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Archives of Toxicology (v.83, #5)

Mechanisms of telomere maintenance and attrition: linking cancer and ageing by Jan G. Hengstler; Rosemarie Marchan; Hermann M. Bolt (pp. 405-406).

Telomere maintenance: all’s well that ends well by Li Phing Liew; Chris J. Norbury (pp. 407-416).

The nucleoprotein structures termed telomeres serve to prevent the mis-identification of eukaryotic chromosome ends as sites of DNA damage, but are also among the genomic regions that pose the most problems during DNA replication. Here, we summarize some of the apparent difficulties encountered by the DNA replication machinery when it approaches the chromosome ends. Eukaryotic cells have evolved diverse mechanisms to overcome these problems, underlining the importance of telomere maintenance for a number of aspects of chromosome function. Of particular interest in this respect are the ways in which telomere-binding proteins and components of the DNA damage response machinery may facilitate replication fork progression through telomeres.

Keywords: Telomeres; DNA replication; Telomere-binding proteins; DNA damage; Recombination

Oxidative stress and apoptotic changes in primary cultures of rat proximal tubular cells exposed to lead by Lin Wang; Heng Wang; Maozhi Hu; Jin Cao; Dawei Chen; Zongping Liu (pp. 417-427).

Lead is a known nephrotoxic element. In this study, primary cultures of rat proximal tubular (rPT) cells were treated with different concentrations of lead acetate (0.25, 0.5 and 1 μM) to investigate its cytotoxic mechanism. A progressive loss in cell viability together with a significant increase in the number of apoptotic and necrotic cells and lactate dehydrogenase release were seen in the experiment. Simultaneously, elevation of reactive oxygen species levels and intracellular [Ca²⁺]i, depletion of mitochondrial membrane potential and intracellular glutathione were revealed during the lead exposure. In addition, apoptotic morphological changes induced by lead exposure in rPT cells were demonstrated by Hoechst 33258 staining. The apoptosis was markedly prevented by N-acetyl-l-cysteine, while the necrosis was not affected. Moreover, catalase and superoxide dismutase activities in the living cells rose significantly. In conclusion, exposure of rPT cells to low-concentration lead led to cell death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death induced by oxidative stress was the chief mechanism. Meanwhile, a group of cells survived lead action, mediated by their ability to activate antioxidant defense systems.

Keywords: Lead; Oxidative stress; Apoptosis; Proximal tubular cells; Primary cell culture

Extrapulmonary translocation of intratracheally instilled fine and ultrafine particles via direct and alveolar macrophage-associated routes by Akiko Furuyama; Sanae Kanno; Takahiro Kobayashi; Seishiro Hirano (pp. 429-437).

Translocation of inhaled ultrafine particles from the lungs into the blood may impair cardiovascular function. We administered ultrafine (20-nm) and fine (200-nm) gold colloid or fluorescein-labeled polystyrene particles to mice intratracheally and examined their localization in the lung and extrapulmonary organs. Fifteen minutes after instillation, dispersed and agglomerated 20-nm gold colloid particles were observed on the surface of endothelial cells, on the alveolar surface, in endocytotic vesicles of alveolar epithelial cells, and in the basement membrane of the lung. A small but noteworthy amount of gold was detected in the liver, kidney, spleen, and heart by inductively coupled plasma-mass spectrometry. After administration of 20- or 200-nm fluorescent particles, free particles were detected infrequently in blood vessels, on the endocardial surface, and in the kidney and liver only in the mice that received 20-nm particles, whereas phagocytes containing 20- or 200-nm particles were found in the extrapulmonary tissues. Fluorescent particle-laden alveolar macrophages administered intratracheally translocated from alveoli to extrapulmonary organs via the blood circulation. Thus, small amounts of ultrafine particles are transported across the alveolar wall into the blood circulation via endocytotic pathways, but particle-laden alveolar macrophages translocate both ultrafine and fine particles from the lungs to the extrapulmonary organs.

Keywords: Ultrafine particle; Fine particle; Intratracheal administration; Endocytosis; Alveolar wall; Alveolar macrophage

Evidence of early involvement of matrix metalloproteinase-2 in lead-induced hypertension by Elen Rizzi; Michele M. Castro; Karla Fernandes; Fernando Barbosa Jr; Gabriel Maisonnave Arisi; Norberto Garcia-Cairasco; Lusiane M. Bendhack; Jose E. Tanus-Santos; Raquel F. Gerlach (pp. 439-449).

