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Archives of Toxicology (v.71, #6)

Protective effect of deferoxamine on chromium(VI)-induced DNA single-strand breaks, cytotoxicity, and lipid peroxidation in primary cultures of rat hepatocytes by Nobuyuki Susa; Shunji Ueno; Yoshinori Furukawa; Masayasu Sugiyama (pp. 345-350).

Incubation of primary cultures of rat hepatocytes with K₂Cr₂O₇ and deferoxamine (DFO), an iron chelator, resulted in a marked decrease in cellular levels of DNA single-strand breaks caused by K₂Cr₂O₇. Cellular treatment with DFO also suppressed both dichromate-induced cytotoxicity – evaluated by the leakage of lactate dehydrogenase, and lipid peroxidation – as monitored by malondialdehyde formation. In addition, treatment with DFO attenuated the suppression of the levels of vitamin E and C as well as the inhibition of alkaline phosphatase and glutathione peroxidase activity attributed to K₂Cr₂O₇. However, DFO had no influence on the cellular level of glutathione or the activity of glutathione reductase and superoxide dismutase suppressed by dichromate. Under the same experimental conditions, cellular uptake and distribution of chromium were not affected by DFO. These results indicate that DFO protects cells from chromium(VI)-induced DNA strand breaks, cytotoxicity, lipid peroxidation, vitamin E and C depression, and glutathione peroxidase inhibition. The role of antioxidants in chromium(VI)-induced cytotoxicity, DNA breaks, and lipid peroxidation is discussed.

Keywords: Key words Chromium(VI) ; Deferoxamine ; DNA breaks ; Cytotoxicity ; Lipid peroxidation

Quantitation of α2-microglobulin after administration of structurally divergent chemical compounds by H. Hildebrand; E. Hartmann; A. Popp; E. Bomhard (pp. 351-359).

α₂-Microglobulin-induced nephropathy is a phenomenon which is exclusively found in adult male rats. Various chemicals are able to bind to α₂-microglobulin thus inhibiting its proteolytic degradation in lysosomes of the P2 segment of the rat nephron. The accumulation of this protein in `protein droplets' or `hyaline droplets' leads to necrosis, followed by regeneration which possibly later results in the formation of tumours. Here we report the development of a monoclonal antibody which is specific for α₂-microglobulin. It was utilized to measure α₂-microglobulin concentrations in plasma and tissues, and to stain α₂-microglobulin in fixed tissue slides. In two studies we administered to adult male Wistar rats two groups of compounds: (1) one group of structurally diverse compounds, which give an overview of chemical entities capable of inducing the accumulation of α₂-microglobulin; and (2) another group of structurally closely related compounds (i.e. substituted benzene derivatives) for the purpose of elucidating possible structure-activity relationships. The degree of α₂-microglobulin-induced nephropathy was determined by immunohistochemical staining of kidney sections. In addition, liver and kidney tissue and plasma concentrations of α₂-microglobulin were measured by competitive ELISA. Liver tissue and plasma concentrations of α₂-microglobulin were not found to be elevated whereas kidney tissue concentrations were higher than the controls. The increase over control values ranged␣from 154% (1,4-dichloromethyl-benzene) to 321% [α-methyl-4-(1-methylethyl)-cyclohexanemethanol]. Comparing structurally related benzene derivatives, the hyaline droplet accumulating (HDA) potential was found to depend both on the type of substituent and its position at the aromatic ring. In general HDA activity increased in the order benzene ≅ phenol ≅ alkylated phenols < halogenated phenols < halogenated␣benzenes. Further QSAR studies are needed to provide a theoretical base for these observations.

Keywords: Key words Hyaline droplet nephropathy ; Plasma concentrations ; Monoclonal antibody ; Xenobiotics ; Structure-activity relationship

Toxicity and cytotoxicity of nigrin b, a two-chain ribosome-inactivating protein from Sambucus nigra : comparison with ricin by Maria Giulia Battelli; Lucía Citores; Laura Buonamici; J. Miguel Ferreras; F. M. de Benito; Fiorenzo Stirpe; Thomás Girbés (pp. 360-364).

Nigrin b, a lectin isolated from the bark of elderberry (Sambucus nigra L.), has structure and enzymatic activity similar to that of ricin and other type 2 ribosome-inactivating proteins (RIPs), and yet is much less toxic to cells and animals. In an attempt to explain this difference, we studied (1) the cytotoxicity of both lectins at 18 and 37 °C, and in the presence of substances interfering with intracellular routing, and (2) the binding of nigrin b to, and its uptake and degradation by HeLa cells, in parallel with ricin. As compared with the latter, (1) less nigrin b was bound and more was degraded by cells, with a resulting lower concentration remaining inside the cells, and (2) there is evidence for a different intracellular routing followed by the two lectins. These results may explain at least partly the different cytotoxicity and consequently the lower toxicity to mice of nigrin b compared with ricin.

