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Archives of Toxicology (v.80, #9)

Repeated 28-day oral toxicity study of vinclozolin in rats based on the draft protocol for the “Enhanced OECD Test Guideline No. 407” to detect endocrine effects by Jae-Ho Shin; Hyun Ju Moon; Tae Sung Kim; Il Hyun Kang; Ho Yeon Ki; Kwang Sik Choi; Soon Young Han (pp. 547-554).

We performed a 28-day repeated-dose toxicity study of vinclozolin, a widely used fungicide, based on the draft protocol of the “Enhanced OECD Test Guideline 407” (Enhanced TG407) to investigate whether vinclozolin has endocrine-mediated properties according to this assay. Seven-week-old SD rats were administered with vinclozolin daily by oral gavage at dose rates of 0, 3.125, 12.5, 50 and 200 mg/kg/day for at least 28 days. The vinclozolin-treated male rats showed a reduction of epididymis and accessory sex organ weights and an alteration of hormonal patterns. A slight prolongation of the estrous cycle and changes in the estrogen/testosterone ratio and luteinizing hormone level were observed in vinclozolin-treated female rats. Thyroxin concentrations were decreased and thyroid-stimulating hormone concentrations were increased in both sexes; however, there were no compound-related microscopic lesions in the thyroid gland or changes in the thyroid weight. The endocrine-related effects of vinclozolin could be detected by the parameters examined in the present study based on the OECD protocol, suggesting the Enhanced TG407 protocol should be a suitable screening test for the detection of endocrine-mediated effects of chemicals.

Keywords: Vinclozolin; Rat; Enhanced OECD Test Guideline 407; Endocrine effects

In vitro toxicological evaluation of a dinuclear platinum(II) complex with acetate ligands by Georgi Momekov; Dilyan Ferdinandov; Adriana Bakalova; Maya Zaharieva; Spiro Konstantinov; Margarita Karaivanova (pp. 555-560).

In the present study the toxicological potential of a tumor-inhibiting dinuclear platinum(II) complex (bis(acetato)diammine-bis-μ-acetato diplatinum(II) dihydrate (BAP)) was evaluated, utilizing in vitro models of nephrotoxicity, myelosuppression and neurotoxicity. Regarding the discrepancies between the hallmark toxicity of the clinically utilized platinum drugs, we used three distinct referent compounds as follows cisplatin for the assessment of in vitro nephrotoxicity, carboplatin in case of cultured bone marrow cells and oxaliplatin for the determination of the in vitro neurotoxicty, respectively. The results obtained indicate that the investigated dinuclear complex is endowed by a lower potential to induce detrimental effects upon these typically susceptible platinum toxicity cellular populations as compared to the corresponding referent drugs. These findings, together with the previously encountered profound cytotoxic efficiency of this dinuclear platinum(II) complex against human tumor cell lines, recall for a further detailed evaluation of BAP as potential antineoplastic agent.

Keywords: Bis(acetato)diammine-bis-μ-acetato diplatinum(II) dihydrate; Dinuclear platinum(II) complexes; Cisplatin; Carboplatin; Oxaliplatin

Alveolar macrophages suppress non-specific inflammation caused by inhalation challenge with trimellitic anhydride conjugated to albumin by Dingena L. Valstar; Marcel A. Schijf; Josje H. E. Arts; C. Frieke Kuper; Frans P. Nijkamp; Gert Storm; Nanne Bloksma; Paul A. J. Henricks (pp. 561-571).

Occupational exposure to low molecular weight chemicals, like trimellitic anhydride (TMA), can result in occupational asthma. Alveolar macrophages (AMs) are among the first cells to encounter these inhaled compounds and were previously shown to affect TMA-induced asthma-like symptoms in the Brown Norway rat (Valstar et al., Toxicol. Appl. Pharmacology 211:20–29, 2006). TMA is a hapten that will bind to endogenous proteins upon entrance of the body. Therefore, in the present study we determined if TMA conjugated to albumin is able to induce asthma-like symptoms and if these are affected by AM depletion. Female Brown Norway rats were sensitized by dermal application of TMA or received vehicle alone on days 0 and 7. One day prior to the inhalation challenge the rats were treated intratracheally with either empty liposomes or liposomes containing clodronate (dichloromethylene diphosphonate) to specifically deplete the lungs of AMs. On day 21, all groups of rats were challenged by inhalation of TMA-BSA. Breathing frequency, tidal volume, and minute ventilation were measured before, during, within 1 h, and 24 h after challenge and the gross respiratory rate score was determined during challenge. Total and TMA-specific IgE levels were determined in serum and lung lavage fluid and parameters of inflammation and tissue damage were assessed in lung lavage fluid and/or lung tissue 24 h after challenge. Sensitization with TMA had no effect on the lung function before challenge, but TMA-BSA challenge resulted in an early asthmatic response as compared to the non-sensitized rats, irrespective of AM depletion. AM depletion had no effect on the sensitization-induced serum and lung lavage fluid IgE levels. TMA-BSA inhalation did not induce airway inflammation and tissue damage irrespective of sensitization, unless AM were depleted. Data indicate that AMs inhibit immunologically non-specific damage and inflammatory cell influx into the lungs as caused by TMA-BSA inhalation. Since effects of inhalation challenge with TMA-BSA are partly different from those of TMA, challenge with the latter is to be preferred for hazard identification.

