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Archives of Toxicology (v.71, #9)

The inhalation acute toxic class method: test procedures and biometric evaluations by W. Diener; D. Kayser; E. Schlede (pp. 537-549).

A method of inhalation acute toxic (ATC) classification is presented with the use of significantly fewer animals in comparison with the classical LC₅₀ test. The principle of the inhalation ATC method is based on the oral ATC method, which has been adopted in 1996 as an official test guideline of the OECD and the European Union. The inhalation ATC method, like the oral ATC method, is a stepwise procedure; three animals of each sex are used simultaneously for each tested concentration, and not, as in the oral ATC method, three animals of each sex separately for each dose. The method was developed for three starting concentrations and two reference systems (based on ppm and mg/l). Depending on the LC₅₀, slope, classification system and starting concentration, on average 50 to 80% fewer animals will be used in comparison to at least 30 animals with the classical LC₅₀ test. The method was biometrically evaluated with the use of the probit model for dose-response relationships. At present, there are 12 different international classification systems based on LC₅₀ values: 6 systems referring to mg/litre and 6 systems based on ppm values, and exposure time varying from 1 to 4 h. The test procedures and the calculations of the classification probabilities demonstrate that the inhalation ATC method is a reliable alternative to the classical LC₅₀ test with the use of significantly fewer animals. Classification probabilities are presented for all classification systems currently in use, and expected numbers of experimental and of moribund/dead animals are demonstrated for one system of each reference system and for all three starting concentrations. The conclusion is justified that there is no need to validate the inhalation ATC method with the use of experimental animals.

Keywords: Key words Acute toxic class (ATC) method (inhalation); LC50 test; Probit model; Classification probabilities; Animal welfare

Hepatotoxicity of diisopropyl ester of malonic acid and chloromalonic acids, disinfection by-products of the fungicide isoprothiolane by H. Jinno; N. Hanioka; S. Nishikawa; R. Yoda; T. Toyo'oka; T. Nishimura; M. Ando (pp. 550-555).

The fungicide isoprothiolane (diisopropyl 1,3-dithiolane-2-ylidenemalonate) decomposes to the diisopropyl esters of malonic acid (DM), chloromalonic acid (DCM) and dichloromalonic acid (DDCM) upon aqueous chlorination. In this study, the cytotoxicity of these compounds was examined using rat hepatocytes cultured on Matrigel. DCM and DDCM caused hepatocellular death at concentrations >0.5 mM, while DM had no effect on the cell viability even at the maximum concentration examined (4 mM). Significant lipid peroxidation, measured as 2-thiobarbituric acid reactive substances, was observed in both DCM- and DDCM-treated hepatocyte cultures, and was significantly enhanced by pretreatment with 0.1 mM bis( p-nitrophenyl)phosphate (BNPP), a carboxylesterase inhibitor. When both BNPP and SKF-525A, a cytochrome P450 inhibitor, were present in the medium, DCM-induced cytotoxicity and lipid peroxidation were significantly suppressed compared to cultures with BNPP-treatment alone. By contrast, the DDCM-induced cytotoxicity was not affected by the combined pretreatment of SKF-525A and BNPP. These results indicate that DCM is metabolically activated by cytochrome P450 in an ester form, while DDCM is activated by a mechanism other than one involving cytochrome P450. To further elucidate the cytochrome P450 isozyme involved in the metabolic activation of DCM, microsomal lipid peroxidation was studied in vitro using microsomes from rats treated with β-naphthoflavone, musk xylene, phenobarbital, pyrazole, or dexamethasone. Among these preparations, the microsomes from dexamethasone-treated rats showed the most extensive lipid peroxidation in the presence of DCM, and the lipid peroxidation was enhanced by BNPP as observed in hepatocyte cultures. These findings suggest the possible involvement of cytochrome P450 3A in the metabolic activation of DCM.

Keywords: Key words Diisopropyl chloromalonates; Rat hepatocytes; Lipid peroxidation; Cytochrome P450 Carboxylesterase

Toxic effect of concomitant administration of cyclosporin A and acyclovir on renal function and morphology in rats by Jörg. Hannemann; Werner Wunderle; Tarik Yousif; Stefan Krüger; Karl Baumann (pp. 556-562).

