inhibition, while those by 2,4,5-TCP and 2,4,6-TCP were not. Furthermore, the cooperativity of the inhibition by HCP was greater than by PCP. Differing from that by PCP, the cooperativity of inhibition increased depending on the temperature (13, 25, 37 °C) and decreased when the membrane was treated with Triton X-100. The cooperativity was also decreased in the presence of albumin. On a Scatchard plot analysis, erythrocyte membranes appeared to have multiple binding sites of different affinities for HCP; binding of HCP to the low affinity site [dissociation constant (Kd) 4.7 × 10–5 M] seemed to be responsible for the observed cooperative inhibition of AchE activities. Neither neostigmine nor fenitrothion altered the cooperativity. HCP seems to be the most potent cooperative inhibitor of AchE in human erythrocyte membranes known to date. HCP may be useful to examine AchE and milieu in human erythrocyte membranes.
Keywords: Key words Hexachlorophene ; Erythrocyte membrane ; Acetylcholinesterase ; Cooperative inhibition ; Hill coefficient
Modifications of breathing pattern induced by inhaled sulphur mustard in mice by R. Vijayaraghavan (pp. 157-164).
A head-only exposure assembly was used for exposing mice to vapours of sulphur mustard (SM). The respiration was monitored using an on-line computer program, capable of recognizing the breathing pattern as sensory irritation, airflow limitation and pulmonary irritation. SM was dissolved in acetone and vapourized using a compressed air nebulizer. Mice were exposed to the vapours (8.5, 16.9, 21.3, 26.8, 42.3 and 84.7 mg/m3) for 1h in a body plethysmograph fitted with a 20-gauge needle and a microphone for sensing the respiratory flow signals. The signals were amplified, digitized and integrated to give tidal volume, and stored in a computer for further analysis. The respiration of the mice was followed for modifications in the breathing pattern until 7 days post-exposure. SM induced sensory irritation during exposure, and there was a concentration dependent decrease in the respiratory frequency and an increase in tidal volume. Lower concentrations showed recovery after stopping the exposure. RD50, the concentration that depresses 50% of the respiration was estimated to be 27.4 mg/m3. Following exposure to higher concentrations the animals started dying after 6␣days. The LC50 was estimated to be 42.5 mg/m3 (14␣days observation period). The respiratory frequency decreased on subsequent days of exposure depending upon the exposure concentration, and the breathing pattern was characteristic of airflow limitation. The ratio of flow/tidal volume was decreased following exposure to concentrations of 26.8 and 42.3 mg/m3. The ratio of flow/tidal volume may be a better measurement than the measurements based on flow alone for the assessment of airflow limitation. Pulmonary irritation was not observed showing that the lungs were not affected. The body weight of the animals decreased progressively. The present methodology will be useful for identifying the effects of SM on the respiratory system, one of the end-points considered when establishing occupational exposure limits.
Keywords: Key words Sulphur mustard ; Breathing pattern ; Sensory irritation ; Airflow limitation ; Mice
Protective effect of povidone-iodine ointment against skin lesions induced by sulphur and nitrogen mustards and by non-mustard vesicants by U. Wormser; Berta Brodsky; Bernard S. Green; Rina Arad-Yellin; Abraham Nyska (pp. 165-170).
Mustard gas (sulphur mustard, SM) is a powerful vesicant employed as a chemical weapon. The present study demonstrates the effect of povidone iodine (PI) ointment against skin toxicity caused by SM. Gross and histopathological examinations showed that application of PI up to 20 min following exposure to the vesicant resulted in marked skin protection. The shorter the interval between exposure and treatment the better was the protection achieved. PI was also effective against other mustards such as carboxybutyl chloroethyl sulphide (CBCS) and mechlorethamine. The fact that PI protected the skin against agents which cannot be oxidized such as iodoacetic acid, divinylsulphone and cantharidine showed that the antidotal effect of PI was unrelated to oxidation of the nitrogen and sulphur atoms of the mustards. PI ointment is proposed as an efficient protective agent against skin toxicity caused by mustards and other alkylators.
Keywords: Key words Mustard gas; Alkylating agents; Antidote
Synthesis and mass spectrometric identification of the major amino acid adducts formed between sulphur mustard and haemoglobin in human blood by Daan Noort; Albert G. Hulst; H. C. Trap; Leo P. A. de Jong; Hendrik P. Benschop (pp. 171-178).
As part of a program to develop methods for the verification of alleged exposure to sulphur mustard, we synthesized and characterized three amino acid adducts presumably formed by alkylation of haemoglobin: 4-(2-hydroxyethylthioethyl)-l-aspartate, 5-(2-hydroxyethylthioethyl)-l-glutamate and N1- and N3-(2-hydroxyethylthioethyl)-l-histidine. Suitable derivatization methods for GC/MS analysis were developed for these adducts as well as for the cysteine and the N-terminal valine adduct. Incubation of human blood with [35S]sulphur mustard in vitro followed by acidic hydrolysis of isolated globin and derivatization with Fmoc-Cl afforded three major radioactive peaks upon HPLC analysis, one of which coeluted with the synthetic Fmoc derivative of N1/N3-(2-hydroxyethylthioethyl)-l-histidine. After pronase digestion of globin the adducts of histidine, glutamic acid, aspartic acid, cysteine and N-terminal valine could be tentatively identified and quantitated. Final identification was obtained from GC/MS analysis. The most abundant adduct, N1/N3-(2-hydroxyethylthioethyl)-l-histidine, could not be sensitively analysed by GC/MS. A convenient LC-tandem MS procedure was developed for this compound, enabling the detection of exposure of human blood to 10␣μM sulphur mustard in vitro.
