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Archives of Toxicology (v.74, #10)
No Title by Makotoy Shibutani; Kunitoshi Mitsumori; Naoko Niho; Shin-ichi Satoh; Hideaki Hiratsuka; Masahiko Satoh; Masami Sumiyoshi; Motohiro Nishijima; Yasutaka Katsuki; Jin Suzuki; Jun-ichi Nakagawa; Masanori Ando (pp. 571-577).
To determine whether low-dose oral administration of cadmium (Cd) induces renal toxicity, six groups of female Sprague-Dawley rats were fed a diet containing low amounts of CdCl2 or Cd-polluted rice at concentrations up to 40 ppm, and were killed after 12, 18, and 22 months (experiment 1). In addition to the determination of cortical Cd levels and histopathological assessment of kidneys, labeling indices (LIs) for proliferating cell nuclear antigen (PCNA) in the renal cortical tubular epithelium of Cd-treated rats were determined as a measure of regenerative activity. For comparison, the kidneys of rats given diets containing small to large amounts of CdCl2 up to 600 ppm for 4 months were similarly examined (experiment 2). Animals in experiment 1 demonstrated spontaneous chronic nephropathy and fluctuation in the tubular PCNA LI, but these findings were not correlated with renal Cd levels at 22 months. PCNA LI on the other hand, appeared to be linked to the severity of chronic nephropathy. In experiment 2, levels of CdCl2 of 200 ppm or more clearly induced degeneration and apoptosis of proximal tubules with high correlations between renal Cd levels, PCNA LI, and the severity of tubular degeneration. The results demonstrated that, in contrast to high-dose Cd administration, treatment with 40 ppm or less for 22 months did not influence tubular regeneration as a component of nonspecific chronic nephropathy, suggesting that long-term oral administration of low levels of Cd does not injure renal tubules in female rats.
Keywords: Cadmium Low-dose long-term exposure Renal toxicity Tubular regeneration Proliferating cell nuclear antigen
No Title by W.J. Weidner; D.S. Waddell; A.J. Sillman (pp. 578-581).
The effect of cadmium chloride on the immunoprecipitation of cadherin and the associated adherens junctional proteins, α- and β-catenin, was examined in isolated bullfrog (Rana catesbeiana) corneas utilizing Western blot and enhanced chemoluminescent techniques. Application of either 1.0 µM or 75.0 µM CdCl2 to the corneal endothelium for 2 h markedly decreased the immunoprecipitation of cadherins as compared to paired control corneas. Immunoprecipitation of α-catenin was increased in response to both doses of CdCl2, while the immunoprecipitation of β-catenin was little changed by either cadmium dose. There is accumulating evidence that cadmium may increase epithelial paracellular permeability by interfering with cadherin complex activity at intercellular junctions. The present study suggests that inorganic cadmium in low micromolar concentrations may decrease the integrity of the corneal endothelium, at least in part through a similar mechanism involving disruption of junctional cadherin complex function.
Keywords: Cornea Cadmium chloride Endothelium Adhesion molecules Cadherins
No Title by M.M. Iba; J. Fung; F.J. Gonzalez (pp. 582-586).
Neurotoxicity of n-hexane is mediated by its metabolite 2,5-hexanedione (2,5-HD). Cytochrome P4502E1 (CYP2E1) has been suggested but not shown to be involved in the formation of the metabolite. An objective of the current study was to assess the essentiality of CYP2E1 for in vivo 2,5-HD formation from n-hexane. This was accomplished by comparing urinary levels of the γ-diketone in n-hexane-treated mice in which the Cyp2e1 gene has been deleted (Cyp2e1–/–) with that in n-hexane-treated wild-type (Cyp2e1+/+) mice. 2,5-HD was detectable not as the free compound but as further metabolites, at levels that were comparable in both strains of mice, following a daily 200 mg/kg i.p. dose of the alkane for 10 days. Continued daily n-hexane treatment resulted in increased urinary levels of 2,5-HD metabolites in Cyp2e1+/+ but not in Cyp2e1–/– mice. Only in Cyp2e1+/+ mice and only on day 21 of n-hexane treatment was a trace level of unchanged 2,5-HD detected. 3-Hexanol was the only other n-hexane metabolite detected in the mice but its concentration was higher in Cyp2e1–/– than in Cyp2e1+/+ mice. In n-hexane-treated rats, in contrast to mice, multiple metabolites of the alkane, including unchanged 2,5-HD, were detected. The results indicate that substantial in vivo formation of 2,5-HD from n-hexane in the mouse requires CYP2E1, and suggest that further detoxification of the metabolite may be very efficient in this species.
