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Archives of Toxicology (v.72, #4)
Induction by mercury compounds of brain metallothionein in rats: Hg0 exposure induces long-lived brain metallothionein by Akira Yasutake; Atsuhiro Nakano; Kimiko Hirayama (pp. 187-191).
Metallothionein (MT) is one of the stress proteins which can easily be induced by various kind of heavy metals. However, MT in the brain is difficult to induce because of blood-brain barrier impermeability to␣most heavy metals. In this paper, we have attempted to induce brain MT in rats by exposure to methylmercury (MeHg) or metallic mercury vapor, both of which are known to penetrate the blood-brain barrier and cause neurological damage. Rats treated with MeHg (40 μmol/kg per day × 5 days, p.o.) showed brain Hg levels as high as 18 μg/g with slight neurological signs 10␣days after final administration, but brain MT levels remained unchanged. However, rats exposed to Hg vapor for 7 days showed 7–8 μg Hg/g brain tissue 24 h after cessation of exposure. At that time brain MT levels were about twice the control levels. Although brain Hg levels fell gradually with a half-life of 26 days, MT levels induced by Hg exposure remained unchanged for >2␣weeks. Gel fractionation revealed that most Hg was in the brain cytosol fraction and thus bound to MT. Hybridization analysis showed that, despite a significant increase in MT-I and -II mRNA in brain, MT-III mRNA was less affected. Although significant Hg accumulation and MT induction were observed also in kidney and liver of Hg vapor-exposed rats, these decreased more quickly than in brain. The long-lived MT in brain might at least partly be accounted for by longer half-life of Hg accumulated there. The present results showed that exposure to Hg vapor might be a suitable procedure to provide an in vivo model with enhanced brain MT.
Keywords: Key words Methylmercury; Mercury vapor; Metallothionein; Brain; Rat
Methylmercury inhibits gap junctional intercellular communication in primary cultures of rat proximal tubular cells by Minoru Yoshida; Toru Kujiraoka; Masayuki Hara; Hirokazu Nakazawa; Yawara Sumi (pp. 192-196).
Methylmercury (MeHg) causes renal injury in addition to central and peripheral neuropathy. To clarify the mechanism of nephrotoxicity by MeHg, we investigated the effect of this compound on intercellular communication through gap junction channels in primary cultures of rat renal proximal tubular cells. Twenty minutes after exposure to 30 μM MeHg, gap junctional intercellular communication (GJIC), which was assessed by dye coupling, was markedly inhibited before appearance of cytotoxicity. When the medium containing MeHg was exchanged with MeHg-free medium, dye coupling recovered abruptly. However, the dye-coupling was abolished again 30 min after replacement with control medium, and the cells were damaged. Intracellular calcium concentration, [Ca2+]i, which modulates the function of gap junctions, significantly increased following exposure of the cells to 30 μM MeHg and returned to control level following replacement with MeHg-free medium. These results suggest that the inhibiting effect of MeHg on GJIC is related to the change in [Ca2+]i, and may be involved in the pathogenesis of renal dysfunction.
Keywords: Key words Methylmercury; Gap junction; Proximal tubular cells; Calcium; Nitric oxide
Glutathione modifies the toxicity of triethyltin and trimethyltin in C6 glioma cells by M. R. Cookson; N. D. Slamon; V. W. Pentreath (pp. 197-202).
It has been demonstrated that exposure to mercury or cadmium compounds causes alterations in the glutathione system in a model glial cell line, C6. Here we report that two organic tin compounds, triethyltin (TET) and trimethyltin (TMT), are also toxic to these cells with EC50 values for cell death of c. 0.02 μM and 0.8 μM respectively. Exposure for 24 h to either of these compounds at sub-toxic concentrations caused increases in the amount of reduced glutathione (GSH) per cell. Increases in glutathione-S-transferase enzyme activity were also demonstrated after TET or TMT exposure. This suggests that glutathione increases occur in glial cells after toxic insults below that required to cause cell death, possibly acting as a protective mechanism. To test whether GSH plays a role in organotin-induced cell death we manipulated GSH in the culture media or via intracellular GSH and looked at the effects on sensitivity to TET or TMT toxicity. Adding GSH to the culture media did not protect the cells. Depletion of intracellular GSH with buthionine-[S,R] sulphoximine did not alter cytotoxicity of TET or TMT. However, pre-treatment with (−)-2-oxo-4-thiazolidine carboxylic acid (OTC), which increases intracellular GSH levels, protected the cells against both compounds. The EC50 for TMT was increased from 0.77 to 1.8 μM, a 2.3-fold shift, whereas the EC50 for TET was increased >20-fold, from 0.022 to 0.47 μM. One interpretation of these results is that GSH protects cells against the toxicity of organic tin compounds without reacting directly with them to any significant extent. Under conditions where GSH is depleted, additional protective mechanisms may be active.
