IJMS: Molecular Toxicology

IJMS, Vol. 13, Pages 1805-1831: Aggregating Data for Computational Toxicology Applications: The U.S. Environmental Protection Agency (EPA) Aggregated Computational Toxicology Resource (ACToR) System

Richard S. Judson — Thu, 09 Feb 2012 00:00:00 CET

Computational toxicology combines data from high-throughput test methods, chemical structure analyses and other biological domains (e.g., genes, proteins, cells, tissues) with the goals of predicting and understanding the underlying mechanistic causes of chemical toxicity and for predicting toxicity of new chemicals and products. A key feature of such approaches is their reliance on knowledge extracted from large collections of data and data sets in computable formats. The U.S. Environmental Protection Agency (EPA) has developed a large data resource called ACToR (Aggregated Computational Toxicology Resource) to support these data-intensive efforts. ACToR comprises four main repositories: core ACToR (chemical identifiers and structures, and summary data on hazard, exposure, use, and other domains), ToxRefDB (Toxicity Reference Database, a compilation of detailed in vivo toxicity data from guideline studies), ExpoCastDB (detailed human exposure data from observational studies of selected chemicals), and ToxCastDB (data from high-throughput screening programs, including links to underlying biological information related to genes and pathways). The EPA DSSTox (Distributed Structure-Searchable Toxicity) program provides expert-reviewed chemical structures and associated information for these and other high-interest public inventories. Overall, the ACToR system contains information on about 400,000 chemicals from 1100 different sources. The entire system is built using open source tools and is freely available to download. This review describes the organization of the data repository and provides selected examples of use cases.

IJMS, Vol. 13, Pages 1804: Retraction: Zhong Ye; Darya O. Mishchuk; Natasha S. Stephens and Carolyn M. Slupsky. Dextran Sulfate Sodium Inhibits Alanine Synthesis in Caco-2 Cells. Int. J. Mol. Sci. 2011, 12, 2325-2335.

Shu-Kun Lin — Thu, 09 Feb 2012 00:00:00 CET

It has been brought to our attention by the corresponding author that the results presented this article [1] are in error due to the fact that the media supplement glutaMAX was used in place of L-glutamine for culture of the control cells, while L-glutamine was used for culture of the treated cells. All authors have confirmed that the reported result could not be reproduced using the correct culture conditions. We would like to thank the authors for pointing out this error thereby upholding the ethics of scientific publication. The Editorial Team and Publisher have agreed with the authors that this manuscript should be retracted. We apologize for any inconvenience this may have caused.

IJMS, Vol. 13, Pages 1186-1208: Inhibition of AKT2 Enhances Sensitivity to Gemcitabine via Regulating PUMA and NF-κB Signaling Pathway in Human Pancreatic Ductal Adenocarcinoma

Dong Chen — Fri, 20 Jan 2012 00:00:00 CET

Invasion, metastasis and resistance to conventional chemotherapeutic agents are obstacles to successful treatment of pancreatic cancer, and a better understanding of the molecular basis of this malignancy may lead to improved therapeutics. In the present study, we investigated whether AKT2 silencing sensitized pancreatic cancer L3.6pl, BxPC-3, PANC-1 and MIAPaCa-2 cells to gemcitabine via regulating PUMA (p53-upregulated modulator of apoptosis) and nuclear factor (NF)-κB signaling pathway. MTT, TUNEL, EMSA and NF-κB reporter assays were used to detect tumor cell proliferation, apoptosis and NF-κB activity. Western blotting was used to detect different protein levels. Xenograft of established tumors was used to evaluate primary tumor growth and apoptosis after treatment with gemcitabine alone or in combination with AKT2 siRNA. Gemcitabine activated AKT2 and NF-κB in MIAPaCa-2 and L3.6pl cells in vitro or in vivo, and in PANC-1 cells only in vivo. Gemcitabine only activated NF-κB in BxPC-3 cells in vitro. The presence of PUMA was necessary for gemcitabine-induced apoptosis only in BxPC-3 cells in vitro. AKT2 inhibition sensitized gemcitabine-induced apoptosis via PUMA upregulation in MIAPaCa-2 cells in vitro, and via NF-κB activity inhibition in L3.6pl cells in vitro. In PANC-1 and MIAPaCa-2 cells in vivo, AKT2 inhibition sensitized gemcitabine-induced apoptosis and growth inhibition via both PUMA upregulation and NF-κB inhibition. We suggest that AKT2 inhibition abrogates gemcitabine-induced activation of AKT2 and NF-κB, and promotes gemcitabine-induced PUMA upregulation, resulting in chemosensitization of pancreatic tumors to gemcitabine, which is probably an important strategy for the treatment of pancreatic cancer.

IJMS, Vol. 13, Pages 427-452: Adaptation of High-Throughput Screening in Drug Discovery—Toxicological Screening Tests

Paweł Szymański — Thu, 29 Dec 2011 00:00:00 CET

High-throughput screening (HTS) is one of the newest techniques used in drug design and may be applied in biological and chemical sciences. This method, due to utilization of robots, detectors and software that regulate the whole process, enables a series of analyses of chemical compounds to be conducted in a short time and the affinity of biological structures which is often related to toxicity to be defined. Since 2008 we have implemented the automation of this technique and as a consequence, the possibility to examine 100,000 compounds per day. The HTS method is more frequently utilized in conjunction with analytical techniques such as NMR or coupled methods e.g., LC-MS/MS. Series of studies enable the establishment of the rate of affinity for targets or the level of toxicity. Moreover, researches are conducted concerning conjugation of nanoparticles with drugs and the determination of the toxicity of such structures. For these purposes there are frequently used cell lines. Due to the miniaturization of all systems, it is possible to examine the compound’s toxicity having only 1–3 mg of this compound. Determination of cytotoxicity in this way leads to a significant decrease in the expenditure and to a reduction in the length of the study.

IJMS, Vol. 13, Pages 369-382: Potentiation of Anticancer Drugs: Effects of Pentoxifylline on Neoplastic Cells

Miroslav Barancik — Wed, 28 Dec 2011 00:00:00 CET

The drug efflux activity of P-glycoprotein (P-gp, a product of the mdr1 gene, ABCB1 member of ABC transporter family) represents a mechanism by which tumor cells escape death induced by chemotherapeutics. In this study, we investigated the mechanisms involved in the effects of pentoxifylline (PTX) on P-gp-mediated multidrug resistance (MDR) in mouse leukemia L1210/VCR cells. Parental sensitive mouse leukemia cells L1210, and multidrug-resistant cells, L1210/VCR, which are characterized by the overexpression of P-gp, were used as experimental models. The cells were exposed to 100 μmol/L PTX in the presence or absence of 1.2 μmol/L vincristine (VCR). Western blot analysis indicated a downregulation of P-gp protein expression when multidrug-resistant L1210/VCR cells were exposed to PTX. The effects of PTX on the sensitization of L1210/VCR cells to VCR correlate with the stimulation of apoptosis detected by Annexin V/propidium iodide apoptosis necrosis kit and proteolytic activation of both caspase-3 and caspase-9 monitored by Western blot analysis. Higher release of matrix metalloproteinases (MMPs), especially MMP-2, which could be attenuated by PTX, was found in L1210/VCR than in L1210 cells by gelatin zymography in electrophoretic gel. Exposure of resistant cells to PTX increased the content of phosphorylated Akt kinase. In contrast, the presence of VCR eliminated the effects of PTX on Akt kinase phosphorylation. Taken together, we conclude that PTX induces the sensitization of multidrug-resistant cells to VCR via downregulation of P-gp, stimulation of apoptosis and reduction of MMPs released from drug-resistant L1210/VCR cells. These facts bring new insights into the mechanisms of PTX action on cancer cells.

IJMS, Vol. 13, Pages 142-172: Protein Kinases and Transcription Factors Activation in Response to UV-Radiation of Skin: Implications for Carcinogenesis

César López-Camarillo — Fri, 23 Dec 2011 00:00:00 CET

Solar ultraviolet (UV) radiation is an important environmental factor that leads to immune suppression, inflammation, photoaging, and skin carcinogenesis. Here, we reviewed the specific signal transduction pathways and transcription factors involved in the cellular response to UV-irradiation. Increasing experimental data supporting a role for p38, MAPK, JNK, ERK1/2, and ATM kinases in the response network to UV exposure is discussed. We also reviewed the participation of NF-κB, AP-1, and NRF2 transcription factors in the control of gene expression after UV-irradiation. In addition, we discussed the promising chemotherapeutic intervention of transcription factors signaling by natural compounds. Finally, we focused on the review of data emerging from the use of DNA microarray technology to determine changes in global gene expression in keratinocytes and melanocytes in response to UV treatment. Efforts to obtain a comprehensive portrait of the transcriptional events regulating photodamage of intact human epidermis after UV exposure reveals the existence of novel factors participating in UV-induced cell death. Progress in understanding the multitude of mechanisms induced by UV-irradiation could lead to the potential use of protein kinases and novel proteins as specific targets for the prevention and control of skin cancer.

