Journal of Applied Toxicology

Cover image for Vol. 35 Issue 10

Early View (Online Version of Record published before inclusion in an issue)

Editor-in-Chief: Philip W. Harvey

Impact Factor: 2.982

ISI Journal Citation Reports © Ranking: 2014: 27/87 (Toxicology)

Online ISSN: 1099-1263

VIEW

  1. 1 - 88
  1. Research articles

    1. The impact of caffeine on connexin expression in the embryonic chick cardiomyocyte micromass culture system

      Bhavesh K. Ahir and Margaret K. Pratten

      Article first published online: 24 AUG 2015 | DOI: 10.1002/jat.3219

      Cardiomyocytes are electrically coupled by gap junctions (GJs) defined as clusters of low-resistance multisubunit transmembrane channels composed of connexins (Cxs). The expression of Cx43, Cx45 and Cx40, which are present in cardiomyocytes, are known to be developmentally regulated. This study investigates the premise that alterations in GJs are one of the mechanisms by which teratogens may act. The most common human teratogen, caffeine was shown to affect cell-to-cell communication via the Cx43, Cx45 and Cx40, by alteration in expression and distribution in the embryonic chick cardiomyocytes MM culture system.

    2. The toxic effects of indoor atmospheric fine particulate matter collected from allergic and non-allergic families in Wuhan on mouse peritoneal macrophages

      Biao Yan, Jinquan Li, Junhui Guo, Ping Ma, Zhuo Wu, ZhenHao Ling, Hai Guo, Yoshino Hiroshi, U. Yanagi, Xu Yang, Shengwei Zhu and Mingqing Chen

      Article first published online: 24 AUG 2015 | DOI: 10.1002/jat.3217

      The association between allergic symptoms in children and exposure to PM2.5 has not fully elucidated, especially the role of PM2.5 from indoor environment involved in allergy or non-allergy is unknown. In this study, indoor PM2.5 from the homes of schoolchildren with allergic symptoms and those of healthy ones were analyzed. It suggested that oxidative stress may contribute to PM2.5-induced toxicity, and PM2.5 from allergic indoor environment produced more serious toxic effects and inflammatory response than that from non-allergic indoor environment.

    3. Human ketosteroid receptors interact with hazardous phthalate plasticizers and their metabolites: an in silico study

      M. K. Sarath Josh, S. Pradeep, K. S. Vijayalekshmy Amma, R. Sudha Devi, S. Balachandran, M. N. Sreejith and Sailas Benjamin

      Article first published online: 24 AUG 2015 | DOI: 10.1002/jat.3221

      Phthalates are ubiquitous environmental pollutants, known for their adverse health effects in test animals, and of late in humans. Molecular interactions of diphthalates, respective monophthalates, phthalic acid and the known endocrine disruptor, bisphenol A with human ketosteroid receptors were explored using Glide (Schrödinger); and their binding efficiencies were compared with that of the natural steroids. From the in silico evidences, most of these diphthalates and their monophthalates showed potentials for anti-steroidal activity by interacting with human ketosteroid receptors.

    4. Aryl hydrocarbon receptor knockout rats are insensitive to the pathological effects of repeated oral exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin

      Joshua A. Harrill, Debra Layko, Abraham Nyska, Renee R. Hukkanen, Rosa Anna Manno, Andrea Grassetti, Marie Lawson, Greg Martin, Robert A. Budinsky, J. Craig Rowlands and Russell S. Thomas

      Article first published online: 17 AUG 2015 | DOI: 10.1002/jat.3211

      The role of AHR in mediating pathological changes in the liver prior to tumor formation was investigated in a 4-week, repeated-dose study using adult female wild-type (WT) and AHR knockout (AHR-KO) rats treated with varying concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD: 0, 3, 22, 100, 300 and 1000 ng kg−1 day−1). Treatment-related increases in the severity of liver and thymus pathology as well as changes in serum chemistry parameters were observed in WT, but not AHR-KO rats. Dose-dependent accumulation of TCDD was observed primarily in the liver of WT rats and primarily in the adipose tissue of AHR-KO rats.

    5. Sensitive periods for 17β-estradiol exposure during immune system development in sea bass head kidney

      Frauke Seemann, Thomas Knigge, Aurélie Duflot, Sabine Marie, Stéphanie Olivier, Christophe Minier and Tiphaine Monsinjon

      Article first published online: 16 AUG 2015 | DOI: 10.1002/jat.3215

      The influence of 17β-estradiol (E2) on estrogen receptor and cytokine gene expression, leukocyte populations and phagocytic activity was assessed in the juvenile sea bass (Dicentrarchus labrax) head kidney during organ regionalization from 98 to 239 dph. E2 exposure, beginning at 90 dph resulted in indirect and delayed modifications of interleukin 1β and estrogen receptor α gene expression, which may affect B-lymphocyte proliferation. The E2 treatment of 120 dph fish led to an increase in estrogen receptor β2 and a decrease in transforming growth factor β1 gene expression, which coincided with decreased phagocytic activity of head kidney lymphocytes and monocytes/macrophages.

    6. An in vitro human skin test for assessing sensitization potential

      S. S. Ahmed, X. N. Wang, M. Fielding, A. Kerry, I. Dickinson, R. Munuswamy, I. Kimber and A. M. Dickinson

      Article first published online: 7 AUG 2015 | DOI: 10.1002/jat.3197

      Chemical sensitization resulting in allergy is an important health issue. Here we describe a human in-vitro skin explant test for sensitization hazard assessment as an alternative approach to using animal models. This method measures histological damage in human skin as a readout of the immune response induced by the test material. We tested 44 chemicals and compared results to the mouse local lymph node assay LLNA and published human sensitization data with a correlation coefficient of 0.90 and 0.91, respectively.

    7. Perfluorinated chemicals, PFOS and PFOA, enhance the estrogenic effects of 17β-estradiol in T47D human breast cancer cells

      Pacharapan Sonthithai, Tawit Suriyo, Apinya Thiantanawat, Piyajit Watcharasit, Mathuros Ruchirawat and Jutamaad Satayavivad

      Article first published online: 3 AUG 2015 | DOI: 10.1002/jat.3210

      PFOS and PFOA do not possess estrogenic activity in T47D hormone-dependent human breast cancer cells. Both compounds enhance the effects of 17β-estradiol (E2) on estrogen response element (ERE) activation, expression of estrogen-responsive pS2 gene, ERK1/2 activation and cell growth.

    8. In vitro cytotoxicity of superparamagnetic iron oxide nanoparticles on neuronal and glial cells. Evaluation of nanoparticle interference with viability tests

      Carla Costa, Fátima Brandão, Maria João Bessa, Solange Costa, Vanessa Valdiglesias, Gözde Kiliç, Natalia Fernández-Bertólez, Pedro Quaresma, Eulália Pereira, Eduardo Pásaro, Blanca Laffon and João Paulo Teixeira

      Article first published online: 26 JUL 2015 | DOI: 10.1002/jat.3213

      The main objective of this work was to evaluate the cytotoxicity of two iron oxide nanoparticles (ION; magnetite), coated with silica and oleic acid, previously determining their possible interference with the methodological procedures. Effects on SHSY5Y and A172 cells were evaluated by means of the MTT, neutral red uptake and alamar blue assays. Results demonstrated that both ION could alter absorbance readings. Significant decreases in cell viability were observed for both cell lines by all assays. In general, oleic acid-coated ION were less cytotoxic than silica-coated ION.

  2. Review Articles

    1. Intersex in fishes and amphibians: population implications, prevalence, mechanisms and molecular biomarkers

      Ahmed Abdel-moneim, David P. Coulter, Cecon T. Mahapatra and Maria S. Sepúlveda

      Article first published online: 24 JUL 2015 | DOI: 10.1002/jat.3204

      Gonadal intersex is a widespread condition among gonochoristic fishes and amphibians. In this review, we use standardized terminology to describe the different forms of intersex reported in earlier studies and identify the indices used to assess their severities, and summarize reports regarding their frequency of occurrence. We also evaluate the global geographic distribution and chronology of the reported cases and highlight molecular biomarkers that have been used or should be investigated for diagnosing intersex in fishes and amphibians.

  3. Research articles

    1. Antimicrobial agent triclosan is a proton ionophore uncoupler of mitochondria in living rat and human mast cells and in primary human keratinocytes

      Lisa M. Weatherly, Juyoung Shim, Hina N. Hashmi, Rachel H. Kennedy, Samuel T. Hess and Julie A. Gosse

      Article first published online: 23 JUL 2015 | DOI: 10.1002/jat.3209

      Triclosan (TCS) is an antimicrobial agent and mast cells are ubiquitous players in physiological processes and in diseases. TCS disrupts adenosine triphosphate production in mast cells, mouse fibroblasts and primary human keratinocytes and increases the oxygen consumption rate. TCS-methyl (no ionizable proton) affects neither degranulation nor adenosine triphosphate production at non-cytotoxic doses, indicating the effects of TCS are due to its proton ionophore structure. TCS is a mitochondrial uncoupler, affecting numerous cell types and functions via this mechanism.

  4. Research Articles

    1. BDE-209 inhibits pluripotent genes expression and induces apoptosis in human embryonic stem cells

      Lili Du, Wen Sun, Huili Zhang and Dunjin Chen

      Article first published online: 23 JUL 2015 | DOI: 10.1002/jat.3195

      Decabromodiphenyl ether (BDE-209) has been detected in human serum, semen, placenta, cord blood and milk worldwide. In this study, human embryonic stem cell lines FY-hES-10 and FY-hES-26 were used to evaluate the potential effects and explore the toxification mechanisms using low-level BDE-209 exposure. BDE-209 exposure could decrease pluripotent genes expression via epigenetic regulation and induce apoptosis through reactive oxygen species (ROS) generation in human embryonic stem cells in vitro.