Keywords: Cyp2e1-knockout mouse n-Hexane Biotransformation 2,5-Hexanedione
No Title by Karen De Smet; Thomas Brüning; Meinolf Blaszkewicz; Hermann M. Bolt; Antoine Vercruysse; Vera Rogiers (pp. 587-592).
The collagen gel sandwich culture of hepatocytes has been proposed as one of the most suitable culture models available for biotransformation studies of xenobiotics. It is a complex model which imitates the cascade of enzymatic events of in vivo biotransformation and allows investigation of biological endpoints under realistic conditions. The biotransformation of trichloroethylene (TRI) has been studied in this model using rat hepatocytes. Headspace gas chromatographic measurements revealed that hepatocytes, cultured for 4 days in this in vitro system, metabolised TRI into the major oxidative metabolites trichloroacetic acid (TCA) and trichloroethanol (TCE). Cultured hepatocytes were exposed either to TRI, or to TCA and TCE. Endpoints studied were albumin secretion and the cytochrome P450 (CYP)-dependent enzymatic activities ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-depentylase (PROD) and N-nitrosodimethylamine demethylase (NDMA). The results show that both the parent compound and its metabolites exert specific effects on different CYP-dependent mono-oxygenase activities, as seen in vivo. It is suggested that collagen gel sandwich cultures represent a useful in vitro model for the investigation of metabolism-linked toxicity studies.
Keywords: Rat hepatocytes Collagen gel Trichloroethylene In vitro model
No Title by A. Lizette Granberg; Björn Brunström; Ingvar Brandt (pp. 593-601).
Autoradiography was used to investigate the cellular sites of irreversible binding of 3H-labelled 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P) in mice. Autoradiograms obtained from solvent-extracted tape-sections revealed an even distribution of DMBA- and B[a]P-derived radioactivity in control mice lacking sites of selective binding in the tissues. In mice pretreated with a cytochrome P4501A (CYP1A) inducer, β-naphthoflavone (BNF) or 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), a noticeable accumulation of bound radioactivity was observed in the pulmonary alveolar region. Increased labelling was also observed in heart tissue of induced mice. As demonstrated by microautoradiography of tissues from CYP1A-induced mice treated with 3H-DMBA or 3H-B[a]P in vivo, irreversible binding in lung tissue was present in endothelial cells of arteries and veins, in the alveolar septal walls, and in type 2 pneumocytes. In heart tissue, binding was confined to endothelial cells of arteries, capillaries and veins. In liver, binding was found in the hepatocytes as well as in endothelial cells of the portal veins, whereas no binding was seen in endothelial cells of the sinusoids, central veins, or arteries. These findings were confirmed in vitro using 3H-DMBA-exposed precision-cut slices, indicating that reactive intermediates of DMBA and B(a)P were formed in situ. The addition of the CYP1A inhibitor ellipticine abolished binding in the target endothelial cells. Increased endothelial binding in the lungs and liver of CYP1A-induced mice was concomitant with increased 7-ethoxyresorufin O-deethylase (EROD) and DMBA hydroxylase activity. In heart, endothelial binding was positively correlated with EROD, but not with DMBA hydroxylase. The results suggest that endothelial cells may be targets for CYP-dependent activation of such toxicants as polycyclic aromatic hydrocarbons. Consequently, the possibility that chemically induced endothelial dysfunction is a risk factor in the aetiology of cardiovascular disease demands consideration.
Keywords: 7,12-Dimethylbenz[a]anthracene Benzo[a]pyrene PCB 126 Endothelial cells Heart Lung Irreversible binding
No Title by Zhi-Zhong Guan; Kai-Qi Xiao; Xian-Yun Zeng; Yi-Guo Long; Yuan-Hua Cheng; Su-Fen Jiang; Ya-Nan Wang (pp. 602-608).
