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Archives of Toxicology (v.73, #6)

Synergistic effects of some metals contaminating mussels on the cytotoxicity of the marine toxin okadaic acid by Adama Traoré; Michelle Bonini; Sébastien D. Dano; Edmond E. Creppy (pp. 289-295).

Okadaic acid (OA), a marine toxin is cytotoxic and promotes tumours in mouse skin. It is a specific and potent inhibitor of protein synthesis and also inhibits phosphatases A1 and A2 in vitro. In the present study, we investigated the influence of metals found at acceptable levels in mussels as environmental pollutants on the cytotoxicity of OA in Vero cells. Among the metals found in mussels (Mytilus edulis), the most represented, in terms of molar quantities per gram of dried weight are aluminium (230 nmol/g), copper (58 nmol/g), lead (16 nmol/g), mercury (14 nmol/g) and cadmium (7.4 nmol/g). A solution containing these five metals Al³⁺, Cu²⁺, Pb²⁺, Hg²⁺ and Cd²⁺ combined at the concentrations detected in mussels, stimulated protein synthesis (+25%, P < 0.01), whereas different dilutions of this solution in the presence of okadaic acid (15 ng/ml, i.e. 18.7 × 10⁻⁹ M) increased the percentage of protein synthesis inhibition from 35 to 79%. The metals also increased the lactate dehydrogenase (LDH) release into the medium and the lipid peroxidation induced by this algal toxin. In addition, these metals reduced the cell viability for an incubation period of 24 h especially at the two higher concentrations. These results indicate that metals (Al³⁺, Cu²⁺, Pb²⁺, Hg²⁺, Cd²⁺) in concentration ranges largely below the acceptable levels, synergistically increase the cytotoxicity of low concentrations of OA in cultured cells.

Keywords: Key words Okadaic acid; Metals in mussels; Cytotoxicity; Synergistic effects; Vero cells

Organophosphate neuropathy due to methamidophos: biochemical and neurophysiological markers by Rob McConnell; Edgar Delgado-Téllez; Ricardo Cuadra; Edmundo Tórres; Matthew Keifer; Juan Almendárez; Jamilet Miranda; Hassan A. N. El-Fawal; Mary Wolff; David Simpson; Ingvar Lundberg (pp. 296-300).

Neuropathy target esterase (NTE), the putative target enzyme for organophosphate induced delayed polyneuropathy (OPIDP), can be measured in lymphocytes but has rarely been assessed in acute human poisoning. Serum autoantibodies to nervous system proteins develop in hens poisoned with neuropathic insecticides and also have not been studied after human poisoning. Serial lymphocyte NTE (LNTE) was measured in a 16-year-old boy after acute poisoning with methamidophos for evaluation as a predictor of subsequent neuropathy. The profiles of serum autoantibodies to neurofilament triplet proteins, myelin basic protein, and glial fibrillary acidic protein were measured in order to characterize changes occurring as a result of OPIDP. Clinical neuropathy characterized by steppage gate and profound lower extremity weakness, decreased grip and pinch strength, and decreased ulnar and absent tibial compound muscle action potentials developed 2 weeks following poisoning. Sensory examination and nerve conduction studies were normal. On day 3 following poisoning LNTE was depressed (77% compared with subsequent baseline enzyme activity). Marked increases in serum immunoglobulin G (IgG) autoantibodies to glial fibrillary acidic protein and to neurofilament 200 were observed after the development of OPIDP. We conclude that inhibition of lymphocyte NTE is predictive of subsequent OPIDP. Serum autoantibody titers to nervous system proteins may be useful markers of neuropathy.

Keywords: Key words Methamidophos; Peripheral nervous system diseases; Neurofilament proteins; Glial fibrillary acidic protein; Lymphocytes

Modifications in the metabolic pathways of benzene in streptozotocin-induced diabetic rat by Chiara Costa; Calcedonio Pupo; Giuseppina Viscomi; Stefania Catania; Monica Salemi; Claudia Imperatore (pp. 301-306).

