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Archives of Toxicology (v.76, #9)

No Title by Kanji Yamasaki; Yutaka Tago; Kenji Nagai; Masakuni Sawaki; Shuji Noda; Mineo Takatsuki (pp. 495-501).

Two repeated-dose studies of 6-n-propyl-2-thiouracil (PTU) in male rats based on the research protocol 'Pubertal Development and Thyroid Function in Immature Male Rats' (pubertal assay) proposed by the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) and the draft protocol of the 'Enhanced OECD Test Guideline 407' (enhanced TG 407) were performed to investigate the suitability of both assays as screening methods for the detection of endocrine-mediated effects and to compare their sensitivity for the endocrine-mediated effects. In the pubertal assay, PTU at doses of 0, 0.01, or 1 mg/kg per day was orally administered to male Sprague-Dawley rats for 30 days, starting at 23 days of age. In the enhanced TG 407 the same doses of PTU were orally administered to male Sprague-Dawley rats for 28 days, starting at 7 weeks of age. In the pubertal assay, decreased serum thyroxine (T4) and triiodothyronine (T3), increased thyroid and pituitary weights, hypertrophy of follicular epithelial cells in the thyroid, and increased basophilic cells in the pituitary were detected as endocrine-mediated effects of PTU in the 1 mg/kg group. In the enhanced TG 407, decreased T4 and T3 were detected in both the 0.01 and 1 mg/kg groups, together with increased thyroid-stimulating hormone in the 1 mg/kg group, increased thyroid and pituitary weights in the 1 mg/kg group, and hypertrophy of follicular epithelial cells in the thyroid and increased basophilic cells in the pituitary of the 1 mg/kg group. Thus, among the parameters tested, the thyroid hormone levels, organ weight changes, and the histopathological assessment allowed detection of the endocrine-related effects of PTU in both the pubertal assay and the enhanced TG 407, but the sensitivity of the hormone analysis was higher in the latter.

Keywords: 6-n-Propyl-2-thiouracil Enhanced Test Guideline 407 Pubertal assay Rat Endocrine effects

No Title by Yutaka Komatsu; Yasumitsu Ogra; Kazuo T. Suzuki (pp. 502-508).

In an animal model of Wilson disease, Long-Evans rats with cinnamon-colored coat (LEC rats), copper (Cu) accumulates in the liver with age up to the onset of acute hepatitis owing to a hereditary defective transporter for the efflux of Cu, ATP7B. The plasma Cu concentration is low in LEC rats because of the excretion of apo-ceruloplasmin (apo-Cp). However, toward and after the onset of chronic hepatitis, plasma Cu concentration increases in the form of holo-Cp, while the liver Cu concentration is maintained at a constant level without the occurrence of fulminant hepatitis. In the present study, the material balance of Cu was studied in LEC rats with chronic hepatitis in order to elucidate the mechanisms underlying the increase of holo-Cp in plasma and the maintenance of Cu at a constant level in the liver. The relationship between the Cu concentration and ferroxidase activity of Cp was analyzed in the plasma of LEC rats of different ages and of Wistar rats fed a Cu-deficient diet for different durations. Cu was suggested to be delivered to Cp in an all-or-nothing manner, resulting in the excretion of fully Cu-occupied holo-Cp (Cu₆-Cp) or totally Cu-unoccupied Cu₀-Cp (apo-Cp), but not partially Cu-occupied Cu n-Cp (where n=1–5). The increase of holo-Cp in acute and chronic hepatitis in LEC rats was explained by the delivery of Cu, accumulating in the non-metallothionein-bound form, to Cp outside the Golgi apparatus of the liver. The plasma Cu concentration and ferroxidase activity were proposed to be specific indicators of the appearance of non-metallothionein-bound Cu in the liver of LEC rats.

Keywords: Copper Ceruloplasmin LEC rat Hepatitis Copper balance

No Title by Valentina Medici; Alessandro Santon; Giacomo Sturniolo; Renata D'Incà; Sabrina Giannetto; Vincenzo Albergoni; Paola Irato (pp. 509-516).