IJMS, Vol. 12, Pages 9172-9188: Dynamics of Protein Phosphatase Gene Expression in Corbicula fluminea Exposed to Microcystin-LR and to Toxic Microcystis aeruginosa Cells

José Carlos Martins — Thu, 08 Dec 2011 00:00:00 CET

This study investigated the in vivo effects of microcystins on gene expression of several phosphoprotein phosphatases (PPP) in the freshwater clam Corbicula fluminea with two different exposure scenarios. Clams were exposed for 96 h to 5 µg L−1 of dissolved microcystin-LR and the relative changes of gene expression of three different types of PPP (PPP1, 2 and 4) were analyzed by quantitative real-time PCR. The results showed a significant induction of PPP2 gene expression in the visceral mass. In contrast, the cyanotoxin did not cause any significant changes on PPP1 and PPP4 gene expression. Based on these results, we studied alterations in transcriptional patterns in parallel with enzymatic activity of C. fluminea for PPP2, induced by a Microcystis aeruginosa toxic strain (1 × 105 cells cm−3) during 96 h. The relative changes of gene expression and enzyme activity in visceral mass were analyzed by quantitative real-time PCR and colorimetric assays respectively. The clams exhibited a significant reduction of PPP2 activity with a concomitant enhancement of gene expression. Considering all the results we can conclude that the exposure to an ecologically relevant concentration of pure or intracellular microcystins (-LR) promoted an in vivo effect on PPP2 gene expression in C. fluminea.

IJMS, Vol. 12, Pages 8947-8960: The Role of the E2F Transcription Factor Family in UV-Induced Apoptosis

Mehlika Hazar-Rethinam — Tue, 06 Dec 2011 00:00:00 CET

The E2F transcription factor family is traditionally associated with cell cycle control. However, recent data has shown that activating E2Fs (E2F1-3a) are potent activators of apoptosis. In contrast, the recently cloned inhibitory E2Fs (E2F7 and 8) appear to antagonize E2F-induced cell death. In this review we will discuss (i) the potential role of E2Fs in UV-induced cell death and (ii) the implications of this to the development of UV-induced cutaneous malignancies.

IJMS, Vol. 12, Pages 8513-8529: Caffeine Abolishes the Ultraviolet-Induced REV3 Translesion Replication Pathway in Mouse Cells

Jun Takezawa — Tue, 29 Nov 2011 00:00:00 CET

When a replicative DNA polymerase stalls upon encountering a photoproduct on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion. Using an alkaline sucrose density gradient sedimentation technique, we previously classified this process termed UV-induced translesion replication (UV-TLS) into two types. In human cancer cells or xeroderma pigmentosum variant (XP-V) cells, UV-TLS was inhibited by caffeine or proteasome inhibitors. However, in normal human cells, the process was insensitive to these reagents. Reportedly, in yeast or mammalian cells, REV3 protein (a catalytic subunit of DNA polymerase ζ) is predominantly involved in the former type of TLS. Here, we studied UV-TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). In the wild-type MEF, UV-TLS was slow (similar to that of human cancer cells or XP-V cells), and was abolished by caffeine or MG-262. In 2 cell lines of Rev3KO-MEF (Rev3−/− p53−/−), UV-TLS was not observed. In p53KO-MEF, which is a strict control for Rev3KO-MEF, the UV-TLS response was similar to that of the wild-type. Introduction of the Rev3 expression plasmid into Rev3KO-MEF restored the UV-TLS response in selected stable transformants. In some transformants, viability to UV was the same as that in the wild-type, and the death rate was increased by caffeine. Our findings indicate that REV3 is predominantly involved in UV-TLS in mouse cells, and that the REV3 translesion pathway is suppressed by caffeine or proteasome inhibitors.

IJMS, Vol. 12, Pages 8302-8315: Coenzyme Q10 Ameliorates Ultraviolet B Irradiation Induced Cell Death Through Inhibition of Mitochondrial Intrinsic Cell Death Pathway

Li Jing — Thu, 24 Nov 2011 00:00:00 CET

Ultraviolet B (UVB) induces cell death by increasing free radical production, activating apoptotic cell death pathways and depolarizing mitochondrial membrane potential. Coenzyme Q10 (CoQ10), an essential cofactor in the mitochondrial electron transport chain, serves as a potent antioxidant in the mitochondria. The aim of the present study is to establish whether CoQ10 is capable of protecting neuronal cells against UVB-induced damage. Murine hippocampal HT22 cells were treated with 0.01, 0.1 or 1 µM of CoQ10 3 or 24 h prior to the cells being exposed to UVB irradiation. The CoQ10 concentrations were maintained during irradiation and 24 h post-UVB. Cell viability was assessed by counting viable cells and MTT conversion assay. Superoxide production and mitochondrial membrane potential were measured using fluorescent probes. Levels of cleaved caspase-9, caspase-3, and apoptosis-inducing factor (AIF) were detected using immunocytochemistry and Western blotting. The results showed that UVB irradiation decreased cell viability and such damaging effect was associated with increased superoxide production, mitochondrial depolarization, and activation of caspase-9 and caspase-3. Treatment with CoQ10 at three different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide production, normalization of mitochondrial membrane potential and inhibition of caspase-9 and caspase-3 activation. It is concluded that the neuroprotective effect of CoQ10 results from inhibiting oxidative stress and blocking caspase-3 dependent cell death pathway.

IJMS, Vol. 12, Pages 8288-8301: Nanotechnology and Nanotoxicology in Retinopathy

Dong Hyun Jo — Wed, 23 Nov 2011 00:00:00 CET

Nanoparticles are nanometer-scaled particles, and can be utilized in the form of nanocapsules, nanoconjugates, or nanoparticles themselves for the treatment of retinopathy, including angiogensis-related blindness, retinal degeneration, and uveitis. They are thought to improve the bioavailability in the retina and the permeability of therapeutic molecules across the barriers of the eye, such as the cornea, conjunctiva, and especially, blood-retinal barriers (BRBs). However, consisting of multiple neuronal cells, the retina can be the target of neuronal toxicity of nanoparticles, in common with the central and peripheral nervous system. Furthermore, the ability of nanoparticles to pass through the BRBs might increase the possibility of toxicity, simultaneously promoting distribution in the retinal layers. In this regard, we discussed nanotechnology and nanotoxicology in the treatment of retinopathy.

IJMS, Vol. 12, Pages 8181-8207: Effect of Polyphenols on Oxidative Stress and Mitochondrial Dysfunction in Neuronal Death and Brain Edema in Cerebral Ischemia

Kiran S. Panickar — Fri, 18 Nov 2011 00:00:00 CET

Polyphenols are natural substances with variable phenolic structures and are elevated in vegetables, fruits, grains, bark, roots, tea, and wine. There are over 8000 polyphenolic structures identified in plants, but edible plants contain only several hundred polyphenolic structures. In addition to their well-known antioxidant effects, select polyphenols also have insulin-potentiating, anti-inflammatory, anti-carcinogenic, anti-viral, anti-ulcer, and anti-apoptotic properties. One important consequence of ischemia is neuronal death and oxidative stress plays a key role in neuronal viability. In addition, neuronal death may be initiated by the activation of mitochondria-associated cell death pathways. Another consequence of ischemia that is possibly mediated by oxidative stress and mitochondrial dysfunction is glial swelling, a component of cytotoxic brain edema. The purpose of this article is to review the current literature on the contribution of oxidative stress and mitochondrial dysfunction to neuronal death, cell swelling, and brain edema in ischemia. A review of currently known mechanisms underlying neuronal death and edema/cell swelling will be undertaken and the potential of dietary polyphenols to reduce such neural damage will be critically reviewed.

IJMS, Vol. 12, Pages 8063-8085: Chromatin Structure Following UV-Induced DNA Damage—Repair or Death?

