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Archives of Toxicology (v.77, #11)

Effect of chronic accumulation of aluminum on renal function, cortical renal oxidative stress and cortical renal organic anion transport in rats by Stella T. Mahieu; Marisa Gionotti; Néstor Millen; María Mónica Elías (pp. 605-612).

The aim of the present work was to study the nephrotoxicity of aluminum lactate administered for 3 months (0.57 mg/100 g bodyweight aluminum, i.p., three times per week) to male Wistar rats. Renal function was studied after 6 weeks of treatment (urine was obtained from rats in metabolic cages) and at the end of the treatment using clearance techniques. Another group of rats was used as kidneys donors at the end of treatment. The renal cortex was separated and homogenized to determine glutathione (GSH) level, glutathione S-transferase (GST) activity and lipid peroxidation (LPO) level. Renal cortex slices were also used to study the p-aminohippuric acid (PAH) accumulation during steady-state conditions and the kinetics of uptake process. Clearance results, at the end of the treatment, indicated that renal functions in treated-rats were not different from those measured in control rats, although the renal concentration parameters differ when they were measured in treated rats after 24 h of food and water deprivation. Balances of water and sodium were also modified at both 1.5 and 3 months of treatment. The activity of alkaline phosphatase (AP) relative to inulin excreted in urine was significantly impaired: controls 2.2±0.6 IUI/mg, Al-treated 5.1±0.5 IU/mg, P<0.05. These data indicated that proximal tubular cells were loosing apical brush border membranes. Data obtained in cortex homogenates indicated that both GSH and GST activity were significantly decreased, and a significant increase of LPO was noted simultaneously in Al-treated rats. Renal accumulation of PAH, estimated as slice-to-medium ratio, decreased significantly in the Al-treated rats: control rats 3.06±0.02 (n=12), Al-treated rats 2.26±0.04 (n=12), P<0.0001. The maximal rate of uptake was also diminished in treated rats, while the apparent affinity remained unchanged. All these results indicate that aluminum accumulation in renal tissue affects cellular metabolism, promotes oxidative stress and induces alterations in renal tubular PAH transport, together with an impairment in sodium and water balance only detected under conditions of water deprivation, without other evident changes in glomerular filtration rate or other global functions measured by clearance techniques at least at this time of chronic toxicity.

Keywords: Aluminum; Nephrotoxicity; Renal clearance; Oxidative stress; PAH transport; Rats

Oral exposure to inorganic mercury alters T lymphocyte phenotypes and cytokine expression in BALB/c mice by Sang Hyun Kim; Victor J. Johnson; Raghubir P. Sharma (pp. 613-620).

Mercury is a well-recognized health hazard and an environmental contaminant. Mercury modulates immune responses ranging from immune suppression to autoimmunity but the mechanisms responsible for these effects are still unclear. Male BALB/c mice were exposed continuously to 0, 0.3, 1.5, 7.5, or 37.5 ppm mercury in drinking water for 14 days. Body weight was reduced at the highest dose of mercury whereas the relative kidney and spleen weights were significantly increased. The dose range of mercury used did not cause hepatotoxicity as indicated by circulating alanine aminotransferase and aspartate aminotransferase levels. Circulating blood leukocytes were elevated in mice treated with the highest dose of mercury. Mercury ranging from 1.5 to 37.5 ppm dose-dependently decreased CD3⁺ T lymphocytes in spleen; both CD4⁺ and CD8⁺ single-positive lymphocyte populations were decreased. Exposure to 7.5 and 37.5 ppm mercury decreased the CD8⁺ T lymphocyte population in the thymus, whereas double-positive CD4⁺/CD8⁺ and CD4⁺ thymocytes were not altered. Mercury altered the expression of inflammatory cytokines (tumor necrosis factor α, interferon γ, and interleukin-12), c-myc, and major histocompatibility complex II, in various organs. Results indicated that a decrease in T lymphocyte populations in immune organs and altered cytokine gene expression may contribute to the immunotoxic effects of inorganic mercury.

Keywords: Mercury; T lymphocytes; Drinking water; Inflammatory cytokines; Major histocompatibility complex II; Immunotoxicity

Activities of drug metabolizing enzymes in bovine colon epithelial cell cultures by Sascha Birkner; Susanne Weber; Angelika Dohle; Günter Schmahl; Hermann Maximilian Bolt; Wolfram Föllmann (pp. 621-629).

