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Archives of Toxicology (v.75, #2)

No Title by P. Andrews; A. Freyberger; E. Hartmann; R. Eiben; I. Loof; U. Schmidt; M. Temerowski; M. Becka (pp. 65-73).

Groups of five male and five female Wistar rats were treated by gavage with 0, 1, 10, and 100 mg flutamide/kg body weight for at least 28 days to investigate whether proposed enhancements to the current subacute rodent OECD test guideline no. 407 could be included into the testing routine, which of the current and/or additional parameters would detect endocrine-mediated effects of flutamide reliably and sensitively, and to provide information on intra-laboratory variability. Two identical studies were performed concurrently. The enhanced protocol requests the additional determination of the specific hormones triiodothyronine, thyroxine, thyroid stimulating hormone, follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, prolactin, testosterone, corticosterone; of oestrus cyclicity and necropsy of all females in the dioestrus stage; of the number of homogenization-resistant testicular spermatids and the number, motility, viability, and morphology of cauda epididymal spermatozoa; of additional organ weights (pituitary, ovaries, uterus, thyroid, male accessory reproductive organs); and of the histopathology of additional organs (pituitary, epididymides, coagulation glands, pancreas, vagina). From a technical standpoint, it was possible to conduct a study according to the enhanced protocol, however, with substantial additional effort, an increase in costs by some 67%, and logistic problems. In line with the specific pharmacological effect of flutamide, treatment-related changes were mainly found in male rats, while females were hardly affected by 100 mg/kg. In male rats, 100 mg/kg was the maximal tolerated dose resulting in reduced body weight gain, but no or little other effects on clinical, haematological, clinico-chemical, or behavioral parameters, and 1 mg/kg was the no-observed-adverse-effect level. Antagonism of peripheral androgen receptors by flutamide resulted in decreased relative organ weights of male accessory reproductive organs, changes that were reliably detected in both studies at 100 mg/kg, but only in one of both studies at 10 mg/kg. Corresponding histopathological changes were also detected reliably at 100 mg/kg. Antagonism of central androgen receptors by flutamide increased LH and FSH levels. LH stimulation of testicular Leydig cells in turn increased testosterone and estradiol levels. Again, all these changes were detected reliably at 100 mg/kg, but only in one of both studies at 10 mg/kg. Corresponding histopathological alterations (increase of LH- and FSH-secreting cells, Leydig cell hypertrophy) were detected reliably and sensitively at 10 mg/kg. Studies on liver enzymes performed outside the scope of the enhanced protocol showed that flutamide at 100 mg/kg generally induced hepatic enzyme activcities, but decreased the activity of the sex-specific testosterone-dependent liver enzyme CYP2C11 in male rats. The laboratory methods employed yielded reliable results, i.e., 93.6% of the quantitative measurements obtained in both studies were in agreement. Doubling the animal number from five to ten per sex and dose does not increase the sensitivity of detection of endocrine-mediated effects above the level already provided by histopathological examination of groups of five animals. Some of the proposed enhancements evaluated (additional organgravimetry and histopathology) were helpful in detecting the endocrine-mediated effects of flutamide reliably, while others did not contribute towards this aim (spermatology resulted in doubtful effects, female cyclicity was not affected, hormone determinations provided mechanistic information). Ongoing testing according to the revised version of the enhanced OECD test guideline no. 407 protocol and using ten compounds interfering with the endocrine system by different mechanisms will result in the identification of the most appropriate enhancements.

Keywords: Enhanced OECD test guideline no. 407 Feasibility Detection of endocrine effects Flutamide Antiandrogenicity

No Title by Yoshio Nakagawa; Kuniaki Tayama (pp. 74-79).