Lead exposure increases blood pressure (BP) by unknown mechanisms. Many recent studies have shown the involvement of matrix metalloproteinases (MMPs) in hypertension, particularly MMP-2. In this work, we have examined whether MMP-2 levels increase with lead-induced increase in BP. We have also investigated whether doxycycline (an MMP inhibitor) affects these alterations. To this end, rats were exposed to lead (90 ppm) and treated with doxycycline or vehicle for 8 weeks. Similar aortic and whole blood lead levels were found in lead-exposed rats treated with either doxycycline or vehicle. Lead-induced increases in BP and aortic MMP-2 levels (activity, protein, and mRNA) were blunted by doxycycline. Doxycycline also prevented lead-induced increases in the MMP-2/TIMP-2 mRNA ratio. No significant changes in vascular reactivity or morphometric parameters were found. In conclusion, lead exposure increases BP and vascular MMP-2, which is blunted by doxycycline. This observation suggests that MMP-2 may play a role in lead-induced increases in BP.

Keywords: Lead; Toxicity; MMP-2; Hypertension; Vascular dysfunction; Doxycycline

Effects of sodium fluoride treatment in vitro on cell proliferation, apoptosis and caspase-3 and caspase-9 mRNA expression by neonatal rat osteoblasts by Xiaoyan Yan; Cuiping Feng; Qinglin Chen; Wentao Li; Hongwei Wang; Lihua Lv; George W. Smith; Jundong Wang (pp. 451-458).

Long-term excessive fluoride intake is linked to skeletal disease. Skeletal health is influenced by the balance between bone formation and resorption of which osteoblast function is critical. The objectives of this study were to determine the effect of fluoride treatment on osteoblast proliferation, apoptosis and caspase-3 and caspase-9 mRNA expression in vitro. Neonatal rat osteoblasts were cultured in the presence of varying concentrations (0.5–30 mg/l) of sodium fluoride and effects of treatments were determined. Treatment with sodium fluoride inhibited osteoblast proliferation in a dose-dependent fashion and effects were maximal after 120 h incubation. A significant increase in osteoblast apoptosis was observed (after 24 and 72-h treatment) in response to the lowest dose of sodium fluoride (0.5 mg/l) and osteoblast apoptosis was further increased in response to higher doses. Increased-osteoblast caspase-3 and caspase-9 mRNA was also observed in response to sodium fluoride treatment (5 mg/l) for 72 h. Results indicate that negative effects of excess fluoride on skeletal health may be mediated in part by inhibition of osteoblast survival.

Keywords: Fluoride; Osteoblasts; Apoptosis; Caspase-3; Caspase-9

Uterotrophic assay, Hershberger assay, and subacute oral toxicity study of 3-amino-1,2,4-triazole based on the Organization for Economic Co-operation and Development draft protocols by Takaaki Umano; Keiji Shiraishi; Yasushi Minobe; Kanji Yamasaki (pp. 459-467).

We performed a uterotrophic assay, the Hershberger assay, and the 28-day repeated-dose toxicity study (enhanced OECD test guideline no. 407) of 3-amino-1,2,4-triazole based on the OECD draft protocols. In the uterotrophic assay, female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and no changes were observed. In the Hershberger assay, the test chemical was orally administered at doses of 0, 40, 200, and 1,000 mg/kg/day to castrated male Wistar rats for 10 consecutive days beginning on postnatal day 56, and no androgen agonistic and antagonistic changes were observed. Alternatively, when the test chemical was orally administered at doses 0, 5, 25, and 125 mg/kg/day for at least 28 days in the subacute oral toxicity study, thyroid follicular epithelial cell hypertrophy with increased thyroid weights was detected in the male and female rats in 25 and/or 125 mg/kg groups, and hypertrophy of the anterior pituitary cells with increased pituitary weights in male and female rats was also observed in the 125 mg/kg group. Furthermore, serum T3 and T4 values decreased and serum TSH values increased in male and female rats in the 125 mg/kg group. Therefore, 3-amino-1,2,4-triazole was concluded to have anti-thyroid acting as endocrine-mediated effects, but no estrogenic or androgenic effects. In addition, decreased body weight, and abnormal biochemical parameters attributed to thyroid, liver or kidney dysfunction were observed in male and female rats in the 25 and/or 125 mg/kg groups.

Keywords: 3-Amino-1,2,4-triazole; Endocrine effects; Updated test guideline 407; Hershberger assay; Rat; Uterotrophic assay

Effects of macrolides on proinflammatory epitops on endothelial cells in vitro by Michael Millrose; Matthias Kruse; Burkhard Flick; Ralf Stahlmann (pp. 469-476).