Andrew W. Farrell — Thu, 17 Nov 2011 00:00:00 CET

In eukaryotes, DNA is compacted into a complex structure known as chromatin. The unravelling of DNA is a crucial step in DNA repair, replication, transcription and recombination as this allows access to DNA for these processes. Failure to package DNA into the nucleosome, the individual unit of chromatin, can lead to genomic instability, driving a cell into apoptosis, senescence, or cellular proliferation. Ultraviolet (UV) radiation damage causes destabilisation of chromatin integrity. UV irradiation induces DNA damage such as photolesions and subjects the chromatin to substantial rearrangements, causing the arrest of transcription forks and cell cycle arrest. Highly conserved processes known as nucleotide and base excision repair (NER and BER) then begin to repair these lesions. However, if DNA repair fails, the cell may be forced into apoptosis. The modification of various histones as well as nucleosome remodelling via ATP-dependent chromatin remodelling complexes are required not only to repair these UV-induced DNA lesions, but also for apoptosis signalling. Histone modifications and nucleosome remodelling in response to UV also lead to the recruitment of various repair and pro-apoptotic proteins. Thus, the way in which a cell responds to UV irradiation via these modifications is important in determining its fate. Failure of these DNA damage response steps can lead to cellular proliferation and oncogenic development, causing skin cancer, hence these chromatin changes are critical for a proper response to UV-induced injury.

IJMS, Vol. 12, Pages 7996-8012: The Impact of the Fusarium Mycotoxin Deoxynivalenol on the Health and Performance of Broiler Chickens

Wageha A. Awad — Wed, 16 Nov 2011 00:00:00 CET

The aim of the present experiment was to investigate the effects of feeding grains naturally contaminated with Fusarium mycotoxins on morphometric indices of jejunum and to follow the passage of deoxynivalenol (DON) through subsequent segments of the digestive tract of broilers. A total of 45 1-d-old broiler chickens (Ross 308 males) were randomly allotted to three dietary treatments (15 birds/treatment): (1) control diet; (2) diet contaminated with 1 mg DON/kg feed; (3) diet contaminated with 5 mg DON/kg feed for five weeks. None of the zootechnical traits (body weight, body weight gain, feed intake, and feed conversion) responded to increased DON levels in the diet. However, DON at both dietary levels (1 mg and 5 mg DON/kg feed) significantly altered the small intestinal morphology. In the jejunum, the villi were significantly (P < 0.01) shorter in both DON treated groups compared with the controls. Furthermore, the dietary inclusion of DON decreased (P < 0.05) the villus surface area in both DON treated groups. The absolute or relative organ weights (liver, heart, proventriculus, gizzard, small intestine, spleen, pancreas, colon, cecum, bursa of Fabricius and thymus) were not altered (P > 0.05) in broilers fed the diet containing DON compared with controls. DON and de-epoxy-DON (DOM-1) were analyzed in serum, bile, liver, feces and digesta from consecutive segments of the digestive tract (gizzard, cecum, and rectum). Concentrations of DON and its metabolite DOM-1 in serum, bile, and liver were lower than the detection limits of the applied liquid chromatography coupled with mass spectrometry (LC-MS/MS) method. Only about 10 to 12% and 6% of the ingested DON was recovered in gizzard and feces, irrespective of the dietary DON-concentration. However, the DON recovery in the cecum as percentage of DON-intake varied between 18 to 22% and was not influenced by dietary DON-concentration. Interestingly, in the present trial, DOM-1 did not appear in the large intestine and in feces. The results indicate that deepoxydation in the present study hardly occurred in the distal segments of the digestive tract, assuming that the complete de-epoxydation occurs in the proximal small intestine where the majority of the parent toxin is absorbed. In conclusion, diets with DON contamination below levels that induce a negative impact on performance could alter small intestinal morphology in broilers. Additionally, the results confirm that the majority of the ingested DON quickly disappears through the gastrointestinal tract.

IJMS, Vol. 12, Pages 7652-7661: Lidocaine Induces Endoplasmic Reticulum Stress-Associated Apoptosis in Vitro and in Vivo

Dae Young Hong — Tue, 08 Nov 2011 00:00:00 CET

We demonstrated that upregulation of both gene expression of endoplasmic reticulum (ER) stress chaperones (BiP, calnexin, calreticulin, and PDI) and ER stress sensors (ATF6, IRE1 and PERK) was induced by lidocaine, a local anesthetic, in PC12 cells. In addition to gene regulation, lidocaine also induced typical ER stress phenomena such as ART6 proteolytic cleavage, eIF2 alpha phosphorylation, and XBP1 mRNA splicing. In in vivo experiments, while lidocaine downregulated gene expression of anti-apoptotic factors (Bcl-2 and Bcl-xl), pro-apoptotic factor (Bak and Bax) gene expression was upregulated. Furthermore, lidocaine induced apoptosis, as measured histochemically, and upregulated PARP1, a DNA damage repair enzyme. These results are the first to show that lidocaine induces apoptosis through ER stress in vitro and in vivo.

IJMS, Vol. 12, Pages 7469-7480: Development of a Human Physiologically Based Pharmacokinetic (PBPK) Toolkit for Environmental Pollutants

Patricia Ruiz — Mon, 31 Oct 2011 00:00:00 CET

Physiologically Based Pharmacokinetic (PBPK) models can be used to determine the internal dose and strengthen exposure assessment. Many PBPK models are available, but they are not easily accessible for field use. The Agency for Toxic Substances and Disease Registry (ATSDR) has conducted translational research to develop a human PBPK model toolkit by recoding published PBPK models. This toolkit, when fully developed, will provide a platform that consists of a series of priority PBPK models of environmental pollutants. Presented here is work on recoded PBPK models for volatile organic compounds (VOCs) and metals. Good agreement was generally obtained between the original and the recoded models. This toolkit will be available for ATSDR scientists and public health assessors to perform simulations of exposures from contaminated environmental media at sites of concern and to help interpret biomonitoring data. It can be used as screening tools that can provide useful information for the protection of the public.

IJMS, Vol. 12, Pages 7199-7215: Roles of Oxidative Stress, Apoptosis, PGC-1α and Mitochondrial Biogenesis in Cerebral Ischemia

Shang-Der Chen — Fri, 21 Oct 2011 00:00:00 CEST

The primary physiological function of mitochondria is to generate adenosine triphosphate through oxidative phosphorylation via the electron transport chain. Overproduction of reactive oxygen species (ROS) as byproducts generated from mitochondria have been implicated in acute brain injuries such as stroke from cerebral ischemia. It was well-documented that mitochondria-dependent apoptotic pathway involves pro- and anti-apoptotic protein binding, release of cytochrome c, leading ultimately to neuronal death. On the other hand, mitochondria also play a role to counteract the detrimental effects elicited by excessive oxidative stress. Recent studies have revealed that oxidative stress and the redox state of ischemic neurons are also implicated in the signaling pathway that involves peroxisome proliferative activated receptor-γ (PPARγ) co-activator 1α (PGC1-α). PGC1-α is a master regulator of ROS scavenging enzymes including manganese superoxide dismutase 2 and the uncoupling protein 2, both are mitochondrial proteins, and may contribute to neuronal survival. PGC1-α is also involved in mitochondrial biogenesis that is vital for cell survival. Experimental evidence supports the roles of mitochondrial dysfunction and oxidative stress as determinants of neuronal death as well as endogenous protective mechanisms after stroke. This review aims to summarize the current knowledge focusing on the molecular mechanisms underlying cerebral ischemia involving ROS, mitochondrial dysfunction, apoptosis, mitochondrial proteins capable of ROS scavenging, and mitochondrial biogenesis.

IJMS, Vol. 12, Pages 7163-7185: Mitochondrial Peroxiredoxin III is a Potential Target for Cancer Therapy

In-Sung Song — Fri, 21 Oct 2011 00:00:00 CEST

Mitochondria are involved either directly or indirectly in oncogenesis and the alteration of metabolism in cancer cells. Cancer cells contain large numbers of abnormal mitochondria and produce large amounts of reactive oxygen species (ROS). Oxidative stress is caused by an imbalance between the production of ROS and the antioxidant capacity of the cell. Several cancer therapies, such as chemotherapeutic drugs and radiation, disrupt mitochondrial homeostasis and release cytochrome c, leading to apoptosome formation, which activates the intrinsic pathway. This is modulated by the extent of mitochondrial oxidative stress. The peroxiredoxin (Prx) system is a cellular defense system against oxidative stress, and mitochondria in cancer cells are known to contain high levels of Prx III. Here, we review accumulating evidence suggesting that mitochondrial oxidative stress is involved in cancer, and discuss the role of the mitochondrial Prx III antioxidant system as a potential target for cancer therapy. We hope that this review will provide the basis for new strategic approaches in the development of effective cancer treatments.

