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Archives of Toxicology (v.72, #5)

Involvement of reactive oxygen species, protein kinase C, and tyrosine kinase in prostaglandin E2 production in Balb/c 3T3 mouse fibroblast cells by quinolone phototoxicity by Kohji Shimoda; Michiyuki Kato (pp. 251-256).

We examined the effect of an antioxidant and protein kinase inhibitors on prostaglandin E₂ (PGE₂) release from Balb/c 3T3 mouse fibroblast cells induced by quinolone phototoxicity. Simultaneous administration of sparfloxacin (SPFX) or lomefloxacin (LFLX) at 12.5 to 100 μ M and ultraviolet-A (UVA) irradiation for 10 min markedly elevated PGE₂ concentration in the incubation medium, whereas levofloxacin (LVFX) at concentrations up to 100 μ M and UVA irradiation did not increase PGE₂ concentration. Pretreatment with 100 μ M pyrrolidine dithiocarbamate (PDTC), an antioxidant, or 1 μ M calphostin C, a selective inhibitor of protein kinase C (PKC), completely inhibited the elevation of PGE₂ in the 24-h incubation medium; pre-treatment with 10 μ M H7, a cyclic nucleotide-dependent protein kinase, and PKC or 1 μ M herbimycin A, a tyrosine kinase inhibitor, inhibited the PGE₂ elevation by 29 to 39%. Conversely, 25 nM staurosporine significantly augmented the PGE₂ elevation by quinolones plus UVA. Interleukin-1β (IL-1β ) and tumor necrosis factor α (TNFα ) were not detected in the incubation medium of 3T3 cells after quinolone plus UVA, corresponding to the lack of effect of antibodies against IL-1α , IL-1β , and TNFα on PGE₂ release from 3T3 cells. These results suggest that PGE₂ production in 3T3 cells by quinolone phototoxicity is modulated by reactive oxygen species, PKC, and tyrosine kinase, but not by IL-1 or TNFα .

Keywords: Key words Quinolone; Phototoxicity; Fibroblasts; Prostaglandin E2

FK506 (Tacrolimus) decreases the cytotoxicity of cyclosporin A in rat hepatocytes in primary culture: implication of CYP3A induction by Sophie Ellouk-Achard; Chantal Martin; Huynh Thien Duc; Hélène Dutertre-Catella; Marc Thevenin; Jean-Michel Warnet; Jean Roger Claude (pp. 257-263).

Tacrolimus (FK506) and cyclosporin A (CsA) are two potent immunosuppressants mainly metabolized by hepatic cytochrome P-450 3A (CYP3A) monooxygenase. The aim of this study was to compare the toxic effects of the two drugs on hepatocytes in primary culture as a function of their metabolism and to explore the variations of cytotoxicity when both drugs are associated. The cytotoxicity of FK506 and CsA, as expressed by their IC₅₀ values, was of the same order but with a switch according to whether hepatocytes were induced or uninduced by dexamethasone, CsA being more toxic in its native form and FK506 through its metabolism. Similar results were obtained with the intracellular calcium content. When both drugs were associated at their IC₅₀ values, the expected additive cytotoxic effect was not observed. Moreover, when small quantities of FK506 were added to CsA at its IC₅₀, cell viability improved in the induced cultures. It is hypothesized that the interaction between the two drugs relies on a mechanism involving both competition of FK506 and CsA for CYP3A and of their immunophilin complexes for a common site on the calcineurin-calmodulin complex.

Keywords: Key words FK506; Cyclosporin A; Cytochrome P-450 3A; Toxicity; In vitro

Oxidative DNA damage and cell proliferation in kidneys of male and female rats during 13-weeks exposure to potassium bromate (KBrO3) by Takashi Umemura; Atsuya Takagi; Kimie Sai; Ryuichi Hasegawa; Yuji Kurokawa (pp. 264-269).