Keywords: Key words C6 cells; Astrocytes; Triethyltin; Trimethyltin; Glutathione
An immunological method for the detection of captopril-protein conjugate by Ryuichi Narazaki; Hiroshi Watanabe; Toru Maruyama; Ayaka Suenaga; Masaki Otagiri (pp. 203-206).
To clarify the mechanism of cytotoxicity induced by captopril (Cp), interactions between tissue protein and Cp were studied by the enzyme-linked immunosorbent assay (ELISA) method. The amide-linked adducts [hemocyanin from keyhole limpet-Cp adduct (KLH-Cp) and poly-l-Lys-Cp adduct (pLys-Cp)] using carbodiimide, and a disulfide-linked adduct [ovalbumin-Cp adduct (OVA-Cp)] were prepared. To determine the formation of the protein-Cp adduct, rabbit antisera against KLH-Cp was employed. The immunoreactivities with pLys-Cp and OVA-Cp against anti KLH-Cp sera were elevated when compared with the unmodified protein. With the inhibition ELISA, this antisera was useful for detecting the Cp disulfide-linked conjugate. In kidney cytosol, a high level of immunoreactivity was observed. Plasma and liver cytosol reactivities were similar, and ∼40% against kidney cytosol. Thus, a method for the detection of the Cp-protein adduct using ELISA has been established. Formation of the Cp-protein adduct was observed in rat plasma and in liver and kidney cytosol. These findings suggest the possibility that the formation of Cp-protein adduct is partially related to cytotoxicity in liver.
Keywords: Key words Thiol drug; Covalent binding; Cytotoxicity; ELISA
Oxidation of methyl- and ethyl- tertiary-butyl ethers in rat liver microsomes: role of the cytochrome P450 isoforms by Alessandra Turini; Giada Amato; Vincenzo Longo; Pier Giovanni Gervasi (pp. 207-214).
Methyl t-butyl ether (MTBE) and ethyl t-butyl ether (ETBE) are commonly used in unleaded gasoline to increase the oxygen content of fuel and to reduce carbon monoxide emissions from motor vehicles. This study was undertaken to investigate: (1) the effect of administration to rats of ETBE and its metabolite, t-butanol, on the induction and/or inhibition of hepatic P450 isoenzymes; (2) the oxidative metabolism of MTBE and ETBE by liver microsomes from rats pretreated with selected P450 inducers and purified rat P450(s), (2B1, 2E1, 2C11, 1A1). ETBE administration by gavage at a dose of 2 ml/kg for 2 days induced hepatic microsomal P4502E1-linked p-nitrophenol hydroxylase and the P4502B1/2-associated PROD and 16β-testosterone hydroxylase, verified by immunoblot experiments. t-Butanol treatments at doses of 200 and 400 mg/kg i. p. for 4 days did not alter any liver microsomal monoxygenases. Both MTBE and ETBE were substrates for rat liver microsomes and were oxidatively dealkylated to yield formaldehyde and acetaldehyde, respectively. The dealkylation rates of both MTBE and ETBE were increased c. fourfold in phenobarbital (PB)-treated rats. In rats pretreated with pyrazole, an inducer of 2E1, only the demethylation of MTBE was increased (c. twofold). When the oxidations of MTBE and ETBE were investigated with purified P450(s) in a reconstituted system, it was found that P4502B1 had the highest activities towards both solvents, whereas 1A1 and 2C11 were only slightly active; P4502E1 had an appreciable activity on MTBE but not against ETBE. Metyrapone, a potent inhibitor of P450 2B, consistently inhibited both the MTBE and ETBE dealkylations in microsomes from PB-treated rats. Furthermore, 4-methylpyrazole (a probe inhibitor of 2E1) and anti-P4502E1 IgG showed inhibition, though modest, only on MTBE demethylation, but not on ETBE deethylation. Inhibition experiments have also suggested that rat 2A1 may exert an important role in MTBE and ETBE oxidation. Taken together, these results indicate that 2B1, when expressed, is the major enzyme involved in the oxidation of these two solvents and that 2E1 may have a role, although minor, in MTBE demethylation. The implications of these data for MTBE and ETBE toxicity remain to be established.
Keywords: Key words Methyl tert-butyl ether metabolism; Ethyl tert-butyl ether metabolism; Cytochrome P450
High frequency of CYP1A1 mutations in a Turkish population by A. Sükrü Aynacioglu; Ingolf Cascorbi; Przemyslaw M. Mrozikiewicz; Ivar Roots (pp. 215-218).
