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Archives of Toxicology (v.76, #2)

No Title by Kanji Yamasaki; Masakuni Sawaki; Shuji Noda; Nobuya Imatanaka; Mineo Takatsuki (pp. 65-74).

We performed a 28-day repeated-dose toxicity study of ethynylestradiol (EE) and bisphenol A (BPA) based on the draft protocol of the 'Enhanced OECD Test Guideline 407', and assessed the sensitivity of a list of parameters for detecting endocrine-related effects of endocrine disruption. Doses of EE at 0, 10, 50 or 200 µg/kg per day, or BPA at 0, 40, 200 or 1000 mg/kg per day were orally administered to Sprague-Dawley rats. The highest dose of BPA was decreased to 600 mg/kg per day from the second week of administration because a male rat given 1000 mg/kg BPA had died within 1 week with toxic clinical signs. In the assay using EE, the decrease of prostate, seminal vesicle and pituitary weights, increase of the testis weight, atrophic changes of the prostate, seminal vesicle and mammary gland, and degenerative changes in the testes were detected in male rats in the 50 and/or 200 µg/kg groups. In females of the 200 µg/kg group, decrease of the ovary weight, increase of the uterine weight, atrophy of the ovary, hypertrophy or squamous metaplasia of the uterine epithelial cells and mucification in the vagina were observed. Furthermore, diestrous, estrous or the unknown stage was prolonged in the 50 and 200 µg/kg groups of rats. Endocrine-mediated effects of EE were not detected in general observations, hematology, serum biochemistry, or hormonal or spermatological examinations. In the assay using BPA, the diestrous stages were prolonged at the highest dose, but changes related to endocrine effects were not detected in other examinations. Thus, among the parameters tested, the weight of endocrine-linked organs and their histopathological assessment and estrous cycle stage allowed the detection of the endocrine-related effect of EE, whereas the estrous cycle stage was only a useful parameter to detect the effect of BPA.

Keywords: Ethynylestradiol Bisphenol A Estrogen agonist Enhanced Test Guideline 407 Rat Endocrine effects

No Title by Peter H. Roos (pp. 75-82).

We have analyzed the induction of the cytochrome P450 enzyme CYP1A1 as a biomarker of effect in duodenum, liver and kidney of rats after oral intake of contaminated soil particles. The soil samples originated from industrial sites and were contaminated with polycyclic aromatic hydrocarbons (PAH) to variable extents, ranging from 60 to 4700 mg PAH/kg soil. Soil samples were administered for one week as a mixture with commercial rodent diets. After exposure, microsomes of several organs were prepared and analyzed for CYP1A1, enzymatically and by Western blots. All contaminated soils led to induction of CYP1A1 in duodenal mucosa cells, regardless of their extent of contamination, showing that relevant doses were mobilized in the gastrointestinal tract and adsorbed. Subsequent distribution of non-metabolized compounds is indicated by induction of CYP1A1 in the liver. However, some samples did not lead to a response in the liver, due to their quantitative and qualitative contaminant composition. In accordance with previous results, there is a sigmoidal dose-response relationship between induction of hepatic CYP1A1 levels and the soil contamination with higher condensates of PAH. In contrast, the response in the duodenum appeared to be hyperbolic and correlated well with the amounts of total PAH. Highly contaminated soil, being nearly devoid of higher condensates of PAH, led to pronounced induction in the duodenum but failed to induce CYP1A1 in the liver. Successful passage of contaminants through the intestinal barrier and the liver compartment is shown by increased CYP1A1 expression in the kidney. Compared with enzyme levels induced in the liver, those of the kidney are much lower and amount to only about 1/20 of the liver values for soils with high induction potential. Hence, oral PAH intake leads to differential induction patterns of CYP1A1 in duodenum, liver and kidney of rats. The observations raise questions concerning the role of the primary duodenal PAH metabolism in preventing contaminant-dependent hazardous effects, and of the significance of differential CYP1A1 expressions for carcinogenic processes in several tissues.

Keywords: CYP1A1 Polycyclic aromatic hydrocarbons Contaminated soil Risk assessment Biomarker Liver Duodenum Kidney Enzyme induction

No Title by Daan Noort; Albert G. Hulst; Rob Jansen (pp. 83-88).

Covalent binding of various clinically important nitrogen mustards to the cysteine-34 residue of human serum albumin, in vitro and in vivo, is demonstrated. A rapid method for detection of these adducts is presented, based on liquid chromatography-tandem mass spectrometry analysis of the adducted tripeptide Cys*-Pro-Phe after digestion of the protein with Pronase.

Keywords: Nitrogen mustards Adducts Albumin Tandem mass spectrometry Biomonitoring Chemotherapy

No Title by E. Weidauer; W. Mörke; H. Foth; H. Brömme (pp. 89-95).