IJMS, Vol. 12, Pages 7114-7162: Manganese Superoxide Dismutase: Guardian of the Powerhouse

Aaron K. Holley — Fri, 21 Oct 2011 00:00:00 CEST

The mitochondrion is vital for many metabolic pathways in the cell, contributing all or important constituent enzymes for diverse functions such as β-oxidation of fatty acids, the urea cycle, the citric acid cycle, and ATP synthesis. The mitochondrion is also a major site of reactive oxygen species (ROS) production in the cell. Aberrant production of mitochondrial ROS can have dramatic effects on cellular function, in part, due to oxidative modification of key metabolic proteins localized in the mitochondrion. The cell is equipped with myriad antioxidant enzyme systems to combat deleterious ROS production in mitochondria, with the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) acting as the chief ROS scavenging enzyme in the cell. Factors that affect the expression and/or the activity of MnSOD, resulting in diminished antioxidant capacity of the cell, can have extraordinary consequences on the overall health of the cell by altering mitochondrial metabolic function, leading to the development and progression of numerous diseases. A better understanding of the mechanisms by which MnSOD protects cells from the harmful effects of overproduction of ROS, in particular, the effects of ROS on mitochondrial metabolic enzymes, may contribute to the development of novel treatments for various diseases in which ROS are an important component.

IJMS, Vol. 12, Pages 6894-6918: Metal-Induced Oxidative Stress and Plant Mitochondria

Els Keunen — Tue, 18 Oct 2011 00:00:00 CEST

A general status of oxidative stress in plants caused by exposure to elevated metal concentrations in the environment coincides with a constraint on mitochondrial electron transport, which enhances ROS accumulation at the mitochondrial level. As mitochondria are suggested to be involved in redox signaling under environmental stress conditions, mitochondrial ROS can initiate a signaling cascade mediating the overall stress response, i.e., damage versus adaptation. This review highlights our current understanding of metal-induced responses in plants, with focus on the production and detoxification of mitochondrial ROS. In addition, the potential involvement of retrograde signaling in these processes will be discussed.

IJMS, Vol. 12, Pages 6668-6684: Principal Component Analysis Coupled with Artificial Neural Networks—A Combined Technique Classifying Small Molecular Structures Using a Concatenated Spectral Database

Steluţa Gosav — Tue, 11 Oct 2011 00:00:00 CEST

In this paper we present several expert systems that predict the class identity of the modeled compounds, based on a preprocessed spectral database. The expert systems were built using Artificial Neural Networks (ANN) and are designed to predict if an unknown compound has the toxicological activity of amphetamines (stimulant and hallucinogen), or whether it is a nonamphetamine. In attempts to circumvent the laws controlling drugs of abuse, new chemical structures are very frequently introduced on the black market. They are obtained by slightly modifying the controlled molecular structures by adding or changing substituents at various positions on the banned molecules. As a result, no substance similar to those forming a prohibited class may be used nowadays, even if it has not been specifically listed. Therefore, reliable, fast and accessible systems capable of modeling and then identifying similarities at molecular level, are highly needed for epidemiological, clinical, and forensic purposes. In order to obtain the expert systems, we have preprocessed a concatenated spectral database, representing the GC-FTIR (gas chromatography-Fourier transform infrared spectrometry) and GC-MS (gas chromatography-mass spectrometry) spectra of 103 forensic compounds. The database was used as input for a Principal Component Analysis (PCA). The scores of the forensic compounds on the main principal components (PCs) were then used as inputs for the ANN systems. We have built eight PC-ANN systems (principal component analysis coupled with artificial neural network) with a different number of input variables: 15 PCs, 16 PCs, 17 PCs, 18 PCs, 19 PCs, 20 PCs, 21 PCs and 22 PCs. The best expert system was found to be the ANN network built with 18 PCs, which accounts for an explained variance of 77%. This expert system has the best sensitivity (a rate of classification C = 100% and a rate of true positives TP = 100%), as well as a good selectivity (a rate of true negatives TN = 92.77%). A comparative analysis of the validation results of all expert systems is presented, and the input variables with the highest discrimination power are discussed.

IJMS, Vol. 12, Pages 6502-6516: 3D-QSAR Studies on Thiazolidin-4-one S1P1 Receptor Agonists by CoMFA and CoMSIA

Chuiwen Qian — Wed, 28 Sep 2011 00:00:00 CEST

Selective S1P1 receptor agonists have therapeutic potential to treat a variety of immune-mediated diseases. A series of 2-imino-thiazolidin-4-one derivatives displaying potent S1P1 receptor agonistic activity were selected to establish 3D-QSAR models using CoMFA and CoMSIA methods. Internal and external cross-validation techniques were investigated as well as some measures including region focusing, progressive scrambling, bootstraping and leave-group-out. The satisfactory CoMFA model predicted a q2 value of 0.751 and an r2 value of 0.973, indicating that electrostatic and steric properties play a significant role in potency. The best CoMSIA model, based on a combination of steric, electrostatic, hydrophobic and H-bond donor descriptors, predicted a q2 value of 0.739 and an r2 value of 0.923. The models were graphically interpreted using contour plots which gave more insight into the structural requirements for increasing the activity of a compound, providing a solid basis for future rational design of more active S1P1 receptor agonists.

IJMS, Vol. 12, Pages 6469-6501: Metabolomics of Oxidative Stress in Recent Studies of Endogenous and Exogenously Administered Intermediate Metabolites

Jia Liu — Wed, 28 Sep 2011 00:00:00 CEST

Aerobic metabolism occurs in a background of oxygen radicals and reactive oxygen species (ROS) that originate from the incomplete reduction of molecular oxygen in electron transfer reactions. The essential role of aerobic metabolism, the generation and consumption of ATP and other high energy phosphates, sustains a balance of approximately 3000 essential human metabolites that serve not only as nutrients, but also as antioxidants, neurotransmitters, osmolytes, and participants in ligand-based and other cellular signaling. In hypoxia, ischemia, and oxidative stress, where pathological circumstances cause oxygen radicals to form at a rate greater than is possible for their consumption, changes in the composition of metabolite ensembles, or metabolomes, can be associated with physiological changes. Metabolomics and metabonomics are a scientific disciplines that focuse on quantifying dynamic metabolome responses, using multivariate analytical approaches derived from methods within genomics, a discipline that consolidated innovative analysis techniques for situations where the number of biomarkers (metabolites in our case) greatly exceeds the number of subjects. This review focuses on the behavior of cytosolic, mitochondrial, and redox metabolites in ameliorating or exacerbating oxidative stress. After reviewing work regarding a small number of metabolites—pyruvate, ethyl pyruvate, and fructose-1,6-bisphosphate—whose exogenous administration was found to ameliorate oxidative stress, a subsequent section reviews basic multivariate statistical methods common in metabolomics research, and their application in human and preclinical studies emphasizing oxidative stress. Particular attention is paid to new NMR spectroscopy methods in metabolomics and metabonomics. Because complex relationships connect oxidative stress to so many physiological processes, studies from different disciplines were reviewed. All, however, shared the common goal of ultimately developing “omics”-based, diagnostic tests to help influence therapies.

IJMS, Vol. 12, Pages 6226-6239: Sirt3, Mitochondrial ROS, Ageing, and Carcinogenesis

Seong-Hoon Park — Fri, 23 Sep 2011 00:00:00 CEST

One fundamental observation in cancer etiology is that the rate of malignancies in any mammalian population increases exponentially as a function of age, suggesting a mechanistic link between the cellular processes governing longevity and carcinogenesis. In addition, it is well established that aberrations in mitochondrial metabolism, as measured by increased reactive oxygen species (ROS), are observed in both aging and cancer. In this regard, genes that impact upon longevity have recently been characterized in S. cerevisiae and C. elegans, and the human homologs include the Sirtuin family of protein deacetylases. Interestingly, three of the seven sirtuin proteins are localized into the mitochondria suggesting a connection between the mitochondrial sirtuins, the free radical theory of aging, and carcinogenesis. Based on these results it has been hypothesized that Sirt3 functions as a mitochondrial fidelity protein whose function governs both aging and carcinogenesis by modulating ROS metabolism. Sirt3 has also now been identified as a genomically expressed, mitochondrial localized tumor suppressor and this review will outline potential relationships between mitochondrial ROS/superoxide levels, aging, and cell phenotypes permissive for estrogen and progesterone receptor positive breast carcinogenesis.

