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Archives of Toxicology (v.80, #7)

Nephrotoxicity of uranyl acetate: effect on rat kidney brush border membrane vesicles by M. Goldman; A. Yaari; Z. Doshnitzki; R. Cohen-Luria; A. Moran (pp. 387-393).

Since the Gulf war exposure to depleted uranium, a known nephrotoxic agent, there is a renewed interest in the toxic effects of uranium in general and its mechanism of nephrotoxicity which is still largely unknown in particular. In order to investigate the mechanism responsible for uranium nephrotoxicity and the therapeutic effect of urine alkalization, we utilized rat renal brush border membrane vesicles (BBMV). Uranyl acetate (UA) caused a decrease in glucose transport in BBMV. The apparent K _i of uranyl was 139±30 μg uranyl/mg protein of BBMV. Uranyl at 140 μg/mg protein of BBMV reduced the maximal capacity of the system to transport glucose [V _max 2.2±0.2 and 0.96±0.16 nmol/mg protein for control and uranyl treated BBMV (P<0.001), respectively] with no effect on the apparent K _m (1.54±0.33 and 1.54±0.51 mM for control, and uranyl treated BBMV, respectively). This reduction in V _max is at least partially due to a decrease in the number of sodium-coupled glucose transporters as apparent from the reduction in phlorizin binding to the uranyl treated membranes, V _max was reduced from 247±13 pmol/mg protein in control BBMV to 119±3 pmol/mg protein in treated vesicles (P<0.001). The pH of the medium has a profound effect on the toxicity of UA on sodium-coupled glucose transport in BBMV: higher toxicity at neutral pH (around pH 7.0), and practically no toxicity at alkaline pH (7.6). This is the first report showing a direct inhibitory dose and pH dependent effect of uranyl on the glucose transport system in isolated apical membrane from kidney cortex.

Keywords: Brush border membrane vesicles; Uranyl acetate; Glucose transport; Nephrotoxicity

A polymorphism in the delta-aminolevulinic acid dehydratase gene modifies plasma/whole blood lead ratio by Marcelo F. Montenegro; Fernando Barbosa Jr; Valeria C. Sandrim; Raquel F. Gerlach; Jose E. Tanus-Santos (pp. 394-398).

Delta aminolevulinic acid dehydratase (ALAD) plays an important role in lead poisoning. This study was carried out to examine the effects of ALAD gene polymorphism (G177C) on %Pb-P(plasma lead)/Pb-B(whole blood) ratio in 142 subjects environmentally exposed to lead. Genotypes for the ALAD G177C polymorphism were determined by PCR and restriction fragment length digestion. Pb-P and Pb-B were determined by inductively coupled plasma mass spectrometry and by graphite furnace atomic absorption spectrometry, respectively. The allele frequencies for ALAD1 and ALAD2 alleles were 0.897 and 0.103, respectively. We combined both ALAD 1-2 and ALAD 2-2 genotypes together (ALAD 1-2/2-2 group) and compared with the ALAD 1-1 genotype group. While no significant differences were found in Pb-B, subjects from the ALAD 1-2/2-2 genotype group presented significantly higher Pb-P concentrations and %Pb-P/Pb-B ratios (0.89±0.07 μg/l, and 1.45±0.10%, respectively) when compared with subjects from the ALAD 1-1 genotype group (0.44±0.05 μg/l, and 0.48±0.02, respectively; both P<0.0001). The higher %Pb-P/Pb-B ratios in carriers of the ALAD-2 allele compared with noncarriers indicate that ALAD 1-2/2-2 subjects are probably at increased health risks associated with lead exposure.

Keywords: ALAD polymorphism; Delta aminolevulinic acid dehydratase; Lead toxicology; Plasma lead; Whole blood lead; Plasma/whole blood lead ratio

Sequencing and phylogenetic analysis of neurotoxin gene from an environmental isolate of Clostridium sp.: comparison with other clostridial neurotoxins by Aparna Dixit; Syed Imteyaz Alam; Lokendra Singh (pp. 399-404).