  5. Research articles

    1. Proposed human stratum corneum water domain in chemical absorption

      Hanjiang Zhu, Eui-Chang Jung, Xiaoying Hui and Howard Maibach

      Article first published online: 23 JUL 2015 | DOI: 10.1002/jat.3208

      To better understand mechanisms of percutaneous absorption and skin decontamination, 21 compounds were studied for their affinities to stratum corneum (SC) and its subunits using a facile method. Differences between chemical absorption to intact SC and total contribution of protein and lipid domains suggest the possibility and significance of a water domain. A longer lag time of absorption into intact SC than to delipidized SC or SC lipid suggests the water domain may delay chemical binding to protein and lipid domains.

    2. Automated swimming activity monitor for examining temporal patterns of toxicant effects on individual Daphnia magna

      Simon Bahrndorff, Thomas Yssing Michaelsen, Anne Jensen, Laurits Faarup Marcussen, Majken Elley Nielsen and Peter Roslev

      Article first published online: 21 JUL 2015 | DOI: 10.1002/jat.3212

      We have evaluated an automated monitor for recording the swimming activity of Daphnia magna to establish temporal patterns of toxicant and temperature effects. The sensitivity of the monitor was evaluated by exposing D. magna to K2Cr2O7 and 2,4-dichlorophenol. Significant effects of toxicant concentrations, exposure time and incubation temperatures were observed. The results demonstrated that the monitor is capable of detecting sublethal behavioural effects that are toxicant and temperature dependent and can serve as a high-throughput screening tool in toxicity testing.

    3. Cytotoxicity and apoptosis induced by silver nanoparticles in human liver HepG2 cells in different dispersion media

      Yuying Xue, Ting Zhang, Bangyong Zhang, Fan Gong, Yanmei Huang and Meng Tang

      Article first published online: 21 JUL 2015 | DOI: 10.1002/jat.3199

      This study evaluated the proliferation and apoptosis of Ag NPs suspended in different solvents in HepG2 cells. The degree of Ag NPs ionization differed with dispersion media. Ag NPs exposure resulted in increased cytotoxicity, induction of apoptosis, generation of ROS and mitochondria injury, which may be directly involved in cellular oxidative stress. The cytotoxicity of Ag NPs to HepG2 cells is concentration- and time-dependent and could be, in partially, attributed to Ag ions released from the nanoparticles in the dispersion.

    4. Cytochrome P450 induction response in tethered spheroids as a three-dimensional human hepatocyte in vitro model

      Lei Xia, Xin Hong, Rashidah Binte Sakban, Yinghua Qu, Nisha Hari Singh, Michael McMillian, Shannon Dallas, Jose Silva, Carlo Sensenhauser, Sylvia Zhao, Heng Keang Lim and Hanry Yu

      Article first published online: 21 JUL 2015 | DOI: 10.1002/jat.3189

      We constructed tethered spheroids on RGD/galactose-conjugated membranes as an in vitro three-dimensional (3D) model using cryopreserved human hepatocytes. CYP3A4 mRNA expression in the tethered spheroids was induced to a significantly greater extent than those in the collagen sandwich cultures, indicating the transcriptional regulation was more sensitive to the CYP inducers in the 3D model. Induction of CYP1A2, CYP2B6 and CYP3A4 activities in the tethered spheroids were comparable to, if not higher than that observed in the collagen sandwich cultures.

    5. An improved model of predicting hepatocarcinogenic potential in rats by using gene expression data

      Fumihiro Yamada, Kayo Sumida and Koichi Saito

      Article first published online: 21 JUL 2015 | DOI: 10.1002/jat.3184

      The prediction system, which estimates the carcinogenicity of a compound with gene expression data, was improved by adding more gene expression data. The total data we used were from a short-term (28-day) study using 41 hepatocarcinogens and 52 non-hepatocarcinogens. Compared with the older version, the improved system had a higher concordance rate with the training data and a good performance with the external test data. we expect to apply it to early stages of new compound development.

    6. Protein profiles of cardiomyocyte differentiation in murine embryonic stem cells exposed to perfluorooctane sulfonate

      Ying-Ying Zhang, Lei-Lei Tang, Bei Zheng, Ren-Shan Ge and Dan-Yan Zhu

      Article first published online: 15 JUL 2015 | DOI: 10.1002/jat.3207

      PFOS was classified as a weak embryotoxic chemical by using embryonic stem cell test procedure and it could block cardiomyocyte differentiation. In total, 176 differential proteins were identified by quantitative proteomics analysis. The differential proteins were classified by GO and connectivity analysis. PFOS affected 32 signaling pathways and mostly influenced the metabolism pathways. Five differentially expressed proteins were confirmed by western blotting. These results revealed potential new targets of PFOS on the developmental cardiovascular system.

    7. Defensive and adverse energy-related molecular responses precede tris (1, 3-dichloro-2-propyl) phosphate cytotoxicity

      Jinkang Zhang, Timothy D. Williams, James K. Chipman and Mark R. Viant

      Article first published online: 15 JUL 2015 | DOI: 10.1002/jat.3194

      To investigate the potential adverse effects of human exposure to tris (1, 3-dichloro-2-propyl) phosphate (TDCIPP), the cytotoxicity of this flame retardant to HepG2/C3A and A549 cells was evaluated by CCK-8 assays. The molecular responses to TDCIPP exposure were investigated using transcriptomic and metabolomic approaches. Defensive responses (e.g. xenobiotic metabolism) as well as energy-related changes were observed, which preceded the cytotoxic effects of TDCIPP in HepG2/C3A cells.

    8. Effects of sulpiride and ethylene glycol monomethyl ether on endometrial carcinogenicity in Donryu rats

      Yoshikazu Taketa, Kaoru Inoue, Miwa Takahashi, Yohei Sakamoto, Gen Watanabe, Kazuyoshi Taya and Midori Yoshida

      Article first published online: 14 JUL 2015 | DOI: 10.1002/jat.3206

      Sulpiride and ethylene glycol monomethyl ether (EGME) stimulate prolactin (PRL) secretion. Here, the effects of PRL on endometrial carcinogenicity were evaluated in rats. Sulpiride (200 ppm) inhibited the uterine carcinogenesis whereas EGME (1250 ppm) did not. Sulpiride prevented the onset of persistent estrus and induced high PRL and progesterone (P4) serum levels. These results suggest that disruption of the estrous cycle with a decrease in estradiol-17β to P4 ratio may explain the inhibitory effects of sulpiride on uterine carcinogenesis.

    9. The comparative toxicity of a reduced, crude comfrey (Symphytum officinale) alkaloid extract and the pure, comfrey-derived pyrrolizidine alkaloids, lycopsamine and intermedine in chicks (Gallus gallus domesticus)

      Ammon W. Brown, Bryan L. Stegelmeier, Steven M. Colegate, Dale R. Gardner, Kip E. Panter, Edward L. Knoppel and Jeffery O. Hall

      Article first published online: 14 JUL 2015 | DOI: 10.1002/jat.3205

      Comfrey (Symphytum officinale), a commonly used herb, contains pro-toxic dehydropyrrolizidine alkaloids, including lycopsamine and intermedine, and has consequently been internationally regulated with respect to its use. To help further define the toxicity of S. officinale, male, California White chicks were used to compare the toxicity of a crude, reduced comfrey alkaloid extract to purified lycopsamine and intermedine. Based on clinical, serum biochemical, tissue adduct concentrations and histopathological analysis, the reduced comfrey extract was more toxic than either pure lycopsamine or intermedine. This suggests a cautionary note when estimates of herbal toxicity are based upon the observed toxicity of isolated toxins.

    10. Kupffer cell-mediated exacerbation of methimazole-induced acute liver injury in rats

      Sho Akai, Yasuaki Uematsu, Koichi Tsuneyama, Shingo Oda and Tsuyoshi Yokoi

      Article first published online: 14 JUL 2015 | DOI: 10.1002/jat.3202

      Methimazole (MTZ) is known to cause liver injury in humans. It has been demonstrated that MTZ-induced liver injury in Balb/c mice is accompanied by T helper 2 cytokine-mediated immune responses; however, there is little evidence for immune responses associated with MTZ-induced liver injury in rats. We found that Kupffer cell-mediated immune responses are crucial factors for the exacerbation of MTZ-induced liver injury in rats, indicating apparent species differences in the immune-mediated exacerbation of liver injury between mice and rats.

    11. Effects of urban particulate matter with high glucose on human monocytes U937

      Yue Zhang, Yiqun Mo, Aihua Gu, Rong Wan, Qunwei Zhang and David J. Tollerud

      Article first published online: 14 JUL 2015 | DOI: 10.1002/jat.3198

      This study examined the effects of urban particulate matter (U-PM) with or without high glucose on human monocytes U937. The results showed that exposure of monocytes to U-PM alone caused ROS generation, increased p38 phosphorylation, up-regulation of MMP-2, MMP-9 and proinflammatory cytokines IL-1β and IL-8, and increased activity of pro-MMP-2 and pro-MMP-9. These effects were enhanced significantly when cells were exposed to U-PM with high glucose. Our findings have important implications in understanding the health effects of PM on diabetics.