IJMS, Vol. 12, Pages 6116-6134: High-Density Real-Time PCR-Based in Vivo Toxicogenomic Screen to Predict Organ-Specific Toxicity

Gabriella Fabian — Mon, 19 Sep 2011 00:00:00 CEST

Toxicogenomics, based on the temporal effects of drugs on gene expression, is able to predict toxic effects earlier than traditional technologies by analyzing changes in genomic biomarkers that could precede subsequent protein translation and initiation of histological organ damage. In the present study our objective was to extend in vivo toxicogenomic screening from analyzing one or a few tissues to multiple organs, including heart, kidney, brain, liver and spleen. Nanocapillary quantitative real-time PCR (QRT-PCR) was used in the study, due to its higher throughput, sensitivity and reproducibility, and larger dynamic range compared to DNA microarray technologies. Based on previous data, 56 gene markers were selected coding for proteins with different functions, such as proteins for acute phase response, inflammation, oxidative stress, metabolic processes, heat-shock response, cell cycle/apoptosis regulation and enzymes which are involved in detoxification. Some of the marker genes are specific to certain organs, and some of them are general indicators of toxicity in multiple organs. Utility of the nanocapillary QRT-PCR platform was demonstrated by screening different references, as well as discovery of drug-like compounds for their gene expression profiles in different organs of treated mice in an acute experiment. For each compound, 896 QRT-PCR were done: four organs were used from each of the treated four animals to monitor the relative expression of 56 genes. Based on expression data of the discovery gene set of toxicology biomarkers the cardio- and nephrotoxicity of doxorubicin and sulfasalazin, the hepato- and nephrotoxicity of rotenone, dihydrocoumarin and aniline, and the liver toxicity of 2,4-diaminotoluene could be confirmed. The acute heart and kidney toxicity of the active metabolite SN-38 from its less toxic prodrug, irinotecan could be differentiated, and two novel gene markers for hormone replacement therapy were identified, namely fabp4 and pparg, which were down-regulated by estradiol treatment.

IJMS, Vol. 12, Pages 5373-5389: p66Shc Aging Protein in Control of Fibroblasts Cell Fate

Jan M. Suski — Mon, 22 Aug 2011 00:00:00 CEST

Reactive oxygen species (ROS) are wieldy accepted as one of the main factors of the aging process. These highly reactive compounds modify nucleic acids, proteins and lipids and affect the functionality of mitochondria in the first case and ultimately of the cell. Any agent or genetic modification that affects ROS production and detoxification can be expected to influence longevity. On the other hand, genetic manipulations leading to increased longevity can be expected to involve cellular changes that affect ROS metabolism. The 66-kDa isoform of the growth factor adaptor Shc (p66Shc) has been recognized as a relevant factor to the oxygen radical theory of aging. The most recent data indicate that p66Shc protein regulates life span in mammals and its phosphorylation on serine 36 is important for the initiation of cell death upon oxidative stress. Moreover, there is strong evidence that apart from aging, p66Shc may be implicated in many oxidative stress-associated pathologies, such as diabetes, mitochondrial and neurodegenerative disorders and tumorigenesis. This article summarizes recent knowledge about the role of p66Shc in aging and senescence and how this protein can influence ROS production and detoxification, focusing on studies performed on skin and skin fibroblasts.

IJMS, Vol. 12, Pages 5213-5237: Mechanisms of Mycotoxin-Induced Neurotoxicity through Oxidative Stress-Associated Pathways

Kunio Doi — Mon, 15 Aug 2011 00:00:00 CEST

Among many mycotoxins, T-2 toxin, macrocyclic trichothecenes, fumonisin B1 (FB1) and ochratochin A (OTA) are known to have the potential to induce neurotoxicity in rodent models. T-2 toxin induces neuronal cell apoptosis in the fetal and adult brain. Macrocyclic trichothecenes bring about neuronal cell apoptosis and inflammation in the olfactory epithelium and olfactory bulb. FB1 induces neuronal degeneration in the cerebral cortex, concurrent with disruption of de novo ceramide synthesis. OTA causes acute depletion of striatal dopamine and its metabolites, accompanying evidence of neuronal cell apoptosis in the substantia nigra, striatum and hippocampus. This paper reviews the mechanisms of neurotoxicity induced by these mycotoxins especially from the viewpoint of oxidative stress-associated pathways.

IJMS, Vol. 12, Pages 4991-5010: Resveratrol Protects against 2-Bromopropane-Induced Apoptosis and Disruption of Embryonic Development in Blastocysts

Wen-Hsiung Chan — Fri, 05 Aug 2011 00:00:00 CEST

2-Bromopropane (2-BP) is used as an alternative to ozone-depleting cleaning solvents. Previously, we reported that 2-BP has cytotoxic effects on mouse blastocysts and is associated with defects in subsequent development. In the present work, we show that 2-BP induces apoptosis in the inner cell mass of mouse blastocysts, and inhibits cell proliferation. Both effects are suppressed by resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties. 2-BP-treated blastocysts displayed lower levels of implantation (compared to controls) when plated on culture dishes in vitro, and a reduced ability to proceed to later stages of embryonic development. Pretreatment with resveratrol prevented 2-BP-induced disruption of embryonic development, both in vitro and in vivo. Further investigation of these processes revealed that 2-BP directly promotes ROS generation, loss of mitochondrial membrane potential (MMP), and activation of caspase-3, whereas resveratrol effectively blocks 2-BP-induced ROS production and the accompanying apoptotic biochemical changes. Our results collectively imply that 2-BP triggers the mitochondrion-dependent apoptotic pathway via ROS generation, and the antioxidant activity of resveratrol prevents 2-BP-induced toxicity.

IJMS, Vol. 12, Pages 4609-4624: Identification and Categorization of Liver Toxicity Markers Induced by a Related Pair of Drugs

Ching-Wei Chang — Fri, 15 Jul 2011 00:00:00 CEST

Drug-induced liver injury (DILI) is the primary adverse event that results in the withdrawal of drugs from the market and a frequent reason for the failure of drug candidates in the pre-clinical or clinical phases of drug development. This paper presents an approach for identifying potential liver toxicity genomic biomarkers from a liver toxicity biomarker study involving the paired compounds entacapone (“non-liver toxic drug”) and tolcapone (“hepatotoxic drug”). Molecular analysis of the rat liver and plasma samples, combined with statistical analysis, revealed many similarities and differences between the in vivo biochemical effects of the two drugs. Six hundred and ninety-five genes and 61 pathways were selected based on the classification scheme. Of the 61 pathways, 5 were specific to treatment with tolcapone. Two of the 12 animals in the tolcapone group were found to have high ALT, AST, or TBIL levels. The gene Vars2 (valyl-tRNA synthetase 2) was identified in both animals and the pathway to which it belongs, the aminoacyl-tRNA biosynthesis pathway, was one of the three most significant tolcapone-specific pathways identified.

IJMS, Vol. 12, Pages 3133-3147: Impaired Mitochondrial Respiratory Functions and Oxidative Stress in Streptozotocin-Induced Diabetic Rats

Haider Raza — Fri, 13 May 2011 00:00:00 CEST

We have previously shown a tissue-specific increase in oxidative stress in the early stages of streptozotocin (STZ)-induced diabetic rats. In this study, we investigated oxidative stress-related long-term complications and mitochondrial dysfunctions in the different tissues of STZ-induced diabetic rats (>15 mM blood glucose for 8 weeks). These animals showed a persistent increase in reactive oxygen and nitrogen species (ROS and RNS, respectively) production. Oxidative protein carbonylation was also increased with the maximum effect observed in the pancreas of diabetic rats. The activities of mitochondrial respiratory enzymes ubiquinol: cytochrome c oxidoreductase (Complex III) and cytochrome c oxidase (Complex IV) were significantly decreased while that of NADH:ubiquinone oxidoreductase (Complex I) and succinate:ubiquinone oxidoreductase (Complex II) were moderately increased in diabetic rats, which was confirmed by the increased expression of the 70 kDa Complex II sub-unit. Mitochondrial matrix aconitase, a ROS sensitive enzyme, was markedly inhibited in the diabetic rat tissues. Increased expression of oxidative stress marker proteins Hsp-70 and HO-1 was also observed along with increased expression of nitric oxide synthase. These results suggest that mitochondrial respiratory complexes may play a critical role in ROS/RNS homeostasis and oxidative stress related changes in type 1 diabetes and may have implications in the etiology of diabetes and its complications.