The Long-Evans Cinnamon (LEC) rat is a mutant animal model for Wilson's disease. It is known that an abnormal accumulation of Cu and Fe in the liver and low concentrations of both ceruloplasmin and Cu in the serum occur in these rats. The accumulation of Cu is explained by the defective expression of the Cu-transporting P-type ATPase gene, homologous to the gene for Wilson's disease (ATP7B). The aim of this work was to clarify the action mechanism of Zn, and to verify the role that this metal plays in LEC rats in short-term treatment experiments (1 and 2 weeks) on concentrations of Cu, Zn, Fe, metallothionein (MT), 8-hydroxy-2'-deoxyguanosine (oh⁸dG) and on the activity of antioxidant enzymes. It is well known that Zn induces MT and has the ability to prevent redox-active metals, Cu and Fe, binding to and causing oxidative damage at active sites of Zn metalloenzymes and nonspecific binding sites on proteins. Zn administration reduces Cu and Fe transport from mucosal to serosal intestinal sides through competitive mechanisms. Our findings show that treatment with zinc acetate increases tissue Zn and MT contents and decreases Cu and Fe concentrations in the liver and kidneys, even if hepatic Zn and MT concentrations decrease with treatment period. Induction of MT synthesis by Zn contributes to the reduction in free radicals produced by Cu and Fe. We also observed that the superoxide dismutase (SOD)activity in liver decreases with treatment duration in association with the Cu and Fe liver decrease. However, the SOD activity in kidney increases in untreated rats at 2 weeks relative to those untreated for 1 week.

Keywords: Metallothionein Antioxidant enzymes Long-Evans Cinnamon rat Copper Zinc

No Title by Uri Wormser; Berta Brodsky; Amnon Sintov (pp. 517-522).

Sulfur mustard (SM, mustard gas) is a chemical warfare vesicant that rapidly penetrates the skin due to its hydrophobicity. This study measured the rate of SM disappearance from the skin after topical application of the vesicant. In both fur-covered and hairless animals, the remaining toxicant levels measured 60 min after exposure to undiluted SM were 0.6% and 0.3%, respectively, of the initially applied SM amount. However, SM concentration reached 0.4% of the initial dose 3 h following exposure in female fur-covered guinea pigs. SM quantities extracted from skin of male fur-covered and hairless guinea pigs immediately after 16 min of exposure to SM vapor were 12.2 and 21.8 µg, respectively; levels declined to 1.6 and 1.7 µg at 30 and 15 min following termination of exposure of male fur-covered and hairless guinea pigs, respectively. Three swabbing treatments of undiluted SM-exposed skin with gauze pads soaked in 0.5% hypochlorite caused 68% reduction in skin SM content. Similar findings were obtained when hypochlorite was replaced by water (64% reduction). SM content in the gauze pads was 59, 38 and 25 µg, respectively, for the first, second and third decontamination processes with water. No SM was detected in the gauze pads soaked with hypochlorite. In vitro studies showed that incubation of SM with 0.5% hypochlorite at a ratio of 10:1 (v/v) did not cause SM inactivation, whereas 4% hypochlorite reduced SM levels by 17%. However, at a decontaminant:SM ratio of 1000:1, 0.5% and 4% hypochlorite reduced SM levels by 92% and 99%, respectively. These findings are important for health authorities and regulatory agencies in planning precautionary steps to be taken in case of emergency and in routine laboratory work.

Keywords: Mustard gas Hypochlorite Toxicokinetics Topical exposure Decontamination

No Title by F. Worek; G. Reiter; P. Eyer; L. Szinicz (pp. 523-529).

Standard treatment of poisoning by organophosphates (OP) includes the administration of an antimuscarinic agent, e.g. atropine, and of an acetylcholinesterase (AChE) reactivator (oxime). The presently available oximes, obidoxime and pralidoxime (2-PAM), are considered to be insufficient for highly toxic OPs, e.g. sarin. In the past decades numerous oximes were prepared and tested for their efficacy in OP poisoning, mostly in animal experiments. However, data indicate that the reactivating potency of oximes may be different in humans and animal species, which may hamper the extrapolation of animal data to humans and may pose a problem in the drug licensing of new compounds. In order to provide data for a better evaluation of the reactivating potency of oximes, experiments were undertaken to determine the reactivation rate constants of several oximes with human, rabbit, rat and guinea-pig AChE inhibited by the OPs sarin, cyclosarin and VX. The results show marked differences among the species, depending on the inhibitor and on the oxime, and indicate that the findings from animal experiments need careful evaluation before extrapolating these data to humans.

Keywords: Organophosphate Acetylcholinesterase; oximes Reactivation kinetics Species differences

No Title by Masafumi Tomita; Tsutomu Nohno; Toshiko Okuyama; Shin-ichiro Nishimatsu; Junko Adachi (pp. 530-537).