    12. Investigating the effect of excess caffeine exposure on placental angiogenesis using chicken ’functional‘ placental blood vessel network

      Zheng-lai Ma, Guang Wang, Wen-hui Lu, Xin Cheng, Manli Chuai, Kenneth Ka Ho Lee and Xuesong Yang

      Article first published online: 14 JUL 2015 | DOI: 10.1002/jat.3181

      In this study, we first demonstrated the morphological similarities between chick yolk sac and chorioallantoic membranes verses the mammalian placenta. Using chick yolk sac and chorioallantoic membrane as a model, we found that 5 - 15µmol/egg of caffeine exposure inhibited angiogenesis. Furthermore, caffeine exposure is able to generate excess ROS that in turn alters the expression of angiogenesis-associated genes. The results implied that the negative impact of caffeine on fetal development was partly attributed to impaired placental angiogenesis.

    13. Angiogenesis is repressed by ethanol exposure during chick embryonic development

      Guang Wang, Shan Zhong, Shi-yao Zhang, Zheng-lai Ma, Jian-long Chen, Wen-hui Lu, Xin Cheng, Manli Chuai, Kenneth Ka Ho Lee, Da-xiang Lu and Xuesong Yang

      Article first published online: 14 JUL 2015 | DOI: 10.1002/jat.3201

      In this study, we investigated the anti-angiogenic effect of ethanol on the YSM during chick embryogenesis. The anti-angiogenic effect of ethanol was found to be associated with the excess ROS production. Both ethanol and AAPH (a ROS inducer) could inhibit cell proliferation, enhance apoptosis and repress expression of angiogenesis-related genes. This further supports our proposal that excess ROS production was central to the anti-angiogenic effect of ethanol.

    14. An assay to determine the sensitive window of embryos to chemical exposure using Xenopus tropicalis

      Lingling Hu, Lijiao Wu, Yingang Xue, Jingmin Zhu and Huahong Shi

      Article first published online: 14 JUL 2015 | DOI: 10.1002/jat.3200

      The Xenopus tropicalis embryos showed great variations of malformation in response to nine tested compounds during four separate 12-h exposure periods. Based on the value of score of malformations, the most sensitive exposure period of embryos was significantly distinguished for eight compounds. A rapid and valid assay was proposed to determine the 12-h sensitive window of embryos to chemical exposure using X. tropicalis.

    15. Investigation of ifosfamide and chloroacetaldehyde renal toxicity through integration of in vitro liver–kidney microfluidic data and pharmacokinetic-system biology models

      Eric Leclerc, Jeremy Hamon and Frederic Yves Bois

      Article first published online: 7 JUL 2015 | DOI: 10.1002/jat.3191

      We have integrated in vitro and in silico data to describe the toxicity of chloroacetaldehyde (CAA) on renal cells via its production from the metabolism of ifosfamide (IFO) by hepatic cells. A pharmacokinetic (PK) model described the production of CAA by the hepatocytes and its transport to the renal cells. A system biology model was coupled to the PK model to describe the production of reactive oxygen species (ROS) and glutathione (GSH) depletion induced by CAA in the renal cells.

    16. Developing Xenopus embryos recover by compacting and expelling single wall carbon nanotubes

      Brian D. Holt, Joseph H. Shawky, Kris Noel Dahl, Lance A. Davidson and Mohammad F. Islam

      Article first published online: 7 JUL 2015 | DOI: 10.1002/jat.3203

      Single wall carbon nanotubes are being developed for wide-ranging applications. However, understanding the direct exposure to Xenopus laevis, a development model, is lacking. We found superficial microinjection of nanotubes suspended with Pluronic F127 into one- to two-cell embryos resulted in the formation and expulsion of compacted, nanotube-filled masses. Expulsion is dramatically different from typical distribution throughout the embryo. Previous studies microinjecting other nanomaterials often report toxicity, yet our results demonstrate recovery, which we speculate results from Pluronic F127's membrane activity and nanotubes’ large aspect ratio.

  6. Review articles

    1. Safety concerns of herbal products and traditional Chinese herbal medicines: dehydropyrrolizidine alkaloids and aristolochic acid

      Bryan L. Stegelmeier, Ammon W. Brown and Kevin D. Welch

      Article first published online: 7 JUL 2015 | DOI: 10.1002/jat.3192

      In many countries, herbal supplements, tisanes and vegetable products, including traditional Chinese medicines, are unregulated and their content is not registered, monitored or verified. Consequently, plant toxins including dehydropyrrolizidine alkaloids can contaminate these products. Dehydopyrrolizidine alkaloids and aristolochic acids are examples that demonstrate the risk and potential toxicity of contaminated, mislabeled or misused products. Work is, therefore, needed to educate consumers of the potential risk and to require the industry to be more responsible to verify content and insure product safety.

    2. Biological effects and toxicity of diluted bitumen and its constituents in freshwater systems

      William A. Dew, Alice Hontela, Stewart B. Rood and Greg G. Pyle

      Article first published online: 7 JUL 2015 | DOI: 10.1002/jat.3196

      Bitumen mined from the oil sands region in Alberta Canada is viscous and difficult to transport by traditional means. Bitumen, therefore, is diluted to form dilbit and other bitumen products that are more easily transported via pipelines or other methods. Little is known about the fate and effects of a dilbit spill in a freshwater ecosystem, which is especially dependent upon whether it floats or sinks. This review summarizes our current understanding of dilbit effects in freshwater ecosystems.

  7. Research Articles

    1. Zebrafish as a model for studying the developmental neurotoxicity of propofol

      Peipei Guo, Zhibin Huang, Tao Tao, Xiaohui Chen, Wenqing Zhang, Yiyue Zhang and Chunshui Lin

      Article first published online: 23 JUN 2015 | DOI: 10.1002/jat.3183

      Whether anesthesia in pregnancy is neurotoxic to the developing fetus is becoming a question of concern. Our goal was to use zebrafish to explore the effect of propofol on embryonic development, apoptosis and MBP expression. We found that embryonic exposure to propofol is neurotoxic, causing increased apoptosis and decreased MBP expression. We believe zebrafish can be used as a novel model to explore the mechanisms of propofol neurotoxicity.

    2. The toxic effects of Bisphenol A on the mouse spermatocyte GC-2 cell line: the role of the Ca2+-calmodulin-Ca2+/calmodulin-dependent protein kinase II axis

      Wenyi Qian, Yixin Wang, Jingying Zhu, Changfei Mao, Qiang Wang, Fei Huan, Jie Cheng, Yanqing Liu, Jun Wang and Hang Xiao

      Article first published online: 18 JUN 2015 | DOI: 10.1002/jat.3188

      The present study investigated whether a perturbation in Ca2+-CaM-CaMKII signaling was involved in Bisphenol A (BPA)-induced injury to mouse spermatocyte GC-2spd (ts) (GC-2) cells. The results showed that BPA decreased cell viability, released mitochondrial cytochrome c and activated caspase-3. However, these processes could be partially abrogated by pretreatment with antagonists of Ca2+, CaM or CaMKII. These results indicate that BPA exposure contributes to male germ cell injury, which may be partially mediated through a perturbation in Ca2+/CaM/CaMKII signaling and the mitochondrial apoptotic process.

    3. Sub-chronic exposure to fluoride impacts the response to a subsequent nephrotoxic treatment with gentamicin

      Mariana Cárdenas-González, Tania Jacobo Estrada, Rafael Rodríguez-Muñoz, Jonatan Barrera-Chimal, Norma A. Bobadilla, Olivier C. Barbier and Luz M. Del Razo

      Article first published online: 17 JUN 2015 | DOI: 10.1002/jat.3186

      The purpose of this work was to investigate if sub-nephrotoxic stimulus induced by fluoride might impact the response to a subsequent nephrotoxic treatment with gentamicin. Rats were exposed to fluoride (0, 15 or 50 ppm) through drinking water during 40 days. Then, rats were co-exposed to gentamicin (40 mg kg–1 day–1) for 7 days. The results suggested that fluoride reduced gentamicin-induced nephrotoxicity by inducing a compensatory response carried out by Hsp72 and by decreasing the gentamicin accumulation.

    4. Distribution and biomarker of carbon-14 labeled fullerene C60 ([14C(U)]C60) in pregnant and lactating rats and their offspring after maternal intravenous exposure

      Rodney W. Snyder, Timothy R. Fennell, Christopher J. Wingard, Ninell P. Mortensen, Nathan A. Holland, Jonathan H. Shannahan, Wimal Pathmasiri, Anita H. Lewin and Susan C. J. Sumner

      Article first published online: 17 JUN 2015 | DOI: 10.1002/jat.3177

      In an investigation of the distribution of radioactivity after administration of [14C(U)]C60 (a 5% PVP-saline suspension) in pregnant and lactating rats after a single tail vein injection, biomarkers of inflammation, cardiovascular injury and oxidative stress were measured. The goals of the investigation were to provide a basic understanding of the distribution, elimination, biological impact and distribution of radioactivity in exposed adult rats, and in their fetuses and pups.

