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Archives of Toxicology (v.81, #5)

OECD validation of the rodent Hershberger assay using three reference chemicals; 17α-methyltestosterone, procymidone, and p,p′-DDE by Jae-Ho Shin; Hyun Ju Moon; Il Hyun Kang; Tae Sung Kim; Su Jung Lee; Ji Youn Ahn; Hoon Bae; Eui Bae Jeung; Soon Young Han (pp. 309-318).

The rodent Hershberger assay is being validated as an in vivo test method for detecting androgenic or antiandrogenic compounds by the Organization for Economic Cooperation and Development (OECD). As part of the international validation work, we studied 17α-methyltestosterone for evaluating androgenic activity, and procymidone and p,p′-DDE for evaluating antiandrogenic activity. Male Sprague–Dawley rats were castrated at postnatal day 42, and only the rats that showed preputial separation were used in this study. Seven days after castration, chemicals were administered daily by gavages to groups of rats for 10 days, as recommended by OECD phase-2 protocol. Administration of 17α-methyltestosterone induced increases of weights of accessory sex tissues and glands in a dose-dependent manner. Administration of procymidone and p,p′-DDE produced a dose-dependent decrease of weights of accessory sex tissues and glands in the rats co-treated with testosterone propionate (0.4 mg/kg/day) subcutaneously. Our data strongly suggested that the current protocol of OECD Hershberger assay (phase-2) should be used as a reliable method for the detection of endocrine related toxicity of other chemicals.

Keywords: OECD evaluation; Hershberger assay; 17α-methyltestosterone; Procymidone; p,p′-DDE

Ultrastructural and metabolic changes in osteoblasts exposed to uranyl nitrate by D. R. Tasat; N. S. Orona; P. M. Mandalunis; R. L. Cabrini; A. M. Ubios (pp. 319-326).

Exposure to uranium is an occupational hazard to workers who continually handle uranium and an environmental risk to the population at large. Since the cellular and molecular pathways of uranium toxicity in osteoblast cells are still unknown, the aim of the present work was to evaluate the adverse effects of uranyl nitrate (UN) on osteoblasts both in vivo and in vitro. Herein we studied the osteoblastic ultrastructural changes induced by UN in vivo and analyzed cell proliferation, generation of reactive oxygen species (ROS), apoptosis, and alkaline phosphatase (APh) activity in osteoblasts exposed to various UN concentrations (0.1, 1, 10, and 100 μM) in vitro. Cell proliferation was quantified by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, ROS was determined using the nitro blue tetrazolium test, apoptosis was morphologically determined using Hoechst 3332 and APh activity was assayed spectrophotometrically. Electron microscopy revealed that the ultrastructure of active and inactive osteoblasts exposed to uranium presented cytoplasmic and nuclear alterations. In vitro, 1–100 μM UN failed to modify cell proliferation ratio and to induce apoptosis. ROS generation increased in a dose-dependent manner in all tested doses. APh activity was found to decrease in 1–100 μM UN-treated cells vs. controls. Our results show that UN modifies osteoblast cell metabolism by increasing ROS generation and reducing APh activity, suggesting that ROS may play a more complex role in cell physiology than simply causing oxidative damage.

Keywords: Uranium; Osteoblasts; Ultrastructure; Oxidative stress; Alkaline phosphatase

Regulation of metal transporters by dietary iron, and the relationship between body iron levels and cadmium uptake by Dong-Won Kim; Ki-Young Kim; Byung-Sun Choi; Pilju Youn; Doug-Young Ryu; Curtis D. Klaassen; Jung-Duck Park (pp. 327-334).

Iron (Fe) plays essential roles in biological processes, whereas cadmium (Cd) is a toxic and non-essential metal. Two metal transporters, divalent metal transporter 1 (DMT1) and metal transporter protein 1 (MTP1), are responsible for Fe transport in mammals. Here, we studied the effect of dietary Fe on the expression of these metal transporters in peripheral tissues, and the uptake by these tissues of Cd. Mice were fed an Fe-sufficient (FeS: 120 mg Fe/kg) or Fe-deficient (FeD: 2–6 mg Fe/kg) diet for 4 weeks. The total Fe levels in the body were evaluated by measuring tissue Fe concentrations. Tissue Cd concentrations were determined 24 h after the mice received a single oral dose of Cd. Animals fed a FeD diet showed depletion of body Fe levels and accumulated 2.8-fold higher levels of Cd than the FeS group. Quantitative real time RT-PCR revealed that whereas DMT1 and MTP1 were both ubiquitously expressed in all FeS peripheral tissues studied, DMT1 was highly expressed in brain, kidney, and testis, whereas MTP1 was highly expressed in liver and spleen. Depletion of the body Fe stores dramatically upregulated DMT1 and MTP1 mRNA expression in the duodenum as well as moderately upregulating their expression in several other peripheral tissues. The iron response element positive isoform of DMT1 was the most prominently upregulated isoform in the duodenum. Thus, DMT1 and MTP1 may play an important role in not only maintaining Fe levels but also facilitating the accumulation of Cd in the body of mammals.