IJMS, Vol. 12, Pages 2783-2796: Mechanistic Investigation of ROS-Induced DNA Damage by Oestrogenic Compounds in Lymphocytes and Sperm Using the Comet Assay

Eduardo Cemeli — Thu, 28 Apr 2011 00:00:00 CEST

Past research has demonstrated that oestrogenic compounds produce strand breaks in the DNA of sperm and lymphocytes via reactive oxygen species (ROS). In the current investigation, sperm and lymphocytes were treated in vitro with oestrogenic compounds (diethylstilboestrol, progesterone, 17β-oestradiol, noradrenaline and triiodotyronine) and several aspects of DNA damage were investigated. Firstly, mediation of DNA damage by lipid peroxidation was investigated in the presence of BHA (a lipid peroxidation blocker). BHA reduced the DNA damage generated by 17β-oestradiol and diethylstilboestrol in a statistically significant manner. No effects were observed for sperm. Secondly, the presence of oxidized bases employing FPG and EndoIII were detected for lymphocytes and sperm in the negative control and after 24 h recovery in lymphocytes but not immediately after treatment for both cell types. The successful detection of oxidized bases in the negative control (untreated) of sperm provides an opportunity for its application in biomonitoring studies. DNA repair at 24 h after exposure was also studied. A nearly complete recovery to negative control levels was shown in lymphocytes 24 h recovery after oestrogenic exposure and this was statistically significant in all cases. Rapid rejoining of DNA, in a matter of hours, is a characteristic of DNA damaged by ROS.

IJMS, Vol. 12, Pages 2502-2517: Global Transcriptomic Profiling Using Small Volumes of Whole Blood: A Cost-Effective Method for Translational Genomic Biomarker Identification in Small Animals

Meagan M. Fricano — Wed, 13 Apr 2011 00:00:00 CEST

Blood is an ideal tissue for the identification of novel genomic biomarkers for toxicity or efficacy. However, using blood for transcriptomic profiling presents significant technical challenges due to the transcriptomic changes induced by ex vivo handling and the interference of highly abundant globin mRNA. Most whole blood RNA stabilization and isolation methods also require significant volumes of blood, limiting their effective use in small animal species, such as rodents. To overcome these challenges, a QIAzol-based RNA stabilization and isolation method (QSI) was developed to isolate sufficient amounts of high quality total RNA from 25 to 500 μL of rat whole blood. The method was compared to the standard PAXgene Blood RNA System using blood collected from rats exposed to saline or lipopolysaccharide (LPS). The QSI method yielded an average of 54 ng total RNA per μL of rat whole blood with an average RNA Integrity Number (RIN) of 9, a performance comparable with the standard PAXgene method. Total RNA samples were further processed using the NuGEN Ovation Whole Blood Solution system and cDNA was hybridized to Affymetrix Rat Genome 230 2.0 Arrays. The microarray QC parameters using RNA isolated with the QSI method were within the acceptable range for microarray analysis. The transcriptomic profiles were highly correlated with those using RNA isolated with the PAXgene method and were consistent with expected LPS-induced inflammatory responses. The present study demonstrated that the QSI method coupled with NuGEN Ovation Whole Blood Solution system is cost-effective and particularly suitable for transcriptomic profiling of minimal volumes of whole blood, typical of those obtained with small animal species.

IJMS, Vol. 12, Pages 2351-2382: Individual Variations in Inorganic Arsenic Metabolism Associated with AS3MT Genetic Polymorphisms

Tetsuro Agusa — Mon, 04 Apr 2011 00:00:00 CEST

Individual variations in inorganic arsenic metabolism may influence the toxic effects. Arsenic (+3 oxidation state) methyltransferase (AS3MT) that can catalyze the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to trivalent arsenical, may play a role in arsenic metabolism in humans. Since the genetic polymorphisms of AS3MT gene may be associated with the susceptibility to inorganic arsenic toxicity, relationships of several single nucleotide polymorphisms (SNPs) in AS3MT with inorganic arsenic metabolism have been investigated. Here, we summarize our recent findings and other previous studies on the inorganic arsenic metabolism and AS3MT genetic polymorphisms in humans. Results of genotype dependent differences in arsenic metabolism for most of SNPs in AS3MT were Inconsistent throughout the studies. Nevertheless, two SNPs, AS3MT 12390 (rs3740393) and 14458 (rs11191439) were consistently related to arsenic methylation regardless of the populations examined for the analysis. Thus, these SNPs may be useful indicators to predict the arsenic metabolism via methylation pathways.

IJMS, Vol. 12, Pages 2325-2335: Dextran Sulfate Sodium Inhibits Alanine Synthesis in Caco-2 Cells

Zhong Ye — Mon, 04 Apr 2011 00:00:00 CEST

To understand and characterize the pathogenic mechanisms of inflammatory bowel disease, dextran sulfate sodium (DSS) has been used to induce acute and chronic colitis in animal models by causing intestinal epithelium damage. The mechanism of action of DSS in producing this outcome is not well understood. In an effort to understand how DSS might impact epithelial cell metabolism, we studied the intestinal epithelial cell line Caco-2 incubated with 1% DSS over 56 hours using 1H NMR spectroscopy. We observed no difference in cell viability as compared to control cultures, and an approximately 1.5-fold increase in IL-6 production upon incubation with 1% DSS. The effect on Caco-2 cell metabolism as measured through changes in the concentration of metabolites in the cell supernatant included a three-fold decrease in the concentration of alanine. Given that the concentrations of other amino acids in the cell culture supernatant were not different between treated and control cultures over 56 hours suggest that DSS inhibits alanine synthesis, specifically alanine aminotransferase, without affecting other key metabolic pathways. The importance of alanine aminotransferase in inflammatory bowel disease is discussed.

IJMS, Vol. 12, Pages 2077-2087: In Vitro Ability of Currently Available Oximes to Reactivate Organophosphate Pesticide-Inhibited Human Acetylcholinesterase and Butyrylcholinesterase

Daniel Jun — Wed, 23 Mar 2011 00:00:00 CET

We have in vitro tested the ability of common, commercially available, cholinesterase reactivators (pralidoxime, obidoxime, methoxime, trimedoxime and HI-6) to reactivate human acetylcholinesterase (AChE), inhibited by five structurally different organophosphate pesticides and inhibitors (paraoxon, dichlorvos, DFP, leptophos-oxon and methamidophos). We also tested reactivation of human butyrylcholinesterase (BChE) with the aim of finding a potent oxime, suitable to serve as a “pseudocatalytic” bioscavenger in combination with this enzyme. Such a combination could allow an increase of prophylactic and therapeutic efficacy of the administered enzyme. According to our results, the best broad-spectrum AChE reactivators were trimedoxime and obidoxime in the case of paraoxon, leptophos-oxon, and methamidophos-inhibited AChE. Methamidophos and leptophos-oxon were quite easily reactivatable by all tested reactivators. In the case of methamidophos-inhibited AChE, the lower oxime concentration (10−5 M) had higher reactivation ability than the 10−4 M concentration. Therefore, we evaluated the reactivation ability of obidoxime in a concentration range of 10−3–10−7 M. The reactivation of methamidophos-inhibited AChE with different obidoxime concentrations resulted in a bell shaped curve with maximum reactivation at 10−5 M. In the case of BChE, no reactivator exceeded 15% reactivation ability and therefore none of the oximes can be recommended as a candidate for “pseudocatalytic” bioscavengers with BChE.

IJMS, Vol. 12, Pages 114-127: Behavioral Impairment and Oxidative Damage Induced by Chronic Application of Nonylphenol

Zhen Mao — Thu, 30 Dec 2010 00:00:00 CET

Nonylphenol (NP) is a degradation product of nonylphenol polyethoxylates, which are widely used in the production of industrial and consumer surfactants. The aim of the present study was to evaluate the effect of NP on the antioxidant capacity and cognitive ability of mice. NP was given orally by gavages at doses of 0, 50, 100, and 200 mg kg−1 d−1 for 90 days. The results showed that NP significantly decreased the activity of superoxide dismutases (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) and at the same time increased malondialdehyde (MDA) levels in mice brains. Exploration, memory function and ability to learn a novel task were significantly decreased in NP fed mice. These results indicate that chronic high dose of NP exposure has the potential to generate oxidative stress and induce the cognitive impairment in male mice.

IJMS, Vol. 11, Pages 4843-4863: 3,4-Methylenedioxymethamphetamine Alters Left Ventricular Function and Activates Nuclear Factor-Kappa B (NF-kB) in a Time and Dose Dependent Manner

David A. Tiangco — Fri, 26 Nov 2010 00:00:00 CET

3,4-Methylenedioxymethamphetamine (MDMA) is an illicit psychoactive drug with cardiovascular effects that have not been fully described. In the current study, we observed the effects of acute MDMA on rabbit left ventricular function. We also observed the effects of MDMA on nuclear factor-kappa B (NF-kB) activity in cultured rat ventricular myocytes (H9c2). In the rabbit, MDMA (2 mg/kg) alone caused a significant increase in heart rate and a significant decrease in the duration of the cardiac cycle. Inhibition of nitric oxide synthase (NOS) by pretreatment with L-NAME (10 mg/kg) alone caused significant dysfunction in heart rate, systolic pressure, diastolic pressure, duration of relaxation, duration of cardiac cycle, and mean left ventricular pressure. Pretreatment with L-NAME followed by treatment with MDMA caused significant dysfunction in additional parameters that were not abnormal upon exposure to either compound in isolation: duration of contraction, inotropy, and pulse pressure. Exposure to 1.0 mM MDMA for 6 h or 2.0 mM MDMA for 12 h caused increased nuclear localization of NF-kB in cultured H9c2 cells. The current results suggest that MDMA is acutely detrimental to heart function and that an intact cardiovascular NOS system is important to help mitigate early sequelae in some functional parameters. The delayed timing of NF-kB activation suggests that this factor may be relevant to MDMA induced cardiomyopathy of later onset.

