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Manganese in Health and Disease
Issues in Toxicology

Series Editors:
Professor Diana Anderson, University of Bradford, UK
Dr Michael Waters, Integrated Laboratory Systems Inc, NC, USA
Dr Timothy C Marrs, Edentox Associates, Kent, UK

Advisor to the Board:

Alok Dhawan, Ahmedabad University, India

Titles in the Series:

1: Hair in Toxicology: An Important Bio-Monitor
2: Male-mediated Developmental Toxicity
3: Cytochrome P450: Role in the Metabolism and Toxicity of Drugs and
other Xenobiotics
4: Bile Acids: Toxicology and Bioactivity
5: The Comet Assay in Toxicology
6: Silver in Healthcare
7: In Silico Toxicology: Principles and Applications
8: Environmental Cardiology
9: Biomarkers and Human Biomonitoring, Volume 1: Ongoing Programs
and Exposures
10: Biomarkers and Human Biomonitoring, Volume 2: Selected Biomarkers
of Current Interest
11: Hormone-Disruptive Chemical Contaminants in Food
12: Mammalian Toxicology of Insecticides
13: The Cellular Response to the Genotoxic Insult: The Question of
Threshold for Genotoxic Carcinogens
14: Toxicological Effects of Veterinary Medicinal Products in Humans:
Volume 1
15: Toxicological Effects of Veterinary Medicinal Products in Humans:
Volume 2
16: Aging and Vulnerability to Environmental Chemicals: Age-related
Disorders and their Origins in Environmental Exposures
17: Chemical Toxicity Prediction: Category Formation and Read-Across
18: The Carcinogenicity of Metals: Human risk through occupational and
environmental exposure
19: Reducing, Refining and Replacing the Use of Animals in Toxicity
Testing
20: Advances in Dermatological Sciences
21: Metabolic Profiling: Disease and Xenobiotics
22: Manganese in Health and Disease

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delivery of each new volume immediately on publication.

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Manganese in Health and
Disease

Edited by

Lucio G. Costa
University of Washington, Seattle, WA, USA
Email: lgcosta@u.washington.edu

Michael Aschner
Albert Einstein College of Medicine, Bronx, NY, USA
Email: michael.aschner@einstein.yu.edu
Issues in Toxicology No. 22

Print ISBN: 978-1-84973-943-6

PDF eISBN: 978-1-78262-238-3
ISSN: 1757-7179

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r The Royal Society of Chemistry 2015

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Preface

Manganese (Mn) is the twelfth most abundant element in the Earth’s crust
and present in drinking water and in food. As an essential trace element, Mn
is required for multiple biochemical and cellular reactions, and is a neces-
sary component for numerous metallo-enzymes, such as Mn superoxide
dismutase, arginase, phosphoenol-pyruvate decarboxylase, and glutamine
synthase, to name a few.
Despite its essentiality, exposures to high levels of Mn from occupational,
iatrogenic, medical, and environmental exposures may contribute to human
morbidity. Excessive Mn accumulation in the brain, primarily in basal
ganglia, may cause clinical signs and morphological lesions analogous to
those seen in Parkinson’s disease (PD). Other tissues may be affected as well.
Mn intoxication cases were originally described over two centuries ago.
Manganism, resulting from exposure to exceedingly high levels of this metal,
was originally described by James Couper (1837), providing insight into the
adverse neurological effects in five Scottish men employed in grinding Mn
dioxide ore. As Mn began to be used more widely in the steel alloy industry,
more cases were recognized, with stronger epidemiological evidence impli-
cating Mn in a number of neurological diseases. Contemporary exposures to
Mn at levels described by Couper are rare, yet concerns about the health
effects of Mn remain, given its abundant occurrence and the potential ex-
posures throughout various life-stages.
This book, to our knowledge, is the first multidisciplinary scientific en-
deavor to address the health effects of Mn. It aims to provide state-of-the-art
information and deepen the understanding of Mn’s adverse health effects. It
commences with a description on various pathways for Mn absorption (lung,
gastrointestinal tract, olfactory pathway), followed by its nutritional needs,
toxicokinetics and toxicodynamics. A large section of the book is devoted to
its adverse effects, emphasizing cellular and molecular mechanisms of