A Clostridium sp. isolated from intestine of decaying fish exhibited 99% sequence identity with C. tetani at 16S rRNA level. It produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) as well as tetanus antitoxin. The gene fragments for light chain, C-terminal and N-terminal regions of the heavy chain of the toxin were amplified using three reported primer sets for tetanus neurotoxin (TeNT). The neurotoxin gene fragments were cloned in Escherichia coli and sequenced. The sequences obtained exhibited approximately 98, 99 and 98% sequence identity with reported gene sequences of TeNT/LC, TeNT/HC and TeNT/HN, respectively. The phylogenetic interrelationship between the neurotoxin gene of Clostridium sp. with previously reported gene sequences of Clostridium botulinum A to G and C. tetani was examined by analysis of differences in the nucleotide sequences. Six amino acids were substituted at four different positions in the light chain of neurotoxin from the isolate when compared with the reported closest sequence of TeNT. Of these, four were located in the β15 motif at a solvent inaccessible, buried region of the protein molecule. One of these substitutions were on the solvent accessible surface residue of α1 motif, previously shown to have strong sequence conservation. A substitution of two amino acids observed in N-terminal region of heavy chain were buried residues, located in the β21 and β37 motifs showing variability in other related sequences. The C-terminal region responsible for binding to receptor was conserved, showing no changes in the amino acid sequence.

Keywords: Clostridium sp.; Tetanus neurotoxin; Phylogeny; Botulinum neurotoxin; Mouse bioassay

Role of oxidative stress, mitochondrial membrane potential, and calcium homeostasis in human lymphocyte death induced by nickel carbonate hydroxide in vitro by Prosper M’Bemba-Meka; Nicole Lemieux; Saroj K. Chakrabarti (pp. 405-420).

When isolated human lymphocytes were treated in vitro with various concentrations of soluble form of nickel carbonate hydroxide (NiCH) (0–1 mM), at 37°C for 4 h, both concentration- and time-dependent effects of NiCH on lymphocyte death were observed. Increased generation of hydrogen peroxide (H₂O₂), superoxide anion (O ₂ ⁻ ), depletion of both no protein (NP-) and protein (P-) sulfhydryl (SH) contents and lipid peroxidation (LPO) were induced by NiCH. Pretreatment of lymphocytes with either catalase (H₂O₂ scavenger), or deferoxamine (DFO) (iron chelator), or excess glutathione (GSH) (an antioxidant) not only significantly reduced the NiCH-induced generation of H₂O₂ and LPO, but also increased the NP-SH and P-SH contents initially reduced by NiCH. NiCH-induced generation of excess O ₂ ⁻ but not excess LPO was significantly reduced by pretreatment with superoxide dismutase (SOD). NiCH-induced lymphocyte death was significantly prevented by pre-treatment with either catalase, or dimethylthiourea/mannitol (hydroxyl radical scavengers), or DFO, or excess GSH/N-acetylcysteine. NiCH-induced lymphocyte death was also significantly prevented by pretreatment with excess SOD. Thus, various types of oxidative stresses play an important role in NiCH-induced lymphocyte death. Cotreatment with cyclosporin A (a specific inhibitor of alteration in mitochondrial membrane potential (ΔΨ_m) not only inhibited NiCH-induced alteration in ΔΨ_m, but also significantly prevented Ni-compound-induced lymphocyte death. Furthermore, NiCH-induced destabilization of cellular calcium homeostasis. As such, NiCH-induced lymphocyte death was significantly prevented by modulating intracellular calcium fluxes such as Ca²⁺ channel blockers and intracellular Ca²⁺ antagonist. Thus, the mechanism of NiCH (soluble form)-induced activation of lymphocyte death signalling pathways involves not only the excess generation of different types of oxidative stress, but also the induction of alteration in ΔΨ_m and destabilization of cellular calcium homeostasis as well.

Keywords: Human lymphocytes; Lymphocyte death; Oxidative stress; Nickel carbonate hydroxide; Mitochondrial membrane potential; Calcium homeostasis

Ethanol hyperpolarizes mitochondrial membrane potential and increases mitochondrial fraction in cultured mouse myocardial cells by Keiko Mashimo; Youkichi Ohno (pp. 421-428).

Cultured mouse heart-derived myocardial and non-muscle cells were exposed to ethanol, stained with cell-permeant fluorescent vital probes, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide) and oxidation-sensitive dihydrorhodamine 123, and analyzed by flow cytometry to elucidate ethanol-induced time-wise alterations in the mitochondrial membrane potential (ΔΨm) and the production of reactive oxygen species (ROS). Ethanol (50 and 200 mM) not only hyperpolarized ΔΨm of both types of cells but also dose-dependently increased ROS production at 24 h, although a 200-mM dose reduced the production until 3 h. These cell pathophysiological reactions suggest the depression of mitochondrial ATPase and mitochondrial respiratory chain. However, differences between these cells appeared after a 24-h exposure to 200 mM ethanol: the increase in ROS production was approximately twice as large for myocardial cells as for non-muscle cells; and the side-scatter parameter of light scattering significantly increased for myocardial cells, but not for non-muscle cells. All these myocyte-specific alterations indicate an increase in the mitochondrial fraction in a cell. This reaction might be a countermeasure against ethanol-induced dysfunction of mitochondrial respiration that is needed to meet the energy requirements of spontaneous myocardial contractions.

