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Archives of Toxicology (v.78, #7)

Subchronic inhalation toxicity of soluble hexavalent chromium trioxide in rats by Hyeon-yeong Kim; Sung-bae Lee; Beom-su Jang (pp. 363-368).

We performed a 90-day repeated-dose inhalation toxicity study of soluble hexavalent chromium trioxide (CrO₃ (VI)). Male Sprague-Dawley (SD) rats were exposed to doses of CrO₃ in the form of 0.5∼5.0 μm aerosol at 0.00, 0.20, 0.50, and 1.25 mg/m³ for 6 h/day, 5 days/week for 13 weeks using inhalation chamber. CrO₃ induced decrease of activity, alopecia and nasal hemorrhage. Body weights of the high-dose 1.25-mg/m³ exposure group were significantly lower than those of the control group. Hematological results revealed the reduction of the number of red blood cell and hematocrit values in the 1.25-mg/m³ exposure group. In addition, the hemoglobin values in the 0.50- and 1.25-mg/m³ exposure groups were significantly decreased compared with those of the control group. Clinical biochemical measurements revealed the reduction in total protein, albumin and alanine aminotransferase (ALT) level of the 0.50- and 1.25-mg/m³ exposure groups. Microscopic examination of the lung showed inflammation reactions caused by Cr exposure. In conclusion, the 13-week repeated exposure with soluble CrO₃ demonstrated the injury in SD rats with the no observed adverse effect level (NOAEL) under 0.20 mg/m³.

Keywords: Anemia; Chromium trioxide; Hexavalent chromium; Inhalation toxicity

Effect of bisphenol A on drug metabolising enzymes in rat hepatic microsomes and precision-cut rat liver slices by Erika Pfeiffer; Manfred Metzler (pp. 369-377).

In order to assess the effects of bisphenol A (BPA) on enzymes of phase I and II biotransformation, studies were conducted in hepatic microsomes and precision-cut liver slices from male Sprague-Dawley rats. A testosterone hydroxylation assay was used for probing the activity of cytochrome P450 (CYP) forms, and an appropriate HPLC method for the separation of testosterone metabolites was developed. BPA markedly inhibited the hydroxylation of testosterone at 2α and 16α but not at 6β or 7α, suggesting a differential inhibition of some CYP forms, in particular CYP2C11. This inhibitory effect was also observed when slices were first exposed to BPA and then incubated with testosterone in the absence of BPA, indicative of an irreversible inhibition of CYP. In liver slices, a differential conjugation of hydroxylated testosterone metabolites was observed, which was significantly decreased in the presence of BPA. BPA also inhibited the conjugation of the model compound umbelliferone. Pretreatment with BPA did not affect the conjugation of testosterone and umbelliferone. No hydroxylation, but extensive conjugation of BPA was observed upon incubation of liver slices with BPA alone or with testosterone or umbelliferone. The rapid and preferred conjugation, however, does not prevent the irreversible inhibition of some CYP forms by BPA. In conclusion, this study has shown that BPA causes a selective and irreversible inhibition of certain CYP forms and interferes with the conjugation of other drugs.

Keywords: Bisphenol A; Inhibition of conjugation; Inhibition of CYP; Testosterone; Umbelliferone

Identification and characterisation of adducts between serum albumin and 4,4’-methylenediphenyl diisocyanate (MDI) in human plasma by G. Johannesson; C. J. Sennbro; P. Willix; C. H. Lindh; B. A. G. Jönsson (pp. 378-383).

Diisocyanates are potent inducers of airways disease. Methylenediphenyl diisocyanate (MDI) is a widely used diisocyanate in the chemical industry. The aim of this study was to identify major and also immunologically relevant protein conjugates of MDI in plasma. Plasma was obtained from an MDI-exposed worker. The plasma was dialysed and then fractionated using ion exchange chromatography (IEC) and gel filtration. These fractions and also aliquots of unfractioned plasma were hydrolysed, derivatised and analysed for isocyanate adduct content using gas chromatography–mass spectrometry. In addition, immunologically relevant proteins were identified through specific IgG immunoblotting using pooled sera from two exposed workers. It was shown by dialysis that 96% of the hydrolysed MDI derivatives were protein bound and that 95% of the MDI adducts co-eluted with serum albumin in plasma using IEC. All MDI–protein adducts co-eluted with serum albumin using gel filtration. IgG immunoblotting showed a major 66 kDa protein and also some intermolecular reactions in serum albumin. This study shows serum albumin to be the major protein in plasma that forms adducts in vivo with MDI. Thus, a quick and simple quantitative method for biological monitoring may be developed for MDI exposure. The results also showed that MDI-specific IgG antibodies preferentially bind to the serum albumin in in-vitro-synthesised MDI–plasma protein conjugates.