It has been assumed that oxidative damage, including formation of 8-hydroxydeoxyguanosine (8-OHdG) adducts in kidney DNA due to potassium bromate (KBrO₃), a renal carcinogen to both sexes of rats, is involved in its mechanisms of tumor induction. However, despite the presumed existence of a repair enzyme(s) for 8-OHdG, there have been no reports demonstrating the changes in adduct levels during medium- or long-term exposure. To elucidate the actual kinetics regarding this parameter during the early stages of KBrO₃ carcinogenesis, we measured 8-OHdG levels in kidney DNA together with cell proliferation in renal tubules in both sexes of rats receiving KBrO₃ at a dose of 500 ppm in the drinking water for 1, 2, 3, 4, and 13 weeks. Rapid elevation of 8-OHdG levels was noted in treated male rats which persisted until the end of the experiment. Increased cell proliferation in the proximal convoluted tubules was also observed throughout the experimental period, concomitant with ₂-globulin accumulation. Increase in 8-OHdG levels in treated females first became apparent 3 weeks after the start of exposure, with cell proliferation only elevated at the 13-week time point. The present study, employing the same route and dose of KBrO₃ known to cause tumors, strongly suggested the requirement of persistent increase of 8-OHdG for neoplastic conversion. Moreover, a clear sex difference in susceptibility to generation of oxidative stress in kidney DNA was found, in addition to ₂-globulin-dependent variation in cell proliferation in the renal tubules.

Keywords: Key words Potassium bromate; 8-Hydroxydeoxy guanosine; Cell proliferation

Oxidative effects in human erythrocytes caused by some oximes and hydroxylamine by Nicole G. M. Palmen; Chris T. A. Evelo (pp. 270-276).

Both oximes and hydroxylamine (HYAM) are compounds with known oxidative capacity. We tested in vitro whether acetaldoxime (AAO), cyclohexanone oxime (CHO), methyl ethyl ketoxime (MEKO) or HYAM affect haemoglobin oxidation (into HbFe³⁺), formation of thiobarbituric acid reactive substances (TBARS), and glutathione (GT) depletion in human haemolysate, erythrocytes or blood. All these parameters are known to be related to oxidative stress. Glutathione S-transferase (GST) activity was measured as it may be affected by oxygen radicals. All three oximes caused a low degree of HbFe³⁺ accumulation in erythrocytes. This was higher in haemolysates indicating that membrane transport may be limiting or that protective mechanisms within erythrocytes are more effective. HbFe³⁺ accumulation was lower for the oximes than for HYAM. AAO and HYAM caused TBARS formation in blood. For HYAM this was expected as free radicals are known to be generated during HbFe³⁺ formation. Free radical generation by AAO and HYAM in erythrocytes was confirmed by the inhibition of GST. For the other two oximes (CHO and MEKO) some special effects were found. CHO did inhibit erythrocyte GST while it did not cause TBARS formation. MEKO was the least potent oxime as it caused no TBARS formation, little HbFe³⁺ accumulation and little GST inhibition in erythrocytes. However, GT depletion was more pronounced for MEKO than for the other oximes, indicating that glutathione conjugation occurs. TBARS formation, GT depletion and GST modulation caused by the oximes and HYAM were also tested in rat hepatocytes. However, no effects were found in hepatocytes. This suggests that a factor present in erythrocytes is necessary for free radical formation. Studies with proposed metabolites of the oximes (i.e. cyclohexanone, acetaldehyde or methylethyl ketone) and addition of rat liver preparations to the erythrocyte incubations with oximes, suggest that metabolism is not a limiting factor in erythrocyte toxicity.

Keywords: Key words Oximes; Hydroxylamine; Human erythrocytes; Oxidizing effects; Rat liver metabolism

Computer-based bioassay for evaluation of sensory irritation of airborne chemicals and its limit of detection by Yves Alarie (pp. 277-282).

