microRNAs in Toxicology and Medicine

By Saura C. Sahu

During the past decade it has become evident that microRNAs regulate gene expressions and control many developmental and cellular processes in eukaryotic organisms. Recent studies suggest that microRNAs play an important role in toxicogenomics and are likely to play an important role in a range of human diseases including cancer. 

microRNAs in Toxicology and Medicine is a comprehensive and authoritative compilation of up-to-date developments in this emerging research area, presented by internationally recognized investigators. It focuses on the role of microRNA in biology and medicine with a special emphasis on toxicology. 

Divided into six parts, topics covered include:

• microRNA and toxicology – including environmental toxicants and perturbation of miRNA signaling; microRNA, and Disease States featuring microRNAs in drug-induced liver toxicity, microRNAs and Inflammation the regulatory role of microRNA in mutagenesis, microRNAs and cancer, and the role of microRNAs in tumor progression and therapy, as well as current understanding of microRNAs as therapeutic targets in cancer
• microRNAs and disease states
• microRNAs and stem cells
• microRNAs and genomics
• microRNAs and epigenomics
• microRNAs and biomarkers – including body fluid microRNAs as toxicological biomarkers, cell-free microRNAs as biomarkers in human diseases, and circulating microRNAs as biomarkers of drug-induced pancreatitis

microRNAs in Toxicology and Medicine is an essential insight into the current trends and future directions of research in this rapidly expanding field for investigators, toxicologists, risk assessors, and regulators in academia, medical settings, industry, and government.

• Language: English
• Publisher: Wiley
• Release date: Aug 23, 2013
• ISBN: 9781118696033

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This edition first published 2014

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Library of Congress Cataloging-in-Publication Data

microRNAs in Toxicology and Medicine / editor, Saura C. Sahu.

pages cm

Includes bibliographical references and index.

ISBN 978-1-118-40161-3 (cloth)

1. Small interfering RNA. 2. Small interfering RNA – Therapeutic use. 3. Genetic regulation. I. Sahu, Saura C., editor of compilation.

QP623.5.S63M536 2014

572.8′8 – dc23

2013020036

A catalogue record for this book is available from the British Library.

ISBN: 9781118401613

I lovingly dedicate this book to:

My parents, Gopinath and Ichhamoni, for their gifts of life, love and living examples

My wife, Jharana, for her life-long friendship, love and support, as well as for her patience and understanding of the long hours spent at home on planning, writing and editing this book.

My children, Megha, Sudhir and Subir, for their love and care

Saura C. Sahu

Laurel, Maryland, USA

List of Contributors

Aamir Ahmad Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, USA

Malin Åkerblom Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, Sweden

Nahid Akhtar Department of Anatomy and Neurobiology, Northeast Ohio Medical University (NEOMED), USA

Azfur S. Ali Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, USA

Shadan Ali Department of Oncology, Karmanos Cancer Institute, Wayne State University School of Medicine, USA

Sumit Arora Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, USA

Kathryn A. Bailey Department of Environmental Sciences and Engineering, UNC Gillings School of Global Public Health, University of North Carolina at Chapel Hill, USA

Arun Bhardwaj Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, USA

Barbara Burwinkel Molecular Epidemiology C080, German Cancer Research Center, Germany and Molecular Biology of Breast Cancer, University Women's Clinic, Germany

Si Chen Division of Biochemical Toxicology, National Center for Toxicological Research/US Food and Drug Administration, USA

Tao Chen Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Food and Drug Administration, USA

Sang-Woon Choi Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, USA and Friedman School of Nutrition Science and Policy, Tufts University, USA

Pierre Cordelier INSERM U1037, Cancer Research Center of Toulouse, France and Université Paul Sabatier Toulouse III, France

Katarina Cuk Molecular Epidemiology C080, German Cancer Research Center, Germany and Molecular Biology of Breast Cancer, University Women's Clinic, Germany

Yang Dai Department of Bioengineering, University of Illinois at Chicago, USA

Christopher J. Davis WWAMI Medical Education Program and Program in Neuroscience, Sleep and Performance Research Center, Washington State University, USA

Joel Fontanarosa Department of Bioengineering, University of Illinois at Chicago, USA

Jennifer L. Freeman School of Health Sciences, Purdue University, USA

Simonetta Friso University of Verona School of Medicine, Italy

Rebecca C. Fry Department of Environmental Sciences and Engineering, UNC Gillings School of Global Public Health, University of North Carolina at Chapel Hill, USA

Luc Gailhouste Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Japan

Marion Gayral INSERM U1037, Cancer Research Center of Toulouse, France and Université Paul Sabatier Toulouse III, France

Samir N. Ghadiali The Ohio State University, Dorothy M. Davis Heart and Lung Research Institute, USA

Lei Guo Division of Biochemical Toxicology, National Center for Toxicological Research/US Food and Drug Administration, USA

Keitaro Hagiwara Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Japan and Department of Biological Sciences, Tokyo Institute of Technology, Japan

Tariq M. Haqqi Department of Anatomy and Neurobiology, Northeast Ohio Medical University (NEOMED), USA

Valerie W. Hu Department of Biochemistry and Molecular Medicine, The George Washington University School of Medicine and Health Sciences, USA

Yan Huang The Ohio State University, Dorothy M. Davis Heart and Lung Research Institute, USA

Brock Humphries Department of Physiology, Michigan State University, USA

Johan Jakobsson Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, Sweden

Matthias Jung Clinic for Psychiatry, Psychotherapy, and Psychosomatic medicine, Martin Luther University, Germany

Daniel B. Kay Department of Psychiatry and Human Behavior University of Mississippi Medical Center, School of Medicine, USA

Nobuyoshi Kosaka Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Japan

Zhenhua Liu School of Public Health and Health Sciences, University of Massachusetts, USA and Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, USA

Yang Luan School of Public Health, Shanghai Jiao Tong University, China

Dharanija Madhavan Molecular Epidemiology C080, German Cancer Research Center, Germany and Molecular Biology of Breast Cancer, University Women's Clinic, Germany

Josephine Malmevik Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, Sweden

William B. Mattes PharmPoint Consulting, USA

Fanxue Meng Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Food and Drug Administration, USA

S. Patrick Nana-Sinkam The Ohio State University, Dorothy M. Davis Heart and Lung Research Institute, USA

Takahiro Ochiya Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Japan

Philip A. Philip Department of Oncology, Karmanos Cancer Institute, Wayne State University School of Medicine, USA

Barry A. Rosenzweig Division of Drug Safety Research, Center for Drug Evaluation and Research, US Food and Drug Administration, USA

Rodney L. Rouse Division of Drug Safety Research, Center for Drug Evaluation and Research, US Food and Drug Administration, USA

Saura C. Sahu Division of Toxicology, Center for Food Safety and Applied Nutrition, Food and Drug Administration, USA

William F. Salminen PAREXEL, USA

Tewarit Sarachana Department of Biochemistry and Molecular Medicine, The George Washington University School of Medicine and Health Sciences, USA

Fazlul H. Sarkar Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, USA and Department of Oncology, Karmanos Cancer Institute, Wayne State University School of Medicine, USA

Insa S. Schroeder Department of Biophysics, GSI Helmholtz Centre for Heavy Ion Research, Germany

Maria S. Sepúlveda Department of Forestry and Natural Resources, Purdue University, USA