Keywords: Adduct; Albumin; Isocyanate; Polyurethane; Antibody

Kinetics of orally administered di(2-ethylhexyl) phthalate and its metabolite, mono(2-ethylhexyl) phthalate, in male pigs by Karl Ljungvall; Bart Tienpont; Frank David; Ulf Magnusson; Karolina Törneke (pp. 384-389).

Di(2-ethylhexyl) phthalate (DEHP) is used as a plastic softener in the polymer industry and is widespread in medical devices. DEHP has been incriminated as an endocrine-disrupting chemical, and the effects of DEHP in various species have included disturbances in the reproductive system. The effects of the chemical have varied, depending upon exposure routes and species. This study was performed in order to characterise the kinetics of DEHP and its metabolite mono(2-ethylhexyl) phthalate (MEHP) in the young male pig, an omnivore model-species for research in reproductive toxicology. Eight pigs were given 1000 mg DEHP/kg bodyweight by oral gavage. The concentrations of DEHP and MEHP were then measured in the plasma and tissues of the pigs at different time points after administration. There was no consistent rise above contamination levels of concentrations of DEHP in the plasma of the pigs. However, the metabolite MEHP reached the systemic blood circulation. The half-life of MEHP in the systemic blood circulation was calculated to be 6.3 h. Absorption from the intestine was biphasic in six of the eight pigs and the mono-exponential elimination-phase started 16 h after the after the administration of DEHP. To conclude, MEHP consistently reaches the systemic circulation in the pig when DEHP is administered orally. The kinetic pattern of the parent substance on the other hand is more difficult to characterise.

Keywords: Di(2-ethylhexyl) phthalate (DEHP); Oral administration; Sus scrofa domestica ; Pharmacokinetics; Chromatography

Reliability of non-invasively acquired human genomic DNA as a substrate for real-time PCR-assisted analysis of genetic polymorphisms by T. Neuhaus; G. Geisen; H. M. Bolt; V. Janzen; A. Kraemer; H. Vetter; Y. Ko (pp. 390-396).

Molecular epidemiological studies require high numbers of participants. The combination of an non-invasive access to human DNA with a rapid genotyping analysis, e.g. by use of LightCycler assisted real-time polymerase chain reaction (PCR), can be helpful in conducting such trials. The aim of our study was to define, for the first time, the use of LightCycler technology in analysis of non-invasively derived DNA. DNA extracted from blood, mouthwash and buccal cytobrush samples from 100 volunteers was analyzed for the genotypes of cytochrome P450 CYP1B1, and glutathione S-transferases GSTT1, GSTM1 and GSTP1. The median amounts of DNA isolated from blood, mouthwash and buccal cytobrush samples were 95, 11 and 8 µg, respectively. While genotyping for CYP1B1 codon 432 polymorphism and GSTP1 codon 105 polymorphism resulted in a complete correspondence for all three modes of sampling, the identification of individuals with null-genotype for GSTT1 or GSTM1 failed in some cases due to atypical courses of the corresponding melting curves, leading to high false-positive rates in the group of non-invasively derived samples. Thus, the results presented here call for caution in using LightCycler assisted real-time PCR in non-invasively collected samples, at least when appropriate control strategies are not implemented.

Keywords: Real-time PCR; Non-invasive DNA; Polymorphisms

Detection of acrolein–lysine adducts in plasma low-density lipoprotein and in aorta of cyclophosphamide-administered rats by Devi Arikketh; Sivasithambaram Niranjali; Halagowder Devaraj (pp. 397-401).

Cyclophosphamide (CY) is an alkylating agent used for the treatment of various types of cancer and is also used as a potent immunosuppressant. Acrolein, a metabolite of CY is cytotoxic and has the ability to covalently bind with proteins in vitro to form acrolein–protein adducts. These protein adducts are considered to be putative markers of oxidative stress and cause damage to protein in aging, atherosclerosis and diabetes. We have, for the first time, detected acrolein–lysine adducts in plasma low-density lipoprotein (LDL) and in the aorta of CY-treated animals by agarose gel electrophoresis, immunoblot and immunohistochemical methods. The extent of lipid peroxidation caused by the metabolite acrolein in plasma LDL was also measured quantitatively by using high-performance liquid chromatography. These results confirm the role of acrolein–lysine adducts in the development of atherosclerosis or atherogenesis.

Keywords: Cyclophosphamide; Acrolein; Low-density lipoprotein; Acrolein–lysine adducts

Changes in the glutathione system of lung cell lines after treatment with hydrocortisone by Udo I. Walther (pp. 402-409).

