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Archives of Toxicology (v.72, #1)

Biotransformation, excretion and nephrotoxicity of haloalkene-derived cysteine S-conjugates by Gerhard Birner; Ulrike Bernauer; Michael Werner; Wolfgang Dekant (pp. 1-8).

The formation of cysteine S-conjugates is thought to play an important role in the nephrotoxicity of haloalkenes such as trichloroethene, tetrachloroethene and hexachlorobutadiene. Glutathione S-conjugates formed from these haloalkenes in the liver are processed to the corresponding cysteine S-conjugates, which may be N-acetylated to mercapturic acids and may be accumulated in the kidney. Haloalkene-derived cysteine S-conjugates are also substrates for cysteine conjugate β-lyases and reactive intermediates are formed in this reaction. The equilibrium between cysteine S-conjugate and mercapturic acid thus influences the extent of β-lyase dependent bioactivation and subsequently the nephrotoxicity of S-conjugates. In this study,␣we compared the rates of N-acetylation in vitro and the biotransformation, excretion and nephro‐ toxicity of S-(1,2-dichlorovinyl)-l-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)-l-cysteine (2,2-DCVC), S-(1,2,2-trichlorovinyl)-l-cysteine (TCVC) and S-(1,2,3,4,4-pentachlorobutadienyl)-l-cysteine (PCBC) in rats after i.v. injection (40 μmoles/kg). Marked differences in the extent of enzymatic N-acetylation were observed; N-acetylation was most efficient with 2,2-DCVC and least efficient with 1,2-DCVC. In urine, within 48 h, most of the given 2,2-DCVC (77% of the recovered dose) and 1,2-DCVC (92%) were recovered as the corresponding mercapturic acids. In contrast, a higher percentage of cysteine S-conjugate and less of the mercapturic acid were recovered in urine after administration of PCBC and TCVC (50 and 23% of dose as mercapturic acid), respectively. Histopathological examination of the kidneys and urine clinical chemistry showed marked differences in the extent of renal damage. Necroses of the proximal tubules were found after TCVC, PCBC and 1,2-DCVC administration in male, but not in female rats. These differences in nephrotoxicity do not correlate with the balance of acetylation/deacetylation. The higher toxicity observed in male rats may indicate the involvement of other parameters such as uptake mechanisms.

Keywords: Key words Haloalkenes; Nephrotoxicity; Biotransformation; Glutathione; Mercapturic acids

Changes in cytochrome P450 enzymes by 1,1-dichloroethylene in rat liver and kidney by Nobumitsu Hanioka; Hideto Jinno; Tetsuji Nishimura; Masanori Ando (pp. 9-16).

We examined the effect of 1,1-dichloroethylene (1,1-DCE) on microsomal cytochrome P450 (P450) enzymes in rat liver and kidney. Rats were treated intraperitoneally with 1,1-DCE daily for 4 days, at doses of 200, 400, and 800 mg/kg. Among the P450-dependent monooxygenase activities in liver microsomes, testosterone 2α-hydroxylase (T2AH), which is associated with CYP2C11 activity, was remarkably decreased by 800 mg/kg 1,1-DCE. The level relative to control activity was <10%. Furthermore, immunoblotting showed that 1,1-DCE (≥400 mg/kg) significantly decreased CYP2C11/6 protein levels in liver microsomes. In addition, 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), benzphetamine N-demethylase (BZND), chlorzoxazone 6-hydroxylase (CZ6H), and testosterone 6β-hydroxylase (T6BH) activities were significantly decreased by the highest dose of 1,1-DCE (by 40–70%). However, the activities of other P450-dependent monooxygenases, namely 7-ethoxyresorufin O-deethylase (EROD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), erythromycin N-demethylase (EMND), lauric acid ω-hydroxylase (LAOH), and testosterone 7α-hydroxylase (T7AH) were not affected by 1,1-DCE at any dose. Immunoblotting showed CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A2/1 protein levels were significantly decreased by 60–66% by 1,1-DCE (800 mg/kg), whereas that of CYP4A1/2 was not affected by any dose of 1,1-DCE. By contrast, among the P450-dependent monooxygenase activities in kidney microsomes, only CZ6H activity was increased by 1,1-DCE (1.6-fold at 800 mg/kg). Also, it was␣observed that 1,1-DCE (800 mg/kg) significantly increased CYP2E1 protein levels by immunoblotting (∼1.5-fold). These results suggest that 1,1-DCE changes the constitutive P450 isoforms in the rat liver and kidney, and that these changes closely relate to the toxicity of 1,1-DCE.