Leming Shi School of Pharmacy, Fudan University, China

Qiang Shi Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, USA

Ajay P. Singh Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, USA and Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, USA

Seema Singh Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, USA

Geir Skogerbø National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, China

Stephanie A. Tammen Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, USA and Friedman School of Nutrition Science and Policy, Tufts University, USA

Karol L. Thompson Division of Drug Safety Research, Center for Drug Evaluation and Research, US Food and Drug Administration, USA

Jérome Torrisani INSERM U1037, Cancer Research Center of Toulouse, France and Université Paul Sabatier Toulouse III, France

Andrey Turchinovich Molecular Epidemiology C080, German Cancer Research Center, Germany and Molecular Biology of Breast Cancer, University Women's Clinic, Germany

Zhishan Wang Department of Physiology, Michigan State University, USA

Gregory J. Weber School of Health Sciences, Purdue University, USA

Zuquan Weng Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, USA

Yaguang Xi Mitchell Cancer Institute, University of South Alabama, USA

Jiekun Xuan Division of Biochemical Toxicology, National Center for Toxicological Research/US Food and Drug Administration, USA

Dongsheng Yan School of Ophthalmology and Optometry, Wenzhou Medical College, China

Jian Yan Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Food and Drug Administration, USA

Chengfeng Yang Department of Physiology, Michigan State University, USA and Center for Integrative Toxicology, Michigan State University, USA

Xi Yang Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, USA

Bin Yi Mitchell Cancer Institute, University of South Alabama, USA

Preface

During the past decade it has become increasingly obvious that microRNAs regulate gene expressions and control many developmental and cellular processes in the eukaryotic organisms. Recent studies strongly suggest that they are likely to play important roles in a wide range of human diseases including cancer. As a result they have become an important component of the molecular mechanisms of the disease processes. Also, published reports strongly suggest that they are expected to play important roles in cellular response to xenobiotic stress affecting expression of microRNA as a mechanism of adaptation and, therefore, they have attracted great interest in toxicology. Thus microRNAs play an important role in toxicogenomics.

The importance of this field of research is evidenced by the increasing number of contributions published each year. It becomes increasingly clear that developments in this field are moving so rapidly that new means are needed to report the status of current ongoing research activities. The contributions presented in this monograph represent a collaborative effort by international experts working in this emerging field of science.

The main purpose of this book is to assemble up-to-date, state-of-the-art information on microRNAs presented by internationally recognized experts in a single edition. Therefore, I sincerely hope that this book will provide an authoritative source of current information on microRNA research and prove useful to the scientists interested in this scientific discipline throughout the world. It is my sincere hope that the information presented in this book will serve as a stimulus to all the investigators interested in this area of research. Also it should be of interest to a variety of other scientific disciplines including toxicology, medicine, and pharmacology, as well as food, drug, and other regulatory sciences.

Saura C. Sahu

Laurel, Maryland, USA

Acknowledgments

Editing this book has been a challenging journey. I express my sincere gratitude to all the individuals who have helped me, directly or indirectly, on this journey.

I am indebted to the internationally recognized experts, who shared my enthusiasm for this field of science and contributed generously to this book. They were selected from academia, industry, and government for their expertise in their own areas of research. Their work speaks for itself and I am grateful to them for their strong commitment, cooperation and excellent contributions in their own areas of expertise.

I thank the staff of the publisher, John Wiley & Sons, Ltd, especially Rebecca Ralf and Sarah Tilley for their excellent help, cooperation, support, and editorial assistance in the timely publication of this book.

Saura C. Sahu

Laurel, Maryland, USA

Part I

microRNAs and Toxicology

Chapter 1

Introduction

Saura C. Sahu

Division of Toxicology, Center for Food Safety and Applied Nutrition, Food and Drug Administration, USA.

The microRNA, found in eukaryotic cells, belongs to a family of small, single-stranded noncoding regulatory ribonucleic acid (RNA) molecules with an average of 22 nucleotides conserved by evolution (Christodoulou et al., 2010). Discovered in 1993 (Lee et al., 1993), they regulate gene expressions, and control many developmental and cellular processes in eukaryotic organisms. The physiological function of the majority of microRNAs is unknown. However, recent studies strongly suggest that they likely to play important roles in a wide range of human diseases, including cancer. As a result they have become an important component to study in the molecular mechanisms of disease processes. However, challenges remain in the understanding of their involvement in various disease processes. Therefore, microRNA research has become a hot new discipline in biology and medicine: microRNAs are promising important biomarkers of diseases.

The microRNAs have attracted great interest in toxicology. Published reports provide evidence that toxic exposures and cellular stress can affect microRNAs (Lema and Cunningham, 2010). Therefore, they are expected to play an important role in cellular responses to xenobiotic exposure. They bind to target messenger RNAs (mRNA) and suppress their translation into proteins. Exposure of cells to xenobiotics leads to altered microRNA expressions, as do other genes that play important roles in toxicology. Altered microRNA expression affects protein translation, which alters cellular physiology leading to adverse biological effects. Also cellular stress affects expression of microRNAs as a mechanism of adaptation (Lema and Cunningham, 2010). Thus microRNAs play an important role in toxicogenomics. Their potential as biomarkers of toxicity appears to be promising.

It is becoming increasingly clear from the rate of published literature that developments in microRNA research are moving rapidly. Therefore, new means are needed to report the current status of this new developing area of research. The purpose of this book, microRNAs in Toxicology and Medicine, is the timely dissemination of information on current interests in this emerging field of science. As the Editor, it gives me great pride to introduce this unique book which encompasses many aspects of microRNA research never published together before. It is only recently that this exciting area of research has attracted the attention of toxicologists. This book deals with information on microRNAs at a level designated to take the reader to the borderline of research in this newly developing scientific discipline. The microRNA research work, actively pursued throughout the world, will lead to major discoveries of fundamental importance and of great clinical significance. This book brings together the ideas and work of investigators of international reputation who have pioneered this exciting area of research in toxicology and medicine. The book provides up-to-date information as well as new challenges in this exciting research area, and reflects the remarkable blossoming of this research in recent years. New ideas and new approaches are brought to bear on exploration of the role played by microRNAs in toxicology and medicine. Therefore, exciting times are ahead for future research. The up-to-date techniques, ideas, applications, and bibliographies are presented in this book in sufficient detail to enable newcomers to this scientific discipline to apply them in their studies and pursue them to any depth. I sincerely hope that the book will provide authoritative information as well as new ideas and challenges on microRNA research for stimulating the creativity of graduate students and investigators who are actively engaged in this rapidly developing field. The extensive collection of current information presented here will make it a valuable reference source for all scientists working in the microRNA area.

References

Christodoulou F, Raible F, Tomer R, Simakov O, Trachana K, Klaus S, et al. 2010. Ancient animal microRNAs and the evolution of tissue identity. Nature 463, 1084–1088.

Lee RC, Feinbaum RL, Ambros V. 1993. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75(5):843–854.

Lema C and Cunningham MJ. 2010. MicroRNAs and their implications in toxicological research. Toxicol Lett. 198(2):100–105.

Chapter 2

Environmental Toxicants and Perturbation of miRNA Signaling

Kathryn A. Bailey and Rebecca C. Fry

Department of Environmental Sciences and Engineering, UNC Gillings School of Global Public Health, University of North Carolina at Chapel Hill, USA.