    5. Complement C5a–C5aR interaction enhances MAPK signaling pathway activities to mediate renal injury in trichloroethylene sensitized BALB/c mice

      Jia-xiang Zhang, Wan-sheng Zha, Liang-ping Ye, Feng Wang, Hui Wang, Tong Shen, Chang-hao Wu and Qi-xing Zhu

      Article first published online: 10 JUN 2015 | DOI: 10.1002/jat.3179

      We have shown previously complement activation as a possible mechanism for trichloroethylene (TCE) sensitization, leading to kidney damages. A Pretreatment with the C5aR antagonist attenuated TCE-induced tissue damage and renal dysfunction, attenuated increase of expression of P-p38, P-ERK and P-JNK proteins and also consistently reduced TCE sensitization-induced increase of IL-2, TNF-α and IFN-γ. Our finding identify C5a binding to C5aR, MAP kinase activation and inflammatory cytokine release as a novel mechanism for complement-mediated renal injury by sensitization with TCE.

  8. Review Articles

    1. Hepatotoxicity mechanisms of isoniazid: A mini-review

      Hozeifa M. Hassan, Hong-li Guo, Bashir A. Yousef, Zhang Luyong and Jiang Zhenzhou

      Article first published online: 10 JUN 2015 | DOI: 10.1002/jat.3175

      Isoniazid (INH) is an antituberculosis drug associated with idiosyncratic liver injury in susceptible patients. INH-induced hepatotoxicity remains a significant clinical problem, but the underlying mechanisms are still unclear, despite the growing evidence that INH and/or its major metabolite, hydrazine, play an important role in hepatotoxicity.

  9. Research Articles

    1. Bik subcellular localization in response to oxidative stress induced by chemotherapy, in Two different breast cancer cell lines and a Non-tumorigenic epithelial cell line

      Aglaé Trejo-Vargas, Elisa Hernández-Mercado, Rosa María Ordóñez-Razo, Roberto Lazzarini, Diego Julio Arenas-Aranda, María Concepción Gutiérrez-Ruiz, Mina Königsberg and Armando Luna-López

      Article first published online: 9 JUN 2015 | DOI: 10.1002/jat.3173

      Bik is a pro-apoptotic protein, which becomes deregulated in cancer. Our objective was to determine Bik subcellular localization after H2O2 or cisplatin treatment. Experiments were performed in human transformed mammary gland cell-lines MDA-MB-231 and MCF-7, and one non-tumorigenic epithelial cell-lineMCF-10A. We found that in MCF-7, Bik is localized within the cytosol and after treatment it translocates into the nucleus. However in MDA-MB-231, Bik localizes in the nucleus and traslocates to the cytosol, and in MCF10A Bik doesn't change location.

    2. Bisphenol A affects placental layers morphology and angiogenesis during early pregnancy phase in mice

      Sabrina Tait, Roberta Tassinari, Francesca Maranghi and Alberto Mantovani

      Article first published online: 9 JUN 2015 | DOI: 10.1002/jat.3176

      Placental development is a potential target of bisphenol A (BPA). Pregnant CD-1 mice were administered with vehicle, 0.5 (BPA05) or 50 mg kg−1 (BPA50) bodyweight day−1 of BPA, from gestational day (GD) 1 to GD11. At GD12, morphological and transcriptomic analyses showed that BPA05 promoted and that BPA50 inhibited blood vessel development and branching. Embryonic structures (head, forelimb) were divergently altered upon BPA05 or BPA50 exposure. Data support CREB and AhR/ARNT complex as the main regulators of BPA05 and BPA50 action, respectively.

    3. Probabilistic hazard assessment for skin sensitization potency by dose–response modeling using feature elimination instead of quantitative structure–activity relationships

      Thomas Luechtefeld, Alexandra Maertens, James M. McKim, Thomas Hartung, Andre Kleensang and Vanessa Sá-Rocha

      Article first published online: 5 JUN 2015 | DOI: 10.1002/jat.3172

      Supervised learning methods promise to improve integrated testing strategies. We curated a skin sensitization data set and evaluated the information from in silico, in chemico and in vitro assays by recursive variable selection. Cell assays consistently ranked high in the feature elimination. A hidden Markov model taking advantage of monotonicity between local lymph node assay and dose is shown to outperform dose-naïve models on all data sets and strongly reduce extreme sensitizers classified as non-sensitizers.

    4. You have full text access to this OnlineOpen article
      Non-clinical safety evaluation of repeated intramuscular administration of the AS15 immunostimulant combined with various antigens in rabbits and cynomolgus monkeys

      N. Garçon, J. Silvano, C. F. Kuper, N. Baudson, C. Gérard, R. Forster and L. Segal

      Article first published online: 1 JUN 2015 | DOI: 10.1002/jat.3167

      The aim of the current paper was to assess the safety profile of vaccine candidates containing the AS15 immunostimulant combined with different antigens in two animal models. Several antigens were tested for this purpose: WT1 (rabbits), p501, dHER2 and recPRAME (cynomolgus monkeys). Only transient differences in hematology and biochemical parameters could be observed, while pathology testing revealed no safety concerns. Our findings support the use of AS15 for clinical development of potential immunotherapeutic cancer vaccines.

    5. Short-term, low-dose cadmium exposure induces hyperpermeability in human renal glomerular endothelial cells

      Liqun Li, Fengyun Dong, Dongmei Xu, Linna Du, Suhua Yan, Hesheng Hu, Corrinne G. Lobe, Fan Yi, Carolyn M. Kapron and Ju Liu

      Article first published online: 25 MAY 2015 | DOI: 10.1002/jat.3168

      Cadmium enters the human body and circulates for a short period in the bloodstream. In this study, we found that short-term, low-dose cadmium exposure increases permeability without cytotoxic effects in human renal glomerular endothelial cells. The hyperpermeability might be caused by membrane dissociation of vascular endothelial cadherin and is partially mediated by p38 mitogen-activated protein kinase pathway.

    6. Onset of hepatocarcinogen-specific cell proliferation and cell cycle aberration during the early stage of repeated hepatocarcinogen administration in rats

      Masayuki Kimura, Hajime Abe, Sayaka Mizukami, Takeshi Tanaka, Megu Itahashi, Nobuhiko Onda, Toshinori Yoshida and Makoto Shibutani

      Article first published online: 22 MAY 2015 | DOI: 10.1002/jat.3163

      This study aimed to determine the onset time of hepatocarcinogen-specific cellular responses. Rats were treated orally with hepatocarcinogens for 3, 7 or 28 days. For comparison, some animals were subjected to partial hepatectomy or treated with noncarcinogenic hepatotoxicants. Obtained results suggest that it may take 28 days to induce hepatocarcinogen-specific disruption of G1/S checkpoint function, early withdrawal of proliferating cells from M phase due to disruptive spindle checkpoint function and aberrant p21Cip1 activation to cause apoptosis reflecting DNA damage accumulation.

    7. RNA sequencing provides insights into the toxicogenomic response of ZF4 cells to methyl methanesulfonate

      Zhouquan Li, Yong Long, Liqiao Zhong, Guili Song, Xiaohua Zhang, Li Yuan, Zongbin Cui and Heping Dai

      Article first published online: 22 MAY 2015 | DOI: 10.1002/jat.3147

      RNA-seq identified 6,637 differentially expressed genes (DEGs). GO enrichment revealed that RNA-associated processes were the most up-regulated, while cell cycle and adhesion were the most repressed, neuron-related processes were the most down-regulated developmental process. KEGG pathway enrichment identified DNA damage repair, cell cycle, apoptosis and spliceosome overrepresented. There were 1,156 AS DEGs specifically expressed after MMS treatment, many of which belonged to metabolism and catabolic process. Cluster analysis of orthologs was able to extrapolate toxicotranscriptomics data between zebrafish and yeast.

    8. A method for estimating the glomerular filtration rate in conscious monkeys

      Hiroshi Satoh, Nana Nomiya, Daiki Imai, Shigeru Sato, Ken Sakurai, Kiyoshi Takasuna and Kazuhisa Furuhama

      Article first published online: 21 MAY 2015 | DOI: 10.1002/jat.3178

      To establish a method for estimating the GFR in monkeys, the radiographic contrast medium iodixanol was administered as tracers to monkeys as a bolus injection; blood was collected after 60, 90 and 120 min. An equation based on a single-blood-sample method derived from Jacobsson's formula was prepared using the data from healthy and gentamicin-treated monkeys by a multisample strategy with iodixanol. The GFR using the equation with iodixanol was in agreement with that from the multisample method with iodixanol.

    9. Developmental toxicity and endocrine disruption of naphthenic acids on the early life stage of zebrafish (Danio rerio)

      Jie Wang, Xiaofeng Cao, Yi Huang and Xiaoyan Tang

      Article first published online: 21 MAY 2015 | DOI: 10.1002/jat.3166

      Naphthenic acids (NAs) have been considered as the primary toxic components of oil sands process-affected water (OSPW). The developmental toxicity and endocrine disruption on the early life stage of zebrafish were investigated using oil sands NAs (OS-NAs) and commercial NAs (C-NAs). Both OS-NAs and C-NAs exposure resulted in a variety of developmental lesions in the fish larvae. Significant up-regulated gene expressions of CYP19b, ERα and VTG were observed in both OS-NAs and C-NAs groups, which indicated the deleteriously estrogenic potential of NAs.

    10. Directional and color preference in adult zebrafish: Implications in behavioral and learning assays in neurotoxicology studies

      Zachary A. Bault, Samuel M. Peterson and Jennifer L. Freeman

      Article first published online: 20 MAY 2015 | DOI: 10.1002/jat.3169

      While a number of zebrafish neurobehavioral assays are recently reported in the literature, many are still being refined. The initial purpose of this study was to apply a published T-maze assay for adult zebrafish that measures how quickly an organism can discriminate between different color stimuli after receiving reinforcement to measure learning in a study investigating the later life impacts of a developmental lead exposure. The original results were inconclusive as a directional and color preference was observed.