Keywords: Key words Nigrin b ; Ribosome-inactivating proteins ; Lectins ; HeLa cells ; Toxicity to mice

Induction of micronuclei with ochratoxin A in ovine seminal vesicle cell cultures by G. H. Degen; M. M. Gerber; S. Obrecht-Pflumio; G. Dirheimer (pp. 365-371).

The genotoxic potential of the carcinogenic mycotoxin ochratoxin A (OTA) has been investigated by means of an in vitro micronucleus assay, an endpoint for genotoxicity which has not been studied previously for OTA. OTA was found to induce dose-dependently micronuclei (MN) in cytokinesis-blocked binucleated ovine seminal vesicle (OSV) cell cultures, which had been treated with the mycotoxin (12–30 μM) for 6 h in medium containing 10% fetal calf serum. For comparison, OSV cells were treated with colcemid (0.02–0.06 μg/ml), or 4-nitroquinoline N-oxide (NQO; 0.5 μM), a typical aneugen and clastogen, respectively. All test compounds increased the frequency of MN in OSV cells, the highest level being induced by 30 μM OTA. When MN were characterized by indirect immunofluorescence microscopy using anti-kinetochore (CREST) antibodies, the majority of MN in colcemid-treated cells was CREST-reactive (>70% kinetochore positive); as expected, this fraction was <10% for the NQO-treatment group. In cells treated with OTA the fraction of kinetochore positive MN was similar (33–40%) to that observed in solvent controls (38%). These data indicate that OTA induces MN apparently by a mixed, although predominantly clastogenic mode of action. OSV cells lack monooxygenase activity but express high prostaglandin H synthase (PGHS) activity. When cells were treated with OTA in the presence of indomethacin (10 and 50 μM), a well known inhibitor of PGHS, the frequency of MN induced by OTA was not decreased, but rather increased. This indicates that metabolic activation of OTA by PGHS seems not to be required for genotoxicity. The increased MN induction in OSV cell cultures is most likely due to competition of indomethacin with OTA for binding to serum proteins thus raising the fraction of free mycotoxin.

Keywords: Key words Ochratoxin A ; Genotoxicity ; Micronuclei ; Ovine seminal vesicle cells ; Clastogen

Effect of subchronic 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on immune system and target gene responses in mice: calculation of benchmark doses for CYP1A1 and CYP1A2 related enzyme activities by Christoph Vogel; Susanne Donat; Olaf Döhr; Joachim Kremer; Charlotte Esser; Markus Roller; J. Abel (pp. 372-382).

The dose-effect relationships were analysed for several noncarcinogenic endpoints, such as immunological and biochemical responses at subchronic, low dose exposure of female C57BL/6 mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The animals were treated i.p. with TCDD according to the initial- and maintenance-dose principle for a period of 135 days. The initial doses were 1, 10 and 100 ng TCDD/kg, the weekly maintenance doses were 0.2, 2 and 20 ng TCDD/kg, respectively. At days 23, 79 and 135 of TCDD treatment 10 animals of each dose group were killed. As immunological parameters the number of thymocytes and the pattern of thymocyte subpopulations were determined. In liver, lung and thymus, mRNA expression of TGF-α, TGF-β₁, TGF-β₂, TGF-β₃, TNF-α, IL-1β and different CYP1 isoforms (CYP1A1, CYP1A2, CYP1B1) was analysed. In the livers, activities of 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-demethylase (MROD) were measured. TCDD content in the liver was determined. The main results are summarized as follows: (1) The TCDD doses were not sufficient to elicit dose-dependent changes of pattern of thymocyte subpopulation. (2) TCDD failed to change the mRNA expression of TGF-α, TGF-β and TNF-α, but led to an increase of IL-1β mRNA expression in liver, lung and thymus. The results show that the TCDD induced IL-1β mRNA increase is at least as sensitive a marker as the induction of CYP1A isoforms. (3) The expression of CYP1B1 mRNA remained unchanged at the doses tested, while CYP1A1 and CYP1A2 mRNA expression was dose-dependently enhanced. EROD and MROD activities in the liver paralleled the increases of CYP1A1 and CYP1A2 mRNA expression. (4) Regression analysis of the data showed that most of the parameters tested fit a linear model. (5) From the data, a benchmark dose for EROD/MROD activities in the livers of female C57BL/6 mice of about 0.03 ng TCDD/kg per day was calculated.

Keywords: Key words TCDD ; Cytochrom P450 ; Cytokines ; Immunesystem ; Benchmark dose

2,3,7,8-Tetrachlorodibenzo-p -dioxin (TCDD) and congeners in infants. A toxicokinetic model of human lifetime body burden by TCDD with special emphasis on its uptake by nutrition by P. E. Kreuzer; G. A. Csanády; C. Baur; W. Kessler; O. Päpke; H. Greim; J. G. Filser (pp. 383-400).