2.1 Introduction

In 2011, the World Health Organization reported that non-communicable diseases (NCDs) such as diabetes mellitus, cardiovascular disease, obesity, and cancer were the top causes of death worldwide (WHO, 2011). Particularly in developing countries, up to 67% of all premature deaths, that is, those occurring before the age of 60, can be attributed to NCDs. While the risk of developing NCDs is dependent on both genetic and environmental factors, mounting evidence suggests that, for most NCDs, environmental factors have a far greater impact than genetic factors (Rappaport and Smith, 2010). In developed countries such as the United States, physical inactivity and high caloric intake are well-established lifestyle factors that contribute to NCD development. However, it is likely that environmental toxicants also play a significant role in NCD development, and due to the latent nature of many toxic effects, the impact of these toxicants has been under-recognized. For instance, the President's Cancer Panel found in its 2010 report that the impact of chemicals on cancer development in Americans has been grossly underestimated and demanded immediate action to overhaul the government's chemical management program (Reuben, 2010). It is becoming increasingly recognized that environmental toxicant exposure, especially early in life, may contribute significantly to other NCDs as well including diabetes, obesity, cardiovascular disease, and respiratory disease (Jardim, 2011).

The long-term or persistent nature of toxicant-induced effects is clearly evident as toxicant exposures during critical developmental windows play a particularly important role in the development of NCDs later in life. In some cases, the persistent effects induced by toxicants have been shown to be multigenerational. For example, in utero exposure to maternal cigarette smoke is associated with increased risk of asthma (Gilliland et al., 2001) and lung function deficits (Gilliland et al., 2003) in children, and this risk is further increased if both the mother and grandmother smoked during pregnancy (Li, Gilliland et al., 2000; Jardim, 2011). Historically, toxicant-associated disease risks such as these have been estimated based on the capacity of a toxicant to damage DNA and/or alter DNA sequence (Weisburger and Williams, 1983; Hou et al., 2011). However, increasing evidence suggests genetic mutations are relevant to the etiology of a small number of toxicant-associated diseases (Foley et al., 2009). As discussed in more detail below, animal studies have indicated that perturbations to the epigenome are likely key events in the development of several diseases associated with toxicant exposure.

The epigenome, which literally means above the genome, is defined as potentially heritable biological information contained outside the DNA sequence (Dolinoy and Jirtle, 2008). Components of the epigenome include DNA methylation, histone post-translational modifications (PTMs), and microRNAs (miRNAs) (Baccarelli and Bollati, 2009). Each of these factors has the potential to play a critical role in regulating gene expression, either at the transcriptional level (DNA methylation and histone PTMs) or at the post-transcriptional level (miRNAs) (Haluskova, 2010). Histone PTMs and especially DNA methylation patterns are the most extensively-studied and best-characterized components of the epigenome. The association of transcriptional competency with particular epigenetic marks, that is, particular histone PTMs and/or DNA methylation patterns, is well-established, especially in the context of gene promoters. For example, the chromatin of transcriptionally inactive gene promoters generally has highly methylated DNA and de-acetylated histones relative to the chromatin of transcriptionally active promoters (see Fuks, 2005; Li et al., 2008; Cedar and Bergman, 2009 for reviews).

miRNAs are the most recently discovered component of the epigenome. As discussed in more detail later, miRNAs are non-protein-encoding RNAs ∼21 nucleotides (nt) in length that regulate gene expression at the post-transcriptional level by binding and inhibiting particular mRNA target(s). Therefore, the capacity of a particular miRNA to control and fine-tune the expression of protein-encoding gene(s) is highly dependent on the expression level of that miRNA.

As with the genome, proper maintenance of the epigenome is essential for normal cellular function. The epigenome plays an essential role in controlling time- and stage-specific gene expression patterns during metazoan development and differentiation and maintains genomic stability by preventing aberrant gene expression (Haluskova, 2010; Wu and Zhang, 2010). The epigenome also plays a key role in mediating adaptive gene expression changes in response to external signals, thus serving as an important link between the environment and gene function (Aguilera et al., 2010). As a key mediator of environmental and developmental signals, the epigenome must be flexible and dynamic. It is also subject to alterations by environmental factors, which is perhaps best illustrated in the case of monozygotic (MZ) twins who have identical DNA methylation patterns and histone PTMs in early life but considerably different epigenetic profiles and gene expression patterns in later life (Fraga et al., 2005).

As mentioned previously, some epigenetic alterations are associated with deleterious effects. In experimental animals, the transient toxicant exposure during particular developmental periods is associated not only with altered disease risk in adulthood, but perturbed DNA methylation patterns and gene expression patterns as well (Jirtle and Skinner, 2007). For instance, neonatal male rats exposed to bisphenol A (BPA) have permanent alterations in the DNA methylation patterns of several cell-signaling and apoptosis-related genes and an increased risk of developing prostate cancer later in life (Ho et al., 2006). Rats transiently exposed to dioxin in utero also have altered DNA methylation patterns associated with the development of various diseases in adulthood including prostate disease and polycystic ovary disease (Manikkam et al., 2012). As demonstrated in these studies, it is believed that DNA methylation patterns are particularly sensitive to the effects of toxicants during certain life stages such as embryogenesis in which the erasure and re-establishment of many DNA methylation marks occurs (Dolinoy and Jirtle, 2008). These altered DNA methylation profiles and disease susceptibilities after transient gestational exposure have been shown in several instances to be transgenerational. For instance, the increased risk of adult onset diseases associated with in utero dioxin exposure was observed through the F3 generation (Manikkam, et al., 2012) and transient in utero exposure to BPA has been shown to cause transgenerational effects in brain mRNA levels and social behavior in mice through the F4 generation (Wolstenholme et al., 2012). Since early-life exposure to environmental toxicants is also associated with altered, potentially multi-generational NCD risk in humans, the results of these animal studies suggest epigenetic alterations provide a plausible and likely critical link between environmental toxicant exposure and human disease (Lillycrop and Burdge, 2012).

Compared to environmental toxicant-associated DNA methylation changes, the relationship between environmental toxicant exposure, miRNA alterations, and disease risk is understudied. Aberrant miRNA levels have been implicated in the development of several diseases including hematological and solid malignancies, neurological disorders, and diabetes mellitus (Lema and Cunningham, 2010; Esteller, 2011; Nana-Sinkam and Croce, 2011; Kumar et al., 2012). Altered miRNA profiles in various cancers have been the subject of particular interest and some miRNAs have been identified as potential biomarkers with diagnostic and/or prognostic value (Lu et al., 2005; Allegra et al., 2012). Although a general down-regulation of mature miRNAs has been reported in human cancers versus normal tissues (Lu, et al., 2005), both up- and down-regulated miRNAs have been observed in cancers and some have been identified as oncogenes or tumor suppressors, respectively, indicating their potential for mechanistic importance in disease development (Esquela-Kerscher and Slack, 2006; Hammond, 2007). In many cases, the mechanisms that lead to the aberrant miRNA levels associated with disease states are unknown. However, it has been suggested that miRNA genes may be particularly susceptible to DNA damage by toxicants as miRNA genes are located at sites of the genome frequently altered in cancers such as fragile sites, sites of minimal loss of heterozygosity, and sites of amplification (Calin et al., 2004; Russ and Slack, 2012).