Keywords: Iron (Fe); Cadmium (Cd); Divalent metal transporter 1 (DMT1); Metal transporter protein 1 (MTP1); Duodenum

Human experimental exposure study on the uptake and urinary elimination of N-methyl-2-pyrrolidone (NMP) during simulated workplace conditions by Michael Bader; Renate Wrbitzky; Meinolf Blaszkewicz; Christoph van Thriel (pp. 335-346).

A human experimental study was carried out with 16 volunteers to examine the elimination of N-methyl-2-pyrrolidone (NMP) after exposure to the solvent under simulated workplace conditions. The NMP concentrations were 10, 40 and 80 mg/m³ for 2 × 4 h with an exposure-free interval of 30 min. Additionally, a peak exposure scenario (25 mg/m³ baseline, 160 mg/m³ peaks for 4 × 15 min, time-weighted average: 72 mg/m³) was tested. The influence of physical activity on the uptake and elimination of NMP was studied under otherwise identical exposure conditions but involving moderate workload on a bicycle ergometer (75 W for 6 × 10 min). The peak times and biological half-lives of urinary NMP and its main metabolites 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) and 2-hydroxy-N-methylsuccinimide (2-HMSI) in urine were analysed as well as the interrelationships between exposure and biomarkers. All analytes showed a close correlation between their post-shift peak concentrations and airborne NMP. An exposure to the current German workplace limit value of 80 mg/m³ under resting conditions resulted in urinary peak concentrations of 2,400 μg/L NMP, 117 mg/g creatinine 5-HNMP and 32 mg/g creatinine 2-HMSI (workload conditions: 3,400 μg/L NMP, 150 mg/g creatinine 5-HNMP, 44 mg/g creatinine 2-HMSI). Moderate workload enhanced the total uptake of NMP by approximately one third. Differences between the estimated and the observed total amount of urinary metabolites point to a significant contribution of dermal absorption on the uptake of NMP. This aspect, together with the influence of physical workload, should be considered for the evaluation of a biological limit value for NMP.

Keywords: N-Methyl-2-pyrrolidone; Biomonitoring; Experimental exposure

Induced secretion of insulin-like growth factor binding protein-1 (IGFBP-1) in human hepatoma cell HepG2 by rubratoxin B by Hitoshi Nagashima; Kumiko Maeda-Nakamura; Keiko Iwashita; Tetsuhisa Goto (pp. 347-351).

The induction of insulin-like growth factor binding protein-1 (IGFBP-1) secretion by rubratoxin B was investigated using human hepatoma cell line HepG2; we also documented the involvement of stress-activated MAP kinases [c-Jun-N-terminal kinases (JNKs) and p38s] in this process. Rubratoxin B dramatically enhanced IGFBP-1 secretion, which peaked at a concentration of 40 μg/ml. The amount of IGFBP-1 mRNA increased with time and plateaued at 6 h. Compared with the amounts of IGFBP-1 secreted, the induction ratios of transcription were much smaller, indicating that IGFBP-1 secretion is regulated chiefly post-transcriptionally. The result of concomitant treatment with rubratoxin B and JNK inhibitor indicated that JNKs do not affect rubratoxin B-induced IGFBP-1 secretion. Alternatively, rubratoxin B-associated induction of IGFBP-1 secretion was marked in the absence of p38 inhibitor but attenuated in its presence. Therefore, p38s appear to stimulate rubratoxin B-induced IGFBP-1 secretion. Treatment with p38 inhibitor slightly increased the amount of rubratoxin B-induced IGFBP-1 mRNA. However this induction ratio was smaller than that of rubratoxin B-induced secretion, suggesting that p38s regulate IGFBP-1 secretion both transcriptionally and post-transcriptionally. In this study, we showed that rubratoxin B induces IGFBP-1 levels in HepG2 cells and p38s contribute to this process.

Keywords: Rubratoxin B; Insulin-like growth factor binding protein-1 (IGFBP-1); Stress-activated MAP kinase (SAPK)

Extrapolating from animal studies to the efficacy in humans of a pretreatment combination against organophosphate poisoning by Aharon Levy; Giora Cohen; Eran Gilat; Joseph Kapon; Shlomit Dachir; Shlomo Abraham; Miriam Herskovitz; Zvi Teitelbaum; Lily Raveh (pp. 353-359).