The estrogenic activity of benzophenone and its metabolites, benzhydrol and p-hydroxybenzophenone, were investigated in vitro by estrogen receptor (ER) competitive ligand binding assay and in vivo by uterotrophic assay in juvenile female Sprague-Dawley (SD) rats. p-Hydroxybenzophenone as well as diethylstilbestrol and bisphenol A, known xeno-estrogenic compounds, competed with fluorescein-labeled 17β-estradiol to bind human recombinant ERα in a concentration-dependent manner. Fifty percent inhibitory values (IC₅₀) of diethylstilbestrol, bisphenol A, and p-hydroxybenzophenone were approximately 10^–8, 10^–5, and 5×10^–5 M, respectively. However, neither the parent compound nor benzhydrol at concentrations from 10^–9 to 5×10^–4 M impaired the binding of 17β-estradiol to ERα. Juvenile female rats (21-days-old) were given s.c. injections of benzophenone, its metabolites, and 17β-estradiol for 3 days. Administration of p-hydroxybenzophenone (100–400 mg/kg) elicited an increase in absolute and relative uterine weights in a dose-dependent manner and 17β-estradiol (10 µg/kg) increased uterine weight approximately fourfold relative to control. The uterine response caused by both compounds was accompanied by an increase in luminal epithelial height and stromal cells in the uterus and an increase in thickness of vaginal epithelial cell layers with cornification. In contrast, benzophenone and benzhydrol at a dose of 400 mg/kg affected neither uterine weight nor histological changes of the uterus and vagina. These results indicate that p-hydroxybenzophenone, a metabolite of benzophenone, exhibits intrinsic xeno-estrogenic activity in the female reproductive tract.

Keywords: Benzophenone Metabolites Uterotrophic assay Competitive binding assay Female rats

Toxicity of PCB-126 in European flounder (Platichthys flesus) with emphasis on histopathology and cytochrome P4501A induction in several organ systems by G.C.M. Grinwis; E.J. van den Brandhof; M.Y. Engelsma; R.V. Kuiper; M.A. Vaal; A.D. Vethaak; P.W. Wester; J.G. Vos (pp. 80-87).

A series of experiments was set up to elucidate the effects of pollution on marine and estuarine fish health, since the European flounder (Platichthys flesus) has shown a relatively high prevalence of (pre)neoplastic liver lesions and lymphocystis virus disease in Dutch coastal and estuarine waters. The hypothesis of a causal relationship between pollution and the above-mentioned diseases was supported by results from semi-field experiments. Therefore several laboratory experiments were carried out to substantiate causality further and to identify the xenobiotics that may play a major role in the field. The present study focuses on polychlorinated biphenyls (PCBs). European flounders (Platichthys flesus) were orally exposed to a single dose of 0, 0.5, 5 or 50 mg PCB-126/kg body weight under controlled laboratory conditions. The effects on liver, gills, gastrointestinal tract, gonads, spleen and mesonephros were examined histologically after 16 days. Induction and localization of cytochrome P4501A (CYP1A) immunoreactivity, and effects on hepatocyte proliferation were visualized immunohistochemically. Effects on thymus size were examined by morphometric analysis of serial sections. Three out of five animals of the highest dose group showed haemorrhages in the fins and tail after 16 days. All animals showed reduced activity in the later stages of the experiment, and some animals of the highest dose group discontinued feeding 14 days after exposure. Strong and exposure-related induction of CYP1A immunoreactivity was noted in hepatocytes, endothelium in all organs examined, and epithelium of the digestive tract and mesonephros at PCB-126 levels of 0.5, 5 and 50 mg/kg. In addition, the strong induction of CYP1A immunoreactivity in a distinct population of haematopoietic cells in the mesonephros and in circulating blood is remarkable, and has not been described previously in other fish species. Furthermore, a morphometrically determined significant reduction in relative thymus size was noted in animals exposed to 50 mg PCB-126/kg. Although the functional implications for the immune system of this reduction need to be further investigated, an impact on the specific resistance against infectious diseases as observed in the field, e.g. viral lymphocystis disease, is not implausible. In addition, a significant increase in absolute liver weight, in hepatosomatic index, and in number of proliferating hepatocytes [measured as immunoreactivity against proliferating cell nuclear antigen (PCNA)] was noted in animals of the highest dose group. From these findings we suppose that PCB-126 (and related chemicals) may play a role in the promotion of tumour development in the liver of European flounders as observed in the field. The results of the present experiment show relatively stronger effects than effects previously reported from experiments with TCDD, suggesting that the TEF of 0.005 assigned to PCB-126 from early life stage mortality experiments in rainbow trout (Oncorhynchus mykiss), underestimates the toxic potential of PCB-126.