An animal model of chronic fluorosis was produced by subjecting Wistar rats to high doses of fluoride in drinking water for a prolonged period. Phospholipid and neutral lipid contents in rat kidney were then analyzed by high-performance liquid chromatography (HPLC), and fatty acid compositions from individual phospholipids were measured by gas chromatography. Lipid peroxidation was detected by the thiobarbituric-acid-reactive substance assay. Results showed that the total phospholipid content significantly decreased in the kidney of the rats treated with high doses of fluoride and the main species influenced were phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Decreased proportions of polyunsaturated fatty acids were observed in PE and PC in kidney of fluoride-treated animals compared to controls. No changes could be detected in the amounts of cholesterol and dolichol in kidneys between the rats treated with fluoride and controls. A significant decrease of ubiquinone in rat kidney was observed in the groups treated with excessive fluoride. High levels of lipid peroxidation were detected in kidney of the rats with fluorosis. It is plausible that the specific modification of lipid composition results from lipid peroxidation. The oxidative stress and modification of cellular membrane lipids may be involved in the pathogenesis of chronic fluorosis and provide a possible explanation for the gross system damage observed in the body, especially in soft tissues and organs.
Keywords: Lipid Fatty acid Fluorosis Lipid peroxidation Kidney
No Title by C. Michielsen; S. Boeren; I. Rietjens; F. van Mil; J. Vos; N. Bloksma (pp. 609-617).
Involvement of the mercapturic acid pathway in the induction of splenomegaly and skin and lung pathology by hexachlorobenzene (HCB) in the rat was investigated by seeking to determine whether pentachloronitrobenzene (PCNB) has the same inflammatory effects as HCB, since both compounds are directly conjugated to glutathione, and further processed into the same mercapturic acid metabolites which are excreted via the urine. Female Brown Norway (BN/SsNOlaHsd) rats at 3 to 4 weeks of age were orally exposed to diets with or without supplementation with 450 mg HCB or equimolar (467 mg) or higher (934 mg) amounts of PCNB per kilogram of diet over 4 weeks. Gross skin lesion development and body weight gains were assessed during exposure and spleen and liver weights as well as histopathologic changes in skin and lung were assessed after exposure. After 3 weeks of exposure, urinary metabolites of the mercapturic acid and oxidative biotransformation pathways were identified using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Oral exposure of the rats to 450 mg/kg HCB resulted in an increase in relative spleen and liver weights as well as in the development of skin and lung pathology in the absence of overall liver toxicity. Equimolar or higher concentrations of PCNB caused none of these effects. Urinary levels of the mercapturic acid N-acetyl-S-(pentachlorophenyl)-cysteine (PCP-NAC), were comparable in HCB- and PCNB-treated rats. Levels of closely related methylsulfide derivatives of PCP-NAC, also generated via the same mercapturic acid pathway, appeared to be significantly higher in PCNB- than in HCB-treated rats, whereas the reverse was true for the urinary levels of the oxidative metabolite pentachlorophenol (PCP). Thus, results indicate that metabolites of the mercapturic acid pathway are not involved in the induction of splenomegaly and skin and lung pathology caused by HCB exposure in BN rats and that the main urinary metabolite of HCB in these BN rats is PCP. Since PCP itself, as well as other cytochrome P450-derived metabolites from HCB, are not likely to be involved in the induction of splenomegaly and skin and lung pathology, it is suggested that either the parent compound HCB or as-yet-unidentified non-P450-generated metabolites are involved in these inflammatory effects of HCB.
Keywords: Hexachlorobenzene Pentachloronitrobenzene Brown Norway rat Skin pathology Lung pathology Splenomegaly Mercapturic acid pathway Biotransformation HPLC LC-MS Pentachlorophenol
No Title by Angelika Dörrenhaus; Jill I.F. Müller; Klaus Golka; Peter Jedrusik; Harald Schulze; Wolfram Föllmann (pp. 618-626).