We expanded a previously described rule-based computerized method to recognize the sensory irritating effect of airborne chemicals. Using 2-chlorobenzylchloride (CBC) as a sensory irritant, characteristic modifications of the normal breathing pattern of exposed mice were evaluated by measuring the duration of braking (TB) after inspiration and the resulting decrease in breathing frequency. From the measurement of TB, each breath was then classified as normal (N) or sensory irritation (S). Using increasing exposure concentrations, the classification S increased from ≤2% (equivalent to sham-exposure) to 100% within a narrow exposure concentration range. The potency of CBC was then evaluated by calculating the concentration necessary to produce 50% of the breaths classified as S, i.e., S₅₀. This approach is easier to use than obtaining RD₅₀ (decrease in respiratory frequency by 50%) when high exposure concentrations are difficult to achieve. Detection limits were also established for this bioassay and experiments were conducted to obtain a level of response just around these limits, in order to delineate the practicality of using this bioassay at low exposure concentrations. Using this approach, sensory irritation was the only effect induced by CBC at low exposure concentrations. However, bronchoconstriction and pulmonary irritation were superimposed on this effect at higher exposure concentrations.

Keywords: Key words Sensory irritation; Chlorobenzylchloride; Breathing pattern recognition

Assessment of early acute lung injury in rodents exposed to phosgene by Alfred M. Sciuto (pp. 283-288).

Phosgene is a highly reactive oxidant gas used in the chemical industry. Phosgene can cause life-threatening pulmonary edema by reacting with peripheral lung compartment tissue components. Clinical evidence of edema is not usually apparent until well after the initial exposure. This study was designed to investigate early signs of acute lung injury in rodents within 45–60 min after the start of exposure. Male mice, rats, or guinea pigs were exposed to 87 mg/m³ (22 ppm) phosgene or filtered room air for 20 min followed by room air washout for 5 min. This concentration-time exposure causes a doubling of lung wet weight within 5 h. After exposure, animals were immediately anesthetized i.p., with pentobarbital. Bronchoalveolar lavage (BAL) was performed and fluid analyzed for total glutathione (GSH), lipid peroxidation thiobarbituric acid reactive substances (TBARS), and protein concentration. Lungs were perfused with saline to remove blood, freeze-snapped in liquid N₂, analyzed for tissue GSH, and TBARS. Lung edema was assessed gravimetrically by measuring tissue wet/dry (W/D) weight ratios and tissue wet weights (TWW). W/D and TWW were significantly higher in mice for phosgene vs air (P=0.001, P<0.0001, respectively), but not in rats or guinea pigs. Tissue TBARS was significantly higher in phosgene-exposed guinea pigs, P=0.027; however, BAL TBARS was higher in both rats and guinea pigs, P=0.013 and P=0.006, respectively. Tissue GSH was significantly lower in phosgene-exposed rats and guinea pigs but not mice, whereas BAL GSH was higher in rats, P<0.0001. There were significantly higher BAL protein levels in all phosgene-exposed species: mice, P<0.0001; rats, P<0.0001; and guinea pigs, P=0.002. Although there appears to be a species-specific biochemical effect of phosgene exposure for some biochemical indices, measurement of BAL protein in all three species is a better indicator of ensuing edema formation.

Keywords: Key words Acute lung injury; Rodents; Phosgene

Quinuclidinium-imidazolium compounds: synthesis, mode of interaction with acetylcholinesterase and effect upon Soman intoxicated mice by Vera Simeon-Rudolf; Elsa Reiner; Mira Škrinjarić-Špoljar; Božica Radić; Ana Lucić; Ines Primožič; Srđanka Tomić (pp. 289-295).