The extrapolation from animal data to therapeutic effects in humans, a basic pharmacological issue, is especially critical in studies aimed to estimate the protective efficacy of drugs against nerve agent poisoning. Such efficacy can only be predicted by extrapolation of data from animal studies to humans. In pretreatment therapy against nerve agents, careful dose determination is even more crucial than in antidotal therapy, since excessive doses may lead to adverse effects or performance decrements. The common method of comparing dose per body weight, still used in some studies, may lead to erroneous extrapolation. A different approach is based on the comparison of plasma concentrations at steady state required to obtain a given pharmacodynamic endpoint. In the present study, this approach was applied to predict the prophylactic efficacy of the anticholinergic drug caramiphen in combination with pyridostigmine in man based on animal data. In two species of large animals, dogs and monkeys, similar plasma concentrations of caramiphen (in the range of 60–100 ng/ml) conferred adequate protection against exposure to a lethal-dose of sarin (1.6–1.8 LD₅₀). Pharmacokinetic studies at steady state were required to achieve the correlation between caramiphen plasma concentrations and therapeutic effects. Evaluation of total plasma clearance values was instrumental in establishing desirable plasma concentrations and minimizing the number of animals used in the study. Previous data in the literature for plasma levels of caramiphen that do not lead to overt side effects in humans (70–100 ng/ml) enabled extrapolation to expected human protection. The method can be applied to other drugs and other clinical situations, in which human studies are impossible due to ethical considerations. When similar dose response curves are obtained in at least two animal models, the extrapolation to expected therapeutic effects in humans might be considered more reliable.

Keywords: Caramiphen; Clearance; Pharmacokinetics; Prophylaxis; Pyridostigmine; Steady state

Ethylbenzene: 4- and 13-week rat oral toxicity by Werner Mellert; Klaus Deckardt; Wolfgang Kaufmann; Bennard van Ravenzwaay (pp. 361-370).

Ethylbenzene was administered to groups of male and female Wistar rats by gavage for 4 (n = 5/dose/sex) and 13 weeks (n = 10/dose/sex) (OECD 408) at doses of 0 (vehicle control), 75, 250, and 750 mg/kg bodyweight/day (mg/kg bw/day), administered am/pm as half doses. In the 4-week study, ≥250 mg/kg increased serum alanine aminotransferase, total bilirubin and cholesterol, liver weights and centrilobular hepatocyte hypertrophy, and kidney weights; males also had post-dose salivation, increased urinary epithelial cell casts and cells, and hyaline droplet nephropathy. In the 13-week study, ≥250 mg/kg increased water consumption and produced post-dose salivation. Liver-related effects: increased serum alanine aminotransferase, gamma-glutamyltransferase, bilirubin, total protein, albumin and globulins, cholesterol, liver weights and centrilobular hepatocyte hypertrophy, and reduced prothrombin times. Kidney-related effects: increased serum potassium, calcium, magnesium, kidney weights, and (males only) urea and hyaline droplets in renal tubular epithelium, and reduced sodium (females only); creatinine was reduced in 750 mg/kg males. The NOAEL of ethylbenzene in these studies, based on hepatocyte hypertrophy and liver- and kidney-related clinical chemistry changes, was 75 mg/kg bw/day.

Keywords: CAS 100-41-4; Ethylbenzene; Repeat dose toxicity; Rat; Oral; Gavage

Protective role of sulphated polysaccharides in abating the hyperlipidemic nephropathy provoked by cyclosporine A by Anthony Josephine; Coothan Kandaswamy Veena; Ganapathy Amudha; Sreenivasan P. Preetha; Palaninathan Varalakshmi (pp. 371-379).

Cyclosporine A (CsA)-induced nephrotoxicity hampers the immense therapeutic potential of such a powerful immunosuppressant. The present study was conducted with an aim to explicate the contribution of sulphated polysaccharides (SPS) in abating the lipid abnormalities induced by CsA in the rat kidney. Hyperlipidemia associated with nephrotic syndrome may play a role in the worsening of renal function. Male albino Wistar rats sorted into four groups were used for the study. CsA was given at a dose of 25 mg/kg body weight, orally for 21 days. Significant alterations in the lipid profile as well an increase in the activity of cholesterol ester synthase, coupled with a decrease in cholesterol ester hydrolase and lipoprotein lipase enzyme activities were noted in the plasma and kidneys of CsA-administered rats. A marked increase in the lipoprotein fractions, low-density lipoprotein (LDL) and very low density lipoprotein (VLDL), along with a decrease in the HDL level were found in CsA-administered rats. The degree of nephrotoxicity allied with lipid discrepancies was evident from augmented urinary excretion of urea, uric acid and creatinine. Further, an enhanced susceptibility of the apo B-containing lipoproteins (LDL + VLDL) to oxidation in vitro, induced by copper ions was also found in the plasma of CsA given groups. While SPS co-treated groups (5 mg/kg body weight, subcutaneously) revealed a normalized lipid profile and lipid metabolizing enzymes, the supplementation of SPS also brought back the elevated urinary constituents close to that of the controls and substantially minimized the oxidative changes. With these observations, it may be concluded herein that SPS may be an ideal choice as a renoprotective and hypolipidemic agent against CsA-induced hyperlipidemic nephropathy.

Keywords: Hyperlipidemia; Cyclosporine A; Sulphated polysaccharides; Lipid profile; LDL oxidation; Urinary constituents

Dye-manufacturing workers and bladder cancer in South Korea by Yangho Kim; Jungsun Park; Yong Chul Shin (pp. 381-384).

The need for a new toxicity testing and risk analysis paradigm to implement REACH or any other large scale testing initiative by Bas J. Blaauboer; Melvin E. Andersen (pp. 385-387).

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Archives of Toxicology (v.81, #5)