    11. You have free access to this content
      Effect-directed identification of endocrine disruptors in plastic baby teethers

      Elisabeth Berger, Theodoros Potouridis, Astrid Haeger, Wilhelm Püttmann and Martin Wagner

      Article first published online: 18 MAY 2015 | DOI: 10.1002/jat.3159

      We combined bioassays and chemical analysis to identify endocrine disrupting chemicals in plastic teethers for babies. One of the tested samples contained methyl-, ethyl- and propylparaben, another leached unidentified antiandrogens. This demonstrates that plastic teethers can be a source of infant exposure to endocrine disrupting chemicals.

    12. You have full text access to this OnlineOpen article
      Acute and subchronic toxicity of 20  kHz and 60  kHz magnetic fields in rats

      Izumi Nishimura, Atsushi Oshima, Kazumoto Shibuya, Takashi Mitani and Tadashi Negishi

      Article first published online: 17 MAY 2015 | DOI: 10.1002/jat.3161

      Despite increasing use of intermediate frequency (IF) magnetic fields (MFs) in occupational and domestic settings, scientific evidence is insufficient for IF MF health risk assessments. Rats were exposed to 20 kHz or 60 kHz sinusoidal MFs for 22 h day−1 for 14 days (acute) or 13 weeks (subchronic). MF-exposed rats did not exhibit significant and reproducible changes in body and organ weights, hematology, clinical chemistry and histopathology. Our results indicate that IF MF exposure does not carry a significant health risk to mammals.

    13. Silver nanoparticles affect the neural development of zebrafish embryos

      Qi Xin, Jeanette M. Rotchell, Jinping Cheng, Jun Yi and Qiang Zhang

      Article first published online: 15 MAY 2015 | DOI: 10.1002/jat.3164

      Silver nanoparticles (AgNPs) have been widely used in commercial products. This study aims to understand the impact of AgNPs on the early developmental stages in zebrafish (Danio rerio) embryos. Embryos were exposed to two sizes of AgNPs at three dose levels, as well to free Ag+ ions, for a range of 4–96 h post-fertilization (hpf)

    14. Toxicological effects of pet food ingredients on canine bone marrow-derived mesenchymal stem cells and enterocyte-like cells

      M. T. Ortega, B. Jeffery, J. E. Riviere and N. A. Monteiro-Riviere

      Article first published online: 14 MAY 2015 | DOI: 10.1002/jat.3158

      Short abstract

      Food ingredients clove leave oil, eugenol, guanosine monophosphate (GMP), GMP + inosine monophosphate, sorbose, ginger root extract, cinnamon bark oil, cinnamaldehyde, thyme oil, thymol, lemongrass oil and citric acid showed differential toxicity in canine bone marrow-derived mesenchymal stem cells, which differentiated into enterocyte-like cells in vitro. The most toxic ingredients were thymol, eugenol, cinnamon bark oil, cinnamaldehyde, clove leave oil and lemongrass oil (0.002–0.54 mg ml–1). GMP, GMP + inosine monophosphate and sorbose were the least toxic ingredients (56.8–116.7 mg ml–1).

    15. Safety data on 19 vehicles for use in 1 month oral rodent pre-clinical studies: administration of hydroxypropyl-ß-cyclodextrin causes renal toxicity

      Guy Healing, Tabassum Sulemann, Peter Cotton, Jayne Harris, Adam Hargreaves, Rowena Finney, Sarah Kirk, Carolin Schramm, Clare Garner, Perrine Pivette and Lisa Burdett

      Article first published online: 10 MAY 2015 | DOI: 10.1002/jat.3155

    16. Maternal exposure to hexachlorophene targets intermediate-stage progenitor cells in the hippocampal neurogenesis involving myelin vacuolation of cholinergic and glutamatergic inputs in mice

      Mizuho Kato, Hajime Abe, Megu Itahashi, Yoh Kikuchihara, Masayuki Kimura, Sayaka Mizukami, Toshinori Yoshida and Makoto Shibutani

      Article first published online: 5 MAY 2015 | DOI: 10.1002/jat.3162

      This study investigated the effect of maternal hexachlorophene exposure on hippocampal neurogenesis in mouse offspring. Pregnant mice were supplemented with hexachlorophene in diet during gestation and lactation. Offspring displayed reversible decrease of type 2 intermediate-stage progenitor cells in the subgranular zone. Myelin vacuolation might be responsible for changes in neurogenesis possibly by reducing nerve conduction velocity of cholinergic inputs from the septal–hippocampal pathway to granule cell lineages and/or GABAergic interneurons, and of glutamatergic inputs to granule cell lineages.

    17. mRNAs and miRNAs in whole blood associated with lung hyperplasia, fibrosis, and bronchiolo-alveolar adenoma and adenocarcinoma after multi-walled carbon nanotube inhalation exposure in mice

      Brandi N. Snyder-Talkington, Chunlin Dong, Linda M. Sargent, Dale W. Porter, Lauren M. Staska, Ann F. Hubbs, Rebecca Raese, Walter McKinney, Bean T. Chen, Lori Battelli, David T. Lowry, Steven H. Reynolds, Vincent Castranova, Yong Qian and Nancy L. Guo

      Article first published online: 29 APR 2015 | DOI: 10.1002/jat.3157

      This study determined global mRNA and miRNA profiles in whole blood from mice exposed by inhalation to MWCNT that correlated with the presence of lung hyperplasia, fibrosis, and bronchiolo-alveolar adenoma and adenocarcinoma. At 17 months post-exposure, whole blood was collected and analyzed using microarray analysis for global mRNA and miRNA expression. The changes in miRNA and mRNA expression, and their respective regulatory networks, identified in this study may potentially serve as blood biomarkers for MWCNT-induced lung pathological changes.

    18. In vitro neurotoxicity evaluation of piperazine designer drugs in differentiated human neuroblastoma SH-SY5Y cells

      M. D. Arbo, R. Silva, D. J. Barbosa, D. Dias da Silva, S. P. Silva, J. P. Teixeira, M. L. Bastos and H. Carmo

      Article first published online: 20 APR 2015 | DOI: 10.1002/jat.3153

      Piperazine designer drugs act as substrates at dopaminergic and serotonergic receptors and/or transporters in the brain. This work aimed to investigate the cytotoxicity of N-benzylpiperazine, 1-(3-trifluoromethylphenyl)piperazine, 1-(4-methoxyphenyl)piperazine and 1-(3,4-methylenedioxybenzyl)piperazine in the differentiated human neuroblastoma SH-SY5Y cell line. Complete cytotoxicity curves were obtained after 24 h incubations with each drug. A significant decrease in intracellular total glutathione content was noted for the drugs. All drugs caused an increase of intracellular free Ca2+ levels, accompanied by mitochondrial hyperpolarization.

    19. Preclinical safety evaluation of low molecular weight heparin–deoxycholate conjugates as an oral anticoagulant

      Ji-young Kim, Ok-Cheol Jeon, Hyun Tae Moon, Seung Rim Hwang and Youngro Byun

      Article first published online: 20 APR 2015 | DOI: 10.1002/jat.3146

      The preclinical safety of an oral anticoagulant (OH09208) was assessed by a comprehensive evaluating program in compliance with standard guidelines. As a result, OH09208 demonstrated acceptable values in single dose acute toxicity studies in rats, repeat dose toxicity studies in rats and dogs, systemic anaphylaxis test, both in vitro and in vivo mutagenicity and genotoxicity studies, and safety pharmacology studies. Overall, there were no unexpected toxicities found in this study that might have precluded the safe administration of OH09208 to humans.

    20. Establishment of a mouse model for amiodarone-induced liver injury and analyses of its hepatotoxic mechanism

      Shohei Takai, Shingo Oda, Koichi Tsuneyama, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi

      Article first published online: 20 APR 2015 | DOI: 10.1002/jat.3141

      In this study, an in vivo mouse model of amiodarone-induced liver injury was developed with co-administration of dexamethasone and possible mechanisms were investigated. It was suggested that amiodarone and/or desethylamiodarone contribute to the pathogenesis of amiodarone-induced liver injury by producing mitochondrial and oxidative stress and Kupffer cell activation. This study provides a new perspective on drug-induced liver injury and is useful for achieving an in vivo hepatotoxicity assay in nonclinical drug development.

    21. Bisphenol A inhibits duodenal movement ex vivo of rat through nitric oxide-mediated soluble guanylyl cyclase and α-adrenergic signaling pathways

      Kaushik Sarkar, Panchali Tarafder and Goutam Paul

      Article first published online: 16 APR 2015 | DOI: 10.1002/jat.3154

      We report here the effect of bisphenol A (BPA) on the duodenal movement of the rat. We found significant depression of duodenal movement by BPA. Furthermore, we observed significant counteractions of BPA-induced inhibition by N-ω-nitro- L-arginine methyl ester (nitric oxide [NO] synthase inhibitor), methylene blue (soluble guanylyl cyclase blocker) and phentolamine (α-adrenergic receptor blocker). The results indicate that NO and norepinephrine secreting intrinsic neurons might be involved in BPA-induced changes. We may conclude that BPA inhibits the duodenal movement by promoting NO- and/or norepinephrine-mediated signaling mechanisms in duodenal smooth muscle cells.