Issues in Toxicology No. 22

Manganese in Health and Disease
Edited by Lucio G. Costa and Michael Aschner
r The Royal Society of Chemistry 2015
Published by the Royal Society of Chemistry, www.rsc.org

vii
viii Preface

toxicity in a host of tissues and organs, particularly the nervous system, with
emphasis on sensitivity to Mn at various life-stages. We conclude with a list
of research needs that will further improve our understanding of the role of
Mn both in health and disease.
We called upon internationally recognized experts on Mn to address
and facilitate the understanding of its role in health and disease, making
a valiant attempt to provide as broad and multidisciplinary approach as
possible. Our goal was to assemble a series of chapters that advance the
latest developments and scientific breakthroughs in this fast-paced research
area, and to provide information that should be of interest to risk assessors,
neurobiologists, and neurotoxicologists, as well as metal and trace element
biologists. We are hopeful that the book offers the reader appreciation and
renewed sense on contemporary issues in Mn research. We are indebted to
the authors for their contributions and hope that, as a reader, whether you
are a novice or a seasoned Mn researcher, the knowledge amassed herein
will stimulate and transform your novel ideas into better understanding on
the role of this unique metal in health and disease.

Michael Aschner
Lucio G. Costa
Contents

Chapter 1 Manganese Transport, Trafficking and Function in

Invertebrates 1
Amornrat Naranuntarat Jensen and Laran T. Jensen

1.1 Introduction 1
1.2 Function of Manganese in Biological Systems 2
1.2.1 Manganese Metalloenzymes 2
1.2.2 Non-Protein Manganese Antioxidants 2
1.2.3 Manganese and Bacterial Virulence 4
1.3 Manganese Transport in Bacteria 4
1.3.1 Bacterial Manganese Uptake Systems 4
1.3.2 Bacterial Manganese Efflux 8
1.3.3 Regulation of Bacterial Manganese Transport 8
1.4 Manganese Transport in Yeast 10
1.4.1 Yeast High Affinity Manganese Uptake,
Smf1p and Smf2p 10
1.4.2 Manganese and Phosphate Coupled Uptake
in Yeast, Pho84p 12
1.4.3 Intracellular Manganese Distribution in Yeast 13
1.4.4 Regulation of Yeast Manganese Transporters 15
1.5 Manganese Transport in the nematode
Caenorhabditis elegans 19
1.5.1 Nramp Manganese Transporters in C. elegans 19
1.5.2 Regulation of C. elegans Nramp Transporters
by Manganese 20
1.5.3 Intracellular Manganese Transporters in
C. elegans 20
1.6 Conclusions 21
References 21

Issues in Toxicology No. 22

Manganese in Health and Disease
Edited by Lucio G. Costa and Michael Aschner
r The Royal Society of Chemistry 2015
Published by the Royal Society of Chemistry, www.rsc.org

ix
x Contents
Chapter 2 Nutritional Requirements for Manganese 34
Jeanne H. Freeland-Graves, Tamara Y. Mousa and
Namrata Sanjeevi

2.1 Introduction 34
2.2 Food Sources 35
2.3 Absorption, Transport and Excretion 36
2.4 Approaches to Assessing Mn Requirements 36
2.4.1 Metabolic Balance 37
2.4.2 Blood Levels of Mn 42
2.4.3 Other Biomarkers 43
2.4.4 Extrapolation to Usual Diet Intake 44
2.5 Deficiencies 45
2.6 Nutritional Recommendations for Mn 50
2.6.1 Life Stage and Gender 50
2.6.2 Infants 51
2.6.3 Children and Adolescents 52
2.6.4 Pregnancy 53
2.6.5 Lactation 54
2.6.6 International Variability of Requirements
and Dietary Levels for Mn 55
2.7 Influence of Bioavailability 56
2.7.1 Fiber and Phytate 57
2.7.2 Mineral Interactions 58
2.7.3 Fat and Protein 61
2.7.4 Polyphenolic Compounds 61
2.8 Toxicity 61
2.8.1 Parenteral Nutrition 62
2.9 Conclusions 63
References 64