Keywords: Key words Cytochrome P450; 1; 1-Dichloroethylene; Cytochrome P450 isoforms; Rat liver microsomes; Rat kidney microsomes

A quantitative property-property relationship (QPPR) approach to estimate in vitro tissue-blood partition coefficients of organic chemicals in rats and humans by Joost DeJongh; Henk J. M. Verhaar; Joop L. M. Hermens (pp. 17-25).

The present study describes quantitative property-property relationships (QPPRs) for the partitioning of organic chemicals between blood and tissue homogenates from both rats and humans. The n-octanol/water partition coefficient (K _ow) is used as a non-biological descriptor. QPPRs for human tissue-blood partition coefficients (PCs) were derived from a dataset of 24 volatile organic compounds in blood, liver, muscle, fat, kidney and brain tissue homogenates. QPPRs were also derived for the PCs of rat tissues, using a dataset of 42 volatile organic compounds in blood, liver, muscle and fat tissue homogenates. These QPPRs were evaluated using a test set of 10 compounds for human tissues and a test set of 14 compounds for rat tissues. For both human and rat test sets, it was generally observed that most estimated PCs were within a range of 50–200% of their experimental values. The present approach is concluded to offer a rapid means for the estimation of tissue-blood PCs of compounds on the basis of K _ow values. In addition, indications for a possible role of tissue components other than lipid and water in the tissue-blood partitioning process of compounds were observed from the calibration results of the model.

Keywords: Key words Partition coefficients; Physiologically based-pharmacokinetics (PB-PK); Distribution; Modelling

Quinolone-induced arthropathy: exposure of magnesium-deficient aged rats or immature rats, mineral concentrations in target tissues and pharmacokinetics by C. Förster; R. Schwabe; E. Lozo; U. Zippel; J. Vormann; T. Günther; H. J. Merker; R. Stahlmann (pp. 26-32).

Quinolone treatment or magnesium deficiency induce identical cartilage lesions in juvenile rats and show additive arthropathogenic effects. It has been shown previously that neither condition is arthropathogenic in 8-week-old rats. Joint cartilage from aged individuals is rather prone to pathological alterations but information on prolonged quinolone treatment and/or dietarily induced magnesium deficiency in aged animals is not available. We treated magnesium-deficient (n = 9) aged Wistar rats (age 15 months) and age-matched controls with daily doses of 600 mg ofloxacin/kg body wt. by gastric intubation for 28 days. Further groups of magnesium-deficient and control rats (n = 9 and n = 10, respectively) received the vehicle only. Peak plasma concentrations of ofloxacin in adult rats were 20.5 ± 5.6 mg/l (mean ± SD) following treatment with a single dose of 600 mg/kg body wt. At the end of the experiment the degree of magnesium deficiency was most pronounced in plasma (Mg²⁺-def., 0.33 ± 0.12 mmol/l; control, 0.97 ± 0.08 mmol/l) and less pronounced in sternal cartilage (Mg²⁺-def., 10.8 ± 3.6 mmol/kg dry wt; control, 13.3 ± 2.8 mmol/kg dry wt), whereas the magnesium concentration in femoral bone remained unchanged (Mg²⁺-def., 201 ± 13 mmol/kg dry wt; control, 204 ± 11 mmol/kg dry wt). Histological investigation of the knee joints revealed no cartilage lesions following ofloxacin treatment, magnesium deficiency or a combination of both conditions. By contrast, cartilage lesions such as scars and erosions of the joint surface, chondrocyte clusters within acellular areas of the cartilage matrix and persisting clefts were detectable in knee joints from 7 of 10 adult rats (age 9 months) which had been treated with 4 × 600 mg fleroxacin/kg body wt. at 5 weeks of age. Mean plasma concentration of fleroxacin in juvenile rats was approx. 50 mg/l between 1.5 and 6 h after dosing and the drug was still detectable in plasma 48 h after dosing (0.4 ± 0.1 mg/l). Our data indicate that joint cartilage in aged rats is not altered by a 4-week quinolone treatment, even during magnesium deficiency. Cartilage lesions in adult rats were only detectable if the animals had been treated during the sensitive phase at 5 weeks postnatally.