    22. Microminipigs as a new experimental animal model for toxicological studies: comparative pharmacokinetics of perfluoroalkyl acids

      Keerthi S. Guruge, Michiko Noguchi, Koji Yoshioka, Eriko Yamazaki, Sachi Taniyasu, Miyako Yoshioka, Noriko Yamanaka, Mitsutaka Ikezawa, Nobuhiko Tanimura, Masumi Sato, Nobuyoshi Yamashita and Hiroaki Kawaguchi

      Article first published online: 15 APR 2015 | DOI: 10.1002/jat.3145

      A single oral dose of a mixture of 10 PFAAs was given to Microminipigs. The blood depuration half-lives of PFAAs ranged from 1.6 to 86.6 days. PFOS and other long-chain carboxyl acids remained in the tissues for a long period. Persistence of PFAAs in edible tissues raises concerns about the safety of swine products. MMPigs can be excellent novel experimental animals for toxicological studies.

    23. Acrylamide induces locomotor defects and degeneration of dopamine neurons in Caenorhabditis elegans

      Jia Li, Dan Li, Yongsheng Yang, Tiantian Xu, Ping Li and Defu He

      Article first published online: 15 APR 2015 | DOI: 10.1002/jat.3144

      In Caenorhabditis elegans, we showed that 48 h exposure to 10-625 mgl−1 acrylamide resulted in significant declines in locomotor behavior and decrease of crawling speeds and body bending angles, which indicated locomotor defects, along with Parkinsonian-like impairment. Acrylamide also affected chemotaxis plasticity and reduced learning ability. Moreover, acrylamide induced down-expression of Pdat-1 and enhanced expression of unc-54, which indicated degeneration of dopaminergic neurons and aggregation of α-synuclein. It suggests that the neurotoxicity of acrylamide is associated with Parkinson's disease.

    24. PM2.5-induced oxidative stress increases adhesion molecules expression in human endothelial cells through the ERK/AKT/NF-κB-dependent pathway

      Wei Rui, Longfei Guan, Fang Zhang, Wei Zhang and Wenjun Ding

      Article first published online: 15 APR 2015 | DOI: 10.1002/jat.3143

      We explored the underlying mechanisms of PM2.5-induced endothelial dysfucntion in EA.hy926 cells. PM2.5 exposure triggered reactive oxygen species (ROS) generation, phosphorylation of Jun N-terminal kinase (JNK), extracellular signal regulatory kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (AKT), activation of nuclear factor kappa B (NF-κB), and increase in expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) as well as adhesion of THP-1 cells. However, ERK, AKT and NF-κB inhibitors significantly down-regulated PM2.5-induced ICAM-1 and VCAM-1 expression, but not JNK inhibitor and p38 MAPK inhibitor. The results suggest that PM2.5-induced ROS trigger ICAM-1 and VCAM-1 expressions via activation of ERK/AKT/NF-κB pathway.

    25. Particulate matter phagocytosis induces tissue factor in differentiating macrophages

      M. Milano, P. Dongiovanni, A. Artoni, S. Gatti, L. Rosso, F. Colombo, V. Bollati, M. Maggioni, P. M. Mannucci, P. A. Bertazzi, S. Fargion and L. Valenti

      Article first published online: 8 APR 2015 | DOI: 10.1002/jat.3156

      Here we show that in human differentiating macrophages, particulate matter with diameter < 10 mcM (PM10) exposure induces tissue factor (TF) and a procoagulant phenotype. This process is dependent on phagocytosis and the activation of stress pathways, and is abolished in differentiated anti-inflammatory macrophages. In rats, alveolar exposure to PM10 causes the recruitment of inflammatory cells and results in local induction of TF, increasing circulating TF levels. TF induction by differentiating lung macrophages, activated following phagocytosis, contributes to the increased risk of thromboembolic complications associated with PM10 exposure.

    26. Impaired aquaporins expression in the gastrointestinal tract of rat after mercury exposure

      Cinzia Bottino, Marta Vázquez, Vicenta Devesa and Umberto Laforenza

      Article first published online: 8 APR 2015 | DOI: 10.1002/jat.3151

      This study aims to evaluate the effects of mercury species exposure on aquaporin (AQP) expression in the gastrointestinal tract of rats treated during 4 days. Both HgCl2 and CH3HgCl reduced AQP expression in stomach, jejunum and colon. In particular, AQP3 and AQP4 are downregulated in stomach and AQP3 and AQP7 in jejunum and colon. This effect on AQP expression could be one of the causes of the gastrointestinal symptoms observed after mercury exposure (diarrhea, luminal water accumulation, fluid loss).

    27. Co-treatment with the non-steroidal anti-androgen drug, flutamide and the natural estrogen, 17β-estradiol does not lead to additive reproductive impairment in juvenile Murray rainbowfish (Melanotaenia fluviatilis)

      Harpreet Bhatia, Anupama Kumar, Jun Du, John C. Chapman and Mike J. McLaughlin

      Article first published online: 8 APR 2015 | DOI: 10.1002/jat.3135

      The aim of this study was to investigate if the anti-androgen, flutamide, and the estrogen, 17β-estradiol work together to feminize juvenile Murray rainbowfish (Melanotaenia fluviatilis). Fish (60 days post-hatch) were exposed to 25 ng/L 17β-estradiol (E2), 25 µg/L flutamide (Flu low), 250 µg/L flutamide (Flu high), E2+Flu low and E2+Flu high. After 35 days of exposure, concentrations of sex steroid hormones, 17β-estradiol and 11-keto testosterone (11-KT), were determined in the head; and vitellogenin (VTG) concentration was measured in the tail.

    28. Augmenting effects of gestational arsenite exposure of C3H mice on the hepatic tumors of the F2 male offspring via the F1 male offspring

      Keiko Nohara, Kazuyuki Okamura, Takehiro Suzuki, Hikari Murai, Takaaki Ito, Keiko Shinjo, Shota Takumi, Takehiro Michikawa, Yutaka Kondo and Kenichiro Hata

      Article first published online: 30 MAR 2015 | DOI: 10.1002/jat.3149

      Gestational exposure can affect the F2 generation through exposure of F1 germ cells. We assessed tumor incidence in the F2 males obtained by reciprocal crossing between the control and gestationally arsenite-exposed F1 males and females in C3H mice. The results demonstrated that the F2 males born to arsenite-F1 males developed tumors at a significantly higher rate than the F2 males born to the control F1 males. We also characterized gene expression of several hepatocellular carcinoma markers in the F2 tumors.

    29. Gene expression profiling of the hippocampal dentate gyrus in an adult toxicity study captures a variety of neurodevelopmental dysfunctions in rat models of hypothyroidism

      Ayako Shiraki, Fumiyo Saito, Hirotoshi Akane, Yumi Akahori, Nobuya Imatanaka, Megu Itahashi, Toshinori Yoshida and Makoto Shibutani

      Article first published online: 30 MAR 2015 | DOI: 10.1002/jat.3140

      Global gene expression profiling was performed in four brain regions in 6-propyl-2-thiouracil (PTU)-administered young adult rats with hypothyroidism. Among the brain regions, gene expression alterations related to neural development and myelination were most profound in the hippocampal dentate gyrus. Because the gene expression profile of the adult dentate gyrus closely related to neurogenesis, 28-day toxicity studies looking at gene expression changes in adult hippocampal dentate gyrus may also detect possible developmental neurotoxic effects.

    30. You have full text access to this OnlineOpen article
      Development of toxicity values and exposure estimates for tetrabromobisphenol A: application in a margin of exposure assessment

      Daniele Wikoff, Chad Thompson, Camarie Perry, Matthew White, Susan Borghoff, Lauren Fitzgerald and Laurie C. Haws

      Article first published online: 30 MAR 2015 | DOI: 10.1002/jat.3132

      Data from the National Toxicology Program (NTP) were utilized to develop an oral reference dose (RfD) and oral cancer slope factor (OSF) for tetrabromobisphenol A (TBBPA). Comparison of exposure estimates based on breast milk consumption, dietary intake, soil/dust ingestion and drinking water ingestion, resulted in margins of exposure > 800 000 in infants, young children, older children and adults. These data collectively indicate a low level of health concern associated with exposures to TBBPA.

    31. Evaluation of combinations of in vitro sensitization test descriptors for the artificial neural network-based risk assessment model of skin sensitization

      Morihiko Hirota, Shiho Fukui, Kenji Okamoto, Satoru Kurotani, Noriyasu Imai, Miyuki Fujishiro, Daiki Kyotani, Yoshinao Kato, Toshihiko Kasahara, Masaharu Fujita, Akemi Toyoda, Daisuke Sekiya, Shinichi Watanabe, Hirokazu Seto, Osamu Takenouchi, Takao Ashikaga and Masaaki Miyazawa

      Article first published online: 30 MAR 2015 | DOI: 10.1002/jat.3105

      The skin sensitization potential of chemicals has been determined with the use of the murine local lymph node assay (LLNA). However, in recent years public concern about animal welfare has led to a requirement for non-animal risk assessment systems for the prediction of skin sensitization potential, to replace LLNA. Selection of an appropriate in vitro test or in silico model descriptors is critical to obtain good predictive performance

    32. Test battery with the human cell line activation test, direct peptide reactivity assay and DEREK based on a 139 chemical data set for predicting skin sensitizing potential and potency of chemicals

      Osamu Takenouchi, Shiho Fukui, Kenji Okamoto, Satoru Kurotani, Noriyasu Imai, Miyuki Fujishiro, Daiki Kyotani, Yoshinao Kato, Toshihiko Kasahara, Masaharu Fujita, Akemi Toyoda, Daisuke Sekiya, Shinichi Watanabe, Hirokazu Seto, Morihiko Hirota, Takao Ashikaga and Masaaki Miyazawa

      Article first published online: 29 MAR 2015 | DOI: 10.1002/jat.3127

      To develop a testing strategy incorporating the human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA) and DEREK, we created an expanded data set of 139 chemicals (102 sensitizers and 37 non-sensitizers) by combining the existing data set of 101 chemicals through the collaborative projects of Japan Cosmetic Industry Association. Of the additional 38 chemicals, 15 chemicals with relatively low water solubility (log Kow > 3.5) were selected to clarify the limitation of testing strategies regarding the lipophilic chemicals.