The immunosuppressive agent cyclosporin A (CyA) and the antiviral drug acyclovir may cause renal functional impairment. CyA-induced immunosuppression increases the rate of viral infections. Therefore we were interested to determine whether short-term co-administration of CyA and acyclovir involves an increased nephrotoxic risk. Male Wistar rats were treated with CyA (20 mg/kg body wt., s.c., once daily for 8 days), acyclovir (15 mg/kg body wt., s.c., 3-times daily for the last 5 days) or a combination of CyA and acyclovir. Blood levels of CyA were determined after a single dose. Urine was monitored for volume, osmolality, total protein and N-acetyl-β-d-glucosaminidase (β-NAG). Concentrations of blood urea nitrogen (BUN) and plasma-creatinine were determined (day 9). Renal cortical slices were monitored for accumulation of weak organic acids (para-aminohippurate, PAH) and bases (tetra-ethylammonium, TEA) and for malondialdehyde (MDA) content. Renal histology was also examined. CyA induced a decrease in body and kidney weight, in urine osmolality and in the excretion of total protein. Plasma-creatinine and BUN as well as MDA content of renal tissues were increased by CyA. Acyclovir alone did not induce significant changes. In comparison to CyA values, urine volume and β-NAG excretion were enhanced and TEA accumulation depressed by the concomitant administration of CyA and acyclovir. CyA- or acyclovir-treatment alone did not result in significant morphological changes. In the group co-administered CyA/acyclovir, the kidneys showed mild to moderate signs of tubulopathy. Short-term co-administration of CyA and acyclovir was concluded to have possibly increased nephrotoxic potential.

Keywords: Key wordsCo-administration; Cyclosporin A; Acyclovir; Nephrotoxicity; Rat

Impaired cellular immune response in rats exposed perinatally to Baltic Sea herring oil or 2,3,7,8-TCDD by Peter S. Ross; Rik L. de Swart; Helen van der Vliet; Linette Willemsen; Arja de Klerk; Geert van Amerongen; Jan Groen; Abraham Brouwer; Ineke Schipholt; Dennis C. Morse; Henk van Loveren; Albert D. M. E. Osterhaus; Joseph G. Vos (pp. 563-574).

While the immunotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been well established, the effects of complex environmental mixtures of polyhalogenated aromatic hydrocarbons (PHAHs) are poorly understood. Many PHAHs, including the polychlorinated-biphenyls (PCBs), -dibenzofurans (PCDFs), and dibenzo-p-dioxins (PCDDs), possess `dioxin-like' activities, and accumulate in the aquatic food chain. Organisms occupying high trophic levels may therefore be exposed to concentrations which may present an immunotoxic risk. In this study, pregnant PVG rats were administered a daily oral dose of 1 ml of the following during pregnancy and lactation: (1) oil extracted from herring caught in the relatively uncontaminated Atlantic Ocean; (2) oil extracted from herring caught in the contaminated Baltic Sea; or (3) the Atlantic herring oil extract spiked with 2,3,7,8-TCDD. The daily intakes of aryl hydrocarbon (Ah)-receptor dependent toxic equivalents (TEQ) for mothers were 0.3 in the Atlantic group, 2.1 in the Baltic group, and 134 ng/kg body wt. in the 2,3,7,8-TCDD positive control group. Immune function and host resistance to rat cytomegalovirus (RCMV) were assessed in offspring aged 11, 25, 46 or 59 days. Rat pups in the positive control TCDD-spiked group exhibited immunosuppression characterized by reduced thymus weight and cellularity, reduced thymocyte and splenocyte proliferative responses to T-dependent mitogens in vitro, reduced virus-associated natural killer (NK) cell and specific antibody responses. While less pronounced, a similar pattern of effects was observed in the rat pups exposed only to the Baltic Sea herring oil. These immunotoxic effects were transient in both exposure groups, with a time-related recovery in immune function possibly due to the half-life of TCDD in rats and the waning exposure levels in the rapidly growing pups. We previously demonstrated that the same Baltic Sea herring led to impaired natural killer cell and T-lymphocyte function in harbour seals during the course of a long-term captive feeding study. The collective results of these studies in rats and seals indicate the immunotoxic potential of environmental mixtures at current levels in the aquatic environment, and suggest that the developing immune system of young mammals may be at particular risk.