There is evidence that miRNAs may play a particularly important role in the cellular defense response to toxicants. It was recently proposed that miRNAs are predicted to target 43% of human protein-encoding genes with mRNA levels known to change upon exposure to at least one environmental toxicant (Wu and Song, 2011). In this analysis, environmental toxicant-responsive genes were twice as likely to be targets of miRNAs as non-responsive genes. Therefore, experimental evidence suggests that miRNAs may play a particularly important role in the development of toxicant-associated disease both as potentially sensitive targets of genotoxic agents and as important mediators of cellular defense responses (Wu and Song, 2011).

In the following sections, we highlight various aspects of miRNAs, including their description, history, biological significance, expression and processing, and their interactions with their mRNA targets. We then discuss the current state of knowledge regarding the effect of various environmental toxicants on miRNA profiles. A comprehensive literature search revealed that for many environmental toxicants, there is no information or very little information available in terms of their effects on miRNA expression. The chemicals we discuss here are divided into four major classes and are notable for their toxic effects, known or suspected roles in human disease development, and potential for widespread exposure. These classes are (1) carcinogenic metals, namely arsenic and cadmium; (2) air toxicants, namely formaldehyde, diesel exhaust particles (DEPs), and cigarette smoke; (3) the polycyclic aromatic hydrocarbon (PAH) benzo(a)pyrene [B(a)P]; and (4) endocrine disruptors, namely bisphenol A (BPA), dichlorodiphenyltricholoroethane (DDT), fludioxonil, fenhexamid, and nonylphenol (NP).

This review includes studies that represent primary areas of research examining miRNA alterations associated with each toxicant, and focuses on those that provide insight into the mechanisms involved in miRNA dysregulation and how such perturbation may lead to disease development. As is evident, the reviewed studies demonstrate that for some toxicants, the current understanding of the impact on miRNA expression is quite advanced while for others, this knowledge is quite limited. Together, the research discussed here reveals the complexity of toxicant-miRNA interactions and highlights that there is much to learn about these relationships.

2.2 miRNAs: Description and Biological Significance

microRNAs (miRNAs) are endogenous, evolutionarily-conserved, non-coding RNAs 21–24 nt in length that regulate gene expression through interference with mRNA function, either by targeting mRNAs for degradation or interfering with mRNA translation into protein (He and Hannon, 2004; Lema and Cunningham, 2010). The first miRNA was identified in 1993 in Caenorhabditis elegans (Lee et al., 1993). This miRNA, lin-4, was shown to play a vital role in the temporal regulation of C. elegans larval development as a post-transcriptional regulator of the protein levels of the lin-14 gene, which must be controlled at specific developmental times for proper cell lineage development (Wightman et al., 1991; Wightman et al., 1993). Analysis of the lin-4 locus revealed it did not produce an mRNA that encoded a protein, but instead a 22 nt RNA molecule that bound with partial complementarity to the 3′ untranslated region (3′ UTR) of the lin-14 mRNA and interfered with its translation into protein (Lee, et al., 1993; Wightman, et al., 1993; Olsen and Ambros, 1999). It was not until 2000 that a second miRNA was identified. Like lin-4, this gene (let-7) was a temporal regulator of C. elegans larval development, and also acted in a similar mechanism as lin-4 by producing a small, non-coding RNA that interfered with gene expression by binding to the 3′UTR of its target mRNA (Reinhart et al., 2000; Vella et al., 2004; Bagga et al., 2005; Pillai et al., 2005). Although both lin-4 and let-7 were initially identified in C. elegans, let-7 was found to be perfectly conserved throughout metazoans (Pasquinelli et al., 2000), suggesting it may play a role as a universal developmental regulator (Grosshans et al., 2005).

Together, these initial results suggested miRNAs may be classified as a new group of conserved molecules that regulate gene expression at the post-transcriptional level, causing a search for miRNA genes across species to begin (Lagos-Quintana et al., 2001). Since this time, >25 000 distinct mature miRNAs have been identified in >190 species including plants, animals, unicellular organisms, and viruses (Kozomara and Griffiths-Jones, 2011). In animals, miRNAs have been shown to regulate the expression of genes involved in virtually every cellular process, underscoring their essential role in the maintenance of proper cell function (Lema and Cunningham, 2010; Osman, 2012).

2.2.1 miRNA Biosynthesis and Processing

miRNA levels are controlled in cell-, tissue-, and species-specific manners at both the transcriptional and post-transcriptional levels (Hudder and Novak, 2008). MiRNA genes have been identified throughout the genome and on all chromosomes (Ro et al., 2007; Yuan et al., 2011). They may be clustered together or isolated and have been found in intergenic regions, in the introns or exons of protein-encoding genes, and in the introns of non-protein-encoding genes (Rodriguez et al., 2004; Shukla et al., 2011). Therefore, regulation of miRNA expression is complex as they may be transcriptionally regulated with protein-encoding host genes, transcribed individually, or co-transcribed with other miRNAs (Lagos-Quintana, et al., 2001; Lau et al., 2001; Baskerville and Bartel, 2005; Hudder and Novak, 2008). The promoters of miRNA genes often resemble the promoters of protein-encoding genes (Zhou et al., 2007). For instance, most, but not all, miRNA gene promoters contain cis-acting targets of RNA polymerase II such as TATA box elements and the expression of some miRNAs is controlled epigenetically by DNA methylation and histone PTMs (Chuang and Jones, 2007; Zhou, et al., 2007).

Most miRNAs are transcribed by RNA polymerase II in the form of a long, primary transcript (pri-miRNA) with a 5′ 7-methylguanosine cap and a 3′ polyadenylated tail (Cai et al., 2004; Lee et al., 2004). Some pri-miRNAs do not contain a cap or polyadenylated tail and are transcribed by RNA polymerase III (Borchert et al., 2006; Canella et al., 2010). Pri-miRNAs, which may range from hundreds of bases to several kilobases in length, contain hairpin structures and may produce several mature miRNAs after processing. Complete pri-miRNA processing to produce mature miRNAs involves two sequential steps, each of which involves a ribonuclease IIII (RNase III) endonuclease and a double-stranded RNA-binding domain (dsRBD) protein (Du and Zamore, 2005). In the first step, the pri-miRNA is cleaved in the nucleus by the RNAase III enzyme Drosha and the dsRBD protein DGCR8/Pasha to produce a ∼70–100 hairpin structure with a 5′ phosphate group and a 2-nucleotide overhang at the 3′ end (pre-miRNA) (Lee et al., 2003; Yeom et al., 2006). This cleavage by Drosha will determine either the 5′ or 3′ end of the mature miRNA (Du and Zamore, 2005). The pre-miRNA is transported to the cytoplasm to undergo the second processing step which involves the RNAseIII endonuclease Dicer and the human immunodeficiency virus transactivating response (TAR) RNA-binding protein (TRBP) (Chendrimada et al., 2005; Haase et al., 2005). This cleavage step defines the other end of the mature miRNA and produces a RNA duplex of ∼21–22 base pairs (bp) (Du and Zamore, 2005). One strand of this duplex, known as the guide strand, becomes the mature miRNA and is incorporated into a ribonucleoprotein complex known as the miRNA-induced silencing complex (miRISC) (Choudhuri, 2010). The other duplex strand, known as the passenger strand (miRNA*), is commonly degraded, although several miRNA*s have been shown to have a functional role in regulating mRNA targets as well (Okamura et al., 2008; Choudhuri, 2010; Guo and Lu, 2010). Often, miRISC formation is coupled with the second processing step that produces the ∼21 RNA duplex. Components of the miRISC include members of the Argonaute (Ago) family of proteins, Dicer, TRBP, and proteins belonging to the glycine-tryptophan repeat-containing protein of 182 kDa (GW182) family (Ding and Han, 2007; Hudder and Novak, 2008; MacRae et al., 2008).