Contents of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and of 16 further congeners – polychlorinated dibenzodioxins and dibenzofuranes (PCDD/PCDF) – were determined in lipids of adipose tissue and of livers of 3 stillborns and of 17 infants (0.43–44 weeks old) who died from sudden infant death syndrome. International toxic equivalents (I-TEq) calculated for the sum of TCDD together with all of the 16 congeners (1.55–29.63 ng/kg lipids of adipose tissue, n = 20; 2.05–57.73 ng/kg liver lipids, n = 19) were within the range of or lower than the values published for adults. TCDD concentrations in lipids of breast-fed infants were higher (0.38–4.1 ng/kg lipids of adipose tissue, n = 9; 0.49–3.9 ng/kg liver lipids, n = 8) compared to non breast-fed subjects (0.16–0.76 ng/kg lipids of adipose tissue, n = 8; 0.29–0.71 ng/kg liver lipids, n = 7). Neither I-TEq values nor TCDD concentrations exceeded values published for adults. Since even in stillborns PCDD/PCDF were found (I-TEq, 9.70–10.83 ng/kg lipids of adipose tissue, 6.17–8.83 ng/kg liver lipids; TCDD, 1.3–2.1 ng/kg lipids of adipose tissue, 0.76–1.5 ng/kg liver lipids; n = 3), transplacental exposure has to be deduced. All of the findings concerning TCDD concentrations in the organism become intelligible on the basis of a physiological toxicokinetic model which was developed to describe the body burden of TCDD for the entire human lifetime in dependence of TCDD uptake from contaminated nutrition. The model reflects sex and age dependent changes in the following parameters: body weight, volumes of liver, adipose and muscle tissue, food consumption, and excretion of faeces. TCDD is supposed to be taken up orally, to be distributed freely in lipids of the organism and to be eliminated unchanged by excretion in lipids of faeces as well as by metabolism in the liver. The model was used to predict the half-life of elimination of TCDD (4 months in newborns increasing to ∼5 years in adults) and concentrations of this compound in lipids of adipose tissue, blood, liver and faeces at different ages. Furthermore, the influence of breast-feeding on the TCDD burden of a mother, her milk and her child was simulated. The model was validated by means of own data gained in adipose tissue and livers of infants and also using a series of values measured by other authors in mother's milk and in tissues and faeces of infants and adults. Predictions as well as experimental findings demonstrate a distinct increase in the TCDD body burden of breast-fed infants. Generally, it can be concluded for the excretion of unchanged, non volatile, non protein bound highly lipophilic compounds that their half-life is short in infants (∼5 months) and increases to ∼10 years reached between 40 and 60 years of age.

Keywords: Key words Polychlorinated dibenzodioxins ; 2; 3; 7; 8-Tetrachlorodibenzo-p-dioxin; Polychlorinated dibenzofuranes; Toxicokinetics; Infant Human; Adipose tissue; Liver

Cytochrome P450-dependent drug oxidation activities in liver microsomes of various animal species including rats, guinea pigs, dogs, monkeys, and humans by Tsutomu Shimada; Mayumi Mimura; Kiyoshi Inoue; Sei-ichi Nakamura; Hajime Oda; Shigeru Ohmori; Hiroshi Yamazaki (pp. 401-408).

Levels of cytochrome P450 (P450 or CYP) proteins immunoreactive to antibodies raised against human CYP1A2, 2A6, 2C9, 2E1, and 3A4, monkey CYP2B17, and rat CYP2D1 were determined in liver microsomes of rats, guinea pigs, dogs, monkeys, and humans. We also examined several drug oxidation activities catalyzed by liver microsomes of these animal species using eleven P450 substrates such as phenacetin, coumarin, pentoxyresorufin, phenytoin, S-mephenytoin, bufuralol, aniline, benzphetamine, ethylmorphine, erythromycin, and nifedipine; the activities were compared with the levels of individual P450 enzymes. Monkey liver P450 proteins were found to have relatively similar immunochemical properties by immunoblotting analysis to the human enzymes, which belong to the same P450 gene families. Mean catalytic activities (on basis of mg microsomal protein) of P450-dependent drug oxidations with eleven substrates were higher in liver microsomes of monkeys than of humans, except that humans showed much higher activities for aniline p-hydroxylation than those catalyzed by monkeys. However, when the catalytic activities of liver microsomes of monkeys and humans were compared on the basis of nmol of P450, both species gave relatively similar rates towards the oxidation of phenacetin, coumarin, pentoxyresorufin, phenytoin, mephenytoin, benzphetamine, ethylmorphine, erythromycin, and nifedipine, while the aniline p-hydroxylation was higher and bufuralol 1′-hydroxylation was lower in humans than monkeys. On the other hand, the immunochemical properties of P450 proteins and the activities of P450-dependent drug oxidation reactions in dogs, guinea pigs, and rats were somewhat different from those of monkeys and humans; the differences in these animal species varied with the P450 enzymes examined and the substrates used. The results presented in this study provide useful information towards species-related differences in susceptibilities of various animal species regarding actions and toxicities of drugs and xenobiotic chemicals.

Keywords: Key words Cytochrome P450 ; CYP ; Species ; Drug metabolism ; Liver microsomes

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Archives of Toxicology (v.71, #6)