The frequency distribution of four cytochrome P4501A1 (CYP1A1) gene mutations was investigated in 271 Turks from southeast Anatolia by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) assay. Allelic linkage of those mutations was proven by peptide nucleic acid-mediated PCR clamping. Mutation m1 (T6235C) forming an MspI restriction site in the 3′-flanking region occurred with 18.1% frequency (95% confidence interval 14.9–21.6%), m2 (A4889G) leading to an Ile/Val exchange in exon 7 had a frequency of 8.9% (6.6–11.6%), and m4 (C4887A; Thr/Asn-exchange also in exon 7) occurred with 5.7% (3.9–8.0%). T5639C (m3) in the 3′-flanking region was not detected. m2 was exclusively found linked with m1 forming allele CYP1A1*2B. The frequency of this allele supposedly at-risk for lung cancer was significantly higher than in Middle European populations, but lower than in the Far East.
Keywords: Key words Cytochrome P4501A1; CYP1A1; Molecular epidemiology; Peptide nucleic acids; PNA; Turkish population
Phosphotriesterase activity identified in purified serum albumins by Miguel A. Sogorb; Nuria Díaz-Alejo; María A. Escudero; Eugenio Vilanova (pp. 219-226).
The phosphotriesterase in chicken serum that hydrolyses O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) was purified in three chromatographic steps. The activity copurified to apparent homogeneity with albumin monitoring by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS/PAGE) and by SDS-capillary electrophoresis in the purified fractions. Commercial chicken serum albumin was further purified and the phosphotriesterase activity remained associated with albumin. Capillary electrophoresis established a molecular weight of 59 ± 4 kDa for both purified proteins (chicken serum and commercial chicken serum albumin). The purified samples were assayed for hydrolytic activity against several carboxylesters, organophosphates and phosphoramidates. From carboxylesters, only p-nitrophenylbutyrate ( p-NPB) hydrolysing activity was found to copurify with the phosphotriesterase. The purified human, chicken, rabbit and bovine serum albumins and recombinant human serum albumin obtained from commercial sources hydrolysed HDCP and p-NPB. Serum albumin also hydrolysed O-butyl O-2,5-dichlorophenyl phosphoramidate, O-ethyl O-2,5-dichlorophenyl phosphoramidate and O-2,5-dichlorophenyl ethylphosphonoamidate but not other organophosphates and phosphoramidates.
Keywords: Key words Phosphotriesterase; Phosphoramidate; Serum albumin; Hen chicken
An algorithm for nasal pungency thresholds in man by Michael H. Abraham; Rachel Kumarsingh; J. Enrique Cometto-Muniz; William S. Cain (pp. 227-232).
Nasal pungency thresholds (NPT) in man have been determined by Cometto-Muniz and Cain for 44 varied compounds, including esters, aldehydes, ketones, alcohols, carboxylic acids, aromatic hydrocarbons and pyridine. With the exclusion of acetic acid, 43 of these NPT values are well correlated through the general linear free energy equation of Abraham, leading to the algorithm, where the independent variables are solute descriptors: 2 H is the dipolarity/polarizability, Σ2 H and Σ2 H are the overall or effective hydrogen-bond acidity and basicity, and L 16 is the solute Ostwald solubility coefficient on hexadecane at 25 °C. Surprisingly, the aliphatic aldehydes and carboxylic acids fit the correlation and with respect to nasal pungency thresholds in man for brief (1–3 s) presentations must be regarded as `nonreactive' compounds. It is suggested mere transport of the compound from the air stream to the receptor area largely determines the potency to produce pungency. Various chemical properties of the receptor area are deduced from the coefficients in Eq. i.
Keywords: Key words Nasal pungency; Sensory irritation; Volatile organic compounds; Hydrogen bonding; Linear free energy equation
Cytotoxicity of fumonisin B1: implication of lipid peroxidation and inhibition of protein and DNA syntheses by Karine Abado-Becognee; Théophile Amondo Mobio; Rachid Ennamany; Francis Fleurat-Lessard; W. T. Shier; F. Badria; Edmond Ekué Creppy (pp. 233-236).
The effects of fumonisin B1 (FB1) from Fusarium moniliforme on lipid peroxidation and protein and DNA syntheses were studied in monkey kidney cells (Vero cells). FB1 was found to be a potent inducer of malondialdehyde (MDA), one of the secondary products formed during lipid peroxidation. At 0.14 μM (0.1 μg/ml), FB1 induced 0.496 ± 0.1 nmoles of MDA/mg protein, compared to the control level 0.134 ± 0.01 nmoles of MDA/mg protein (P < 0.005). No inhibition of protein or DNA synthesis was observed at this concentration of FB1. Inhibition of protein and DNA syntheses was observed at FB1 concentrations >14 μM (10 μg/ml) with an IC50 of 33 μM for both protein synthesis and DNA synthesis. These results indicate that lipid peroxidation is a very sensitive cellular response to the mycotoxin fumonisin B1 observed at concentrations lower than that required to inhibit cellular synthesis of macromolecules, protein and DNA. This oxidative damage induced by FB1 concentrations encountered in naturally contaminated foodstuffs and feed might lead to mutagenicity and genotoxicity.