Keywords: Myocardial cells; Ethanol; JC-1; Dihydrorhodamine 123; Mitochondrial membrane potential

Dual activity of triterpenoids: apoptotic versus antidifferentiation effects by Lubos Cipak; Lubica Grausova; Eva Miadokova; Ladislav Novotny; Peter Rauko (pp. 429-435).

Triterpenoids are natural, biologically active compounds extracted from many plants. They possess antiinflammatory, anticancer, and antioxidant properties. In the report presented, antiproliferative effects and leukemia cell growth and apoptosis modulating activities of ursolic acid (UA) and oleanolic acid (OA) were investigated. Both triterpenoids are inhibitors of leukemia cell growth and inductors of apoptosis. However, when applied in combination with anthracycline antitumor antibiotic doxorubicin (Dox), UA and OA diversely modulate therapeutic efficacy of Dox, due to different antioxidant activities. Compare to OA showing synergism/additive effect with Dox, UA (stronger antioxidant) acts antagonistically and reduces leukemia cell growth inhibiting and differentiation effects induced by Dox. In conclusion, these findings suggest that although triterpenoids UA and OA can induce apoptosis, their antioxidant activities can interfere with the therapeutic effect of antitumor antibiotic Dox which mechanism of action is attributed to the production of reactive oxygen species.

Keywords: Apoptosis; Differentiation; Triterpenoid; Doxorubicin; Antioxidant

Isolation and biological characterization of neurotoxic compounds from the sea anemone Lebrunia danae (Duchassaing and Michelotti, 1860) by J. Sánchez-Rodríguez; Karina Cruz-Vazquez (pp. 436-441).

This paper describes two neurotoxic proteins obtained from the Caribbean sea anemone Lebrunia danae. To assess the neurotoxic activity of the venom of L. danae, several bioassays were carried out, and to evaluate the effect of the toxin, Median Lethal Doses (LD₅₀) were determined in vivo using sea crabs (Ocypode quadrata) and Artemia salina nauplii with the crude extract of the proportion of 2.82 mg/m. The proteins with neurotoxic effects were isolated using low-pressure liquid chromatography. The fractions containing the neurotoxic activity were analyzed by SDS-PAGE and showed protein bands with an apparent molecular weight of 62.50 kDa (LdNt1) and 58 kDa (LdNt2). To demonstrate that these proteins were indeed responsible for the neurotoxic activity observed, we injected a small fraction of the purified protein into the third walking leg of a crab and observed the typical convulsions, paralysis and death provoked by neurotoxins. Hemolytic activity was also tested for 0.238 mg of crude extract; the hemolytic value was 39.5, 49.6 and 50.1% for cow, sheep and pig erythrocytes, respectively.

Keywords: Cnidaria; Lebrunia danae ; Nematocysts; Neurotoxin; Cytolysins

Study on the mechanism of photosensitive dermatitis caused by ketoprofen in the guinea pig by Takashi Nakazawa; Takeo Shimo; Noriko Chikamatsu; Takako Igarashi; Osamu Nagata; Masaharu Yamamoto (pp. 442-448).

To investigate the mechanism on photosensitive dermatitis caused by ketoprofen (KP) in humans, the following experiments were performed by topical application on guinea pigs. The phototoxicity study involving treatment with 10% solution of KP, its enantiomers (R-KP and S-KP), loxoprofen, and flurbiprofen revealed no phototoxic reactions. In the photoallergenicity study, KP and its enantiomers (0.5–2% solution) induced skin reaction at all dosages; however, loxoprofen and flurbiprofen (1–5% solution) did not induce such a photoallergenic reaction. These results suggest that the chemical structure of the benzophenone chromophore in KP would be one of the important factors for induction of the photoallergy since both loxoprofen and flurbiprofen do not possess this structure and hence lack photoallergenic potential. Furthermore, to assess time profiles of KP concentration in the skin and plasma, guinea pigs received a repeated topical application of R-KP and S-KP at a dosage of 40 mg/kg over a period of 3 days. Plasma KP concentrations were extremely low as compared to skin KP concentrations and were not detected at 72 h after the final dosing. At 24 h after the final dosing, KP concentrations in the skin with R-KP and S-KP treatment were 187.4 and 254.7 μg/g, respectively, and their half-lives were 80.5 and 84.4 h, respectively. KP concentrations at 336 h after final dosing were 11.3 μg/g for R-KP and 15.7 μg/g for S-KP treatment. The acylglycerol-combined KP concentrations at 336 h were 2% or less as compared to KP concentrations with R-KP and S-KP treatment. There were no differences in KP concentrations in the skin between R-KP and S-KP and in combined KP concentrations between the enantiomers. The present study indicates that photosensitive dermatitis after topical application of KP in humans, caused by photoallergenicity and not phototoxicity, can be reproduced in the animal testing, and suggests that the skin reaction may be caused by the long period of retention of KP in the skin.