Keywords: Key words Sulphur mustard ; Adducts ; Haemoglobin; LC tandem MS ; Multiple reaction monitoring
Permethrin absorption not detected in single-pass perfused rabbit ear, and absorption with oxidation of 3-phenoxybenzyl alcohol by G. E. Bast; D. Taeschner; H. G. Kampffmeyer (pp. 179-186).
Isolated rabbit ears were single-pass perfused with a protein-free medium. Permethrin (0.05–23.5%, w/w) was applied in four distinct ointments. Permethrin, 3-phenoxybenzyl alcohol, 3-phenoxybenzaldehyde, and 3-phenoxybenzoic acid were analysed by HPLC. Permethrin was not detected in the effluent. The permeation coefficient, calculated from the detection limit was <7.3 × 10−12 (cm/sec). The appearance rate of the 3-phenoxybenzyl moieties in the effluent agreed with the absorption of the corresponding impurities in the various ointments. In supernatant of homogenised skin, the hydrolysis rate of permethrin was linear; about 4 pmol/min per cm² at 10 μM substrate concentration. The proportion of 3-phenoxybenzoic acid, a further metabolite of 3-phenoxybenzyl alcohol increased when an oxidizing co-factor system was added. The appearance rate in the effusate of 3-phenoxybenzyl alcohol following the lipophobic ointment was five times faster than from isopropyl myristate. The formation rate of 3-phenoxybenzoic acid followed saturation kinetics. Occupational systemic poisoning by dermal absorption of permethrin seems very unlikely since humans bear more epithelial cell layers than rabbits. These experiments do not contradict, however, possible paraesthesia during systemic poisoning after inhalation or ingestion of the pyrethroid- containing aerosols used in agriculture.
Keywords: Key words Permethrin; 3-Phenoxybenzyl alcohol; Skin; Rabbit
Transplacental kinetics of lead in pregnant mini-pigs by Jing Lü; Georg Petroianu; Benny Widjaja; Roderich Rüfer (pp. 187-192).
This study examined the maternal and fetal blood kinetics of lead in pregnant, near term mini-pigs. A lead dose of 1 mg/kg was administered to the animals by i.v. injection as bolus. During the 5-h sampling period, the two-compartment maternal pharmacokinetics demonstrated a rapid phase T 1/2 of 8 min and a slower phase T1/2 of 199 min. Lead reached the fetus with a time lag of 81 min. At 24 h after administration the ratio of fetal to maternal blood lead concentration seemed to become stable. When lead was injected directly into the fetal blood circulation, the decay of fetal blood lead fitted a one-compartment model. The disappearance half-life was 92 min. Lead can obviously accumulate in fetal liver; the lead level in fetal brain showed no detectable changes. This study confirmed that lead can also pass through the epitheliochorial placenta.
Keywords: Key words Lead acetate ; Placental transfer ; Pharmacokinetics ; Pregnant pig ; Fetus
In vivo genotoxicity of selected herbicides in the mouse bone-marrow micronucleus test by T. Gebel; S. Kevekordes; K. Pav; R. Edenharder; H. Dunkelberg (pp. 193-197).
The herbicides alachlor, atrazine, terbuthylazine, gluphosinate-ammonium, isoproturon, pendimethaline and trifluralin were tested for genotoxicity in the mouse bone-marrow micronucleus test (MNT). Both atrazine and trifluraline caused a significant increase in the number of micronuclei at doses of 1400 mg/kg body weight in female mice only. Alachlor, terbuthylazine, gluphosinate-ammonium, isoproturon and pendimethaline did not have any genotoxic effect in the mouse bone-marrow micronucleus test in either female or male animals.
Keywords: Key words Mouse bone-marrow micronucleus test ; Herbicides; Atrazine; Trifluraline
Nitrogen mustard-mediated mutagenesis in human T-lymphocytes in vitro by L. S. Olsen; Birgitte Korsholm; Bjørn A. Nexø; Karsten Wassermann (pp. 198-201).
The cytotoxic and mutagenic effect of the bifunctional alkylating agent nitrogen mustard (HN2) was examined. Primary human lymphocytes were exposed to graded doses of HN2 in vitro and relative survival was determined. Mutation induction at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus was measured by cloning the exposed T-cells in microtitre plates in the presence and absence of 6-thioguanine (TG). The IC50-value determined for 30 min exposure to HN2 was 1.34 μM. The mutant frequencies (MF) in exposed T-cell cultures were 10-fold (2 μM HN2) to 32-fold (4 μM HN2) higher than those of unexposed cultures (median values). Nitrogen mustard-mediated mutagenesis is discussed in terms of the current ideas about DNA damage and repair.
Keywords: Key words Nitrogen mustard ; T-cell cloning ; hprt mutant frequency ; Toxicity ; Human T-lymphocytes