Manganese: Toxicokinetics and Toxicodynamics

Chapter 3 Manganese Superoxide Dismutase 79

Kinsley K. Kiningham

3.1 Introduction 79
3.2 Manganese Incorporation into SOD2 82
3.3 Manganese Superoxide Dismutase is Essential for Life 82
3.4 Post-Translational Modification of MnSOD 85
3.4.1 Nitration of MnSOD Compromises
Mitochondrial Function in Various Disease
States 85
3.4.2 Phosphorylation of MnSOD can Enhance
Activity and Stability 88
3.4.3 Acetylation of MnSOD Reduces Enzymatic
Activity 88
Contents xi
3.5 MnSOD and Redox Signaling 89
3.6 Transcriptional Regulation of MnSOD Expression 92
3.7 MnSOD and Disease 95
3.7.1 Cancer 95
3.7.2 Cardiovascular Disease 97
3.7.3 Neurodegenerative Disorders 98
3.7.4 MnSOD Polymorphisms and Disease 99
3.8 Future Directions 100
Abbreviation List 101
References 102

Chapter 4 Olfactory Transport of Manganese: Implications

for Neurotoxicity 119
David C. Dorman and Melanie L. Foster

4.1 Introduction 119

4.2 Anatomical Features of the Olfactory System 120
4.3 Scientific Evidence in Support of Olfactory Transport
of Manganese 121
4.3.1 Manganese Transport Kinetics 124
4.4 Manganese-Enhanced Magnetic Resonance Imaging
(MEMRI) of the Olfactory System 125
4.5 Toxicological Significance of Olfactory Transport of
Manganese 125
4.5.1 Olfactory System Pathology 126
4.5.2 Biochemical Effects 126
4.5.3 Olfactory Function 127
4.5.4 Species Differences 127
4.6 Conclusions 127
References 128

Chapter 5 Manganese Transport Across the Pulmonary

Epithelium 133
Khristy J. Thompson, Jonghan Kim and
Marianne Wessling-Resnick

5.1 The Air–Blood Barrier 133

5.1.1 Microanatomy of the Lungs 133
5.1.2 Lung Manganese Exposures 134
5.2 Overview of Manganese Transport 135
5.2.1 Divalent Metal Transporter-1 (Slc11a2) 135
5.2.2 Ferroportin (Slc40a1) 136
5.2.3 Transferrin/Transferrin Receptor (CD71) 137
5.2.4 Other Manganese Transporters:
Non-Selective Ion Channels 137
5.2.5 Hypothetical Model for Pulmonary
Manganese Transport 138
xii Contents
5.3 Interplay Between Manganese and Iron Status 138
5.3.1 Pulmonary Manganese Uptake and Iron
Deficiency 140
5.3.2 Iron Overload and Lung Manganese
Absorption 142
5.3.3 Roles for Tf and the Tf Receptor in the Lungs 142
5.4 Non-Selective Ion Channels 143
5.5 Toxic Effects of Manganese on Lung Epithelial Cells 144
5.6 Infection and Manganese in the Lungs 145
5.7 Future Directions 146
Acknowledgements 148
References 148

Chapter 6 Are There Distinguishable Roles for the Different

Oxidation States of Manganese in Manganese Toxicity? 158
Thomas E. Gunter

6.1 Introduction 158

6.2 A Brief Review of the Inorganic Chemistry of Mn21
and Mn31 159
6.3 Current Physical Techniques Useful in Mn
Speciation 160
6.3.1 UV-Visible Spectroscopy 160
6.3.2 XANES Spectroscopy 161
6.3.3 Electron Paramagnetic Resonance
Spectroscopy (EPR) 163
6.4 Studies Most Relevant to Mn Speciation 164
6.5 Studies of Biological Effects of Exposure to Mn21 or
Mn31 Complexes 166
6.6 Is Mn21 Oxidized to Mn31 within Cells or
Mitochondria? 170
6.7 Transport of Mn31 via the Transferrin Mechanism 171
6.8 The Toxicologically Important Steps of a
Mn21-Inhibited Process 172
6.9 Effects of Exposure to Nanoparticles Containing a
Range of Mn Oxidation States 173
6.10 Conclusions 174
Acknowledgements 176
References 176