2.2.2 Interaction of miRNAs with mRNA Targets

Binding of a miRNA to its target mRNA causes gene silencing by two major mechanisms, namely: targeting the mRNA for degradation and/or interfering with its translation into protein (Zhang et al., 2007). There is also experimental evidence that suggests miRNAs may regulate gene expression via the proteolysis of nascent polypeptides (Nottrott et al., 2006; Vimalraj and Selvamurugan, 2012).

The degradation of mRNAs may occur via endonucleolytic cleavage or by promoting mRNA degradation through deadenylation, de-capping, and exonucleolytic cleavage (Petersen et al., 2006; Garneau et al., 2007; Mathonnet et al., 2007; Standart and Jackson, 2007; Zdanowicz et al., 2009; Fabian et al., 2010). Generally, endonucleolytic mRNA cleavage only occurs by endonuclease Argonaute 2 (Ago2) within the miRISC when there is perfect or near-perfect complementarity between the miRNA and its mRNA target (Hutvagner and Zamore, 2002; Khvorova et al., 2003; Liu et al., 2004; Rand et al., 2004; Chendrimada, et al., 2005; Ding and Grosshans, 2009). Therefore, the mechanism of miRNA-mediated inhibition is generally dependent on the degree of complementarity between the miRNA:mRNA complex (Zhang, et al., 2007). In plants, a single miRNA generally binds to the coding region (and less frequently, the 3′ UTR) of a single mRNA target with perfect or almost perfect complementarity, in which the miRNA-containing RISC functions as an endonuclease (Rhoades et al., 2002; Bartel, 2004; Du and Zamore, 2005). In animals, miRNAs generally bind the 3′ UTR of mRNA targets with a greater degree of mismatches than in plants. In animals, near-perfect complementarity is limited to the 5′ end of the miRNA, corresponding to nucleotides ∼2–7, known as the seed region (Ambros, 1989; Lewis et al., 2003; Lewis et al., 2005). The remainder of the miRNA binds to its target with variable levels of complementarity and the miRNA:mRNA complex may include bulges and non-Watson–Crick base-pairing (Hudder and Novak, 2008). In animals, this imperfect complementarity between the miRNA and the 3′ UTR of its mRNA target generally results in translational repression as opposed to mRNA degradation or endonucleolytic cleavage (Jones-Rhoades et al., 2006; Brodersen et al., 2008; Zhang and Su, 2009). As a result of this imperfect base pairing, a particular miRNA may be capable of binding several similar mRNA sequences. Therefore in animals, each miRNA may bind several different mRNA targets or several regions within the same miRNA transcript, and each mRNA may bind multiple different miRNAs (Lema and Cunningham, 2010).

Over 2000 mature miRNAs have been identified in humans (Kozomara and Griffiths-Jones, 2011), and through the analyses of the 3′ UTR of human genes, it is estimated that >60% of protein-encoding genes have sequences that are conserved targets of miRNAs (Friedman et al., 2009).

2.3 Environmental Toxicant-Associated miRNA Perturbations

2.3.1 Toxicant Class 1: Carcinogenic Metals (Arsenic and Cadmium)

2.3.1.1 Arsenic

Chronic exposure to the metalloid arsenic (As) is associated with cancers of the skin, urinary bladder, prostate, liver, and lung (IARC, 1987; IARC, 2004a). Chronic exposure to arsenic is also associated with a variety of other adverse health effects in humans that are collectively known as arsenicosis. Hallmark signs of arsenicosis include characteristic skin lesions but may also include diabetes mellitus, peripheral vascular disease, atherosclerosis, neurological effects, and the aforementioned cancers (Das and Sengupta, 2008; Sengupta et al., 2008a). The major source of As exposure worldwide is drinking water contaminated with inorganic forms of As (iAs), which can exist in either a trivalent (+3) or pentavalent (+5) state. It is estimated that millions of people worldwide are exposed to iAs levels in their drinking water that exceed the World Health Organization's recommended limit of 10 ppb (Uddin and Huda, 2011). Inorganic As is metabolized in humans and rodents to yield a series of trivalent and pentavalent monomethylated and dimethylated arsenicals (MMAs and DMAs, respectively) (Thomas et al., 2001). This biotransformation is important for several reasons. First, the toxicity of arsenicals is dependent on both methylation status and oxidation state. In general, the trivalent forms of MMAs and DMAs are the most toxic forms of As and, along with iAs(III), are implicated in As-associated disease development (Styblo et al., 2000; Hughes, 2002; Kitchin and Ahmad, 2003). Second, since the enzymes required for DNA methylation and iAs biotransformation utilize the same intracellular methyl donor, S-adenosyl methionine (SAM) (Zhao et al., 1997; Ren et al., 2011), iAs biotransformation is believed to have a considerable impact on the DNA methylation landscape (Smeester et al., 2011; Bailey and Fry, 2012; Bailey et al., 2013).

The first study that investigated the effects of As exposure on miRNAs identified four up-regulated miRNAs (miR-22, miR-34a, miR-221, miR-222) and one down-regulated miRNA (miR-210) that were significantly modulated in human lymphoblasts after exposure to 2 µM iAs(III) for six days (Niculescu and Zeisel, 2002). These miRNAs were modulated in these cells in a similar manner under folate-limiting conditions. Dietary folate is used as a source of methyl groups required for DNA methylation (Niculescu and Zeisel, 2002), and these results are interesting in light of theories that suggest iAs exposure/metabolism may alter DNA methylation patterns by perturbing methyl donor availability (Pilsner and University, 2007).

Since this initial study, altered miRNA profiles have been observed with several effects associated with As exposure including vascular injury (Li, Shu et al., 2012) and angiogenesis (Cui et al., 2012). However, most studies that have examined the effects of arsenic on miRNA levels can be divided into two major categories, namely the role miRNAs play in (1) iAs(III)-induced carcinogenesis and (2) arsenic trioxide (ATO)-mediated apoptosis.

Studies investigating the role of miRNAs in As-mediated carcinogenesis have identified several miRNAs with tumor suppressive or oncogenic functions. For instance, the iAs(III)-mediated malignant transformation of tumor protein 53 (TP53)-deficient human bronchial epithelial cells was dependent on the significant reduction of miR-200b levels (Wang et al., 2011). These iAs(III)-transformed cells exhibited highly migratory and invasive characteristics, but stable re-expression of miR-200b in these cells reversed these characteristics and eliminated their transformed phenotypes in nude mice (Wang, et al., 2011; Wang, Yang et al., 2012). Interestingly, the iAs-mediated miR-200b reduction was accompanied by increased methylation of the miR-200 promoter, suggesting alterations in DNA methylation patterns may play a role in aberrant miR-200b expression (Wang, et al., 2011).