Benzene is a ubiquitous environmental pollutant primarily metabolized by a cytochrome P-450 (CYP-450) isoenzyme, CYP-450 IIE1. A consistent induction of CYP450 IIE1 has been observed in both rat and human affected by diabetes mellitus. The aim of this study was to evaluate whether streptozotocin (STZ)-induced diabetes determines modifications in the metabolic pathways of benzene in rat. Benzene (100 mg/kg per day, dissolved in corn oil) was administered i.p. once a day for 5 days. Urine samples were collected every day in STZ-treated and normoglycaemic animals, treated and untreated with benzene (n = 10). Urinary levels of trans,trans-muconic acid and of phenol, catechol and hydroquinone (free and conjugated with sulphuryl and glucuronic group) were measured by high-performance liquid chromatography (HPLC). In normoglycaemic rats during the 5 days of treatment with benzene we observed a progressive and significant decrement in the urinary excretion of phenol, phenyl sulphate and glucuronide, catechol, catechol glucuronide, hydroquinone, hydroquinone glucuronide and t,t-muconic acid (P < 0.05). In the diabetic animals, conversely, the same metabolites showed progressively increasing urinary levels (P < 0.05). Catechol sulphate and hydroquinone sulphate levels were below the instrument's detection limit. In the comparison between diabetic and normoglycaemic benzene treated rats, the inter-group difference was significant (P < 0.05) from day 3 of treatment for t,t-muconic acid, and from day 1 for free and conjugated phenol, free and glucuronide catechol and free hydroquinone. In the normoglycaemic rat exposed to benzene the decreasing trend observed in urinary excretion of free and conjugated metabolites may be due to their capability to reduce cytochromial activity. Conversely, in the diabetic rat, urinary levels of benzene metabolites tended to increase progressively, probably due to the consistent induction of CYP-450 IIE1 observed in diabetes, which would overwhelm the inhibition of this isoenzyme caused by phenolic metabolites. Furthermore, the metabolic switch towards detoxification metabolites observed after administration of high doses of benzene is not allowed in the diabetic because of reduced glutathione-S-transferase activity. As a consequence, higher levels of hydroquinone, phenol and catechol, considered the actual metabolites responsibles for benzene toxicity, will accumulate in the diabetic rat. Extrapolating these data to human, we may thus suggest that occupational exposure to benzene of a diabetic subject poses a higher risk level, as his metabolism tends to produce and accumulate higher levels of reactive benzene catabolites.

Keywords: Key words Benzene; Benzene metabolites; Diabetes; Cytochrome P-450; Rat

Carnosine prevents glyceraldehyde 3-phosphate-mediated inhibition of aspartate aminotransferase by Timothy A. Swearengin; Catherine Fitzgerald; Norbert W. Seidler (pp. 307-309).

Post-mitotic tissues, such as the heart, exhibit high concentrations (20 mM) of carnosine (β-alanyl-l-histidine). Carnosine may have aldehyde scavenging properties. We tested this hypothesis by examining its protective effects against inhibition of enzyme activity by glyceraldehyde 3-phosphate (Glyc3P). Glyc3P is a potentially toxic triose; Glyc3P inhibits the cardiac aspartate aminotransferase (cAAT) by non-enzymatic glycosylation (or glycation) of the protein. cAAT requires pyridoxal 5-phosphate (PyP) for catalysis. We observed that carnosine (20 mM) completely prevents the inhibition of cAAT activity by Glyc3P (5 mM) after brief incubation (30 min at 37 °C). After a prolonged incubation (3.25 h) of cAAT with Glyc3P (0.5 mM) at 37 °C, the protection by carnosine (20 mM) persisted but PyP availability was affected. In the absence of PyP from the assay medium, cAAT activities (plus Glyc3P) were 95 ± 18.2 μmol/min per mg protein (mean ± SD), minus carnosine and 100 ± 2.4, plus carnosine; control activity was 172 ± 3.9. When PyP (1.0 μM) was included in the assay medium, cAAT activities (plus Glyc3P) were 93 ± 14.8, minus carnosine and 151 ± 16.8, plus carnosine, P < 0.001; control activity was 180 ± 17.7. These data, which showed carnosine moderating the effects of both Glyc3P and PyP, suggest that carnosine may be an endogenous aldehyde scavenger.

Keywords: Key words Maillard reaction; Carnosine; Non-enzymatic glycosylation; Glyceraldehyde 3-phosphate; Aspartate aminotransferase

Inflammatory responses of rat alveolar macrophages following exposure to fluoride by Seishiro Hirano; Mitsuru Ando; Sanae Kanno (pp. 310-315).

Inhalation exposure to fluoride compounds has been associated with respiratory failure. We have addressed effects of fluoride on alveolar macrophages and lung responses to intratracheally (i.t.) instilled fluoride in rats. I.t. instillation of fluoride at doses of 200 and 400 μg F/rat caused significant polymorphonuclear leukocyte (PMN) infiltration in the rat lung at 20 h post-administration, while 100 μg fluoride did not recruit a significant number of PMNs in the alveolar space. Total RNA was extracted from the lung lavage cells obtained from 5 h post i.t. instillation and mRNA levels of chemokines and proinflammatory cytokines were semi-quantitatively evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). I.t. instillation of fluoride significantly enhanced mRNA expression of cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory proteins-1α and -2. Fluoride-induced augmentation in IL-1β mRNA expression was also examined by Northern hybridization following in vitro exposure of alveolar macrophages to fluoride. However, the enhancement of IL-1β mRNA expression following in vitro exposure to fluoride was observed only at 500 μM, a dose higher than the 50% lethal concentration (LC₅₀). Non-specific adhesion of alveolar macrophages to the plastic dish was significantly increased following in vitro exposure to fluoride. The fluoride-induced non-specific adhesion was significantly reduced by anti-CD18, suggesting that β₂ integrin played a role in the increase of adherence. Those results suggest that fluoride activates alveolar macrophages, enhances the production of chemokines and proinflammatory cytokines, and causes PMN infiltration in the lung.

