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Archives of Toxicology (v.78, #10)

Animal testing and alternative approaches for the human health risk assessment under the proposed new European chemicals regulation by Thomas Höfer; Ingrid Gerner; Ursula Gundert-Remy; Manfred Liebsch; Agnes Schulte; Horst Spielmann; Richard Vogel; Klaus Wettig (pp. 549-564).

During the past 20 years the EU legislation for the notification of chemicals has focussed on new chemicals and at the same time failed to cover the evaluation of existing chemicals in Europe. Therefore, in a new EU chemicals policy (REACH, Registration, Evaluation and Authorisation of Chemicals) the European Commission proposes to evaluate 30,000 chemicals within a period of 15 years. We are providing estimates of the testing requirements based on our personal experiences during the past 20 years. A realistic scenario based on an in-depth discussion of potential toxicological developments and an optimised “tailor-made” testing strategy shows that to meet the goals of the REACH policy, animal numbers may be significantly reduced below 10 million if industry would use in-house data from toxicity testing, which are confidential, if non-animal tests would be used, and if information from quantitative structure activity relationships (QSARs) would be applied in substance-tailored testing schemes. The procedures for evaluating the reproductive toxicity of chemicals have the strongest impact on the total number of animals bred for testing under REACH. We are assuming both an active collaboration with our colleagues in industry and substantial funding of the development and validation of advanced non-animal methods by the EU Commission, specifically in reproductive and developmental toxicity.

Keywords: Animal experiments; EU chemicals policy; Hazard assessment; Regulatory toxicology; Risk assessment

Mitochondrial viability and apoptosis induced by aluminum, mercuric mercury and methylmercury in cell lines of neural origin by Tarja Toimela; Hanna Tähti (pp. 565-574).

Mercury and aluminum are considered to be neurotoxic metals, and they are often connected with the onset of neurodegenerative diseases. In this study, mercuric mercury, methylmercury and aluminum were studied in three different cell lines of neural origin. To evaluate the effects, mitochondrial cytotoxicity and apoptosis induced by the metals were measured after various incubation times. SH-SY5Y neuroblastoma, U 373MG glioblastoma, and RPE D407 retinal pigment epithelial cells were subcultured to appropriate cell culture plates and 0.01–1,000 µM concentrations of methylmercury, mercuric and aluminum chloride were added into the growth medium. In the assay measuring the mitochondrial dehydrogenase activity, WST-1, the cultures were exposed for 15 min, 24 or 48 h before measurement. Cells were allowed to recover from the exposure in part of the study. Apoptosis induced by the metals was measured after 6-, 24- and 48-h exposure times with the determination of activated caspase 3 enzyme. Mitochondrial assays showed a clear dose-response and exposure time-response to the metals. The most toxic was methylmercury (EC50 ~0.8 µM, 48 h), and the most sensitive cell line was the neuroblastoma cell line SH-SY5Y. Furthermore, there was marked mitochondrial activation, especially in connection with aluminum and methylmercury at low concentrations. This activation may be important during the initiation of cellular processes. All the metals tested induced apoptosis, but with a different time-course and cell-line specificity. In microscopic photographs, glioblastoma cells formed fibrillary tangles, and neuroblastoma cells settled along the fibrilles in cocultures of glial and neuronal cell lines during aluminum exposure. The study emphasized the toxicity of methylmercury to neural cells and showed that aluminum alters various cellular activities.

Keywords: Aluminum; Apoptosis; Mercury; Mitochondrial activity; Neural cell lines

Genotoxicity of inorganic mercury salts based on disturbed microtubule function by Daniela Bonacker; Thomas Stoiber; Minsheng Wang; Konrad J. Böhm; Irina Prots; Eberhard Unger; Ricarda Thier; Hermann M. Bolt; Gisela H. Degen (pp. 575-583).

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg²⁺ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg²⁺ inhibits microtubule assembly at concentrations above 1 µM, and inhibition is complete at about 10 µM. In this range, the tubulin assembly is fully (up to 6 µM) or partially (~6–10 µM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect-concentration for inhibition of microtubule assembly in vitro was 1 µM Hg²⁺, the IC₅₀ 5.8 μM. Mercury(II) salts at the IC₅₀ concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a “gliding assay” mimicking intracellular movement and transport processes in vitro, HgCl₂ affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 µM and a complete inhibition is reached at 1 μM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 µM HgCl₂. Between 15 and 20 µM HgCl₂there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 µM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg²⁺, with some preponderance of aneugenicity. A morphological effect of high Hg²⁺ concentrations (100 µM HgCl₂) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg²⁺ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg²⁺ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

Keywords: Chromosomal damage; Cytoskeleton; Genotoxicity; Kinesin; Mechanisms; Mercury; Micronuclei; Microtubules; Tubulin

Transplacental passage of Pt after treatment with the new triamine complex cis-diaminechloro-[2-(diethylamino) ethyl 4-amino-benzoate, N⁴]-chloride platinum (II) monohydrochloride monohydrate by Emanuela Ognio; Barbara Chiavarina; Gabriele Caviglioli; Maddalena Lapide; Maurizio Viale (pp. 584-588).