In a separate study, the up-regulation of miR-21 was implicated in the iAs(III)-mediated transformation of human embryo lung fibroblast cells (Ling et al., 2012). Using specific inhibitors, it was found that the iAs-mediated induction of miR-21 was dependent on the reactive oxygen species (ROS)-mediated activation of pathways involving extracellular signal-regulated kinase mitogen activated protein kinase (ERK MAPK) and transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). It was also determined that while miR-21 promoted anchorage-independent cell growth during iAs(III)-mediated cellular transformation, its overexpression alone was insufficient to induce the transformed phenotype.

Arsenic was also found to induce miR-190 in a human bronchial cell line in a dose-dependent manner (Beezhold et al., 2011). In this case, miR-190 overexpression alone was found to allow the cells to acquire malignant characteristics, including increased proliferation and anchorage-independent growth. MiR-190 was found to target the mRNA of the PH domain leucine-rich repeat protein phosphatase (PHLPP) gene, which encodes a protein with tumor suppressive functions including the inhibition of protein kinase B (PKB; Akt) and the promotion of apoptosis (Gao et al., 2005). Therefore, it is believed that miR-190 may allow cells to acquire self-sustaining growth signals during iAs-mediated carcinogenesis (Beezhold, et al., 2011).

Arsenic trioxide (ATO; As2O3) is a potent inducer of apoptosis that has shown promise in the treatment of relapsed/refractory acute promyelocytic leukemia (APL) (Niu et al., 1999; Soignet et al., 2001). At the pharmacological dose (2 µM) that induces considerable apoptosis in an APL cell line, 88 miRNAs were shown to be differentially expressed compared to untreated cells (Ghaffari et al., 2012). Functional analysis of the 23 most dysregulated miRNAs were performed through an examination of the expression of these miRNAs in solid and/or hematological tumors and by predicting functions of their validated targets. These analyses revealed that most of the 22 up-regulated miRNAs had tumor suppressor or metastatic suppressor functions, whereas the one down-regulated miRNA had an oncogenic function. A pathway-level analysis revealed that the target genes of these miRNAs were involved in functions such as cell cycle, apoptosis, TP53-response, and cell signaling pathways such as those involving MAPKs. The authors suggested these miRNAs likely play a mediatory role in the action of ATO, but point out that the mechanism by which these miRNAs are altered by ATO remains unknown.

Other studies have identified specific miRNAs that play important roles in ATO action. For instance, several modulated miRNAs were found to be likely critical mediators of ATO-mediated apoptosis of malignant cell lines, namely miR-29a in hepatocellular carcinoma cells (Meng et al., 2011) and miR-19a in human bladder carcinoma cells (Cao et al., 2011). In chronic myeloid leukemia (CML) cells, miR-153 was identified as a likely important mediator of ATO-mediated apoptosis (Liu et al., 2012). In this study, miR-153, which induces apoptosis by targeting the anti-apoptotic b-cell lymphoma 2 (BCL2) transcript (Xu et al., 2010), was found to be down-regulated in an ATO-resistant CML line, and ATO-mediated apoptosis was restored by the exogenous expression of miR-153 (Liu, et al., 2012).

One study identified two miRNAs as important mediators of cardiotoxicity associated with ATO treatment. Patients who have received ATO treatment have an increased risk of developing a cardiac electrical disorder known as long QT syndrome that is associated with sudden death (Ohnishi et al., 2000). The mechanism of ATO-associated cardiac toxicity is unclear in humans, but it was found in guinea pigs that ATO exposure was associated with the up-regulation of two muscle-specific miRNAs (miR-1 and miR-133) that were associated with cardiac electrical disorders (Shan et al., 2012). Inhibition of miR-1 and miR-133 by antisense inhibitors eliminated the cardiac electrical disorders in ATO-exposed guinea pigs, suggesting these miRNAs may be promising therapeutic targets to manage ATO-associated cardiotoxicity.

Together, these studies have identified specific miRNAs that likely play crucial roles in mediating various effects of As, namely iAs(III)-mediated malignant transformation, ATO-mediated apoptosis, and ATO-mediated cardiotoxicity. These studies have also provided evidence suggesting that As may perturb miRNA levels via alterations in DNA methylation patterns.

2.3.1.2 Cadmium

Cadmium (Cd) is alternately classified as a heavy metal (Valko et al., 2005) or transition metal (Waalkes, 2000). It exists primarily in the (+2) oxidation state and is usually combined with oxygen to form cadmium oxide (CdO), chlorine to produce cadmium chloride (CdCl2) or sulfur and oxygen to produce cadmium sulfate (CdSO4) (Bridges and Zalups, 2005; Valko, et al., 2005). Cd has a widespread presence in the environment (e.g., plastics, cigarette smoke, nickel-cadmium batteries) and certain occupations (e.g., smelting-related occupations) may be significant sources of human Cd exposure (Valko, et al., 2005). Cd exposure is particularly concerning due to its particularly long biological half-life of ∼15–20 years in humans (Jin et al., 1998; Bernard, 2008). Cd may occur through ingestion of food and water but mainly occurs through inhalation (e.g., cigarette smoke) and is primarily associated with cancers of the lung (Waalkes, 2000). Cd exposure is also associated with the development of cancers at other internal sites (pancreas, prostate, and kidney, and urinary bladder) and bone disease (Jin, et al., 1998; Waalkes, 2000). Cd has also been described as an endocrine disruptor (Henson and Chedrese, 2004) and can produce teratogenic effects in animals (Samarawickrama and Webb, 1981; Menoud and Schowing, 1987).

Much of the research on miRNA perturbations associated with Cd exposure have been conducted in plants such as rapeseed and rice (Huang et al., 2009; Ding et al., 2011; Zhou et al., 2012), and studies exploring the effects of Cd exposure in mammals have been limited. To our knowledge, there is one study that examines miRNA perturbations in humans exposed to Cd. In this study, analysis of peripheral blood leukocytes (PBLs) from Italian foundry workers exposed to a wide range of levels of metal-rich particulate matter (PM) revealed a significant negative relationship between the levels of miR-146a and Cd (as well as miR-146a levels and lead (Pb) (Bollati et al., 2010). As miR-146a is believed to limit inflammatory responses activated by the innate immune system (Williams et al., 2008), the authors suggested that the down-regulation of miR-146a may be a mechanism by which the metal components of PM (i.e., Cd and Pb) may exacerbate inflammatory responses in the lung (Bollati, et al., 2010).

The effects of Cd exposure on miRNA levels have been explored in vitro as well. Twelve miRNAs were found to be down-regulated in human hepatoblastoma cells after exposure to 10 µM CdCl2 for 24 h (Fabbri et al., 2012). Pathways and functions of predicted targets of these miRNAs included those involved in cytoskeletal remodeling, MAPK signaling, and TP53 signaling. Interestingly, four out of these 12 miRNAs were part of the let-7 family of miRNAs. As previously mentioned, let-7 was the second miRNA identified (Reinhart, et al., 2000) and the let-7 family regulates many genes with carcinogenesis-associated functions such as cell cycle progression and apoptosis (Boyerinas et al., 2010). In general, let-7 members are considered tumor suppressors and are often down-regulated in cancers (Boyerinas, et al., 2010; Fabbri, et al., 2012). Therefore, the dysregulation of miRNAs with tumor suppressive functions may represent early events that play important roles in Cd-mediated carcinogenesis.