Keywords: Key words Fluoride; Alveolar macrophage; Polymorphonuclear leukocyte; Cytokine; Adhesion

Effects of solvent on DNA adduct formation in skin and lung of CD1 mice exposed cutaneously to benzo(a)pyrene by Ewan D. Booth; Robert W. Loose; William P. Watson (pp. 316-322).

The effect of solvent polarity and lipophilicity on DNA adduct formation by polycyclic aromatic hydrocarbons in skin and lung has been studied in CD1 mice exposed cutaneously in vivo to benzo(a)pyrene (∼0.01–7.0 μg/animal) in either tetrahydrofuran or n-dodecane. The nature and amounts of DNA adducts, measured as 7R,8S,9R-trihydroxy-10S-(N²-deoxyguanosyl-3′-phosphate)-7,8,9,10-tetrahydrobenzo(a)pyrene, in relation to exposure dose and treatment regime was determined by ³²P-postlabelling. In skin DNA there was a linear relationship between exposure dose and adduct formation with both solvents, though the amount of adduct formed was significantly lower from treatment with benzo(a)pyrene in n-dodecane than in tetrahydrofuran. The amounts of adducts measured in skin DNA ranged from 67 amol adducts/μg DNA at the lowest exposure dose of benzo(a)pyrene in n-dodecane to 3.5 fmol adducts/μg DNA (1 adduct in 5 × 10⁷ nucleotides to 1 adduct in 9 × 10⁵ nucleotides) at the highest dose. In tetrahydrofuran the corresponding levels were 89 amol adducts/μg DNA (1 adduct in 3 × 10⁷ nucleotides) to 16.9 fmol adducts/μg DNA (1 adduct in 2 × 10⁵ nucleotides). DNA adducts could not be detected in lung tissue following cutaneous treatment of animals with benzo(a)pyrene in n-dodecane. Cutaneous treatment of animals with benzo(a)pyrene in tetrahydrofuran, however, resulted in adducts in lung DNA at a level of 88 amol/μg DNA from exposures only at the highest dose (6.72 μg/animal). The difference in octanol-water partition coefficient, log P_ow between n-dodecane compared to tetrahydrofuran is considered to be the most likely reason for the reduction in the bioavailability of benzo(a)pyrene and/or its metabolites and hence the degree of genotoxicity in tissues. The results suggest that other paraffinic hydrocarbon solvents may moderate the genotoxicity of polycyclic aromatic hydrocarbons in vivo. The assessment of the genotoxicity in vivo of mixtures of compounds should be carried out on complete mixtures of substances of interest in order to take account of these possible antagonistic or synergistic effects.

Keywords: Key words Benzo(a)pyrene; n-Dodecane; Tetrahydrofuran; DNA adduct; Mouse skin

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on liver phosphoenolpyruvate carboxykinase (PEPCK) activity, glucose homeostasis and plasma amino acid concentrations in the most TCDD-susceptible and the most TCDD-resistant rat strains by Matti Viluksela; Mikko Unkila; Raimo Pohjanvirta; Jouni T. Tuomisto; Bernhard U. Stahl; Karl K. Rozman; Jouko Tuomisto (pp. 323-336).

Reduced gluconeogenesis due to decreased activity of key gluconeogenic enzymes in liver, together with feed refusal, has been suggested to play an important role in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced lethality in rats. This study was carried out to further analyse the toxicological significance of reduced gluconeogenesis by comparing dose-responses and time-courses of effects of TCDD on the activity of phosphoenolpyruvate carboxykinase (PEPCK) in liver, liver glycogen concentration as well as plasma concentrations of glucose and amino acids in both genders of TCDD-sensitive Long-Evans (L-E) rats and TCDD-resistant Han/Wistar (H/W) rats. A dose-dependent decrease in PEPCK activity was observed in H/W rats, but in L-E rats the activity was not decreased. However, TCDD impaired the strong increase in liver PEPCK activity observed in pair-fed controls of the L-E strain. Liver glycogen concentrations were severely decreased in L-E rats and moderately in H/W rats. This effect seems to be secondary to reduced feed intake, since a similar decrease was seen in pair-fed controls. Decreases in plasma glucose concentrations were also more profound in L-E rats than in H/W rats, but pair-fed controls were generally less affected. Circulating concentrations of amino acids were markedly increased in TCDD-treated L-E rats, which is likely to reflect increased mobilization of amino acids and their decreased metabolism in liver. Reduction of liver PEPCK activity cannot account for the sensitivity difference of these two strains of rats in terms of mortality. Nevertheless, the response of both strains of TCDD-treated rats regarding gluconeogenesis is different from that seen in pair-fed controls and suggesting that impairment of this pathway contributes to the development of the wasting syndrome.