Exfoliated human urinary tract epithelial cells and renal tubular cells from urinary sediments of healthy adults, of urological patients and of internal patients were isolated and cultured. Cells started proliferating within 1 week after seeding a sediment. Proliferating cells formed colonies of different morphologies, designated as type-1 or type-2 cell colonies. Type-1 cell colonies showed irregular contours and spindle-like cells within the colonies. Subcultivation of type-1 cells for up to six passages was possible. Type-2 cell colonies showed smooth-edged contours and subcultivation was not possible. The epithelial character of type-1 cells was demonstrated by positive immunohistochemical staining for cytokeratin-7. In contrast to carbonic anhydrase-positive stained Madin Darby canine kidney cells (MDCK), which were used as positive controls for renal tubular cells, type-1 cells were carbonic anhydrase-negative on staining with the cobalt phosphate method. This indicates that type-1 cells were not of renal tubular origin. Type-2 cells were positively stained for carbonic anhydrase, indicating that type-2 cells were renal tubular cells. Type-2 cell colonies could be assigned to two subgroups with different cell forms. Colonies of cobblestone-like cells more often occurred than type-2 cell colonies with spindle-like cells, which are described in this study for the first time. Colonies with cobblestone-like cells formed domes (hemicysts), whereas spindle-like type-2 cell colonies did not. Cultures of urinary sediments from healthy adults, elderly multimorbid patients treated with furosemide, and urological patients with urolithiasis treated with sulfamethoxazole/trimethoprim and/or with a percutaneous nephrostomy catheter were compared. In 52% of all cultured sediments from healthy adults, in 30% of those from multimorbid patients, and in 75–80% of those from urological patients cells proliferated to colonies. The ratios of type-1 to type-2 cell colonies were 3.3:1 (healthy adults), 1.4:1 (urological patients with urolithiasis), and 1.8:1 (urological patients with urolithiasis, urine was directly collected from the renal pelvis with a percutaneous nephrostomy catheter). Successful cultures of the urinary sediments from these three groups revealed means of 3 or 4 colonies, 14 colonies, and 21 colonies, respectively. Differences in the number of colonies in relation to sex were observed only for the group of urological patients. It was shown that type-1 cells were urothelial cells, which did not show morphological differences due to their locations of origin within the urinary tract, whereas type-2 cells were probably renal tubular cells. These findings offer new aspects in the culturing of human urothelial or kidney epithelial cells with a method based on noninvasive collecting of specimens and requiring only minimal culture effort. The cultures obtained by this method can be used for in vitro studies in toxicological and clinical research.
Keywords: Transitional cells Renal tubular cells Human urinary sediment Percutaneous nephrostomy catheter Kidney stone
No Title by Osamu Yamamoto; Yoshiaki Doi; Hideaki Kudo; Mitsuaki Yoshizuka; Sunao Fujimoto (pp. 627-631).
Acute toxicity of bis (tributyltin) oxide in the sweat glands in the rat footpad was investigated by electron microscopy and an energy-dispersive X-ray microanalyzer. Male Wistar rats received an intramuscular injection of 0.5 ml/kg bis (tributyltin) oxide. After 6–8 h, swelling of mitochondria appeared in the secretory cells of the sweat glands. After 12 h, the secretory cells began to show intracytoplasmic edema. After 16–20 h, secretory cells in some sweat glands showed marked hydropic degeneration with swollen cytoplasm. Using X-ray microanalysis, tin peaks were preferentially obtained from the swollen mitochondria of the affected secretory cells. Mitochondria dysfunction due to the toxic effects of bis (tributyltin) oxide induced changes in the secretory cells of rat sweat glands. After 24–48 h, the secretory portion of the sweat glands contained three types of cells: degenerating dark cells, regenerating cells carrying injured mitochondria, and light cells which were morphologically very similar to the cells in the transitional portion of the sweat gland. These light cells appeared to differentiate into active secretory cells after settling down in the secretory portion. Based on these observations, we concluded that the cells in the transitional portion could play an important role at least as reserve cells against secretory cell toxicity. In association with the regenerating process of the damaged secretory portions, increased mitotic activities were seen in different areas of all the dermal sweat ducts. The above-mentioned morphological observations for cell damage and subsequent regeneration and renewal of secretory cells in sweat gland intoxication have not been reported so far.
Keywords: Bis (tributyltin) oxide Sweat gland toxicity Ultrastructure X-ray microanalysis Sweat gland kinetics
No Title by Akira Harazono; Makoto Ema (pp. 632-637).
In our previous studies, tributyltin chloride (TBTCl) at doses of 16.3 mg/kg and above caused implantation failure (preimplantation embryonic loss) and postimplantation embryonic loss in rats following administration on gestational day (GD) 0 through GD 3 and GD 4 through GD 7, respectively. This study was designed to assess the effects of TBTCl on uterine function as a cause of early embryonic loss in pseudopregnant rats. TBTCl was given orally to pseudopregnant rats at doses of 4.1, 8.1, 16.3 and 32.5 mg/kg on pseudopregnant day (PPD) 0 to PPD 3 or 8.1, 16.3, 32.5 and 65.1 mg/kg on PPD 4 to PPD 7. The decidual cell response was induced by bilateral scratch trauma on PPD 4. The uterine weight on PPD 9 served as an index of uterine decidualization. Uterine weight and serum progesterone levels on PPD 9 were significantly decreased after administration of TBTCl at doses of 16.3 mg/kg and above on PPD 0 to PPD 3 or PPD 4 to PPD 7. Administration of TBTCl at doses of 8.1 mg/kg and above on PPD 0 to 3 also significantly decreased serum progesterone levels on PPD 4. TBTCl had no effect on ovarian weight and number of corpora lutea. It can be concluded that TBTCl suppresses the uterine decidual cell response and decreases progesterone levels, and these effects are responsible for early embryonic loss due to TBTCl exposure.