Four compounds were prepared: 3-oxo-1- methylquinuclidinium iodide (I), 2-hydroxyiminomethyl-1,3-dimethylimidazolium iodide (II) and two conjugates of I and II linked by -(CH₂)₃- (III) and -CH₂-O-CH₂- (IV). The aim was to evaluate separately the properties of I and II as opposed to III and IV, which contain both moieties in the same molecule. All four compounds were reversible inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7). The enzyme/inhibitor dissociation constants for the catalytic site ranged from 0.073 mM (II) to 1.6 mM (I). The dissociation constant of I for the allosteric (substrate inhibition) site was 4.8 mM. Possible binding of the other compounds to the allosteric site could not be measured because II, III and IV reacted with the substrate acetylthiocholine (ATCh) and at high ATCh concentrations the non-enzymic reaction interfered with the enzymic hydrolysis of ATCh. The rate constants for the non-enzymic ATCh hydrolysis were between 23 and 37 l/mol per min. All four compounds protected AChE against phosphorylation by Soman and VX. The protective index (PI) of I (calculated from binding of I to both, catalytic and allosteric sites in AChE) agreed with the measured PI; this confirms that allosteric binding contributes to the decrease of phosphorylation rates. The PI values obtained with III and IV were higher than those predicted by the assumption of their binding to the AChE catalytic site only. The toxicity (i.p. LD₅₀) of compounds I, II, III and IV for mice was 0.21, 0.68, 0.49 and 0.77 mmol/kg body wt. respectively. All four compounds protected mice against Soman when given (i.p.) together with atropine 1 min after Soman (s.c.). One-quarter of the LD₅₀ dose fully protected mice (survival of all animals) against 2.52 (IV), 2.00 (I and III) and 1.58 (II) LD₅₀ doses of Soman.

Keywords: Keywords Acetylcholinesterase; Quinuclidine-imidazolium compounds; Reversible inhibition and protection of acetylcholinesterase against phosphorylation; Antidotal effect against Soman poisoned mice; Soman

Involvement of apoptosis in the rat germ cell degeneration induced by nitrobenzene by Kazutoshi Shinoda; Kunitoshi Mitsumori; Kazuo Yasuhara; Chikako Uneyama; Hiroshi Onodera; Kiyoshi Takegawa; Michihito Takahashi; Takashi Umemura (pp. 296-302).

Nitrobenezene (NB) produces germ cell degeneration, especially of spermatocytes in rats. To examine the possible involvement of apoptosis in this process, the extent and nature of nuclear DNA fragmentation after NB dosing were assessed using both terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and DNA gel electrophoresis, in addition to conventional histological and electron microscopic procedures. Adult Sprague Dawley rats were treated with a single oral dose of NB (250 mg/kg) and euthanized subsequently at 6, 12, and 24 h and 2, 3, 5, and 7 days. The earliest morphological signs of germ cell degeneration in testes were found in pachytene spermatocytes 24 h after dosing. Electron micrographs of degenerating spermatocytes showed marked nuclear chromatin condensation at the nuclear periphery and crowding of cytoplasmic constituents, which are characteristic of apoptosis. Coincident with the appearance of such morphological changes, degenerating spermatocytes contained fragmented DNA as revealed by TUNEL. The presence of DNA laddering, a hallmark of apoptosis on gel electrophoresis, was first apparent and most prominent at 24 h, gradually becoming less detectable. No such changes were observed up to 12 h after dosing or in control animals. These results demonstrated unequivocal involvement of apoptosis in the induction of germ cell degeneration caused by NB.

Keywords: Key words Nitrobenzene; Apoptosis; Testicular toxicity

The disposition and metabolism of 1,3,5-[U-14C]trioxane in male Wistar albino rats by Danuta Ligocka; Andrzej Sapota; Marek Jakubowski (pp. 303-308).