IJMS, Vol. 11, Pages 4796-4813: Functional Toxicogenomics: Mechanism-Centered Toxicology

Matthew North — Wed, 24 Nov 2010 00:00:00 CET

Traditional toxicity testing using animal models is slow, low capacity, expensive and assesses a limited number of endpoints. Such approaches are inadequate to deal with the increasingly large number of compounds found in the environment for which there are no toxicity data. Mechanism-centered high-throughput testing represents an alternative approach to meet this pressing need but is limited by our current understanding of toxicity pathways. Functional toxicogenomics, the global study of the biological function of genes on the modulation of the toxic effect of a compound, can play an important role in identifying the essential cellular components and pathways involved in toxicity response. The combination of the identification of fundamental toxicity pathways and mechanism-centered targeted assays represents an integrated approach to advance molecular toxicology to meet the challenges of toxicity testing in the 21st century.

IJMS, Vol. 11, Pages 4771-4781: Identification of Compounds in the Essential Oil of Nutmeg Seeds (Myristica fragrans Houtt.) That Inhibit Locomotor Activity in Mice

Muchtaridi — Tue, 23 Nov 2010 00:00:00 CET

The present study was designed to evaluate the inhibitory effect of nutmeg (Myristica fragrans Houtt.) seed essential oil on the locomotor activity of mice in a wheel cage. Active compounds in the essential oil were identified by off-line solid phase extraction (SPE-C18) and GC/MS analysis. The essential oil was administered by inhalation at doses of 0.1, 0.3, and 0.5 mL/cage. The results showed that inhalation of nutmeg seed essential oil at a dose of 0.5 mL/cage decreased locomotion by 68.62%; and inhalation of 0.1 and 0.3 mL/cage inhibited locomotion by 62.81% and 65.33%, respectively. Generally, larger doses and longer administrations of nutmeg seed essential oil exhibited greater locomotor inhibition. Subsequently, the plasma concentrations of essential oil compounds were measured. The most concentrated compound in the plasma was myristicin. Half an hour after the addition of 1 mL/cage of nutmeg seed oil, the plasma concentration of myristicin was 3.7 mg/mL; one and two hours after the addition, the blood levels of myristicin were 5.2 mg/mL and 7.1 mg/mL, respectively. Other essential oil compounds identified in plasma were safrole (two-hour inhalation: 1.28 mg/mL), 4‑terpineol (half-hour inhalation: 1.49 mg/mL, one-hour inhalation: 2.95 mg/mL, two-hour inhalation: 6.28 mg/mL) and fatty esters. The concentrations of the essential oil compounds in the blood plasma were relatively low (mg/mL or ppm). In conclusion, the volatile compounds of nutmeg seed essential oil identified in the blood plasma may correlate with the locomotor-inhibiting properties of the oil when administered by inhalation.

IJMS, Vol. 11, Pages 4697-4714: Comparison of TNFα to Lipopolysaccharide as an Inflammagen to Characterize the Idiosyncratic Hepatotoxicity Potential of Drugs: Trovafloxacin as an Example

Michael J. Liguori — Thu, 18 Nov 2010 00:00:00 CET

Idiosyncratic drug reactions (IDRs) are poorly understood, unpredictable, and not detected in preclinical studies. Although the cause of these reactions is likely multi-factorial, one hypothesis is that an underlying inflammatory state lowers the tolerance to a xenobiotic. Previously used in an inflammation IDR model, bacterial lipopolysaccharide (LPS) is heterogeneous in nature, making development of standardized testing protocols difficult. Here, the use of rat tumor necrosis factor-α (TNFα) to replace LPS as an inflammatory stimulus was investigated. Sprague-Dawley rats were treated with separate preparations of LPS or TNFα, and hepatic transcriptomic effects were compared. TNFα showed enhanced consistency at the transcriptomic level compared to LPS. TNFα and LPS regulated similar biochemical pathways, although LPS was associated with more robust inflammatory signaling than TNFα. Rats were then codosed with TNFα and trovafloxacin (TVX), an IDR-associated drug, and evaluated by liver histopathology, clinical chemistry, and gene expression analysis. TNFα/TVX induced unique gene expression changes that clustered separately from TNFα/levofloxacin, a drug not associated with IDRs. TNFα/TVX cotreatment led to autoinduction of TNFα resulting in potentiation of underlying gene expression stress signals. Comparison of TNFα/TVX and LPS/TVX gene expression profiles revealed similarities in the regulation of biochemical pathways. In conclusion, TNFα could be used in lieu of LPS as an inflammatory stimulus in this model of IDRs.

IJMS, Vol. 11, Pages 4511-4525: The Role of Molecular Biology in the Biomonitoring of Human Exposure to Chemicals

Balam Muñoz — Fri, 12 Nov 2010 00:00:00 CET

Exposure to different substances in an occupational environment is of utmost concern to global agencies such as the World Health Organization and the International Labour Organization. Interest in improving work health conditions, particularly of those employees exposed to noxious chemicals, has increased considerably and has stimulated the search for new, more specific and selective tests. Recently, the field of molecular biology has been indicated as an alternative technique for monitoring personnel while evaluating work-related pathologies. Originally, occupational exposure to environmental toxicants was assessed using biochemical techniques to determine the presence of higher concentrations of toxic compounds in blood, urine, or other fluids or tissues; results were used to evaluate potential health risk. However, this approach only estimates the presence of a noxious chemical and its effects, but does not prevent or diminish the risk. Molecular biology methods have become very useful in occupational medicine to provide more accurate and opportune diagnostics. In this review, we discuss the role of the following common techniques: (1) Use of cell cultures; (2) evaluation of gene expression; (3) the “omic” sciences (genomics, transcriptomics, proteomics and metabolomics) and (4) bioinformatics. We suggest that molecular biology has many applications in occupational health where the data can be applied to general environmental conditions.

IJMS, Vol. 11, Pages 4361-4380: Hazardous Apoptotic Effects of 2-Bromopropane on Maturation of Mouse Oocytes, Fertilization, and Fetal Development

Wen-Hsiung Chan — Wed, 03 Nov 2010 00:00:00 CET

2-Bromopropane (2-BP) is used as an alternative to ozone-depleting cleaning solvents. Previously, we reported that 2-BP has cytotoxic effects on mouse blastocysts and is associated with defects in subsequent development. Here, we further investigate the effects of 2-BP on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, 2-BP induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with 2-BP during in vitro maturation (IVM) resulted in increased resorption of postimplantation embryos and decreased fetal weights. Experiments with a mouse model disclosed that consumption of drinking water containing 20 μM 2-BP led to decreased oocyte maturation in vivo and fertilization in vitro, as well as impairment of early embryonic development. Interestingly, pretreatment with a caspase-3-specific inhibitor effectively prevented 2-BP-triggered hazardous effects, suggesting that embryonic impairment by 2-BP occurs via a caspase-dependent apoptotic process. A study using embryonic stem cells as the assay model conclusively demonstrated that 2-BP induces cell death processes through apoptosis and not necrosis, and inhibits early embryo development in mouse embryonic stem cells. These results collectively confirm the hazardous effects of 2-BP on embryos derived from pretreated oocytes.