Increased formation of reactive oxygen species is a cause of paraquat (PQ)-induced injury and also provides a link between the signaling pathways and transcriptional events that regulate the expression of a large number of genes. However, the molecular mechanisms involved in PQ-induced injury remain unclear. To investigate the changes in gene expression at the onset of PQ injury, we used the differential display-polymerase chain reaction (PCR) method. Rats were treated intraperitoneally with 20 mg/kg PQ, and after 3 h the lungs were immediately excised. Samples of mRNA from normal and treated rats were used to prepare radiolabeled cDNAs, which were electrophoresed. Then the transcription levels were compared. We isolated 26 fragments of cDNA that were potentially affected by PQ, and determined their nucleotide sequences. Six clones of interest were selected and analyzed further. The reverse transcript-PCR based on their sequence information confirmed the differential expression for five clones: four clones were up-regulated and one was down-regulated. We were particularly interested in two genes that had homology with the known gene: TATA box-binding protein-associated factor, RNA polymerase II, B, 150 kDa (TAFIIB), and a candidate gene for lipodystrophy, Lpin2. Both genes were significantly up-regulated within 3 h of PQ intake and the stimulation continued during our 24-h observation period. In addition, up-regulation of Lpin2 was observed in the lungs, but not in the liver and kidneys. In situ hybridization using lung sections showed that the expression of both genes was strongly visualized in Clara cells and in alveolar macrophages. These findings suggest a stimulation of transcription levels and changes in lipid metabolism in Clara cells and in macrophages in the lungs, which result in their playing a crucial role at the onset of PQ-driven pulmonary injury.

Keywords: Paraquat Gene expression Differential display Clara cell Rats

No Title by H.-W. Vohr; J. Pauluhn; H. Ahr (pp. 538-544).

The elicitation of respiratory allergy in animal models is exquisitely complex and interpretation of results from different laboratories cannot readily be compared due to variability in testing protocols, biomarkers and techniques used to identify 'positive' responses. On the one hand, guinea-pigs have been proposed as a good model with which to study allergic and irritant bronchial hyperresponsiveness. On the other hand, considerable efforts have been made to develop animal models that take the immunological mechanisms into account to reduce the complexity as well as duration of the guinea-pig assays. In principle, local skin reactions can easily be determined by the local lymph node assay (LLNA) introduced by Kimber and Weisenberger. In contrast to lung sensitization there are already simplified and reliable models available to test for and discriminate contact sensitizers from skin irritants, i.e. the modified local lymph node assay IMDS (integrated model for the differentiation of skin reactions). Modifications of this assay verified that methods other than radioactive labelling may be comparably sensitive, and that it is possible to eliminate 'false positive' results induced by irritants (IMDS). Thus, we asked whether there could be a similar simplified model like the modified LLNA or IMDS for investigations of respiratory allergens. Therefore, we analysed immune reactions induced by the dermal and respiratory route, respectively. Analyses of the draining lymph nodes of the lung and the ear were carried out before and after challenge via the pulmonary tract. The results clearly support that (1) the reactions in the lung draining lymph nodes could be used as early indicators of respiratory sensitization, and (2) the specificity of the immune competent cells seem to be dependent of the route of administration during induction.

Keywords: Lung-draining lymph nodes Trimellitic anhydride Respiratory allergy Bronchoalveolar lavage Route-dependent activation Modified integrated model for the differentiation of skin reactions (IMDS) Brown Norway rats

No Title by K. Chitra; C. Latchoumycandane; P. Mathur (pp. 545-551).

Nonylphenol, an environmental contaminant, has been shown to induce reproductive abnormalities in male rats. The nature and mechanism of action of nonylphenol on the epididymal sperm has not been elucidated. In the present study we have sought to investigate whether administration of nonylphenol induces oxidative stress in rat epididymal sperm. Nonylphenol was administered orally to male rats at 1, 10 and 100 µg/kg body weight per day for 45 days. Twenty-four hours after the last treatment, rats were weighed and killed using anaesthetic ether. The body weight of the animals treated with nonylphenol did not show any significant change. The weights of the testes and epididymides decreased significantly whereas the weights of seminal vesicles and ventral prostate remained unchanged at all doses of nonylphenol in treated rats. Epididymal sperm were collected by cutting the epididymides into small pieces in Ham's F-12 medium at 32°C. Administration of nonylphenol decreased the epididymal sperm counts in a dose-dependent manner. The activities of antioxidant enzymes superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase decreased significantly while the levels of H₂O₂ generation and lipid peroxidation increased significantly in the animals treated with nonylphenol when expressed in terms of milligram protein and milligram DNA. The activity of α-glucosidase, a negative control against antioxidant enzymes, in the sperm of nonylphenol-treated rats did not show any significant change at any of the doses. The results suggest that graded doses of nonylphenol elicit depletion of antioxidant defence system in sperm, indicating nonylphenol-induced oxidative stress in the epididymal sperm of rats.

Keywords: Nonylphenol Antioxidant enzymes Sperm Oxidative stress Lipid peroxidation Rats

No Title by Danko Škare; Božica Radić; Ana Lucić; Maja Peraica; Ana-Marija Domijan; Sanja Milković-Kraus; Vlasta Bradamante; Ivan Jukić (pp. 552-552).

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Archives of Toxicology (v.76, #9)

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Archives of Toxicology (v.76, #9)