Keywords: Occupational asthma; Alveolar macrophages; Trimellitic anhydride; Early asthmatic response; Airway inflammation

Changes in hepatic cytosolic glutathione S-transferase activity and expression of its class-P during prenatal and postnatal period in rats treated with aflatoxin B1 by Faezeh Fatemi; Abdolamir Allameh; Abolfazl Dadkhah; Mehdi Forouzandeh; Somayeh Kazemnejad; Roya Sharifi (pp. 572-579).

The effect of aflatoxin B1 (AFB1) on the expression of glutathione S-transferase-P (GST-P) which is the major isoform of GST in developmental stages has been investigated in rat liver during prenatal and postnatal stages. Following administration of AFB1 (0, 0.5, 1.0, 2.0, 3.0 or 4.0 mg/kg bw) injected I.P on day 8.5 of gestation the number of dead or reabsorbed fetuses and malformed embryos were recorded. Then the fetal livers were processed for measurement of total GST and GST-P activities, using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) as substrates respectively. RT-PCR using rat GST-P specific primers was performed on mRNA extracted from livers. Besides, the effects of AFB1 on hepatic GST and GST-P were assessed in groups of suckling rats directly injected with the toxin. The results show that a single dose of AFB1 (1.0 or 2.0 mg/kg bw) caused approximately 50–60% depletion in fetal liver GST towards CDNB. Postnatal experiments revealed that liver GST (using CDNB as substrate) was significantly induced (~40%) in suckling rats injected with a single dose of AFB1 (3.0 mg AFB1/kg) 24 h before killing. Liver GST-P expression was unaffected due to AFB1 exposures of rats before and after the birth. This finding was substantiated by western blotting and RT-PCR techniques. These data suggest that AFB1-related induction in rat liver total GST after birth may be implicated in protective mechanisms against AFB1. In contrast, inhibition of this enzyme in fetal liver following placental transfer of the carcinogen may explain high susceptibility of fetal cells to transplancental aflatoxins. Furthermore, lack of influence of AFB1 on GST-P expression in developmental stages can role out the involvement of this class of GST in AFB1 biotransformation.

Keywords: Aflatoxin B1; Glutathione transferase; Prenatal; Postnatal; mRNA expression

High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening by P. J. O’Brien; W. Irwin; D. Diaz; E. Howard-Cofield; C. M. Krejsa; M. R. Slaughter; B. Gao; N. Kaludercic; A. Angeline; P. Bernardi; P. Brain; C. Hougham (pp. 580-604).