An inflammatory reaction at the site of infusion is a common clinical problem that is observed after the intravenous application of antibiotics and other drugs. The pathomechanism of this infusion-related phlebitis is not fully understood. We analyzed the effects of the three macrolide antibiotics erythromycin, clarithromycin and azithromycin on human endothelial cells in vitro. As a positive control quinupristin/dalfopristin was studied. The cytotoxicity of all substances was analyzed by a modified MTT cytotoxicity assay with 3T3-fibroblasts and EA.hy 926 endothelial cells. Cells were incubated for 10 days with the antibiotics. After adding MTT the optical density was measured which correlates with cell death. Clarithromycin exhibited the strongest cytotoxic effect on EA.hy 926 cells (EC₅₀ 30 mg/L), followed by azithromycin (EC₅₀ 40 mg/L), a cytotoxic effect of erythromycin could only be observed at much higher concentrations (EC₅₀ 310 mg/L). The reaction of the endothelial cells was further analyzed in detail by means of flow cytometry. For these experiments the endothelial cell line EA.hy 926 as well as primary cells (HUVEC) were used. The antigens were stained with fluoresceinisothiocyanat- or phycoerythrin-conjugated monoclonal antibodies for the following surface antigens: CD34, E-selectin (CD62E), ICAM-1 (CD54) and VCAM-1 (CD106). Cells were incubated with the antibiotics at concentrations ranging from 100 to 800 mg/L (clarithromycin and azithromycin) and from 200 to 1,200 mg/L (erythromycin). These concentrations occur under therapeutic conditions at the site of infusion. Cells were incubated for 2 h and analysis was carried out after an additional culture period of 22 h without test compounds. A significantly enhanced expression of all four antigens was observed which was most pronounced at 800 mg/L (erythromycin), 600 mg/L (azithromycin) and 400 mg/L (clarithromycin). A concentration of 800 mg/L erythromycin medium caused an increase of the expression of CD34 (+6%), E-selectin (+5%), ICAM-1 (+14%) and VCAM-1 (+5%). At lower concentrations (600 mg/L) azithromycin provokes a stronger upregulation of the proinflammatory antigens: CD34 (+17%), E-selectin (+18%), ICAM-1 (+27%) and VCAM-1 (+17%). At a concentration of 400 mg/L medium clarithromycin induced a similar effect as erythromycin at twice this concentration: CD34 (+5%), E-selectin (+7%), ICAM-1 (+23%) and VCAM-1 (+4%). Reactions of the HUVECs were less pronounced than those of the EA.hy 926 cells. Cell surface markers involved in interactions between endothelial cells and leukocytes proved to be useful markers to study differences in the proinflammatory potential of the three macrolides. By analysing the upregulation of these antigens on EA.hy 926 cells in vitro the risk of phlebitis could be predictable for other drugs as well.

Keywords: Quinupristin; Dalfopristin; Erythromycin; Clarithromycin; Azithromycin; Infusion phlebitis; Cell adhesion molecules; CD34; Endothelial cells

Effect of α-tocopherol on carbon tetrachloride intoxication in the rat liver by Chinatsu Iida; Kozue Fujii; Eriko Koga; Yukiko Washino; Yuko Kitamura; Ikuyo Ichi; Kouichi Abe; Tatsuya Matsura; Shosuke Kojo (pp. 477-483).

Carbon tetrachloride (1 ml/kg body weight as a 1:1 mixture of CCl₄ and mineral oil) was orally administered to rats. After 12 h, the activity of plasma ALT (alanine aminotransferase) was significantly higher than that of the control group, and plasma ALT and AST (aspartate aminotransferase) activities significantly increased 24 h after CCl₄ administration. These results indicated that the necrotic process had initiated at about 12 h and developed thereafter. After 6–24 h of CCl₄ administration, the hepatic level of vitamin C, the most sensitive indicator of oxidative stress, decreased significantly, indicating that oxidative stress was significantly enhanced 6 h after CCl₄ intoxication and thereafter. Oral administration of vitamin E (1 ml/kg body weight as a 1:1 mixture of α-tocopherol and mineral oil) 12 h before CCl₄ administration caused a significant elevation of liver vitamin E level and ameliorated liver necrosis 24 h after CCl₄ intoxication based on plasma AST and ALT. Vitamin E also significantly restored the hepatic vitamin C concentration 12 and 24 h after CCl₄ intoxication, demonstrating that vitamin E functioned as an antioxidant. The liver vitamin E concentration was not changed by vitamin E supplementation to rats that did not receive CCl₄. This result indicated that vitamin E accumulated in the damaged liver. The activation of JNK, ERK1/2 and p38 MAPK took place 1.5 h after CCl₄ administration. Co-administration of α-tocopherol with CCl₄ did not affect these early changes in MAPKs.

Keywords: Antioxidant; Ascorbic acid; Carbon tetrachloride; CCl₄ ; MAPK; Necrosis; Oxidative stress; α-Tocopherol

Exposure to diphenyl ditelluride, via maternal milk, causes oxidative stress in cerebral cortex, hippocampus and striatum of young rats by Eluza Curte Stangherlin; Ana Paula Ardais; Joao Batista Teixeira Rocha; Cristina Wayne Nogueira (pp. 485-491).