Keywords: Ketoprofen; Nonsteroidal anti-inflammatory drug; Pharmacokinetics of R- and S-ketoprofen; Photoallergenicity; Guinea pigs

Effects of cypermethrin and methyl parathion mixtures on hormone levels and immune functions in Wistar rats by Ping Liu; Xiaoxiao Song; Weihong Yuan; Weihua Wen; Xinan Wu; Jian Li; Xuemin Chen (pp. 449-457).

To study interaction and dose-related effects of mixed cypermethrin and methyl parathion on endocrine and immune functions, 120 Wistar rats were divided randomly into six groups of ten male and ten female rats, respectively, at the age of 2 months. All groups were force-fed every 2 days for 30 days with cypermethrin 0.0, 8.0, 0.0, 8.0, 1.8, 0.4 mg/kg bw and methyl parathion 0.0, 0.0, 0.23, 0.23, 0.0518, 0.0115 mg/kg bw. Controls received vehicle solvent only. Body weight gain and organ weights were measured. Serum or blood were used to test luteinizing hormone, follicle stimulating hormone, estradiol (E₂), testosterone, thyroid hormones (T₃, T₄, TSH), IgG, IgA, rate of neutrophil phagocytosis and of lymphocyte transformation. The effects on relative weights of ovaries and adrenals, IgA and rate of lymphocyte transformation were antagonistic interaction; the effect on estradiol was synergistic in female, whereas addictive in male rats; and the other indices indicated addictive interaction. Organ weights were similar in exposed and control animals except for adrenal (heavier in exposed rats, P<0.01). Serum levels of FSH and estradiol were higher in exposed groups than in controls (P<0.01). IgG levels were lower in exposed rats than in controls (P<0.01), and IgA levels were higher in exposed females than in controls (P<0.01). Lymphocyte transformation rates were lower (P<0.01) and neutrophil phagocytosis rates were higher (P<0.01) in exposed rats than in controls. Our results showed that exposure to low-dose mixtures of cypermethrin and methyl parathion may affect hormone levels (especially estradiol) and immune function in rats, and the NOAELs of combined compounds were located at 1/600 LD₅₀.

Keywords: Cypermethrin; Methyl parathion; Reproductive hormones; Immune function; Mixture

Teratogenic effects of nitroimidazopyridazine on the CNS in fetal Wistar (WU) rats by Thomas Woehrmann; Jutta Volland; Klaus Tuch; Gerd Bode; Ulrich Hübel (pp. 458-464).

Teratogenic effects caused by a new nitroimidazopyridazine were examined in Wistar (WU) rats after repeated oral administration of 0, 2.5, 10, and 40 mg/kg, given on days 6–17 post coitum (p.c.) (Day of mating = Day 0) in a regular study on embryo-fetal development according to ICH S5A. At day 20 p.c., fetuses were removed and carefully examined under a dissecting microscope for external, visceral and skeletal malformations. The exposure to the high dose of the test compound during the organogenesis and early histogenesis periods of prenatal development induced prominent CNS malformations (exencephaly, neural tube defects (NTD)) associated with external malformations (hyperflexion of the forelimbs). To support the data from this study additional histological evaluation of the brains was performed with the following results: disorganization of the cerebral cortex associated with ectopic subcommissural organs. Additionally, an in vitro test (whole embryo culture, WEC) showed alterations of the developing neural tube after the incubation of rat embryos with the test compound on gestation days 9.5–11.5. Our data demonstrated that nitroimidazopyridazine caused NTDs and limb malformations during organogenesis. Based on these data the further development of the test compound was stopped.

Keywords: Nitroimidazopyridazine; Teratogenicity; CNS – malformations; Histopathology; Whole embryo culture (WEC); Rat

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Archives of Toxicology (v.80, #7)