Administration of anti-inflammatory glucocorticoids is a drug option in the therapy of acute respiratory distress syndrome (ARDS), according to present pathophysiological concepts. Surprisingly, glucocorticoids failed to show beneficial effects. This failure is not understood. In this investigation changes in the glutathione system due to hydrocortisone were found to consist of glutathione depletion and lowered glutathione reductase activities in alveolar epithelial type II cells, contrasted with unchanged activities in a fibroblast-like lung cell line. The glutathione system is thought to be the most important cellular antioxidative system and therefore alveolar epithelial type II cells might be more susceptible to oxidative stress after glucocorticoid treatment. As alveolar epithelial type II cells may be important targets in ARDS, because of their functions (stem cells of type I epithelial cells; surfactant synthesis), these changes might provide an explanation for the failure of glucocorticoids. In the present experiments the capability of hydrocortisone-treated alveolar epithelial type II cells to synthesise glutathione was found to be cysteine dependent at physiological concentrations. Transposing this observation to the in vivo situation, it might be expected that glucocorticoid efficacy in ARDS therapy requires co-administration of substances that increase glutathione synthesis, e.g. N-acetylcysteine.

Keywords: Glucocorticoids; Lung fibroblasts; Alveolar epithelial type II cells; ARDS

Formic acid excretion in rats and mice exposed to bromodichloromethane: a possible link to renal tubule cell proliferation in long-term studies by Ted Lock; Lisa Cottrell; Tony Soames; Matt Jacobsen; Rebecca Williams (pp. 410-417).

Male F344 rats exposed to bromodichloromethane (BDCM) by gavage at 50 or 100 mg/kg/day for 5 days a week for 28 days excreted large amounts of formic acid in their urine, which was accompanied by a change in urinary pH. Male B6C3F1 mice exposed to BDCM at 25 or 50 mg/kg/day for 5 days a week for 28 days also excreted increased amounts of formic acid in their urine. In rats, formate excretion was dose and time dependant, being markedly elevated after four doses and remaining at that level after 3 weeks of dosing at 100 mg/kg/day BDCM, while at 50 mg/kg/day there was some suggestion of a decline after 3 weeks. In contrast, in mice formate excretion did not start to a major extent until 3 weeks of dosing, with the biggest response at 4 weeks. There was no increase in clinical chemistry markers of liver or kidney injury in either rats or mice following 28-day exposure to BDCM. However, morphological examination of the kidneys showed some mild renal tubule injury in two out of five rats exposed to 100 mg/kg/day BDCM. This was associated with a marked increase in cell proliferation in the renal cortex of all rats exposed to 100 mg/kg/day. No increase in cell proliferation was seen in the renal cortex of rats exposed to BDCM at 50 mg/kg/day, or in mice exposed to 25 or 50 mg/kg/day BDCM for 28 days. Long-term exposure to formic acid is known to cause kidney damage, suggesting that excretion of this acid may be a contributory factor to the increase in cell proliferation and kidney damage seen in the longer-term studies with BDCM.

Keywords: Bromodichloromethane; Formic acid; Nephrotoxicity; Renal cancer; Renal tubule cell proliferation; ¹H-NMR

Effects of ciprofloxacin on joint cartilage in immature dogs immediately after dosing and after a 5-month treatment-free period by Eckhard von Keutz; Christine Rühl-Fehlert; Wolfgang Drommer; Martin Rosenbruch (pp. 418-424).

A study in young beagle dogs was performed in which the animals were treated for 2 weeks with ciprofloxacin at oral doses of 0, 10, 30 or 90 mg/kg per day. Immediately after treatment half of the number of animals were killed and all weight-bearing joints were subject to a thorough gross and histopathological investigation, including special staining of the cartilage matrix, and immunohistochemistry as well as electron microscopy. The remaining animals were maintained for an additional 5-months treatment-free period before being killed. Again, all weight-bearing joints were subject to a thorough gross and histopathological investigation. After 14 days of treatment with ciprofloxacin, oral doses of 30 and 90 mg/kg induced the characteristic arthropathy (blisters, erosions) in juvenile beagle dogs. As expected the lesions persisted while the animals were growing. In contrast, and to our knowledge demonstrated for the first time, an oral dose of 10 mg/kg ciprofloxacin did not induce joint lesions after short-term treatment in juvenile beagle dogs and was also not associated with arthrotoxicity when the dogs became older.

Keywords: Juvenile beagle dogs; Detection of latent arthrotoxicity; Ciprofloxacin

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Archives of Toxicology (v.78, #7)