Keywords: Key words Quinolones; Magnesium; Arthropathy; Rats; Ofloxacin

Propyl gallate-induced DNA fragmentation in isolated rat hepatocytes by Yoshio Nakagawa; Peter Moldéus; Gregory Moore (pp. 33-37).

Incubation of isolated rat hepatocytes with propyl gallate (PG) at concentrations of ≥1 mM induced cell killing, whereas PG at ≤0.5 mM did not cause cell death during a 3-h incubation. PG at ≥0.5 mM elicited the ladder formation of soluble low-molecular weight DNA fragments with integer multiples of approximately 180 bp and specific nuclear DNA cleavages detected cytopathologically by labeling of a digoxigenin-nucleotide complex to new 3′-OH ends. Both of these PG-induced changes observed in hepatocytes are characteristic features of apoptosis. In contrast, the pretreatment of N-acetylcysteine (4 mM), a precursor of intracellular glutathione (GSH) and antioxidant, prevented PG (0.5 mM)-induced formation of soluble DNA fragments and loss of cellular GSH, ATP, and formation of blebbing. These results suggest that when the concentration of PG is decreased, the effects of PG on hepatocytes change from acute necrotic to apoptotic mode, and that the onset of DNA fragmentation is associated with GSH depletion.

Keywords: Key words Propyl gallate; DNA fragmentation; Cytotoxicity; Glutathione; Rat hepatocyte

Spatial distribution of glutathione, glutathione-related and antioxidant enzymes in cultured mouse embryos by Gian Mario Tiboni; Tonino Bucciarelli; Fernanda Amicarelli; Stefania Angelucci; Elisabetta Iammarrone; Umberto Bellati; Paolo Sacchetta; Carmine Di Ilio (pp. 38-44).

The present study was undertaken to evaluate the detoxifying capacity of organogenesis-stage murine concepti cultured in vitro. Investigative attention was particularly focused on the embryonic tissue distribution of cytoprotective pathways. Glutathione (GSH) status, GSH-related and antioxidant enzymes were assayed in the embryo proper (EP), visceral yolk sac (VYS) and ectoplacental cone (EC) of 29.44 ± 1.56 (mean ± SD) somite pairs concepti. All the tissues displayed significant and comparable concentrations of GSH, further supporting this tripeptide as critical in protection against embryotoxicants. The totality of enzymatic activities was detectable in the selected embryonic compartments. In terms of spatial distribution analysis, maximal activities were found in EC (glutathione peroxidase, glutathione reductase, superoxide dismutase and glyoxalase I and II), and VYS (glutathione transferase and catalase). These results indicate: (1) the organogenesis-stage conceptus, in addition to significant amounts of GSH, expresses constitutive activities of GSH-related and antioxidant enzymes; (2) maximal activity levels are detectable in the embryonic sites which, at the developmental stage selected for assay, serve (VYS) or are evolving to serve (EC) embryo/maternal exchange, and thus represent the primary sites of interaction with foreign compounds.

Keywords: Key words Glutathione; Superoxide dismutase; Catalase; Glyoxalase system; Mouse whole-embryo culture

Characterization of the role played by antigen challenge in the suppression of in vivo humoral immunity by 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD) by R. A. Matulka; D. L. Morris; S. W. Wood; N. E. Kaminski; M. P. Holsapple (pp. 45-51).