Keywords: Fish flounder Histopathology Haematopoietic Immunotoxicity Immunocytochemistry Liver Morphometry Platichthys flesus Thymus PCB Toxicology TEF

No Title by Fatih Gultekin; Namik Delibas; Sulhattin Yasar; Ibrahim Kilinc (pp. 88-96).

Reactive oxygen species (ROS) may be involved in the toxicity of chlorpyrifos-ethyl (CE) [O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)phosphorothioate]. We have, therefore, examined the in vivo effects of CE on the rat erythrocyte antioxidant system and evaluated the ameliorating effects of melatonin and a combination of vitamin E and vitamin C on the oxidative damage induced by CE. The experimental groups were: (1) control group, (2) CE-treated group (CE), (3) vitamin E plus vitamin C treatment group (Vit), (4) melatonin-treated group (Mel), (5) vitamin E plus vitamin C plus CE treatment group (Vit+CE), and (6) melatonin plus CE treatment group (Mel+CE). Vitamin E and vitamin C were administered intramuscularly once a day for 6 consecutive days at 150 and 200 mg/kg, respectively, in the Vit and Vit+CE groups. Melatonin was administered intramuscularly at 10 mg/kg per day for 6 consecutive days in the Mel and Mel+CE groups. At the end of the fifth day, the rats of CE, Vit+CE and Mel+CE groups were treated orally with the first of two equal doses of 41 mg/kg CE, the second oral dose being given 21 h later. Blood samples were taken 24 h after the first CE administration. Levels of thiobarbituric acid reactive substance (TBARS), antioxidant defence potential (AOP), and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were determined in erythrocytes. In comparison with the control group, oral administration of CE significantly (P<0.05) stimulated TBARS activity while significantly (P<0.05) inhibiting AOP and the activities of SOD and CAT. However, GSH-Px activity remained unchanged by CE treatment. Treatment with melatonin and vitamins E plus C significantly (P<0.05) reduced the CE-induced increase of TBARS, and overcame the inhibitory effect of CE on SOD and CAT, but not on AOP. Melatonin treatment significantly (P<0.05) increased only GSH-Px activity, irrespective of the effect of CE. These results suggest that CE treatment increases in vivo lipid peroxidation and decreases antioxidant defence by increasing oxidative stress in erythrocytes of rats, and melatonin and a combination of vitamin E and vitamin C can reduce this lipoperoxidative effect.

Keywords: Chlorpyrifos-ethyl Erythrocyte Lipid peroxidation Melatonin Vitamin

No Title by Mehdi Shakibaei; Ralf Stahlmann (pp. 97-102).