IJMS, Vol. 11, Pages 3793-3802: Antigenotoxic Effect of Chamomilla recutita (L.) Rauschert Essential Oil in Mouse Spermatogonial Cells, and Determination of Its Antioxidant Capacity in Vitro

Alejandra Hernández-Ceruelos — Thu, 30 Sep 2010 00:00:00 CEST

Chamomilla recutita (L.) Rauschert (Asteraceae), popularly known as chamomile, is a plant used in traditional medicine for various therapeutic purposes. Chamomile essential oil (CEO) is particularly known to inhibit the genotoxic damage produced by mutagens in mice somatic cells. The aim of this research was to determine the inhibitory potential of CEO on the genotoxic damage produced by daunorubicin (DAU) in mice germ cells. We evaluated the effect of 5, 50, and 500 mg/kg of essential oil on the rate of sister chromatid exchange (SCE) induced in spermatogonia by 10 mg/kg of the mutagen. We found no genotoxicity of CEO, but detected an inhibition of SCE after the damage induced by DAU; from the lowest to the highest dose of CEO we found an inhibition of 47.5%, 61.9%, and 93.5%, respectively. As a possible mechanism of action, the antioxidant capacity of CEO was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging method and ferric thiocyanate assays. In the first test we observed a moderate scavenging potential of the oil; nevertheless, the second assay showed an antioxidant capacity similar to that observed with vitamin E. In conclusion, we found that CEO is an efficient chemoprotective agent against the damage induced by DAU in the precursor cells of the germinal line of mice, and that its antioxidant capacity may induce this effect.

IJMS, Vol. 11, Pages 3760-3768: The Individual and Combined Effects of Deoxynivalenol and Aflatoxin B1 on Primary Hepatocytes of Cyprinus Carpio

Cheng-Hua He — Wed, 29 Sep 2010 00:00:00 CEST

Aflatoxin B1 (AFB1) and deoxynivalenol (DON) are important food-borne mycotoxins that have been implicated in animal and human health. In this study, individual and combinative effects of AFB1 and DON were tested in primary hepatocytes of Cyprinus carpio. The results indicated that the combinative effects of AFB1 and DON (0.01 μg/mL AFB1 and 0.25 μg/mL DON; 0.02 μg/mL AFB1 and 0.25 μg/mL DON; 0.02 μg/mL AFB1 and 0.5 μg/mL DON) were higher than that of individual mycotoxin (P

IJMS, Vol. 11, Pages 3397-3412: Practical Application of Toxicogenomics for Profiling Toxicant-Induced Biological Perturbations

Naoki Kiyosawa — Mon, 20 Sep 2010 00:00:00 CEST

A systems-level understanding of molecular perturbations is crucial for evaluating chemical-induced toxicity risks appropriately, and for this purpose comprehensive gene expression analysis or toxicogenomics investigation is highly advantageous. The recent accumulation of toxicity-associated gene sets (toxicogenomic biomarkers), enrichment in public or commercial large-scale microarray database and availability of open-source software resources facilitate our utilization of the toxicogenomic data. However, toxicologists, who are usually not experts in computational sciences, tend to be overwhelmed by the gigantic amount of data. In this paper we present practical applications of toxicogenomics by utilizing biomarker gene sets and a simple scoring method by which overall gene set-level expression changes can be evaluated efficiently. Results from the gene set-level analysis are not only an easy interpretation of toxicological significance compared with individual gene-level profiling, but also are thought to be suitable for cross-platform or cross-institutional toxicogenomics data analysis. Enrichment in toxicogenomics databases, refinements of biomarker gene sets and scoring algorithms and the development of user-friendly integrative software will lead to better evaluation of toxicant-elicited biological perturbations.

IJMS, Vol. 11, Pages 3375-3386: Cigarette Smoke Affects ABCAl Expression via Liver X Receptor Nuclear Translocation in Human Keratinocytes

Claudia Sticozzi — Fri, 17 Sep 2010 00:00:00 CEST

Cutaneous tissue is the first barrier against outdoor insults. The outer most layer of the skin, the stratum corneum (SC), is formed by corneocytes embedded in a lipid matrix (cholesterol, ceramide and fatty acids). Therefore, the regulation of lipids and, in particular, of cholesterol homeostasis in the skin is of great importance. ABCA1 is a membrane transporter responsible for cholesterol efflux and plays a key role in maintaining cellular cholesterol levels. Among the many factors that have been associated with skin diseases, the environmental stressor cigarette smoke has been recently studied. In the present study, we demonstrate that ABCA1 expression in human cells (HaCaT) was increased (both mRNA and protein levels) after CS exposure. This effect was mediated by the inhibition of NFkB (aldehydes adducts formation) that allows the translocation of liver X receptor (LXR). These findings suggest that passive smoking may play a role in skin cholesterol levels and thus affect cutaneous tissues functions.

IJMS, Vol. 11, Pages 2839-2855: Hazardous Effects of Curcumin on Mouse Embryonic Development through a Mitochondria-Dependent Apoptotic Signaling Pathway

Chia-Chi Chen — Mon, 02 Aug 2010 00:00:00 CEST

In this study, we examined the cytotoxic effects of curcumin, the yellow pigment of Curcuma longa, on the blastocyst stage of mouse embryos, subsequent embryonic attachment, and outgrowth in vitro and in vivo implantation by embryo transfer. Mouse blastocysts were incubated in medium with or without curcumin (6, 12 or 24 μM) for 24 h. Cell proliferation and growth were investigated using dual differential staining, apoptosis was analyzed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and implantation and post-implantation development of embryos were measured by in vitro development analysis and in vivo embryo transfer, respectively. Blastocysts treated with 24 μM curcumin displayed significantly increased apoptosis and decreased total cell number. Interestingly, we observed no marked differences in the implantation success rates between curcumin-pretreated and control blastocysts during in vitro embryonic development through implantation with a fibronectin-coated culture dish. However, in vitro treatment with 24 μM curcumin was associated with decreased implantation rate and increased resorption of postimplantation embryos in mouse uterus, as well as decreased fetal weight in the embryo transfer assay. Our results collectively indicate that in vitro exposure to curcumin triggers apoptosis and retards early postimplantation development after transfer to host mice. In addition, curcumin induces apoptotic injury effects on mouse blastocysts through ROS generation, and further promotes mitochondria-dependent apoptotic signaling processes to impair sequent embryonic development.

IJMS, Vol. 11, Pages 2715-2745: Nitric Oxide: Perspectives and Emerging Studies of a Well Known Cytotoxin

William A. Paradise — Fri, 16 Jul 2010 00:00:00 CEST

The free radical nitric oxide (NO●) is known to play a dual role in human physiology and pathophysiology. At low levels, NO● can protect cells; however, at higher levels, NO● is a known cytotoxin, having been implicated in tumor angiogenesis and progression. While the majority of research devoted to understanding the role of NO● in cancer has to date been tissue-specific, we herein review underlying commonalities of NO● which may well exist among tumors arising from a variety of different sites. We also discuss the role of NO● in human physiology and pathophysiology, including the very important relationship between NO● and the glutathione-transferases, a class of protective enzymes involved in cellular protection. The emerging role of NO● in three main areas of epigenetics—DNA methylation, microRNAs, and histone modifications—is then discussed. Finally, we describe the recent development of a model cell line system in which human tumor cell lines were adapted to high NO● (HNO) levels. We anticipate that these HNO cell lines will serve as a useful tool in the ongoing efforts to better understand the role of NO● in cancer.

IJMS, Vol. 11, Pages 2383-2392: Effects of Titanium Dioxide Nanoparticle Aggregate Size on Gene Expression

Okuda-Shimazaki — Mon, 07 Jun 2010 00:00:00 CEST

Titanium dioxide (titania) nanoparticle aggregation is an important factor in understanding cytotoxicity. However, the effect of the aggregate size of nanoparticles on cells is unclear. We prepared two sizes of titania aggregate particles and investigated their biological activity by analyzing biomarker expression based on mRNA expression analysis. The aggregate particle sizes of small and large aggregated titania were 166 nm (PDI = 0.291) and 596 nm (PDI = 0.417), respectively. These two size groups were separated by centrifugation from the same initial nanoparticle sample. We analyzed the gene expression of biomarkers focused on stress, inflammation, and cytotoxicity. Large titania aggregates show a larger effect on cell viability and gene expression when compared with the small aggregates. This suggests that particle aggregate size is related to cellular effects.

IJMS, Vol. 11, Pages 896-911: Degradation of Microcystin-LR and RR by a Stenotrophomonas sp. Strain EMS Isolated from Lake Taihu, China

Jian Chen — Tue, 02 Mar 2010 00:00:00 CET

A bacterial strain EMS with the capability of degrading microcystins (MCs) was isolated from Lake Taihu, China. The bacterium was tentatively identified as a Stenotrophomonas sp. The bacterium could completely consume MC-LR and MC-RR within 24 hours at a concentration of 0.7 µg/mL and 1.7 µg/mL, respectively. The degradation of MC-LR and MC-RR by EMS occurred preferentially in an alkaline environment. In addition, mlrA gene involved in the degradation of MC-LR and MC-RR was detected in EMS. Due to the limited literature this gene has rare homologues. Sequencing analysis of the translated protein from mlrA suggested that MlrA might be a transmembrane protein, which suggests a possible new protease family having unique function.