The present study evaluated the effect of diphenyl ditelluride [(PhTe)₂] exposure to mothers on the cerebral oxidative status of their offspring. The dams received (PhTe)₂ or canola oil via subcutaneous injection once daily during the first 14 days of lactational period. At post natal day 28, biochemical parameters of oxidative stress were evaluated in cerebral structures—cortex, hippocampus and striatum—of young rats. Exposure to (PhTe)₂ increased lipid peroxidation levels and inhibited δ-ALA-D, catalase and SOD activities in hippocampus and striatum of young rats. (PhTe)₂ induced changes in the levels of non-enzymatic antioxidant defenses in cortex and striatum of young rats. The exposure to (PhTe)₂, via maternal milk, caused oxidative stress in cerebral structures of young rats. Thus, the possible role of disrupted prooxidant/antioxidant balance in (PhTe)₂ toxicity was demonstrated. These results highlighted a possible molecular mechanism involved in toxicity caused by (PhTe)₂.

Keywords: Diphenyl ditelluride; Tellurium; Young rat; Oxidative stress; Brain; Antioxidant

Hypothermic storage of isolated human hepatocytes: a comparison between University of Wisconsin solution and a hypothermosol platform by Alina Ostrowska; Kenan Gu; Donald C. Bode; Robert G. Van Buskirk (pp. 493-502).

Until now little is known about the functional integrity of human hepatocytes after hypothermic storage. In order to address this limitation, we evaluated several commercially available hypothermic preservation media for their abilities to protect freshly isolated hepatocytes during prolonged cold storage. Human hepatocytes were isolated from non-transplantable/rejected donor livers and resuspended in ice-cold University of Wisconsin solution (UW), HypoThermosol-Base (HTS-Base), or HypoThermosol-FRS (HTS-FRS) with or without the addition of fetal bovine serum. Cells were stored at 4°C for 24–72 h, and evaluated for hepatocyte viability (trypan blue exclusion, or labeling with fluorochromes), cell attachment, and function. The energy status of hepatocytes was evaluated by measurement of intracellular adenosine 5′-triphosphate. To determine whether the test cells expressed metabolic functions of freshly isolated cells, the activities of major phase I (cytochromes P450, FMO) and phase II (UGT, ST) drug-metabolizing enzymes were examined. Although hepatocytes are shown to be satisfactory after 24 h storage in all of the tested solutions, the cell viability, energy status, and xenobiotic metabolism following cold preservation in HTS-FRS was consistently and, in some cases, markedly higher when compared with other systems. The same metabolites for each of the tested substrates were detected in all groups of cells. Moreover, the use of HTS-FRS eliminates the need for serum in preservation solutions. HTS-FRS represents an improved solution compared to HTS-Base and UW for extending the shipping/storage time of human hepatocytes.

Keywords: Human hepatocytes; Hypothermia; HypoThermosol; UW; Viability

Involvement of oxidative stress in hepatocellular tumor-promoting activity of oxfendazole in rats by Yasuaki Dewa; Jihei Nishimura; Masako Muguruma; Meilan Jin; Masaomi Kawai; Yukie Saegusa; Toshiya Okamura; Takashi Umemura; Kunitoshi Mitsumori (pp. 503-511).

The tumor-promoting effects of oxfendazole (OX), a benzimidazole anthelmintic, were investigated using a medium-term rat hepatocarcinogenesis model. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) and were given a powdered diet containing 0 or 500 ppm OX for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after OX treatment. The numbers and areas of glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in the livers of rats treated with OX, with concomitantly increased cell proliferation, compared with those in the livers of the DEN alone group. Quantitative real-time RT-PCR analysis revealed that OX induced not only mRNA expression of phase I enzymes Cyp1a1, Cyp1a2, but also Nrf2-regulated phase II enzymes such as Gpx2, Nqo1, Yc2, Akr7a3 and Gstm1, presumably due to an adaptive response against OX-induced oxidative stress. Reactive oxygen species production increased in microsomes isolated from the livers of OX-treated rats. Furthermore, OX enhanced oxidative DNA damage (as assessed by 8-hydroxydeoxyguanosine; 8-OHdG) and lipid peroxidation (as assessed by thiobarbituric acid-reactive substances; TBARS). These results suggest that administration of OX at a high dose and for a long term enhances oxidative stress responses, which may contribute to its tumor-promoting potential in rats.

Keywords: Oxfendazole; Reactive oxygen species; Oxidative stress; Tumor-promotion; Rat

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Archives of Toxicology (v.83, #5)