Keywords: Key words Food chain; Host resistance; Immunotoxicology; Polychlorinated biphenyls (PCBs) 2; 3; 7; 8-tetrachlorodibenzo-p-dioxin (TCDD)

Inhibition instead of enhancement of lipid peroxidation by pretreatment with the carcinogenic peroxisome proliferator nafenopin in rat liver exposed to a high single dose of corn oil by Wolfgang W. Huber; Bettina Grasl-Kraupp; Herbert Stekel; Christina Gschwentner; Harald Lang; Rolf Schulte-Hermann (pp. 575-581).

Oxidative stress is discussed as a possible hepatocarcinogenic mechanism of peroxisome proliferators (PP) in rodents and is suggested to result from the induction of peroxisomal β-oxidation (PBOX) by PP. The induced PBOX is assumed to produce excessive H₂O₂ from the degradation of fatty acids, ultimately leading to oxidative stress and lipid peroxidation. In the present short term-study, we attempted to stimulate lipid peroxidation in male Wistar rats by (1) inducing PBOX enzymes with the peroxisome proliferator nafenopin at 90 mg/kg body weight per day in the diet for 10–11 days, and (2) by supplying the induced PBOX with an abundant amount of fatty acid as substrate, using a corn oil gavage at 20 ml/kg body weight. The corn-oil gavage alone, i.e. without preceding nafenopin treatment, enhanced liver triacylglycerol nine- to tenfold and hepatic lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS), was increased 50% compared with controls. Both observations were made after 18 h when the peak elevations occurred. Upon pretreatment with nafenopin, asssociated with a sevenfold induction of PBOX, the corn oil gavage however caused only a threefold maximal increase in hepatic triacylglycerol, also at the 18 h time-point; TBARS remained almost at control levels, as monitored at seven time points over 24–25 h. These results suggest that nafenopin reduces rather than enhances lipid peroxidation, despite the provision, in a short term study, of high doses of substrate to the induced enzyme system that is hypothetically causing oxidative stress in the liver.

Keywords: Key words Peroxisome proliferator; Nafenopin; Fat; Oxidative stress; Lipid peroxidation; Rat liver

Relationship between acute toxicity of (bis)aziridinylbenzoquinones and cellular pyridine nucleotides by Wingston A. Morgan; Bram Prins; Andries S. Koster (pp. 582-587).

The toxicity of aziridinylbenzoquinones may occur by a number of mechanisms, including oxidative stress caused by redox cycling and the activation of the aziridine groups. Isolated hepatocytes were used to assess the relationship between the redox status of NADP(H) associated with oxidative stress, the level of NAD(H) closely linked with DNA repair and the cytotoxicity of three 2,5-bis(aziridinyl)-1,4-benzoquinones (BABQ). Exposure of hepatocytes to the BABQ TW13 (200 μM) and TW25 (100 μM), which are able to arylate and to redox cycle, resulted in increased intracellular NADP⁺ from <0.3 nmol/mg protein to 1.5 nmol/mg protein within 60 min. The increase in intracellular NADP⁺ was followed by the onset of cell death by 180 min. In contrast, exposure to lower concentrations of TW13 (100 μM), TW25 (50 μM) and carboquone (100–200 μM) (which neither arylates nor redox cycles via a one-electron reduction) resulted in a less pronounced (<1.0 nmol/mg) increase in NADP⁺ and there was no evidence of cell death within the 180 min incubation. BABQ had a concentration dependent effect on intracellular NAD⁺. Exposure of hepatocytes to TW13 (200 μM) and TW25 (100 μM) resulted in a decrease in intracellular NAD⁺ from >2.7 to <1.0 nmol/mg protein within 60 min. At concentrations of the BABQ where the level of NAD⁺ remained >1.0 nmol/mg protein after 30 min, the hepatocytes remained viable at 180 min. These changes in intracellular pyridine nucleotides suggests two mechanisms may be involved in BABQ cytotoxicity. At high concentrations, aziridinylbenzoquinones may cause cytotoxicity via oxidative stress following redox cycling. At lower concentrations, however, the predominant pyridine nucleotide change is a prolonged depletion of NAD⁺, suggesting extensive DNA damage which may lead to delayed cell death.