To develop and validate a practical, in vitro, cell-based model to assess human hepatotoxicity potential of drugs, we used the new technology of high content screening (HCS) and a novel combination of critical model features, including (1) use of live, human hepatocytes with drug metabolism capability, (2) preincubation of cells for 3 days with drugs at a range of concentrations up to at least 30 times the efficacious concentration or 100 μM, (3) measurement of multiple parameters that were (4) morphological and biochemical, (5) indicative of prelethal cytotoxic effects, (6) representative of different mechanisms of toxicity, (7) at the single cell level and (8) amenable to rapid throughput. HCS is based on automated epifluorescence microscopy and image analysis of cells in a microtiter plate format. The assay was applied to HepG2 human hepatocytes cultured in 96-well plates and loaded with four fluorescent dyes for: calcium (Fluo-4 AM), mitochondrial membrane potential (TMRM), DNA content (Hoechst 33342) to determine nuclear area and cell number and plasma membrane permeability (TOTO-3). Assay results were compared with those from 7 conventional, in vitro cytotoxicity assays that were applied to 611 compounds and shown to have low sensitivity (<25%), although high specificity (∼90%) for detection of toxic drugs. For 243 drugs with varying degrees of toxicity, the HCS, sublethal, cytotoxicity assay had a sensitivity of 93% and specificity of 98%. Drugs testing positive that did not cause hepatotoxicity produced other serious, human organ toxicities. For 201 positive assay results, 86% drugs affected cell number, 70% affected nuclear area and mitochondrial membrane potential and 45% affected membrane permeability and 41% intracellular calcium concentration. Cell number was the first parameter affected for 56% of these drugs, nuclear area for 34% and mitochondrial membrane potential for 29% and membrane permeability for 7% and intracellular calcium for 10%. Hormesis occurred for 48% of all drugs with positive response, for 26% of mitochondrial and 34% nuclear area changes and 12% of cell number changes. Pattern of change was dependent on the class of drug and mechanism of toxicity. The ratio of concentrations for in vitro cytotoxicity to maximal efficaciousness in humans was not different across groups (12±22). Human toxicity potential was detected with 80% sensitivity and 90% specificity at a concentration of 30× the maximal efficacious concentration or 100 μM when efficaciousness was not considered. We conclude that human hepatotoxicity is highly concordant with in vitro cytotoxicity in this novel model and as detected by HCS.

Keywords: HCS; Hepatotoxicity; Human; Sublethal; Multi-parameter

Effects of N-nitrosofenfluramine, a component of Chinese dietary supplement for weight loss, on CD-1 mice by Kanako Satoh; Ryouichi Nonaka; Yukie Tada; Nobutaka Fukumori; Akio Ogata; Arisa Yamada; Tsuyoshi Satoh; Fumiko Nagai (pp. 605-613).

Many cases of hepatopathy including deaths have frequently occurred after ingestion of Chinese dietary supplements for weight loss containing N-nitrosofenfluramine (N-fen), a nitroso derivative of fenfluramine (Fen), which was used for the treatment of obesity in the United States. Since Fen decreases appetite by decreasing the serotonin level and exhibits an antibiotic effect, N-fen may have been added, expecting a similar effect. Thus, we synthesized N-fen and orally administered it to mice, and investigated its effect on the liver as well as on the cerebral serotonin nervous system to investigate whether N-fen exhibits an anorectic effect. Three doses of N-fen were orally administered once daily to mice for 1 week. No significant changes in body weight, food intake, and general condition were noted. The liver and kidney weights were significantly increased. On blood chemistry, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities were increased, and total bilirubin and albumin were slightly decreased. On histopathological examination, acidophilic changes and mild cellular swelling were noted in the liver. The liver drug-metabolizing enzyme (P-450) level was significantly higher. The effect of N-fen on the serotonin (5HT) nervous system was examined by quantitative autoradiography of the mouse brain, and it was found that N-fen did not decrease the 5HT nerve activity. Effects of reuptake and release of monoamine neurotransmitters [dopamine (DA), 5HT, and norepinephrine (NE)] were investigated. N-fen inhibited a little 5HT reuptake, and did not inhibit reuptakes of DA and NE. Moreover, N-fen did not affect release of the three monoamines. The above findings suggested that N-fen did not exhibit a serotonin nerve fiber-mediated anorectic effect in mice, but induced hepatopathy.

Keywords: N-nitrosofenfluramine; Fenfluramine; Hepatopathy; Central nervous system; Chinese diet supplement

Effects of nano particles on cytokine expression in murine lung in the absence or presence of allergen by Ken-ichiro Inoue; Hirohisa Takano; Rie Yanagisawa; Takamichi Ichinose; Miho Sakurai; Toshikazu Yoshikawa (pp. 614-619).

Particulate matter (PM) can exacerbate allergic airway diseases. Health effects of PM with a diameter of less than 100 nm, called nano particles, have been focused. We have recently demonstrated that carbon nano particles (14, 56 nm) exaggerate allergic airway inflammation in mice. In the present study, we investigated the effects of repeated pulmonary exposure to carbon nano particles on the expression of a variety of cytokines in the absence or presence of allergen in mice. ICR mice were divided into six experimental groups. Vehicle, two sizes of carbon nano particles, ovalbumin (OVA), and OVA + nano particles were administered intratracheally. Nano particles increased the lung protein levels of thymus and activation-regulated chemokine (TARC), macrophage inflammatory protein (MIP)−1α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the absence or presence of allergen. The enhancement was more prominent with 14 nm of nano particles than with 56 nm of nano particles in overall trend. 14 nm nano particle exposure significantly enhanced the lung expressions of interleukin (IL)-2 and IL-10 in the presence of allergen as compared with allergen exposure. These results suggest that pulmonary exposure to nano particles can induce the lung expression of TARC, MIP-1α, GM-CSF in the absence of allergen and can enhance that of TARC, MIP-1α, GM-CSF, IL-2, and IL-10 in the presence of allergen. The enhancing effects are more prominent with smaller particles.