Chapter 7 Effect of Manganese on Signaling Pathways 182

Tanara V. Peres, Fabiano M. Cordova, Mark W. Lopes,
Ana Paula Costa and Rodrigo Bainy Leal

7.1 Introduction 182

7.2 Manganese may Alter Cell Signaling in the Striatum 183
Contents xiii
7.3 Manganese Modulation of Tyrosine Hydroxylase
Activity 187
7.4 Alteration in MAPK and AKT Signaling Induced by
Manganese 188
7.5 Manganese Action on GSK-3b and the Canonical
Wnt/b-Catenin Pathway 191
7.6 Final Considerations 192
References 192

Chapter 8 Manganese and Oxidative Stress 199

Daiana Silva Ávila, Marcelo Farina,
João Batista Teixeira da Rocha and Michael Aschner

8.1 Introduction 199

8.2 Mechanisms Mediating Mn-Induced Oxidative
Stress and Toxicity 201
8.2.1 Mn and Mitochondria 201
8.2.2 Manganese and Dopamine Oxidation 204
8.2.3 Manganese and Antioxidant Homeostasis 205
8.2.4 Manganese and Protein Aggregates 205
8.3 Antioxidant Approach against Manganism 207
8.4 Concluding Remarks 209
References 210

Chapter 9 Mutual Neurotoxic Mechanisms Controlling Manganism

and Parkisonism 221
Jerome A. Roth

9.1 Introduction 221

9.2 Parkin 224
9.3 DJ-1 226
9.4 PINK1 229
9.5 ATP13A2 230
9.6 LRRK2 233
9.7 a-Synuclein 234
9.8 VPS35 236
9.9 Others 237
9.10 Conclusion 238
References 239

Chapter 10 Mechanism of Manganese-Induced Impairment of

Astrocytic Glutamate Transporters 258
Pratap Karki, Keisha Smith, Michael Aschner and Eunsook Lee

10.1 Introduction 258

xiv Contents
10.2 Mn Neurotoxicity 259
10.2.1 Sources of Human Exposure to Mn and its
Transport to the Central Nervous
System 259
10.2.2 Cellular Mechanisms of Mn Neurotoxicity 260
10.3 Role of Astrocytes in Mn Neurotoxicity 262
10.3.1 Mn-induced astrocyte swelling 262
10.3.2 Glial Cell-derived Neuroinflammation in
Mn Neurotoxicity 263
10.3.3 Astrocytic Glutamate Transporters in
Neurological Disorders 263
10.3.4 Mn Reduces Expression and Function of
Astrocytic Glutamate Transporters 264
10.4 Mechanism of Mn-induced Impairment
of Astrocytic Glutamate Transporters 264
10.4.1 Mn-activated Signaling Pathways in
Astrocytes 264
10.4.2 Mn-induced Transcriptional Regulation of
Glutamate Transporter GLT-1: Role of
Yin Yang 1 265
10.5 Summary 268
Acknowledgements 268
References 268

Chapter 11 Impairment of Glutamine/Glutamate-c-aminobutyric

Acid Cycle in Manganese Toxicity in the Central Nervous
System 279
Marta Sidoryk-Wegrzynowicz and Michael Aschner