    33. A novel in chemico method to detect skin sensitizers in highly diluted reaction conditions

      Yusuke Yamamoto, Haruna Tahara, Ryota Usami, Toshihiko Kasahara, Yoshihiro Jimbo, Takanori Hioki and Masaharu Fujita

      Article first published online: 25 MAR 2015 | DOI: 10.1002/jat.3139

      We modified the ADRA (amino acid derivative reactivity assay) method by reducing the test chemical concentration in the solution 100-fold. We investigated the accuracy of skin sensitization predictions made using this modified method, which was designated the ADRA-dilutional method (ADRA-DM). The predictive accuracy of the ADRA-DM for skin sensitization was 90% for 82 test chemicals, and no precipitation of test compounds was observed. These results show that the ADRA-DM is a versatile and useful method to predict skin sensitization.

    34. Comparative effects of sulfhydryl compounds on target organellae, nuclei and mitochondria, of hydroxylated fullerene-induced cytotoxicity in isolated rat hepatocytes

      Yoshio Nakagawa, Akiko Inomata, Akio Ogata and Dai Nakae

      Article first published online: 23 MAR 2015 | DOI: 10.1002/jat.3137

      The exposure of rat hepatocytes to C60(OH)24 caused cell death accompanied by the formation of plasma membrane blebs, the loss of cellular levels of ATP and GSH, and the induction of DNA fragmentation and reactive oxygen species. C60(OH)24-induced DNA damage and mitochondrial dysfunction were effectively prevented by pretreatment with sulfhydryl compounds, NAC, L-cysteine, and L-methionine. These results suggest that the onset of toxic effects is at least partially attributable to a thiol redox-state imbalance caused by the fullerenol.

    35. Meloxicam inhibits fipronil-induced apoptosis via modulation of the oxidative stress and inflammatory response in SH-SY5Y cells

      Jae Hyeon Park, Youn Sun Park, Je-Bong Lee, Kyung-Hun Park, Min-kyoung Paik, Mihye Jeong and Hyun Chul Koh

      Article first published online: 13 MAR 2015 | DOI: 10.1002/jat.3136

      Oxidative stress and inflammatory responses have been identified as key elements of neuronal cell apoptosis. In this study, we investigated the mechanisms by which inflammatory responses contribute to apoptosis in human neuroblastoma SH-SY5Y cells treated with fipronil (FPN). Based on the cytotoxic mechanism of FPN, we examined the neuroprotective effects of meloxicam against FPN-induced neuronal cell death.

    36. Cellular localization of uranium in the renal proximal tubules during acute renal uranium toxicity

      Shino Homma-Takeda, Keisuke Kitahara, Kyoko Suzuki, Benjamin J. Blyth, Noriyoshi Suya, Teruaki Konishi, Yasuko Terada and Yoshiya Shimada

      Article first published online: 13 MAR 2015 | DOI: 10.1002/jat.3126

      To investigate cellular dynamics and localization of uranium in the renal proximal tubules, high-resolution quantitative in situ measurements by high-energy synchrotron radiation X-ray fluorescence analysis was performed for renal sections from a rat model of uranium-induced acute renal toxicity. Our findings suggest that site-specific accumulation of uranium in micro-regions of the S3 segment of the proximal tubules and retention of uranium in concentrated areas during recovery are characteristics of uranium behavior in the kidney.

    37. Time profiles and toxicokinetic parameters of key biomarkers of exposure to cypermethrin in orally exposed volunteers compared with previously available kinetic data following permethrin exposure

      Mylène Ratelle, Jonathan Coté and Michèle Bouchard

      Article first published online: 13 MAR 2015 | DOI: 10.1002/jat.3124

      Time profiles and toxicokinetic parameters of key biomarkers of exposure to cypermethrin pyrethroid were documented in 6 orally exposed volunteers and compared with previously available kinetic data following permethrin dosing. Blood and urine samples were collected over 72 h postdosing. Trans-DCCA, cis-DCCA and 3-PBA metabolites were quantified. Urinary rate time courses of metabolites showed apparent elimination t1/2 circa 6.4 h. These data confirm that the kinetics of cypermethrin is similar to that of permethrin in humans.

    38. Non-clinical safety assessment of single and repeated intramuscular administration of a human papillomavirus-16/18 vaccine in rabbits and rats

      Lawrence Segal, Danielle Morelle, Kari Kaaber, Eric Destexhe and Nathalie Garçon

      Article first published online: 6 MAR 2015 | DOI: 10.1002/jat.3131

      The human papillomavirus vaccine Cervarix® is for the prevention of cervical cancer. It contains recombinant virus-like particles and AS04 (MPL and aluminium salt). Local and systemic toxic effects of Cervarix® or AS04 were evaluated in repeated-dose studies in rabbits and rats. Treatment-related changes included a slight transient increase in circulating neutrophils and injection site inflammation. Thirteen weeks post-fourth dose, recovery at the injection site was near complete. Therefore in these non-clinical models, Cervarix® and AS04 alone were safe and well tolerated.

    39. Non-clinical safety and biodistribution of AS03-adjuvanted inactivated pandemic influenza vaccines

      Lawrence Segal, Sandrine Wouters, Danielle Morelle, Gaëlle Gautier, Julien Le Gal, Thomas Martin, Frieke Kuper, Eric Destexhe, Arnaud M. Didierlaurent and Nathalie Garçon

      Article first published online: 27 FEB 2015 | DOI: 10.1002/jat.3130

      To support the clinical development of candidate AS03-adjuvanted pandemic-influenza vaccines, local and systemic toxicity of 3–4 intramuscular (i.m.) injections of AS03, or AS03-adjuvanted or unadjuvanted split-A(H5N1) vaccines was evaluated in rabbits. The biodistribution of split-A(H5N1) vaccines and the constituents of AS03 was explored in mice. All test articles were well tolerated. Treatment-related effects were primarily associated with AS03 and were indicative of a transient mild local inflammation. The biodistribution kinetics of AS03 constituents were consistent with AS03 inducing this inflammation.

    40. Toxicity induced by Basic Violet 14, Direct Red 28 and Acid Red 26 in zebrafish larvae

      Bing Shen, Hong-Cui Liu, Wen-Bin Ou, Grant Eilers, Sheng-Mei Zhou, Fan-Guo Meng, Chun-Qi Li and Yong-Quan Li

      Article first published online: 27 FEB 2015 | DOI: 10.1002/jat.3134

      Basic Violet 14, Direct Red 28 and Acid Red 26 are classified as carcinogenic dyes despite insufficient toxicity data. In this paper, the toxicity of these dyes was assessed in a zebrafish model and the underlying toxic mechanisms were investigated. Treatment with these dyes resulted in common developmental abnormalities including delayed yolk sac absorption and swimming bladder deflation. Basic Violet 14 caused hepatotoxicity and Acid Red 26 caused cardiovascular toxicity.

    41. Distribution and biomarkers of carbon-14-labeled fullerene C60 ([14C(U)]C60) in female rats and mice for up to 30 days after intravenous exposure

      Susan C. J. Sumner, Rodney W. Snyder, Christopher Wingard, Ninell P. Mortensen, Nathan A. Holland, Jonathan H. Shannahan, Suraj Dhungana, Wimal Pathmasiri, Li Han, Anita H. Lewin and Timothy R. Fennell

      Article first published online: 27 FEB 2015 | DOI: 10.1002/jat.3110

      A comprehensive investigation of the distribution of polyvinylpyrrolidone-formulated [14C(U)]C60 (suspended in saline to form a 5% polyvinylpyrrolidone–saline suspension) in female rats and mice administered a single or five consecutive daily tail vein injections. Biomarkers of inflammation, cardiovascular injury and oxidative stress were measured to study the biological impact of [14C(U)]C60 exposure. The goals of the investigation were to provide a basic understanding of the distribution, migration, elimination and biological impacts of [14C(U)]C60 in rats and mice.

    42. The effect of a methyl-deficient diet on the global DNA methylation and the DNA methylation regulatory pathways

      Shota Takumi, Kazuyuki Okamura, Hiroyuki Yanagisawa, Tomoharu Sano, Yayoi Kobayashi and Keiko Nohara

      Article first published online: 17 FEB 2015 | DOI: 10.1002/jat.3117

      Methyl-deficient diets are considered to induce DNA methylation changes. However, the contribution of the active DNA demethylation pathways has not been investigated. Here, we investigated the involvement of the active DNA demethylation pathways in the DNA methylation changes by a methyl-deficient diet. Our results suggest that the DNA methylation status is strictly controlled by the balance between the DNA methylation and the active DNA demethylation pathways, despite the upregulation of the active DNA demethylation pathway by a methyl-deficient diet.