The metabolic competence of cultured bovine colon epithelial cells was evaluated by determining activities of phase I and II enzymes in colonocytes cultured for different intervals (maximum of 10 days) compared with activities measured in freshly isolated cells. Cytochrome P450 1A1-associated 7-ethoxyresorufin O-deethylase (EROD) activity was detectable in freshly isolated colonocytes and in colon cells maintained in culture for up to 5 days. In contrast to liver samples, cytochrome P450 3A4-associated 7-benzyloxyresorufin O-debenzylase (BROD) activity was not detectable in bovine colon cells. Prostaglandin H synthase-mediated production of prostaglandin E₂ was found in freshly isolated and also in cultured colonocytes. Both isoenzymes (COX 1 and COX 2) were detected in cultured cells. To examine phase II metabolic potency, activities of N-acetyltransferases 1 and 2, of phenol and amino sulfotransferases, of glutathione S-transferases alpha, mu, pi and theta and of UDP-glucuronyltransferase were measured. N-Acetyltransferase (NAT) activity (substrate p-aminobenzoic acid, PABA, a diagnostic substrate for the human NAT-1 enzyme) was stable under culture conditions and during the observed culture period comparable to that of freshly isolated cells. In contrast, sulfamethazine, a specific substrate for NAT-2, was not acetylated, neither in bovine colon cells nor in bovine liver samples. Whereas activity of amino sulfotransferase (substrate 2-naphthylamine) decreased continuously during the entire culture period, the activity of phenol sulfotransferase (substrate 1-naphthol) decreased only slowly. Activity of total glutathione S-transferases (alpha, mu, and pi) (substrate 1-chloro-2,4-dinitrobenzene) decreased after 2 days in culture, but was stable during the following culture period. Activity of glutathione S-transferase theta (substrate epoxy-3-nitrophenoxypropane) changed during the culture period. At the beginning and the end (after 10 days) of the culture period maximum activity was measured. Activity of UDP-glucuronyltransferase increased during the culture period reaching a maximum after 7 days. The results show that cultured bovine epithelial colon cells express several enzyme activities required for the biotransformation of xenobiotics.

Keywords: Primary cell culture; Xenobiotic metabolism; Phase I enzymes; Phase II enzymes; In vitro model

Sulfhydryl modification by 4,4′-dithiodipyridine induces calcium mobilization in human osteoblast-like cells by Soong-Yu Kuo; Chin-Man Ho; Wei-Chung Chen; Chung-Ren Jan (pp. 630-637).

The effect of oxidants on Ca²⁺ movement in osteoblasts is unclear. In this study, we show that 4,4′-dithiodipyridine (4,4′-DTDP), a reactive disulphide that mobilizes Ca²⁺ in muscle, induces an increase in cytoplasmic free-Ca²⁺ concentrations ([Ca²⁺]_i) in MG63 human osteosarcoma cells loaded with the Ca²⁺-sensitive dye fura-2. 4,4′-DTDP acted in a concentration-dependent manner with an EC₅₀ of 10 μM. Removing extracellular Ca²⁺ reduced the Ca²⁺ signal by 35%. In Ca²⁺-free medium, the 4,4′-DTDP-induced [Ca²⁺]_i increase was not changed by depleting store Ca²⁺ with 50 μM brefeldin A (a Golgi apparatus permeabilizer), by 2 μM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler), by 1 μM thapsigargin (an inhibitor of the endoplasmic reticulum Ca²⁺ pump) or by 5 μM ryanodine. Ca²⁺ signals induced by 4,4′-DTDP in Ca²⁺-containing medium were not affected by modulation of protein kinase C activity or suppression of phospholipase C activity. However, 4,4′-DTDP-induced Ca²⁺ release was inhibited by a thiol-selective reducing reagent, dithiothreitol (0.05-2.5 mM), in a concentration-dependent manner. Collectively, this study shows that 4,4′-DTDP induced [Ca²⁺]_i increases in human osteosarcoma cells via releasing store Ca²⁺ from multiple stores in a manner independent of protein kinase C or phospholipase C activity. The store Ca²⁺ release induced by 4,4′-DTDP appears to be associated with thiol oxidation. Furthermore, overnight incubation with 4,4′-DTDP inhibited cell activity in a concentration-dependent manner.

Keywords: Ca²⁺ stores; 4,4′-Dithiodipyridine; Free radicals; Osteosarcoma cells; Reactive oxygen species

Tachykinin substance P signaling involved in diesel exhaust-induced bronchopulmonary neurogenic inflammation in rats by Simon S. Wong; Nina N. Sun; Ingegerd Keith; Chol-Bum Kweon; David E. Foster; James J. Schauer; Mark L. Witten (pp. 638-650).

This study characterizes the molecular neurotoxicity of diesel exhaust (DE) on the tachykinin substance P (SP) signaling system in the lungs. A total of 96 female Fischer 344/NH rats (~175 g, ~4 weeks old) were randomly assigned to eight groups in a 2×4 factorial design: capsaicin versus non-capsaicin (vehicle) pretreatment, and filtered room air versus two exposure levels of DE with diesel engine room control. The rats were exposed nose-only to room air or low (35.3 µg/m³) and high concentrations (669.3 µg/m³) particulates directly from a Cummins N14 research engine at 75% throttle for 4 h/day, 5 days/week, for 3 weeks. The findings showed that exposure to DE dose-dependently induced bronchopulmonary neurogenic inflammation, both in capsaicin- and vehicle-pretreated rats, as measured by plasma extravasation, edema, and inflammatory cells. DE inhalation affected the SP signaling processes, including stored SP depletion and the gene/protein overexpression for neurokinin-1 receptor. DE also significantly reduced the activity of neutral endopeptidase, a main degradation enzyme for SP. Consequently, these changes may be regarded as critical factors that switched neurogenic pulmonary responses from their protective functions to a detrimental role that perpetuates lung inflammation. These changes may possibly be associated with the mass concentration of DE particles due to their physico-chemical characteristics. Moreover, capsaicin-pretreated rats had more sensitivity to these levels of DE exposure due to stimulation of bronchopulmonary C-fibers. However, the effects of capsaicin treatment were not consistent and apparent in this study. Taken together, our findings suggest that neurokininergic mechanisms may possibly be involved in DE-induced lung inflammation, but that bronchopulmonary C-fibers did not dominate DE-induced inflammatory abnormalities.