The effects of cadmium telluride (CdTe) nanoparticles on miRNA signaling has also been investigated. Quantum dots (QDs) are luminescent CdTe nanoparticles that are used extensively in medical imaging, and concerns over their safety have been raised in light of this extensive use and observations that QDs can induce oxidative damage and apoptosis in vitro (Lovric et al., 2005; Li, Chen et al., 2011; Wang, Li et al., 2012). The viability, metabolism, and miRNA profiles of mouse fibroblast cells were shown to be altered in both time- and dose-responsive manners after short-term exposure (12–24 h) to CdTe QDs (Li, Wang et al., 2011), suggesting miRNAs associated with specific changes in cellular metabolism and viability could be identified. A total of 51 CdTe QD-responsive miRNAs (35 up-regulated/16 down-regulated) were described in the study. By comparing the levels of mature miRNAs to their pri-miRNA precursors, the authors determined that differential miRNA expression induced by CdTe occurred at the level of pri-miRNA transcription and not subsequent processing steps that produce mature miRNAs (Li, Wang et al., 2011).

Although the number of studies that examine Cd-associated miRNA perturbations are limited, they include a study of miRNA perturbations in a human population. As illustrated in this review, studies that address the effects of toxicants in human subjects are often few in number but can provide critical information in terms of identifying risk factors and mechanisms of disease development. Together, the studies discussed here provide groundwork for future work that addresses the roles of miRNAs in Cd-mediated carcinogenesis and CdTe toxicity.

2.3.2 Toxicant Class 2: Air Toxicants (Formaldehyde, Diesel Exhaust Particles, Cigarette Smoke)

Ambient air is a complex mixture of gases, volatile organic compounds, particulate matter (PM), and biological agents such as bacteria and fungal spores (Jardim, 2011). The components of air are dependent on both natural (e.g., water vapor) and anthropogenic (e.g., industrial emissions, vehicle exhaust) factors and therefore air quality is location-dependent. Poor air quality is associated with both acute and long-term detrimental effects on human health. Acute effects include events such as heart attacks or asthma attacks whereas long-term exposure is associated with the development of cardiovascular disease, cancers, and respiratory diseases (Bernstein et al., 2004; Chen et al., 2008). Children, the elderly, and individuals with underlying respiratory disease such as asthma are particularly susceptible to the adverse effects of poor air quality (Gong, 1992). The US Environmental Protection Agency (EPA) has identified six common air pollutants known as criteria pollutants in the United States for which it has set ambient air quality standards. These pollutants are ozone (a primary component of smog), particulate matter (PM) (e.g., smoke), carbon monoxide, nitrogen oxides, sulfur dioxides, and Pb (EPA, 2012).

There are few studies that have investigated the effects of air toxicants on miRNA alterations. As previously mentioned, one study found a negative association between the levels of miR-146a and Cd and miR-146a and Pb in the PBLs of Italian foundry workers exposed to metal-rich PM (Bollati, et al., 2010). In our laboratory and the laboratories of our collaborators, we have investigated the effects of gaseous formaldehyde (Rager et al., 2011; Rager et al., 2013) and diesel exhaust particles (DEPs) (Jardim et al., 2009) on miRNA signaling.

2.3.2.1 Formaldehyde

Gaseous formaldehyde is formed during the combustion of carbon-containing compounds (e.g., automobile exhaust, tobacco smoke, methane) and from the outgassing of various items commonly found indoors due to the widespread use of formaldehyde in the manufacturing, construction, and textile industries (Kim et al., 2011). Formaldehyde levels can therefore be of concern both indoors and outdoors. Long-term exposure to formaldehyde is associated with the development of a wide range of adverse effects including pulmonary disorders (e.g., asthma) and neurotoxicity (Kim, et al., 2011). Formaldehyde has been classified as a known human carcinogen targeting the nasopharynx and is controversially linked to the development of myeloid leukemias (IARC, 2006; Kim, et al., 2011).

Our laboratory was the first to report that environmentally-relevant concentrations of gaseous formaldehyde alter miRNA levels in vitro (Rager, et al., 2011) and in vivo (Rager, et al., 2013). In the in vitro study, a total of 89 miRNAs were down-regulated in human lung epithelial cells after short-term exposure (4 h) to 1 ppm gaseous formaldehyde. Several of these miRNAs had predicted targets that had previously been shown to be transcriptionally altered by formaldehyde exposure (Li et al., 2007), including genes involved in tumorigenesis and inflammation.

In the in vivo study, cynomolgus macaques were exposed to gaseous formaldehyde (2 and 6 ppm) for 6 h per day over a course of two days (Rager, et al., 2013). A dose-dependent response was observed in terms of number of miRNAs altered in the nasal epithelium (3 and 13 miRNAs in the 2 and 6 ppm groups, respectively). Two of these miRNAs (miR-26b and miR-140-5p) were modulated in human airway cells by 1 ppm gaseous formaldehyde in vitro, revealing a level of concordance between these two studies. The most up-regulated miRNA (miR-125b) and most down-regulated miRNA (miR-142-3p) after exposure to 6 ppm formaldehyde in vivo were analyzed at the systems level, revealing predicted targets involved in apoptosis (miR-125) and in integrin linked kinase (ILK) signaling (miR-142-3p). Importantly, the expression of several selected mRNA targets of these miRNAs had an inverse relationship with their respective miRNA levels. In addition, six of the 13 miRNAs altered in the 6 ppm group (miR-142-3p, miR-145, miR-152, miR-203, miR-26b, and miR-29a) had been previously shown to be altered in human nasopharyngeal cancers (Sengupta et al., 2008b; Chen et al., 2009; Li, Chen et al., 2011; Wong et al., 2012; Rager, et al., 2013).

These studies have identified miRNAs that are responsive to formaldehyde exposure in target cells in vitro and in vivo. Importantly, the modulation of miRNAs observed in nasopharyngeal cancers and miRNAs that regulate targets involved in tumorigenesis after the short-term exposures described previously suggest these miRNA alterations may represent early events in the development of formaldehyde-associated cancers.

2.3.2.2 Diesel Exhaust Particles (DEPs)

Diesel exhaust (DE) is a complex mixture of gases (e.g., carbon monoxide) and particulate matter (DEPs) from the incomplete combustion of diesel fuel (Ghio et al., 2012). Adverse health effects are associated with both the gaseous and particulate phases of DE (Westphal et al., 2012). DE exposure is associated with non-cancer health effects such as pulmonary inflammation, cardiovascular disease, exacerbation of asthma, and increased susceptibility to lung infections (Hesterberg et al., 2009; Ghio, et al., 2012). DE has also been recently classified as a human carcinogen in which DE exposure is associated with an increased risk of lung cancer (IARC, 2012).

Exposure of human bronchial epithelial cells to DEPs for 24 h were found to modulate 197 miRNAs compared to controls (130 up-regulated/67 down-regulated). Analyses of the 12 most significantly-altered miRNAs identified predicted targets with functions and networks associated with tumorigenesis and inflammation (Jardim, et al., 2009).