The present study was designed to investigate 1,3,5-[U-¹⁴C]trioxane (TOX) distribution, excretion and metabolism. The experiments were performed on male Wistar albino rats after a single administration of TOX at doses of 40 mg/kg and 400 mg/kg. The exhaled air proved to be the main route of ¹⁴C elimination, mainly as ¹⁴CO₂. During the first 12 h following the administration of 40 mg/kg of TOX the exhalation of ¹⁴CO₂ was monophasic, with a half-life of 3.5 h. After the administration of 400 mg/kg, TOX was eliminated mainly as ¹⁴CO₂ with the exhaled air (77%) and unchanged TOX (8%). About 3% of ¹⁴C was excreted in the urine as unchanged 1,3,5-trioxane. With regards to TOX elimination from blood plasma for the lower dose, a biphasic process was observed, with half-lives of 4.5 and 72 h. The amount of ¹⁴C bound by the erythrocytes was minute compared with the amount in blood plasma. When the higher dose of TOX was administered the efficiency of ¹⁴C binding to the erythrocytes was found to be 10 times higher than the respective value for blood plasma. Among the examined tissues the highest concentration of TOX-derived radioactivity was detected in the liver while the lowest was in fat tissue and brain. A subsequent decay of radioactivity occurred in the tissues. The results of the present study indicate that TOX belongs to the group of compounds, which are rapidly eliminated from the organism; hence TOX should not be expected to accumulate within the tissues. The data obtained confirm the assumed pattern of metabolic transformation, according to which 1,3,5-trioxane undergoes enzymatic transformation to formaldehyde, with carbon dioxide and water being the final products.

Keywords: Key words 1; 3; 5-[U-14C]Trioxane; Blood; Tissue distribution; Excretion; Metabolism

N-methylcarbamoyl adducts at the N-terminal valine of globin in workers exposed to N,N-dimethylformamide by Jürgen Angerer; Thomas Göen; Axel Krämer; Heiko Udo Käfferlein (pp. 309-313).

N,N-dimethylformamide (DMF) is a commonly used industrial solvent. The formation of some metabolites of DMF in humans occurs via N-methylcarbamoylated species (e.g. N-methylcarbamoylated glutathione). The aim of our study was to investigate whether DMF leads to N-methylcarbamoylated adducts at the N-terminal valine of haemoglobin (Hb). Therefore, Hb adduct levels of ten DMF exposed workers and ten controls were analysed by a specific and sensitive detection method using capillary gas chromatography and a mass selective detector (GC/MS). Using this method we were able to show for the first time that Hb adducts are formed during the metabolism of DMF in humans. The general population, however, shows still unidentified background levels of this adduct which are on average lower by a factor of 50. The pathway for the formation of the investigated DMF-Hb adduct in workers exposed to DMF is still unknown. As identical adducts were also found after exposure to methylisocyanate (MIC), our work indicates the formation of MIC during the metabolism of DMF. The formation of Hb adducts with DMF and its relevance for occupational health is a subject of further research.

Keywords: Key words N; N-Dimethylformamide; Biochemical effect monitoring; Haemoglobin adducts; Methylisocyanate; 3-Methyl-5-isopropylhydantoin

The olfactory system as a portal of entry for airborne polychlorinated biphenyls (PCBs) to the brain? by Raimund Apfelbach; Axel Engelhart; Peter Behnisch; Hanspaul Hagenmaier (pp. 314-317).

Ferrets, mammalian carnivores, kept in an indoor enclosure were continuously exposed to low concentrations of polychlorinated biphenyls (PCBs) in the ambient air for 5 years. After that time PCB concentrations were quantified in the olfactory bulbs and in the remaining brain, adipose tissue and liver. The results revealed unexpectedly high PCB concentrations in the olfactory bulbs, surpassing those in the remaining brain and the peripheral tissues. The PCB congener pattern in the olfactory bulbs resembled that found in the ambient air and the less chlorinated volatile PCBs were found in higher concentrations. We, therefore, assume that airborne PCBs enter directly via the olfactory system and are transported through the axons to the olfactory bulbs where they accumulate.

Keywords: Key words Airborne PCBs; Olfactory system; Axoplasmic transport; Ferret

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Archives of Toxicology (v.72, #5)