Keywords: Diesel exhaust; Particulate matter; Substance P; Neutral endopeptidase; Neurokinin-1 receptor; Neurogenic inflammation; Preprotachykinin gene

Dietary elevated sucrose modulation of diesel-induced genotoxicity in the colon and liver of Big Blue rats by Lotte Risom; Peter Møller; Max Hansen; Herman Autrup; Jette Bornholdt; Ulla Vogel; Håkan Wallin; Henrik E. Poulsen; Lars O. Dragsted; Steffen Loft (pp. 651-656).

Earlier studies have indicated that sucrose possesses either co-carcinogenic or tumor-promoter effects in colon carcinogenesis induced by genotoxic carcinogens. In this study we investigated the role of sucrose on diesel exhaust particle (DEP)-induced genotoxicity in the colonic mucosa and liver. Big Blue rats were fed with DEP (0.8 ppm in feed) and/or sucrose (3.45% or 6.85% w/w in feed) for 3 weeks. DEP increased both DNA strand breaks and DNA adducts in colon. Interestingly, sucrose also increased the level of bulky DNA adducts in colon. DEP and sucrose had no effect on DNA strand-breaks and DNA adducts in liver. DEP and sucrose treatment did not have any effect on mutation frequency in colon and liver. Oxidative DNA damage detected as 8-oxodG (8-oxo-7,8-dihydro-2′-deoxyguanosine) and endonuclease III or formamidopyrimidine DNA glycosylase sensitive sites was unaltered in colon and liver. The mRNA expression levels of the DNA repair enzymes N-methylpurine DNA glycosylase (MPG), 8-oxoguanine DNA glycosylase (OGG1) and ERCC1 (part of the nucleotide excision repair complex) measured by reverse transcription-polymerase chain reaction were increased in liver by DEP feeding. In colon, expression was unaffected by DEP or sucrose feeding. Among biomarkers of oxidative stress, including vitamin C, malondialdehyde and protein oxidations (γ-glutamyl semialdehyde and 2-amino adipic semialdehyde) in plasma and liver, only malondialdehyde was increased in plasma by sucrose/DEP feeding. In conclusion, sucrose feeding did not increase DEP-induced DNA damage in colon or liver.

Keywords: Sucrose; Diesel exhaust particles; Big Blue rats; Colon; Liver

Epigenetic properties of the diarrhetic marine toxin okadaic acid: inhibition of the gap junctional intercellular communication in a human intestine epithelial cell line by Adama Traoré; Isabelle Baudrimont; Sebastien Dano; Ambaliou Sanni; Yvan Larondelle; Yves Jacques Schneider; Edmond Ekue Creppy (pp. 657-662).

Okadaic acid (OA) is produced by several types of dinoflagellates (marine plankton) and has been implicated as the causative agent of diarrhetic shellfish syndrome. Previous studies have shown that okadaic acid is a tumor promoter and a specific potent inhibitor of protein phosphatases and protein synthesis. These effects in turn affect intracellular processes such as metabolism, contractility, gene transcription, and the maintenance of cytoskeletal structure. Gap junctional intercellular communication (GJIC) is a means of maintaining cellular homeostasis in organs, the disruption of which favors tumor cell growth. The GJIC involves the transfer of small water-soluble molecules through intercellular channels (gap junctions), composed of proteins called connexins. OA disrupts cellular homeostasis in Caco-2 cells through several mechanisms including protein synthesis inhibition, apoptosis, and clastogenic effects. The aim of this study was then to evaluate the expression of the connexin 43 (Cx 43) mRNA in relation with the cytotoxicity induced by OA (3.75–60 ng/ml) in a human colonic epithelial cell line in culture (Caco-2 cells). OA produced a dose-dependent inhibition of GJIC in Caco-2 cells, along with a parallel decrease in the expression of Cx 43 as shown by immunohistochemistry using anti-Cx 43 antibody. Since Cx 43 is implicated in the suppression of tumors and OA is a tumor promoter, the inhibition of GJIC may play an important role in its carcinogenesis. These data are discussed in relation to the toxicity of OA, total RNA synthesis, and possible specificity of Cx 43 inhibition in the GJIC.

Keywords: Okadaic acid; Intestinal Caco-2 cells; Connexin 43 mRNA; Gap junction inhibition

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Archives of Toxicology (v.77, #11)

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Archives of Toxicology (v.77, #11)