Keywords: Key words 2; 3; 7; 8-Tetrachlorodibenzo-p-dioxin; TCDD; Phosphoenolpyruvate carboxykinase; PEPCK; Glucose; Glycogen; Amino acids

In vitro models to study mechanisms involved in cyclosporine A-mediated glomerular contraction by B. L'Azou; J. Medina; W. Frieauff; A. Cordier; J. Cambar; A. Wolf (pp. 337-345).

The immunosuppressive drug, cyclosporin A (CsA), which is successfully used to prevent rejection in organ transplantation, induces renal side-effects as shown by a decrease in glomerular filtration rate and ultrafiltration coefficient regulated by the tone of mesangial cells.The aim of the present study was to investigate the effect of CsA on isolated glomeruli and mesangial cells, which constitute appropriate in vitro models for renal vasoreactivity studies. The roles of different intracellular and extracellular mediators such as calcium, endothelin-1 (ET-1), prostaglandins (TXA₂and PGI₂) and reactive oxygen intermediates (ROIs) were analysed. CsA caused a concentration- and time-dependent decrease in the planar cross-sectional areas of isolated glomeruli and mesangial cells as determined by image analysis. Intracytosolic free calcium concentration determined by fluorimetric analysis was significantly increased after 30 min CsA (10 μM) incubation. In the contraction experiment, the calcium antagonist verapamil inhibited the CsA response. ET-1, TXB₂ and keto-PGF_1α were determined directly, however no changes were found statistically significantly different from respective controls. In contrast to these results, the ET-1 specific antibody was able to reduce CsA-mediated cell contraction. In the presence of a prostacyclin agonist iloprost, CsA-induced contraction was also modified. The role of ROIs using a 2′7′-dichlorofluorescein diacetate (DCFdAc) fluorimetric method was directly determined by observing, with 10 μM CsA, a significant production of hydrogen peroxide (H₂O₂), which was able alone to induce mesangial cell contraction. Coincubation with the antioxidants led to a significant inhibition of mesangial cell contraction. These results suggest that CsA caused an imbalance in the normal level of all investigated vasoconstrictive and vasodilator mediators, which shifted towards the advantage of vasoconstrictive action.

Keywords: Key words Cyclosporin; Mesangial cells; Isolated glomeruli; Vasoreactivity; Intracellular mediators

Usefulness of the assessment of urinary enzyme leakage in monitoring acute fluoride nephrotoxicity by Kan Usuda; Koichi Kono; Tomotaro Dote; Hiroyuki Nishiura; Teruaki Tagawa (pp. 346-351).

A single oral dose of sodium fluoride (NaF) in aqueous solution was given to male Wistar rats. Twenty-four-hour urine samples were collected and examined to evaluate fluoride-induced acute renal damage. The following parameters were measured in 24-h urine: urine volume and urinary excretion of fluoride, N-acetyl-β-d-glucosaminidase (NAG), α-glutathione-S-transferase (α-GST), and creatinine (CR). Fluoride exposure produced specific, dose-dependent changes of these parameters. Significant increases of fluoride and fluoride-induced polyuria were observed. NAG as specific marker of proximal convoluted tubule (PCT) function showed a significant increase when the lowest dose of fluoride was administered. At this minimal dose, α-GST, a specific marker for the S3 segment, did not show a significant increase but presented the strongest relationship (r = 0.83) to fluoride dose. No significant changes were measured for CR excretion, which showed a low correlation coefficient (r = 0.36) to administered fluoride. The specific differences in the increase pattern of these parameters show that the PCT is more susceptible to damage by low-dose fluoride than the S3 segment or the glomerulus. We concluded that both NAG and α-GST are useful for the diagnosis of fluoride-induced acute nephrotoxicity. Proper evaluation of these urinary indices may be of help to establish the site and extent of kidney injury in acute fluoride toxicity.

Keywords: Key words Fluoride; Acute renal failure; α-Glutathione-S-transferase; N-acetyl-beta-d-glucosaminidase; Creatinine

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Archives of Toxicology (v.73, #6)