Quinolone therapy can be associated with tendon disorders (tendinitis, ruptures), but little is known about possible ultrastructural changes in tendons after exposure to these antimicrobials. We studied the Achilles tendons from fleroxacin-treated adult rats by electron microscopy. Wistar rats were treated orally with single oral doses of 0, 30, 100, 300 or 600 mg fleroxacin/kg body weight (n=6 per group). The animals were killed 4 weeks after treatment, Achilles tendon samples were collected and tangential sections were made from the distal part of the tendon. Subsequently, tendons were cut crosswise for preparation of ultrathin sections. Samples were fixed by using glutaraldehyde, osmium tetroxide, tannic acid and finally contrasted with uranyl acetate/lead citrate before they were examined by transmission electron microscopy. The rats did not show any general effects such as behavioural changes or body weight changes which could be attributed to the treatment. However, we were able to detect pathological changes even at the lowest dose level (30 mg/kg), which increased in incidence and severity with increasing doses. Tenocytes exhibited degenerative changes such as multiple vacuoles and large vesicles in the cytoplasm that resulted from swelling and dilatation of cell organelles (mitochondria, endoplasmic reticulum). The nucleus became dense and the chromatin had clumped to form rough plaques. The cells detached from the extracellular matrix. Other important findings were a general decrease of the fibril diameter and an increase in the distance between the collagenous fibrils. The finding that these rather low single doses of a fluoroquinolone induce ultrastructural changes in Achilles tendons from rats, which were not associated with clinical symptoms and which were still present 4 weeks after treatment, is of concern. Further toxicological as well as clinical studies are needed to characterize the conditions under which quinolone-induced tendon lesions develop.

Keywords: Achilles tendon Rat Fleroxacin Fluoroquinolones Electron microscopy

No Title by M. Dalgaard; A. Hossaini; K. S. Hougaard; U. Hass; O. Ladefoged (pp. 103-109).

In one study, pregnant Wistar rats were exposed to 1200 ppm toluene by inhalation 6 h a day from gestational day (GD) 7 to postnatal day (PND) 18. Sperm analysis was performed in the adult male offspring at PND 110 by using computer-assisted sperm analysis. Toluene did not affect the semen quality of exposed rats. In another study, pregnant rats were exposed to 1800 ppm from GD 7 to GD 20, and the male offspring were killed at PND 11, 21 or 90. Paired testes weight, histopathology and immunoexpression of vimentin in Sertoli cells were used as markers of testis toxicity. In the brain, the number of apoptotic cells in the hippocampus and cerebellum were counted after visualisation by means of the TUNEL assay. Mean body weight in pups of exposed dams was lower than in pups from control litters. This decrease was still statistically significant at PND 11, but at PND 21 and 90 the body weight of toluene-exposed males tended to approach that of the controls. Absolute and relative testes weights were reduced in all three age groups, although not to a statistically significant degree. Histopathological examinations of the testis and immuno-expression of vimentin did not reveal any differences between toluene-exposed animals and control animals. In the hippocampus, almost no apoptosis was observed in any age group, and there were no differences in apoptotic neurodegeneration between male rats exposed to 1800 ppm and control animals at PND 11, 21 or 90. Generally, a marked increase in number of apoptotic cells was observed in cerebellar granule cells at PND 21 compared with the other age groups. Toluene induced a statistically significant increase in the number of apoptotic cells in the cerebellar granule layer at PND 21. The mean was increased from 37 in the control group to 71 in the toluene-exposed group. Thus, the granular cell layer in cerebellum is a highly relevant tissue with which to study toluene-induced apoptosis, because of the continuous migration of neurons and high frequency of neuronal apoptosis during the weaning period. In summary, it is concluded, that neither pre- and postnatal exposure to 1200 ppm toluene nor prenatal exposure to 1800 ppm induced significant effects on the reproductive parameters investigated. However, prenatal exposure to 1800 ppm toluene did increase neuronal apoptosis in the cerebellum of weaned male rats, possibly by delaying postnatal migration of granule cells to their final destination, or by toluene-induced retardation of generalised fetal growth.

Keywords: Toluene Developmental toxicity Apoptosis in the cerebellum Semen quality Testis

No Title by A. Traoré; I. Baudrimont; S. Ambaliou; S.D. Dano; E.E. Creppy (pp. 110-117).