Cis-diaminechloro-[2-(diethylamino) ethyl 4-amino-benzoate, N⁴]-chloride platinum (II) monohydrochloride monohydrate (DPR) is a monofunctional Pt triamine complex synthesized starting from cisplatin and procaine hydrochloride, characterized by a good antitumor activity coupled with low toxic effects and able to impair prenatal development of mice but at doses outside or just in the upper range of therapeutic doses. In the present paper the transplacental passage of DPR-derived Pt was investigated in CD1 mice on days 9, 13, 16 and 18 of pregnancy, 24 h after ip administration of 21 mg/kg DPR. For comparison, groups of mice were treated with an equivalent Pt-containing dose of cisplatin (10.7 mg/kg). Similarly to cisplatin, small amounts of Pt were detected in fetuses on day 9. From day 13 of gestation the concentration of DPR- and cisplatin-derived Pt increased up to the highest fetal concentrations detected on day 16. On day 18 the concentration of Pt decreased. Most importantly, on days 13–18 of pregnancy cisplatin-derived Pt was always significantly higher than that assayed after DPR administration. In addition, on day 13 of pregnancy Pt exposure of fetuses was significantly higher when dams were treated with cisplatin (AUC_0.5–24= 3.40 vs. 4.95 μg·h/g). Finally, it is worth noting that serum decay of Pt after DPR or cisplatin administration in adult female mice was similar with AUC_0.13–2hs of 7.5 and 6.6 μg·h/ml, respectively. When we determined the concentration of Pt into the main organs of fetuses from dams treated with either DPR or cisplatin on day 18 of gestation, we observed a different organ distribution. In fact, while the concentration of DPR-derived Pt was greater in the heart (1.08±0.30 vs. 0.78±0.35 μg/g, p <0.10), an opposite situation was found in the kidney (0.51±0.20 vs. 0.69±0.22 μg/g, p <0.05). In conclusion, our data show that DPR may pass through the placenta with an efficiency significantly lower than that of cisplatin. This finding may represent one of the possible causes of the lower embryotoxic/teratogenic effect of DPR as compared to cisplatin.

Keywords: Atomic absorption spectroscopy; Cisplatin; DPR; Embryotoxicity; Transplacental passage

Embryonic turkey liver: activities of biotransformation enzymes and activation of DNA-reactive carcinogens by Carmen E. Perrone; Hans-Juergen Ahr; Jian Dong Duan; Alan M. Jeffrey; Ulrich Schmidt; Gary M. Williams; Harald H. Enzmann (pp. 589-598).

Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovo turkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using ³²P-postlabeling for DNA adducts. In ovo exposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4’-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had ³²P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity.

Keywords: Avian turkey liver; Biotransformation enzymes; DNA adducts

The effects of enrofloxacin on decorin and glycosaminoglycans in avian tendon cell cultures by Jung Hae Yoon; Randolph L. Brooks Jr.; Jian Zeng Zhao; David Isaacs; Jaroslava Halper (pp. 599-608).

Tendonitis and tendon rupture have been reported to occur during or following therapy with fluoroquinolone antibiotics. Though the pathogenesis is unknown, several studies suggest that fluoroquinolone antibiotics alter proteoglycan content in soft tissues, including tendons, and thereby alter collagen fibrillogenesis. To better understand the mechanism of action of fluoroquinolones, we studied the effects of enrofloxacin, a widely used fluoroquinolone in veterinary medicine, on avian tendon cell cultures established from gastrocnemius tendons from 18-day-old chicken embryos. We found that cell proliferation was progressively inhibited with increasing concentrations of enrofloxacin. This was accompanied by changes in morphology, extracellular matrix content and collagen fibril formation as detected by electron microscopy. We also observed a 35% decrease in the content of total monosaccharides in enrofloxacin-treated cells. The ratio of individual monosaccharides was also altered in enrofloxacin-treated cells. Enrofloxacin also induced the synthesis of small amounts of keratan sulfate in tendon cells. Moreover we observed enrofloxacin-induced changes in glycosylation of decorin, the most abundant tendon proteoglycan, resulting in the emergence of multiple lower molecular bands that were identifiable as decorin after chondroitinase ABC and N-glycanase treatment of extracts from enrofloxacin-treated cells. Medium conditioned by enrofloxacin-treated cells contained less decorin than did medium conditioned by control cells. We hypothesize that enrofloxacin induces either changes in the number of N-linked oligosaccharides attached to the core protein of decorin or changes in decorin degradation process. In conclusion, our data suggest that enrofloxacin affects cell proliferation and extracellular matrix through changes in glycosylation.

Keywords: Cell proliferation; Decorin; Enrofloxacin; Monosaccharides; Morphological changes; Tendon cells

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Archives of Toxicology (v.78, #10)