Keywords: Key words (bis)Aziridinyl-benzoquinones; Pyridine nucleotide; Oxidative stress; Redox cycling

Styrene oxide DNA adducts: in vitro reaction and sensitive detection of modified oligonucleotides using capillary zone electrophoresis interfaced to electrospray mass spectrometry by Wolfgang Schrader; Michael Linscheid (pp. 588-595).

Styrene is one of the most important synthetic chemicals in the world and is subject to investigations concerning carcinogenicity and mutagenicity due to the active metabolite, styrene-7,8-oxide. This epoxide shows a tendency to react, among others, with DNA and DNA constituents. The in vitro reaction of styrene oxide with DNA was investigated by cleaving incubated calf thymus DNA with two different enzymes, namely Benzonase and alkaline phosphatase, to obtain oligonucleotides of the type n-nucleotide-(n− 1)-phosphate with chain length from 2 to 8 bases. Alkylated and non-alkylated nucleotides were separated in groups according to their chain length using capillary zone electrophoresis and were detected with electrospray mass spectrometry. This improvement in sensitivity made it possible to obtain new information about the reaction of styrene oxide with DNA, especially to detect unknown reaction products. The results indicate that primarily purine bases were alkylated by styrene oxide before pyrimidine bases, which react with higher concentrations of styrene oxide. This means that in addition to the already reported adducts in DNA at the N-7-, O⁶- and N²-position of guanine also adducts at the nucleophilic sites of adenine can be found using mass spectrometry. We anticipate for the future this procedure will allow us to investigate base sequence specific reactions as well as interactions from xenobiotics and cytostatic drugs, since reaction products would directly be detectable.

Keywords: Key words DNA adducts; Styrene oxide; Capillary electrophoresis/mass spectrometry; Oligonucleotides; Nucleotide modification

Influence of polymorphisms of GSTM1 and GSTT1 for risk of renal cell cancer in workers with long-term high occupational exposure to trichloroethene by Thomas Brüning; Marga Lammert; Manuela Kempkes; Ricarda Thier; Klaus Golka; Hermann M. Bolt (pp. 596-599).

Suspected nephrocarcinogenic effects of trichloroethene (TRI) in humans are attributed to metabolites derived from the glutathione transferase (GST) pathway. The influence of polymorphisms of GSTM1 and GSTT1 isoenzymes on the risk of renal cell cancer in subjects having been exposed to high levels of TRI over many years was investigated. GSTM1 and GSTT1 genotypes were determined by internal standard controlled polymerase chain reaction. Fourty-five cases with histologically verified renal cell cancer and a history of long-term occupational exposure to high concentrations of TRI were studied. A reference group consisted of 48 workers from the same geographical region with similar histories of occupational exposures to TRI but not suffering from any cancer. Among the 45 renal cell cancer patients, 27 carried at least one functional GSTM1 gene (GSTM1+) and 18 at least one functional GSTT1 gene (GSTT1+). Among the 48 reference workers, 17 were GSTM1+ and 31 were GSTT1+. Odds ratios for renal cell cancer were 2.7 for GSTM1+ individuals (95% CI, 1.18–6.33; P < 0.02) and 4.2 for GSTT1+ individuals (95% CI, 1.16–14.91; P < 0.05), respectively. The data support the present concept of the nephrocarcinogenicity of TRI.

Keywords: Key words Trichloroethene; Renal cell cancer; GSTM1; GSTT1; Glutathione transferases

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Archives of Toxicology (v.71, #9)

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Archives of Toxicology (v.71, #9)