Keywords: Key words Fumonisin B1; Fusarium moniliforme; Lipid peroxidation; Malondialdehyde; Protein and DNA syntheses
Reactivating potency of obidoxime, pralidoxime, HI 6 and HLö 7 in human erythrocyte acetylcholinesterase inhibited by highly toxic organophosphorus compounds by F. Worek; R. Widmann; O. Knopff; L. Szinicz (pp. 237-243).
The treatment of poisoning by highly toxic organophosphorus compounds (nerve agents) is unsatisfactory. Until now, the efficacy of new potential antidotes has primarily been evaluated in animals. However, the extrapolation of these results to humans is hampered by species differences. Since oximes are believed to act primarily through reactivation of inhibited acetylcholinesterase (AChE) and erythrocyte AChE is regarded to be a good marker for the synaptic enzyme, the reactivating potency can be investigated with human erythro‐cyte AChE in vitro. The present study was undertaken to evaluate the ability of various oximes at concentrations therapeutically relevant in humans to reactivate human erythrocyte AChE inhibited by different nerve agents. Isolated human erythrocyte AChE was inhibited with soman, sarin, cyclosarin, tabun or VX for 30 min and reactivated in the absence of inhibitory activity over 5–60 min by obidoxime, pralidoxime, HI 6 or HLö 7 (10 and 30 μM). The AChE activity was determined photometrically. The reactivation of human AChE by oximes was dependent on the organophosphate used. After soman, sarin, cyclosarin, or VX the reactivating potency decreased in the order HLö 7 > HI 6 > obidoxime > pralidoxime. Obidoxime and pralidoxime were weak reactivators of cyclosarin-inhibited AChE. Only obidoxime and HLö 7 reactivated tabun-inhibited AChE partially (20%), while pralidoxime and HI 6 were almost ineffective (5%). Therefore, HLö 7 may serve as a broad-spectrum reactivator in nerve agent poisoning at doses therapeutically relevant in humans.
Keywords: Key words Acetylcholinesterase; Organophosphates; Oxime; Reactivation
Expiration of ethane in rats under variously elevated inspiratory O2-concentrations by Kerstin Kritzler; Gerhard Schöch; Heinrich Topp (pp. 244-246).
Expired ethane is regarded as a noninvasive indicator of lipid peroxidation. As a model of oxidative stress we have investigated in male Wistar rats (body wt. 309 ± 15 g) the effects of various levels of elevated inspiratory oxygen concentrations on the expiration rate of ethane. After 4 days under 21 vol% O2 (basic condition) the rats were exposed for 6 or 5 days to 40, 60 or 80 vol% O2 over 8 or 23 h/day. The variously O2-enriched air was conducted through the cages and expired ethane adsorbed onto charcoal was thermo-desorbed and measured by gas chromatography. Basic ethane expiration was 3.1 ± 0.8 pmol/100 g body wt. per min. At 40 vol% O2 over 8 or 23 h/day no increase or a maximum average 47% increase (P < 0.01) in ethane expiration occurred on day 4; 60 vol% O2 over 8 or 23 h/day led to a corresponding increase of 56 or 87% (P < 0.05 or P < 0.01) on day 3; 80 vol% O2 over 8 or 23 h/day led to a corresponding increase of 81 or 66% (P < 0.01) on days 3 or 2. Our results indicate that with up to 60 vol% O2 a temporary increase in lipid peroxidation occurs in a dose dependent manner. However, at 80 vol% O2 no further increase in the maximum ethane expiration occurred. The latter finding and the finding of only transient increase in ethane expiration in probably due to antioxidative counteraction.
Keywords: Key words Ethane expiration; Lipid peroxidation; Noninvasive methods; Rats; Reactive oxygen species
Induction of single-strand breaks in lymphocyte DNA of rats exposed to hyperoxia by Rao R. Vangala; Kerstin Kritzler; Gerhard Schöch; Heinrich Topp (pp. 247-248).
A modified technique of alkaline filter elution was used to evaluate single-strand breaks (SSB) as a measure of DNA lesions in lymphocytes of male Wistar rats exposed to 60 or 80 vol% oxygen, over 23 h/day and for 6 or 5 days, respectively. An acceleration of elution was observed with samples from experiments at both increased levels of oxygen. With proteinase K treatment, a twofold increase in elution rates after exposure to 60 vol% oxygen and a threefold increase at 80 vol% oxygen were indicative of increased occurrence of SSB. This is explained by an involvement of reactive oxygen species in DNA damage under elevated oxygen levels.
Keywords: Key words DNA damage; Oxidative stress; Rat lymphocytes; Reactive oxygen species