Keywords: Tributyltin chloride Decidual cell response Pseudopregnancy Rat
No Title by Götz A. Westphal; Jürgen Bünger; Thomas G. Schulz; Michael M. Müller; Ernst Hallier (pp. 638-641).
N-Nitrosodiethylamine (NDEA) is carcinogenic in all investigated animal species at relatively low dosages. No threshold has been detected for these carcinogenic effects. The substance has been extensively investigated in various in vitro systems, revealing only weak mutagenicity at relatively high dosages. We reinvestigated NDEA in the Ames test with Salmonella typhimurium TA1535 to establish appropriate modifications of the standard Ames test protocol, to achieve a dose-dependent mutagenic response at a reasonably low dose range. Two main modifications were evaluated. Since the metabolism of dialkylnitrosamines is postulated to be mainly dependent on cytochrome P4502E1, a pyrazole-induced rat liver S9 was applied. The second modification involved a gastight preincubation, since metabolites of NDEA might evaporate from the incubation mixture. Cytochrome P4502E1 induction in Wistar rats was achieved by pyrazole treatment. For comparison, a rat liver S9-fraction produced by β-naphtoflavone/phenobarbital induction was used. N-Nitrosopyrrolidine served as positive control for pyrazole-induced S9-mix with TA1535. NDEA showed no mutagenic response under all test conditions in the presence of pyrazole-induced S9-mix. A strong mutagenic response, exceeding the base rate up to 15-fold at a dose range of 25–1000 µg/plate, was observed using β-naphtoflavone/phenobarbital-induced S9-mix, gastight preincubation and TA1535. In conclusion the Ames test with gastight preincubation can be useful for the testing of volatile compounds or substances leading to gaseous metabolites. The weak response of NDEA in the Ames test observed previously seems mainly to be due to the volatile character of its mutagenic metabolites. Our results do not support the hypothesis that cytochrome P4502E1 is a major toxifying enzyme for the formation of Ames-test-positive metabolites from NDEA.
Keywords: N-Nitrosodiethylamine Ames test Volatile metabolites Cytochrome P4502E1
No Title by E. Eder; D. Schuler (pp. 642-648).
2-Hexenal is formed by plants, and humans are regularly exposed to this mutagenic/genotoxic compound via vegetable foods. 2-Hexenal has not been tested for carcinogenicity, but it forms exocyclic 1,N 2-propanodeoxyguanosine adducts like other carcinogenic α,β-unsaturated carbonyl compounds. To quantify the respective DNA adducts as an approach to a theoretical cancer risk assessment, we used a newly developed 32P-postlabelling technique based on nuclease P1 enrichment, allowing a detection limit of 3 adducts per 108 nucleotides. Adduct levels were measured at different doses and the covalent binding index (CBI) was found to be dose-dependent. This can be explained by glutathione depletion at higher doses. The CBI at low doses was 0.06. A negligible cancer risk of 1–5 per 107 lives was estimated on the basis of TD50 values calculated from the correlation between CBI and TD50 of Lutz and on the daily intake of 2-hexenal via vegetable foods, fruit juices and black tea. A risk of 1.6–8.5 per 106 lives was estimated for the hypothetical case of glutathione depletion, e.g. due to consuming special medicaments. In every case, the benefit from eating fruit and vegetables is clearly higher than a possible low and unavoidable cancer risk. Utilization of 2-hexenal as a flavouring agent or as a fungicide, breeding fungus-resistant plants or technological gene construction of fungus resistance may lead to a high hypothetical cancer risk of 2–6 per 104 lives under certain circumstances which are avoidable and deserves special case-by-case consideration.
Keywords: Exocyclic DNA-hexenal adduct Biomarker Approach to cancer risk