As with Cd, few studies have addressed miRNA pertubations by DEPs and formaldehyde. However, as observed with formaldehyde, this study of the effects of DEPs indicate that even after short-term exposure, the miRNA changes associated with these toxicants in vitro and/or in vivo are consistent with adverse health effects elicited by these toxicants in humans (i.e., inflammation and carcinogenesis) (Ghio, et al., 2012), suggesting miRNAs may play important roles in mediating these effects.

2.3.2.3 Cigarette Smoke

The most extensively-studied air toxicant in terms of impact on miRNA profiles is cigarette smoke. Although we discuss individual toxicants that are components of cigarette smoke in other portions of this review (e.g., Cd, formaldehyde, B(a)P), we describe studies next that examined the effects of cigarette smoke or cigarette smoke condensates (CSCs), which are complex mixtures and therefore represent the simultaneous effects of many toxic chemicals including multiple known carcinogens (Hecht, 2012).

Cigarette smoke contains thousands of chemicals. These chemicals include gases, volatile compounds, and those found in the submicron-sized solid particles of cigarette smoke (Harris, 1996). Many of these chemicals are highly mutagenic and redox active and several have been identified as known carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and tobacco-specific nitrosamines (TSNAs) (Harris, 1996; Pryor, 1997; IARC, 2004b; Hecht, 2012). The exact chemical components of cigarette smoke vary and are dependent on factors such as the type of tobacco smoked, device used for smoking, and whether the smoke is mainstream cigarette smoke (MCS), that is, smoke that is inhaled and exhaled by a smoker; sidestream cigarette smoke (SSCS), which is smoke that is released from the burning end of a cigarette; or environmental cigarette smoke (ECS), which is a mixture of MCS and SSCS (Harris, 1996; IARC, 2004b).

Diverse health effects associated with cigarette smoke exposure are observed both in smokers (exposed mainly to MCS) and non-smokers (exposed to ECS, or secondhand smoke). Both MCS and ECS are classified as known human carcinogens (IARC, 2004b). Although the lung is the major target organ of smoking-related cancers, smoking is also associated with the development of cancers in other sites such as the urinary bladder, esophagus, oral cavity, liver, and prostate (US Public Health Service, 1989; IARC, 2004b). Smoking is also associated with the development of multiple degenerative diseases including cardiovascular disease and chronic obstructive pulmonary disease (COPD) (US Public Health Service, 1989; IARC, 2004b). Children are particularly sensitive to the effects of ECS exposure, which include increased incidence of lower respiratory tract infections and ear infections and an increased risk of asthma development (IARC, 2004b; Hwang et al., 2012). ECS exposure is also associated with diverse effects in non-smoking adults including lung cancer and cardiovascular disease (US Public Health Service, 1989; IARC, 2004b).

Both the duration and number of cigarettes smoked per day are believed to play an important role in the risk of developing smoking-associated lung cancer (Flanders et al., 2003). Smoking cessation is associated with a reduced risk of developing lung cancer and this trend is observed in most other organs that are targets of smoking-related cancers (IARC, 2004b).

Much of the research on the effects of cigarette smoke on miRNA expression has been conducted in the lungs of mice and rats. Several studies have reported that the majority of dysregulated miRNAs in rats and mice are down-regulated in response to cigarette smoke, a trend that is interpreted as the activation of protective responses and/or processes involved in the early stages of pulmonary disease (Izzotti et al., 2009a; Izzotti et al., 2011a; Russ and Slack, 2012). For instance, the expression levels of 484 miRNAs were analyzed in rat lungs after exposure to ECS, and 24 of the 25 dysregulated miRNAs were down-regulated in ECS-exposed lungs versus controls (Izzotti, et al., 2009a). Based on the functions of their predicted targets, these modulated miRNAs were shown to influence many different cellular processes including some associated with carcinogenesis such as stress response, cell proliferation, and angiogenesis. Of note, several of these down-regulated miRNAs included those implicated in lung cancer development. These included let-7, which is frequently down-regulated in lung cancers (Osada and Takahashi, 2011), and this down-regulation was accompanied by the up-regulation of several let-7 mRNA targets involved in cell cycle progression including the Ras oncogene (Johnson et al., 2005; Izzotti, et al., 2009). Another down-regulated gene of interest in the ECS-exposed rat lung included miR-125a-prec (Izzotti, et al., 2009a). MiR-125a-prec targets a gene that is highly expressed in lung cancers, the epidermal growth factor (EGF) receptor v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (Erbb2) (Fujimoto et al., 2005). Interestingly, it has been hypothesized that miRNAs like miR-125a and let-7 may be down-regulated by cigarette smoke because their genes lie in areas of the genome that are particularly susceptible to DNA damage by genotoxic agents (Calin, et al., 2004; Izzotti, Calin et al., 2009a). For instance, the miR-125a gene has a single polymorphism at nucleotide 8 (G/U) in which the U allele is associated with defects in processing of the miR-125a pri-miRNA to its mature form (Slack et al., 2000; Duan et al., 2007; Izzotti, et al., 2009a).

In mice and rats, miRNA alterations mediated by ECS or MCS have been shown to be dependent on age, gender, and co-exposure to other agents such as the oral chemopreventative agents N-acetylcysteine (NAC) and oltipraz (OPZ) (Izzotti et al., 2003; Izzotti et al., 2009b; Izzotti et al., 2010; Izzotti et al., 2010; Izzotti, Larghero et al., 2011a; Izzotti et al., 2011b). Interestingly, NAC and OPZ were shown to attenuate the overall down-regulation of miRNAs in the rat lung after 28 days exposure to ECS and NAC (Izzotti, et al., 2010), and NAC and phenethyl isothiocyante (PEITC) were shown to attenuate this effect in mice exposed to MCS during the first four months of life (Izzotti, Larghero et al., 2011a). The exact effect these chemopreventative agents had on miRNA profiles differed between the agents and indicate that miRNA profiling may be used as a valuable tool to evaluate the effectiveness of potential therapies (Izzotti, et al., 2010).

One study examined the stability of cigarette smoke-altered miRNA profiles (Izzotti, et al., 2011b). In this study, the expression of 697 miRNAs and the production of DNA adducts were examined in mice exposed to varying concentrations of MCS for the first four months of life. Both the number of miRNAs and number of DNA adducts formed, including bulky DNA adducts and DNA lesions indicative of oxidative damage [8-oxo-7,8-dihydro-2′deoxyguanosine (8-oxodGuo)], were altered in a dose-dependent manner (Izzotti, et al., 2011b; Ravanat et al., 2012). Whereas the incidence of DNA adducts generally increased with MCS dose, miRNAs were generally down-regulated with increasing concentrations of MCS. In the case of miRNAs, there appeared to be a threshold dose necessary for miRNA dysregulation to occur. The MCS-induced miRNA alterations and DNA adducts were reversed within weeks after exposure cessation. These included miRNA alterations associated with even the highest sublethal dose of MCS, in which the dysregulation of almost all perturbed miRNAs (including let-7) were reversed 1 week after exposure cessation (Izzotti, et al., 2011b). The authors point out that this reversibility of miRNA patterns and DNA damage are consistent with results that link the risk of smoking-associated cancers with the duration