11.1 Glutamine Content and Regional Distribution and

its Role in the Central Nervous System 279
11.2 Glutamine Transporting Systems in General 280
11.3 Glutamate: Role in Central Nervous System and
Transporters 281
11.4 The Glutamine/Glutamate-g-Aminobutyric Acid 283
11.5 Manganese 284
11.5.1 Essentiality and Toxicity 284
11.5.2 Transporting System 284
11.5.3 Manganese Effects on Astrocytes Function
and Astrocyte–Neuronal Integrity 285
11.5.4 Manganese Involvement in PKCd
Signalling 286
11.6 Manganese and GGC 287
11.6.1 Manganese and Glutamate Transporting
System 287
Contents xv
11.6.2 Manganese and Glutamine Turnover 288
11.6.3 Manganese Involvement in SNAT3
Expression and Function 289
11.7 Summary 290
References 291

Chapter 12 Manganese and Neuroinflammation 297

Kelly A. Kirkley and Ronald B. Tjalkens

12.1 Introduction 297

12.2 Astrocytes 298
12.2.1 Description and Distribution of Astrocytes 298
12.2.2 Functional Roles of Astrocytes 298
12.3 Microglia 300
12.3.1 Description and Distribution of Microglia 300
12.3.2 Functional Roles of Microglia 301
12.4 Neuroinflammation 301
12.4.1 Overview of Neuroinflammation in the
CNS 301
12.4.2 Role of Astrocytes in Neuroinflammation 303
12.4.3 Role of Microglia in Neuroinflammation 303
12.4.4 NF-kB Signaling in Neuroinflammatory
Injury from Manganese 304
12.5 Neuroinflammation in Diseases of the CNS 305
12.5.1 Seizure 305
12.5.2 Parkinson’s Disease 306
12.5.3 Manganism 308
12.6 Conclusion 313
References 313

Chapter 13 Modeling Manganese Kinetics for Human Health Risk

Assessment 322
Miyoung Yoon, Michael D. Taylor, Harvey J. Clewell and
Melvin E. Andersen

13.1 Introduction 322

13.2 Key Findings from Mn Pharmacokinetic Studies 323
13.3 Pharmacokinetic Modeling of Mn 328
13.3.1 Initial Development 328
13.3.2 Mn PBPK Models 329
13.3.3 Inter-species Extrapolation to Non-human
Primates 334
13.3.4 Development of Human PBPK Models
for Mn 336
13.3.5 Life Stage Extrapolation 338
xvi Contents
13.4 Application of PBPK Models in Human Health Risk
Assessment 343
13.5 Suggested Research 345
Acknowledgements 346
References 346

Chapter 14 Significance and Usefulness of Biomarkers of Exposure to

Manganese 355
Perrine Hoet and Harry A. Roels

14.1 Introduction 355

14.2 Biological Matrices and Reference Values 356
14.2.1 Mn in Blood 356
14.2.2 Mn in Urine 357
14.2.3 Mn in Hair and Nail 357
14.2.4 Mn in Brain 358
14.2.5 Reference Values 359
14.3 Occupationally Exposed Subjects 366
14.3.1 External–Internal Exposure Relationship:
Ambient Air-Biomarker 366
14.3.2 Internal Exposure – Effect Relationship 383
14.4 Non-Occupational Exposure 384
14.4.1 Environmental Exposure 384
14.4.2 Other Mn Exposures 385
14.5 Conclusion 386
References 387

Manganese: Health Effects

Chapter 15 Manganese and Parenteral Nutrition 405
Judy L. Aschner and Nathalie L. Maitre

15.1 Parenteral Nutrition and Manganese (Mn)

Supplementation 405
15.2 Existing Guidelines for Parenteral Manganese
Supplementation 406
15.2.1 Adult Guidelines 406
15.2.2 Pediatric Guidelines 407
15.3 Risk Factors for Manganese Excess and Toxicity 410
15.3.1 Disease States 410
15.3.2 Nutritional Iron Status 411
15.4 Consequences of Excessive Parenteral Mn 411
15.4.1 Adults: Manganism and other
Neuropsychiatric Disorders 411
15.4.2 Infants and Children: Cognition and
Neurodevelopment 412
Contents xvii
15.5 Detection of Manganese Body Burden 414
15.5.1 Mn Measurements in Blood 414
15.5.2 Magnetic Resonance Imaging (MRI) 415
15.6 Future Directions for Optimizing Mn in Parenteral
Nutrition 416
15.6.1 Knowledge Gaps and Research Priorities 416
15.6.2 Recommendations for Clinical Practice
Modifications 416
Acknowledgements 417
References 417