The goal of these studies was to characterize the role played by antigen challenge in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunosuppression. The effects of single exposure to TCDD (4.2, 14, and 42 μg/kg) in B6C3F1 mice on the reverse plaque assay (RPA; no sensitization) and the sheep red blood cell (SRBC) antibody response were compared. While the RPA was suppressed in a dose-dependent fashion with significance at the two highest doses, a much more dramatic effect was noted with the primary anti-SRBC response: a suppression was noted at the lowest dose, which was comparable to that observed with the highest dose in the RPA. Subsequent studies compared the RPA in B6C3F1 (Ah-high responder) and DBA/2 (Ah-low responder) mice after both single and repeated exposure to identical cumulative doses of TCDD (4.2, 14, and 42 μg/kg). The repeated exposures consisted of 14 consecutive daily treatments of 0.3, 1.0, and 3.0 μg/kg. The results indicated only a slight difference in the effects of TCDD in the two strains after single exposure, and even less difference after repeated exposure. Moreover, administering TCDD on different days relative to the SRBC challenge indicated a suppression in both strains when given 1, 2, or 3 days before antigen challenge, on the day of antigen challenge, or 1 or 2 days after antigen challenge. The only day of administration where the suppression was attenuated was 3 days after antigen challenge. These results confirm an important relationship between antigen challenge and TCDD exposure on immunosuppression.

Keywords: Key words 2; 3; 7; 8-Tetrachlorodibenzo-p-dioxin (TCDD); Immunotoxicity; Humoral immunity; Antigen challenge

Fluoride mediates apoptosis in osteosarcoma UMR 106 and its cytotoxicity depends on the pH by S. Hirano; Mitsuru Ando (pp. 52-58).

Although an excess intake of fluoride has been reported to cause skeletal fluorosis, very little is known about the mechanism of adverse effects of fluoride on bone. In the present study cytotoxic effects of fluoride were studied using the osteosarcoma cell line, UMR 106. The DNA ladder formation upon agarose electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining revealed that UMR 106 underwent apoptosis following exposure to 5 mM fluoride for 8 h. On the other hand exposure to A23187, a calcium ionophore, caused necrosis while co-exposure to fluoride and A23187 inhibited fluoride-mediated apoptosis in UMR 106. The proliferation of UMR 106 cells cultured for 6 days in the presence of 0.5 mM fluoride was significantly decreased compared to the control culture. The cytotoxic effects of fluoride were modulated by both the cell density and the pH of the culture medium. The fluoride-induced viability loss in UMR 106 was enhanced in culture of high cell-density and inversely correlated with pH of the culture medium. Enhancement of fluoride cytotoxicity at acidic pH was also observed in rat alveolar macrophages and RAW 264, a macrophage cell line. The results suggest that fluoride-mediated apoptosis and culture conditions, including pH of the medium, should be taken into consideration to evaluate toxicity of fluoride in vitro.

Keywords: Key words Fluoride; A23187; Apoptosis; UMR 106; pH

Long-term pulmonary and systemic toxicity following intravenous mercury injection by M. dell'Omo; Giacomo Muzi; Alfred Bernard; Sabrina Filiberto; Robert R. Lauwerys; Giuseppe Abbritti (pp. 59-62).

Long-term pulmonary and systemic toxicity following mercury intravenous injection has rarely been assessed. We present the results of a detailed investigation assessing pulmonary and systemic long-term toxic effects in a subject who had pulmonary and systemic mercury microembolism diagnosed more than 11 years previously. Radiographic examination showed the persistence of mercury microemboli in both lungs and elsewhere␣in the body. Lung function tests revealed a decreased diffusing capacity for carbon monoxide and P o ₂ probably indicative of microscopic inflammation of lung interstitium. Electroneuromyography showed signs of mild axonopathy in both legs. At semen analysis, a high proportion of motionless spermatozoa was present. Urinary excretion of mercury was high. Signs of interstitial lung impairment, peripheral axonopathy and asthenozoospermia in a subject who had mercury microembolism persisting for more than 11 years might be evidence of long-term mercury toxicity.

Keywords: Key words Mercury; Pulmonary embolism; Intravenous injection; Toxicity

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Archives of Toxicology (v.72, #1)