Okadaic acid (OA) is a shellfish toxin produced by dinoflagellates, in mussels. It is a potent tumour promoter and represents a potential threat to human health even at low concentrations. OA targets mainly the gastrointestinal tract in acute poisoning, causing diarrhoea. Therefore the present investigations were designed to study the ability of okadaic acid to induce cytotoxicity and DNA lesions in a human colonic cell line (Caco-2). Incubation of Caco-2 cells with OA (3.75–60 ng/ml, i.e 4.6×10^–3–7.5×10^–2 µM) causes a significant reduction in cell viability. Moreover, okadaic acid inhibits protein and DNA synthesis with, respectively, IC₅₀ of 16 and 6.5 ng/ml after 24 h incubation. It also provokes cell cycle arrest, characterised by an increase in the number of S phase cells, correlated with a significant decrease in G0/G1 phase cells at high concentration. One of the main results obtained in these investigations is the apoptosis induced by OA in Caco-2 cells of intestinal origin, shown by DNA laddering in agarose gel electrophoresis (250–1000 base pairs). OA also induces clastogenic effects evaluated by DNA fragmentation analysis using the method of Higuchi and Aggarwal (52% for 60 ng/ml) and comet assay (increase of the frequency of comets and their tails length). Therefore, the cell death induced by OA seems clearly to be concentration-dependent after 24 h of incubation. The cytotoxic properties of okadaic acid and its ability to damage DNA result in cell death, mainly by apoptosis. Since consumption of shellfish contaminated with acceptable okadaic acid concentrations exposes colonic cells to harmful concentrations of this toxin, the possibility that OA would display its toxic effects on intestinal cells in vivo should be evaluated in human primary intestinal cells and human intestinal slices for cytotoxic effects, DNA fragmentation and apoptosis.

Keywords: Okadaic acid Apoptosis Clastogenicity Caco-2 colonic cell line

No Title by Götz A. Westphal; Michael M. Müller; Claudia Herting; Jürgen Bünger; Ernst Hallier (pp. 118-122).

Dicyclohexylamine × nitrite is classified as an "experimental equivocal tumorigenic agent" by the National Toxicology Program. Since no genotoxic effects of the substance itself are known, the reported tumorigenic potential of dicyclohexylamine × nitrite could be due to generation of N-nitrosodicyclohexylamine (N-NO-DCHA), which occurs under conditions of use and can be detected in foils that contain dicyclohexylamine × nitrite. Therefore, we investigated possible mutagenic properties of N-NO-DCHA in the Ames test and the cytokinesis-block micronucleus assay with human lymphocytes. Since N-NO-DCHA is not commercially available, the substance was synthesized and purified by thin-layer chromatography. Identity was confirmed by gas chromatography/mass spectroscopy (GC/MS) and ¹H- and ¹³C-NMR. More than 97% purity was achieved. Stability and availability in the solvent were checked by GC/MS. N-NO-DCHA induced micronuclei in isolated human lymphocytes at a dose range of 15–100 µg/ml (=71.4–476.2 µM), exceeding the base rate significantly at one or two nontoxic concentrations in four out of six experiments. For the Ames test, arochlor-1254-, β-naphthoflavone/phenobarbital- and pyrazole-induced S9-fractions were used with Salmonella typhimurium TA100, TA1535, TA98 and TA104. No effects were seen in the Ames test, with the exception of microcolony induction at doses higher than 250 µg (=1.2 mmol) N-NO-DCHA/plate using TA104 and 20% arochlor-1254 induced S9 at pH 6.5. In conclusion, N-NO-DCHA was negative in the Ames test using TA98, TA100 and TA1535, inconclusive using TA104, and weakly genotoxic in the in vitro micronucleus test with isolated human lymphocytes. With regard to the tumorigenicity of the majority of nitrosamines, our data underline the necessity of further studies on possible genotoxic effects of N-NO-DCHA.

Keywords: N-Nitrosodicyclohexylamine Ames test Cytokinesis-block micronucleus assay

No Title by Lakshmi K. Akkineni; Magnus Zeisig; Pawel Baranczewski; Lars-Gösta Ekström; Lennart Möller (pp. 123-125).

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Archives of Toxicology (v.75, #2)