Chapter 16 Developmental Effects of Manganese 426

Scott M. Langevin and Erin N. Haynes

16.1 Introduction 426

16.2 Manganese Deficiency and Development 427
16.3 Manganese Toxicity and Development 427
16.3.1 Brain Development 428
16.3.2 Birth Outcomes 430
16.3.3 Onset of Puberty 431
16.4 Conclusions 432
References 433

Chapter 17 The Effects of Manganese on Female Pubertal

Development 437
William L. Dees, Jill K. Hiney and Vinod K. Srivastava

17.1 Introduction 437

17.2 Critical Events Associated with the Normal Onset of
Female Puberty 438
17.3 Acute Effects of Mn on Puberty-Related Hormones:
A Hypothalamic Site of Action 439
17.4 Chronic Effects of Mn on Puberty-Related
Hormones and the Timing of Puberty 440
17.5 Downstream Mechanism(s) of Mn Action on GnRH
Release 442
17.6 Effect of Mn on GnRH Gene Expression 443
17.7 Upstream Mechanisms of Mn Action in the Control
of GnRH Neuronal Activity 447
17.7.1 Mn Action on Kiss-1 Gene Expression 447
17.7.2 A Potential Role for Divalent Metal
Transporter-1 449
17.8 Low Level Mn Exposure and Precocious Puberty 450
17.9 Conclusions 451
Acknowledgements 452
References 453
xviii Contents
Chapter 18 A Decade of Studies on Manganese Neurotoxicity in
Non-Human Primates: Novel Findings and Future
Directions 459
Tomás R. Guilarte

18.1 The Early Studies on Manganese Neurotoxicity in

Non-Human Primates 459
18.2 Early Behavioral and Neuroimaging Findings 461
18.2.1 Behavioral Findings 461
18.2.2 Positron Emission Tomography Findings 462
18.2.3 Magnetic Resonance Spectroscopy and
T1-Weighted Magnetic Resonance Imaging
Findings 463
18.3 Chronic Mn Exposure Impairs Dopamine Neuron
Function in the Striatum and Produces Extensive
Degeneration in the Frontal Cortex 464
18.3.1 Effects of Chronic Mn Exposure on
Dopaminergic Neuron Terminals in the
Striatum Measured by PET and
Confirmation by Ex Vivo Methods 464
18.3.2 Effects of Chronic Mn Exposure on the
Glutamatergic and GABAergic Systems 466
18.3.3 The Frontal Cortex in Mn-Exposed
Non-Human Primates: Alzheimer’s
Disease-like Pathology and
Neurodegeneration 467
18.4 Behavioral Studies Reveal Significant Impairment
in Working Memory and Visuospatial Paired
Associative Learning in Mn-Exposed Non-Human
Primates 469
18.5 Novel Findings and Future Directions 470
Acknowledgements 471
Dedication 472
References 472

Chapter 19 Imaging Modalities for Manganese Toxicity 477

Ulrike Dydak and Susan R. Criswell

19.1 Introduction 477

19.2 Magnetic Resonance Imaging 478
19.2.1 Basics of MRI 478
19.2.2 Manganese as MRI Contrast Agent 479
19.2.3 Morphological Changes Assessed
by MRI 485
19.2.4 Magnetic Resonance Spectroscopy 486
Contents xix
19.2.5 Diffusion Weighted Imaging 489
19.2.6 Functional MRI 490
19.3 PET and SPECT Imaging 491
19.3.1 PET Studies in Non-Human Primates 492
19.3.2 PET Studies in Human Subjects 493
19.3.3 SPECT Studies 496
19.4 X-Ray Fluorescence 497
19.5 Conclusions 500
Acknowledgements 501
References 501