Keywords: Nano particle; Allergen; Cytokine

In utero exposure to nicotine and chlorpyrifos alone, and in combination produces persistent sensorimotor deficits and Purkinje neuron loss in the cerebellum of adult offspring rats by Mohamed B. Abou-Donia; Wasiuddin A. Khan; Anjelika M. Dechkovskaia; Larry B. Goldstein; Sarah L. Bullman; Ali Abdel-Rahman (pp. 620-631).

This study was carried out to investigate the effect of in utero exposure to the cholinotoxicants, nicotine and chlorpyrifos, alone or in combination on neurobehavioral alterations and neuronal morphology latter in adult age. In the present study, 90 days old (corresponding to a human adult age) male and female offspring rats were evaluated for neurobehavioral, and neuropathological alterations following maternal, gestational exposure to nicotine and chlorpyrifos (O,O-diethyl-O-3,5,6-trichloro-2-pyridinyl phosphorothioate), alone and in combination. Female Sprague-Dawley rats (300–350 g) with timed-pregnancy were treated with nicotine (3.3 mg/kg/day, in bacteriostatic water via s.c. implantation of mini osmotic pump), chlorpyrifos (1.0 mg/kg, daily, dermal, in 75% ethanol, 1.0 ml/kg) or a combination of both chemicals, on gestational days (GD) 4–20. Control animals received bacteriostatic water via s.c. implantation of mini osmotic pump and dermal application of 70% ethanol. The offspring at postnatal day (PND) 90 were evaluated for neurobehavioral performance, changes in the activity of plasma butyrylcholinesterase (BChE) and acetylcholinesterase (AChE), and neuropathological alterations in the brain. Neurobehavioral evaluations included beam-walk score, beam-walk time, incline plane performance and forepaw grip time. Male and female offspring from mothers treated with nicotine and CPF, alone or in combination showed impairments in the performance of neurobehavioral tests, indicating sensorimotor deficits. Female offspring from mothers treated with a combination of nicotine and chlorpyrifos showed significant increase in plasma BChE activity. Brain regional AChE activity showed differential increases in male and female offspring. Brainstem and cerebellum of female offspring from mothers treated with nicotine or chlorpyrifos, alone or in combination showed increased AChE activity, whereas brainstem of male offspring from mothers treated with nicotine alone or a combination of nicotine and chlorpyrifos showed increase in AChE activity. Also, male offspring exposed in utero to nicotine exhibited increased AChE activity. Histopathological evaluations using cresyl violet staining showed a decrease in surviving Purkinje neurons in the cerebellum in offspring of all treatments groups. An increase in glial fibrillary acidic protein (GFAP) immuno-staining was observed in cerebellum white matter as well as granular cell layer (GCL) of cerebellum following all exposures. These results indicate that in utero exposure to nicotine and chlorpyrifos, alone and in combination produced significant sensorimotor deficits in male and female offspring, differential increase in brain AChE activity, a decrease in the surviving neurons and an increased expression of GFAP in cerebellum in adult offspring rats at a corresponding human adult age. Collectively, this study demonstrates that maternal exposure to environmental neurotoxic chemicals, i.e., nicotine and chlorpyrifos leads to developmental abnormalities in the offspring that persist latter into adulthood.

Keywords: Chlorpyrifos; Nicotine; Cerebellum; Purkinje cells; Behavioral; Maternal; Developmental; Adult; Offspring; Acetylcholinesterase; Glial fibrillary acidic protein

Inhibition of endothelial nitric oxide synthase activity and suppression of endothelium-dependent vasorelaxation by 1,2-naphthoquinone, a component of diesel exhaust particles by Yang Sun; Keiko Taguchi; Daigo Sumi; Shigeru Yamano; Yoshito Kumagai (pp. 632-632).

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Archives of Toxicology (v.80, #9)