    43. Heterozygous p53 knockout mouse model for dehydropyrrolizidine alkaloid-induced carcinogenesis

      Ammon W. Brown, Bryan L. Stegelmeier, Steven M. Colegate, Kip E. Panter, Edward L. Knoppel and Jeffery O. Hall

      Article first published online: 17 FEB 2015 | DOI: 10.1002/jat.3120

      Dehydropyrrolizidine alkaloids (DHPAs) are a large, structurally diverse, potentially carcinogenic group of plant-derived protoxins that are common food contaminates. We utilized a heterozygous p53 knockout mouse model to compare the carcinogenic potential of various DHPAs. Exposure to riddelliine, a model DHPA, increased the odds of tumor development (odds ratio 2.05 and Wald 95% confidence limits between 1.2 and 3.4). Our research demonstrates the utility of this model for investigation of comparative carcinogenesis of different DHPAs and their N-oxides.

    44. Involvement of mitogen-activated protein kinase and NF-κB signaling pathways in perfluorooctane sulfonic acid-induced inflammatory reaction in BV2 microglial cells

      Jingying Zhu, Wenyi Qian, Yixin Wang, Rong Gao, Jun Wang and Hang Xiao

      Article first published online: 12 FEB 2015 | DOI: 10.1002/jat.3119

      Perfluorooctane sulfonic acid (PFOS) has been used in extensive commercial and industrial applications and is believed to be an emerging persistent organic pollutant. In this paper, we demonstrated that PFOS possesses immunomodulatory potential and exerts its action on inflammatory cytokines, tumor necrosis factor-alpha and interleukin-6, in microglial cells. The inflammatory reaction mediated by PFOS was partially via mitogen-activated protein kinases (MAPK) and NF-κB signals. Thus, these findings offer a potential mechanism for the study of proinflammatory activity of PFOS in the central nervous system.

    45. Two-generation reproduction and teratology studies of feeding aditoprim in Wistar rats

      Xu Wang, Ziqiang Tan, Guyue Cheng, Ihsan Awais, Lingli Huang, Dongmei Chen, Yuanhu Pan, Zhenli Liu and Zonghui Yuan

      Article first published online: 7 FEB 2015 | DOI: 10.1002/jat.3121

      The reproduction/development toxicity of aditoprim was evaluated by feeding diets containing 0~1000 mg kg-1, respectively. At 1000 mg kg-1 group, body weights in F0 and F1 rats, fetal body weight and number of viable fetuses in F0 and F1 generation significantly decreased. Teratogenicity study showed that body weights, fetal body lengths, tail lengths, litter weights and number of viable fetuses significantly decreased at 1000 mg kg-1 group. The NOAEL for reproduction/development toxicity of aditoprim was 100 mg kg-1 diet.

    46. Human bone morphogenetic protein-7 does not counteract aristolochic acid-induced renal toxicity

      Marie-Hélène Antoine, Frédéric Debelle, Julie Piccirilli, Fadoua El Kaddouri, Anne-Emilie Declèves, Eric De Prez, Cécile Husson, Frédérique Mies, Marie-Françoise Bourgeade and Joëlle L. Nortier

      Article first published online: 7 FEB 2015 | DOI: 10.1002/jat.3116

      Aristolochic acids (AA) are nephrotoxic and profibrotic agents, leading to chronic kidney disease. As some controversial studies have reported a nephroprotective effect of exogenous recombinant human bone morphogenetic protein (rhBMP)-7 in several models of renal fibrosis, we investigated the putative effect of rhBMP-7 to prevent progressive tubulointerstitial damage after AA intoxication in vitro and in vivo.

    47. Bisphenol A promotes X-linked inhibitor of apoptosis protein-dependent angiogenesis via G protein-coupled estrogen receptor pathway

      Jian Liu, Xin Jin, Nana Zhao, Xiaolei Ye and Chenjiang Ying

      Article first published online: 7 FEB 2015 | DOI: 10.1002/jat.3112

      Our study investigates the mechanisms underlying the pro-angiogenic effects of bisphenol A (BPA). We demonstrated that BPA markedly induces endothelial cell proliferation, migration and tube formation by activating endothelial nitric oxide synthase. BPA-induced nitric oxide generation appeared to be associated with X-linked inhibitor of apoptosis protein, which competes with endothelial nitric oxide synthase for caveolin-1. BPA was shown to exert its pro-angiogenic effect by upregulating X-linked inhibitor of apoptosis protein expression via G protein-coupled estrogen receptor activation but not via estrogen receptor ERα or ERβ.

    48. Safety assessment of aditoprim acute, subchronic toxicity and mutagenicity studies

      Xu Wang, Ziqiang Tan, Yuanhu Pan, Awais Ihsan, Qianying Liu, Lingli Huang, Guyue Cheng, Dongmei Chen, Yanfei Tao, Zhenli Liu and Zonghui Yuan

      Article first published online: 7 FEB 2015 | DOI: 10.1002/jat.3107

      Aditoprim (ADP) is a new developed dihydrofolate reductase inhibitor. The LD50 was 1400 mg kg-1 body weight (BW) day-1 in rats and 1130 mg kg-1 BW day-1 in mice. In the subchronic study, the main target organ for toxicity of ADP was the liver, and lymphocytic infiltration and hepatocytic necrosis were noted at 1000 mg kg-1 ADP diet group. The genotoxicity of ADP is negative. The NOAEL level for ADP was about 1.44-1.53 mg kg-1 BW day-1 in rats.

    49. Effects of cylindrospermopsin on the phagocytic cells of the common carp (Cyprinus carpio L.)

      Anna Sieroslawska, Anna Rymuszka and Łukasz Adaszek

      Article first published online: 29 JAN 2015 | DOI: 10.1002/jat.3118

      Cylindrospermopsin is a cyanotoxin with cytotoxic activity. In this study, we assessed the potential impact of cylindrospermopsin on the basic functions of phagocytic cells from common carp (Cyprinus carpio L.), including phagocytosis, reactive oxygen and nitrogen species production, and the structure of microfilaments and selected cytokine expression. The results indicated that the cyanotoxin cylindrospermopsin is able to modify basic features of carp phagocytic cells, which might result in adverse consequences for fish health.

    50. The relationship between Cd-induced autophagy and lysosomal activation in WRL-68 cells

      Su-Fang Meng, Wei-Ping Mao, Fang Wang, Xiao-Qian Liu and Luan-Luan Shao

      Article first published online: 29 JAN 2015 | DOI: 10.1002/jat.3114

      The present study shows the relationship between Cd-induced autophagy and lysosomal activation in WRL-68 cells. We found that the activation of lysosomal function was dependent on autophagosome and autophagosome–lysosome fusion. Autophagosome–lysosome fusion was inhibited by a rise of pH of acidic compartments. We also found that the intracellular Ca2+ did not markedly affect lysosomal pH, but lysosomal pH had a profound effect on the intracellular Ca2+. We infer that the intracellular Ca2+ channels or pumps are possibly pH-dependent in WRL-68 cells.

    51. Effects of homocysteine on mesenchymal cell proliferation and differentiation during chondrogenesis on limb development

      Gilian Fernando Bourckhardt, Manuela Sozo Cecchini, Dib Ammar, Karoline Kobus-Bianchini, Yara Maria Rauh Müller and Evelise Maria Nazari

      Article first published online: 23 JAN 2015 | DOI: 10.1002/jat.3111

      We investigated whether homocysteine (Hcy) can affect cell cycle proteins and mesenchymal differentiation during limb development. An increase of p53, which can be activated by DNA damage and a decrease of PCNA and p21 in Hcy-treated embryos were observed. Additionally, Hcy induced an increase of Pax9 and Sox9 proteins. We have described impairments induced by Hcy, which does not change the morphology of the cartilage mold. These findings provide new insights to understand the cellular basis of Hcy toxicity.

    52. Cytotoxicity of luteolin in primary rat hepatocytes: the role of CYP3A-mediated ortho-benzoquinone metabolite formation and glutathione depletion

      Fuguo Shi, Peng Zhao, Xiaobing Li, Hong Pan, Shiping Ma and Li Ding

      Article first published online: 21 JAN 2015 | DOI: 10.1002/jat.3106

      Luteolin, a well-known flavonoid, is widely distributed in the plant kingdom and present in many plant families. It can be found in diets, supplements and herbal medicines. The cytotoxicity of luteolin in hepatocytes was evaluated in this study. An electrophilic ortho-benzoquinone metabolite was identified by liquid chromatography coupled with tandem mass spectrometry in rat liver microsomes and primary rat hepatocytes. The CYP3A-mediated reactive metabolite formation was responsible for the cytotoxicity. Intracellular glutathione depletion by the ortho-benzoquinone metabolite was an initiating event in the cytotoxicity occurrence.

    53. Genomic and gene expression responses to genotoxic stress in PAC2 zebrafish embryonic cell line

      Maja Šrut, Jean-Paul Bourdineaud, Anamaria Štambuk and Göran I. V. Klobučar

      Article first published online: 21 JAN 2015 | DOI: 10.1002/jat.3113

      In this study, we assessed PAC2 cell line responses toward different forms of genotoxic stress by using the battery of tests: the Comet assay, quantitative random-amplified polymorphic DNA, amplified fragment length polymorphism and expression of several DNA repair, oxidative stress response and xenobiotic metabolism genes. PAC2 cell line exhibited genotoxic responses in all used test methods upon direct, and to a lower extent upon indirect acting genotoxicant.

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