Chapter 20 Epidemiological Studies of Parkinsonism in

Welders 513
Harvey Checkoway, Susan Searles Nielsen and
Brad A. Racette

20.1 Parkinsonism: Clinical, Pathological, and

Epidemiological Features 513
20.2 Manganese and Parkinsonism: Historical
Background 514
20.3 Epidemiological Studies of PS/PD among Welders 515
20.4 Discussion 515
References 520

Chapter 21 Cognitive Effects of Manganese in Children and Adults 524

Roberto Lucchini and Silvia Zoni

21.1 Introduction 524

21.2 Cognitive Effects in Children 525
21.2.1 Reduction of IQ 528
21.2.2 Executive Functions 528
21.2.3 Memory 529
21.2.4 Academic Achievement 529
21.2.5 Mental Development 530
21.3 Cognitive Effects in Adults 530
21.3.1 Occupational Studies 530
21.3.2 Environmental Studies 532
21.4 Conclusions 533
References 533

Chapter 22 Manganese and Huntington Disease 540

Andrew M. Tidball, Terry Jo Bichell and Aaron B. Bowman

22.1 Huntington Disease Pathobiology and

Environmental Influence 540
xx Contents
22.2 A History of HD and Metal Ions 541
22.2.1 Manganese and HD 542
22.2.2 Iron Homeostasis and HD 543
22.2.3 Copper and HD 545
22.3 Manganese Essentiality and Toxicity in HD Related
Phenotypes 545
22.3.1 Regulation of Amines and Nitric
Oxide 546
22.3.2 Glutamate Excitotoxicity 547
22.3.3 Mitochondrial Dysfunction: Oxidative
Stress and Energetics 551
22.3.4 IGF/PI3K/AKT Signaling in HD and
Manganese Exposure 554
22.3.5 p53 Pathway 556
22.4 Conclusions 558
References 559

Chapter 23 Manganese and Prion Disease 574

Huajun Jin, Dilshan S. Harischandra, Christopher Choi,
Dustin Martin, Vellareddy Anantharam, Arthi Kanthasamy
and Anumantha G. Kanthasamy

23.1 Introduction 574

23.2 Prion Diseases 576
23.2.1 Creutzfeldt–Jakob Disease (CJD) 576
23.2.2 Kuru 577
23.2.3 Gerstmann–Sträussler–Scheinker
Syndrome 578
23.2.4 Fatal Familial Insomnia 578
23.3 Prion Protein (PrPC) 580
23.3.1 Structure of PrPC 580
23.3.2 Physiological Function of Prion
Protein 581
23.4 Metals and Prion Diseases 583
23.4.1 Manganese 583
23.4.2 Manganese Binding to PrPC 584
23.4.3 Role of Manganese in the Pathogenesis of
Prion Disease 585
23.4.4 Role of Manganese in the Physiological
Function and Expression of PrPC 588
23.4.5 Role of other Metals in Prion
Disease 589
23.5 Conclusions 590
Acknowledgements 590
References 590
Contents xxi
Chapter 24 DNA Damage Induced by Manganese 604
Julia Bornhorst and Tanja Schwerdtle

24.1 Genotoxic Lesions Induced by Manganese 604

24.1.1 Damage Induced by Manganese at the
Chromosomal Level 604
24.1.2 Damage Induced by Manganese at the DNA
Level 608
24.1.3 Mutations Induced by Manganese 609
24.2 Sources of the Genotoxic Potential of Manganese 610
24.3 Consequences of DNA Damage Induced by
Manganese 613
24.4 Conclusions 614
References 614

Post-face 621

Subject Index 624

CHAPTER 1

Manganese Transport,
Trafficking and Function in
Invertebrates
AMORNRAT NARANUNTARAT JENSENa AND
LARAN T. JENSEN*b
a
Department of Pathobiology, Faculty of Science, Mahidol University,
Bangkok, Thailand; b Department of Biochemistry, Faculty of Science,
Mahidol University, Bangkok, Thailand
*Email: laran.jen@mahidol.ac.th

1.1 Introduction
Manganese is a biologically important trace metal and is required for the
growth and survival of most, if not all, living organisms. It is perhaps best
known for its prominent role as a redox-active cofactor in free radical de-
toxifying enzymes.1–8 However, the utilization of manganese in biological
systems is substantially more diverse. The uptake and distribution of man-
ganese is critical for proper function of manganese-requiring enzymes;
however, this same metal can have deleterious effects in biological systems if
homeostasis is disrupted.9–12 In order to prevent toxicity, cells maintain
manganese under tight homeostatic control. Adding complexity to the cel-
lular control of manganese homeostasis is the presence of multiple types of
manganese transporter that participate in the specific transport of manga-
nese or in general divalent metal ion transport.