This in vitro study investigated the formation of hydroxyl radicals (•OH) under anaerobic conditions through the direct reaction between paraquat radicals (PQ⁺•) and hydrogen peroxide (H₂O₂) by quantitative UV-VIS and electron spin resonance (ESR) spectroscopy. PQ⁺• was formed by paraquat reduction using either sodium dithionite or the xanthine/xanthine oxidase reaction as electron donors. The anaerobic formation of PQ⁺• was quantified both by measuring light absorption at 605 nm or by ESR techniques respectively, using either the absorption coefficient or ultramarine as a stable spin standard. Detection of •OH took place with aid of the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). Generation or addition of H₂O₂ to PQ⁺• eliminates the 35-line ESR signal of PQ⁺• and subsequently generates the 8-line ESR signal of the DEPMPO-OH adduct. The elimination of PQ⁺• as well as the formation of OH-DEPMPO adduct was not influenced by 1.0 mM deferoxamine, indicating that iron or other transition metals are, at least under anoxic conditions, not necessarily involved in the generation of the most aggressive reactive oxygen species •OH.

Keywords: Paraquat Hydroxyl radical Electron spin resonance 5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide

No Title by M. Mileva; R. Bakalova; L. Tancheva; A. Galabov (pp. 96-103).

The present study provides a direct experimental evidence that the combination of influenza A/Aichi/2/68 (H3N2) infection with different models of "oxidative stress", such as immobilization, cold and cold-restraint, is associated with graduated oxidative disturbances in the liver of mice, despite the absence of virus and inflammation in this tissue. It was found that experimental influenza virus infection is accompanied with a significant increase of lipid peroxidation products, a decrease of natural antioxidants (vitamin E, glutathione) and cytochrome P-450, an inhibition of cytochrome c reductase and liver monooxygenases (analgin-N-demethylase and amidopyrine-N-demethylase). Immobilization and cold stress, applied separately or in combination (cold-restraint), did not influence significantly any of the analysed parameters compared to those of the control group of non-infected mice. Preliminary exposure of mice to immobilization or cold stress and subsequent inoculation of influenza virus resulted in a significant increase of lipid peroxidation products and a significant decrease of vitamin E and reduced glutathione, compared with levels in control (non-infected) animals. Compared to influenza virus-infected and non-stressed animals, the changes in all these parameters were negligible. Immobilization or cold stress, applied in combination with influenza virus infection, partially prevented the suppressive effect of influenza virus on cytochrome P-450 and liver monooxygenases. A tendency towards normalization of these parameters to the control levels was observed. However, after application of cold-restraint plus influenza virus infection, the level of cytochrome P-450 and activity of cytochrome c reductase stayed markedly lower than in infected and non-stressed animals. The activities of liver monooxygenases were slightly increased compared with those of infected and non-stressed animals, but stayed relatively low compared to control (non-infected) mice. Combination of cold-restraint and influenza virus infection resulted in a greater synergistic increase of lipid peroxidation products and a greater synergistic decrease of vitamin E and reduced glutathione compared to controls, as well as to influenza virus-infected and non-stressed animals.

Keywords: Influenza virus infection Immobilization Cold-restraint stress Lipid peroxidation Antioxidants

No Title by U. Bergman; A. Östergren; A.-L. Gustafson; E. Brittebo (pp. 104-112).

The aim was to study the long-term response in the olfactory mucosa of NMRI mice after exposure to the olfactory toxicants dichlobenil (a herbicide) or methimazole (an antithyroid drug). Three and six months after exposure to dichlobenil (2× or 1×25 mg/kg i.p.), the dorsomedial part of the olfactory region showed a respiratory metaplasia with abundant invaginations and a fibrotic lamina propria. In contrast, 3 months after exposure to a toxic dose of methimazole (2×50 mg/kg i.p.), the olfactory neuroepithelium and lamina propria had been restored. To study the regenerative events, we used an antibody derived against growth-associated protein 43 (GAP-43), which stains immature neurons. To study epithelial differentiation and horizontal basal cells (HBCs) we used an antibody derived against some cytokeratins. Two weeks after methimazole treatment, there was a marked increase of GAP-43-stained cells in the whole olfactory region, which correlated with the observed regeneration at that time. Two weeks after dichlobenil treatment, the damaged atypical epithelium in the olfactory region showed a distinct keratin staining of basal and columnar cells whereas GAP-43-stained cells were not found. Despite a transient increase of GAP-43-stained cells in the border zone between damaged and undamaged olfactory mucosa, an expansion of a normal neuroepithelium into the damaged olfactory region was not detected in the dichlobenil-treated mice. An intact lamina propria is suggested as a prerequisite for repopulation of the neuroepithelium after toxicant-induced injury.

Keywords: Dichlobenil Methimazole Olfactory neuropeithelium Growth-associated protein 43 Neurogenesis Cytokeratin Basal cell

No Title by C. Latchoumycandane; K. Chitra; P. Mathur (pp. 113-118).

The ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce oxidative stress in various tissues of animals has been reported. The nature and mechanism of action of TCDD on the antioxidant system of sperm has not been studied. In the present study we have sought to investigate whether TCDD induces oxidative stress in the epididymal sperm of rats. Subchronic doses of TCDD (1, 10, and 100 ng/kg body weight per day) were administered orally to male Wistar strain rats for 45 days. After 24 h of the last treatment the rats were killed using diethyl ether. The epididymides were removed and cleared from the adhering tissues. Epididymal sperm were collected by cutting the epididymides into small pieces in Ham's F12 medium, and counted using a hemocytometer. The epididymal sperm counts in the TCDD-treated groups decreased in a dose-dependent manner from the control value of 8.2±0.14 × 10⁸ to 5.31±0.15×10⁸. Since a positive correlation (r=0.95; n=24) was observed between sperm count and DNA content of the epididymal sperm, DNA content was routinely used as an indicator of sperm count, and the results were expressed in terms of both protein and DNA. There was a significant decline in the activities of superoxide dismutase (40±2.17 to 27.1±0.76/mg protein and 32.41 to 18.07±0.76/mg DNA), catalase (2.49±0.13 to 2.03±0.05/mg protein and 2.01±0.05 to 1.35±0.05/mg DNA), glutathione reductase (71.2±3.87 to 48±1.79/mg protein and 57.58±1.52 to 31.94/mg DNA) and glutathione peroxidase (22.4±1.43 to 16.9±1.57/mg protein and 18.08±0.61 to 11.38±1.22/mg DNA) while there were increases in the levels of hydrogen peroxide (20.8±1.96 to 55.3±0.88/ mg protein and 16.18±1.88 to 36.87±0.88/ mg DNA) and lipid peroxidation (2.17±0.2 to 6.08/mg protein and 1.75±0.12 to 4.05±0.12/mg DNA) in the epididymal sperm. The results suggest that graded doses of TCDD elicit depletion of antioxidant defense system in sperm, indicating TCDD-induced oxidative stress in the epididymal sperm. In conclusion, the adverse effect on male reproduction in TCDD-treated rats may be due to the induction of oxidative stress in sperm.

Keywords: 2,3,7,8-Tetrachlorodibenzo-p-dioxin TCDD Sperm Antioxidant enzymes Lipid peroxidation Sperm counts Rat

No Title by Uri Wormser; Berta Brodsky; Reuven Reich (pp. 119-121).

Recently we have shown that post-exposure treatment with povidone iodine (PI) protects against nitrogen and sulfur mustard-induced skin lesions. Since proteolytic activity is involved in skin damage caused by chemical irritants, we have studied the effect of iodine on mechlorethamine (HN2)-induced skin collagenolytic activities in the haired guinea pig model. The matrix metalloproteinase-9 (MMP-9) activity increased by 30, 46, 12 and 23% after 3, 24, 48 and 72 h of HN2 exposure, respectively, whereas the MMP-2 was elevated by 8, 65, 8 and 30%, respectively. Topical treatment with PI at 15 and 120 min after HN2 exposure decreased the MMP-9 activity by 67% and 60%, respectively, when skin was analyzed 3 h after exposure. The same trend was observed in the MMP-2 and MMP-1 activities after PI treatment. A stronger effect of PI treatment 15 min following exposure was observed in skin analyzed 24 h after exposure, i.e. a decrease of 83% and 88% in MMP-9 and MMP-2 activities, respectively. Similar findings were observed with an interval of 120 min between HN2 exposure and PI treatment. A much weaker effect was observed on MMP-1 activity. A similar trend of PI-induced reduction in the three types of collagenase activity was found in skin analyzed 48 and 72 h after exposure. Reduced collagenolytic activity may serve as one of the mechanisms by which iodine protects the skin against chemical insult.

Keywords: Nitrogen mustard Iodine Dermatotoxicity Collagenase Matrix metalloproteinase Skin irritation

No Title by Nazira S. Karamova; Olga B. Ivanchenko; Olga N. Ilinskaya; Vladimir V. Zobov; Vladimir S. Reznik (pp. 122-126).

Agent No. 547 (1,3-bis[ω-(diethyl-ortho-nitrobenzylammonio)-pentyl]-6-methyluracil dibromide), a newly synthesized inhibitor of mammalian-specific acetyltcholinesterase (EC 3.1.1.7) was investigated for genotoxicity using the DNA-repair test, Ames test and in vivo micronucleus test with mouse peripheral blood erythrocytes. Agent No. 547 did not cause significant changes in growth of repair-deficient Escherichia coli tester strains. The compound was non-mutagenic in Salmonella typhimurium strains TA98 and TA100 with and without rat microsomal activation mixture. However, we observed a marked increase in number of His⁺ revertants for both tester strains in preincubation assays. The results obtained in the micronucleus test indicate that agent No. 547 possesses significant clastogenic activity. At the high dose tested (0.5 mg/kg), the compound induced a seven-fold increase in the number of micronuclei over the spontaneous background 48 h after treatment. The results suggest that further work should be promoted to identify the metabolic pathways involved in genotoxicity of agent No. 547 in mammalian cells and to evaluate the real risk of its exposure.

Keywords: 1,3-Bis[ω-(diethyl-ortho-nitrobenzylammonio)-pentyl]-6-methyluracil dibromide Acetyltcholinesterase inhibitor Genotoxicity

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Archives of Toxicology (v.76, #2)

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Archives of Toxicology (v.76, #2)