Issues in Toxicology No. 22

Manganese in Health and Disease
Edited by Lucio G. Costa and Michael Aschner
r The Royal Society of Chemistry 2015
Published by the Royal Society of Chemistry, www.rsc.org

1
2 Chapter 1

Cells appear to transport manganese solely as the divalent cation and

several classes of manganese transporters have been characterized. These
include Nramp H1-manganese transporters,13–16 ATP-binding cassette (ABC)
manganese permeases,17–21 manganese transporting P-type ATPases,22,23
cation diffusion facilitators (CDFs),24–26 and inorganic phosphate transpor-
ters with high affinity for Mn–HPO4 complexes.27–29 Bacteria typically con-
tain one or more of these types of transporter, and these classes of
transporter are also present in eukaryotic cells.30,31 These transporters
comprise both high and low affinity manganese uptake systems and the
transporter utilized depends on the concentration of manganese in the en-
vironment. The homeostatic range for manganese is quite wide, with cellular
levels of manganese between 0.04 and 2.0 mM under optimal growth
conditions.10,16,29,32,33 Cells rarely experience optimal environmental
levels of manganese and often face extreme conditions of either manganese
deficiency or excess.34 Cells activate stress response mechanisms in an at-
tempt to return manganese levels to the homeostatic range. The response
typically results in the upregulation or downregulation of cell surface
and intracellular transport systems. The regulation of manganese uptake,
distribution, and efflux can occur at both the transcriptional and post-
translational levels, although the specific route of regulation varies in
different organisms.

1.2 Function of Manganese in Biological Systems

1.2.1 Manganese Metalloenzymes
Manganese metalloenzymes are involved in a wide range of cellular func-
tions, including detoxification of reactive oxygen species, protein glycosyla-
tion, polyamine biosynthesis, DNA biosynthesis, nucleic acid degradation,
phospholipid biosynthesis and processing, polysaccharide biosynthesis,
protein catabolism, the urea cycle, photosynthesis, and sugar catabol-
ism.2,35–45 Manganese-dependent enzymes that participate in these pro-
cesses typically utilize manganese in Lewis acid–base reactions or as a
reduction/oxidation center to facilitate catalysis. These types of reaction
are exemplified by arginase (Lewis acid) and Mn superoxide dismutase
(reduction/oxidation),2,3,46,47 and the role of manganese in these reactions is
shown in Figure 1.1.

1.2.2 Non-Protein Manganese Antioxidants

The importance of manganese in biological systems is not limited to
enzyme-mediated catalysis. Non-enzymatic manganese is involved in the
formation of bacterial products, including secreted antibiotics,48 and con-
tributes to the stabilization of bacterial cell walls.49 In addition, the accu-
mulation of non-protein complexes of manganese can function in the
removal of reactive oxygen species (ROS), especially superoxide.50–53 These
Manganese Transport, Trafficking and Function in Invertebrates 3

Figure 1.1 Typical chemistry performed by manganese in enzymes. (A) Di-nuclear

manganese center of arginase. The manganese cofactor of arginase does
not participate in redox reactions but instead functions as a Lewis acid to
accept a pair of electrons from the bound water molecule, allowing
deprotonation and increasing its reactivity. (B) Catalytic detoxification of
superoxide anions by manganese superoxide dismutase (Mn-Sod) en-
zymes. The catalytic cycle for Mn-Sod has been called a ‘‘ping–pong’’
reaction in which the manganese cofactor alternates between the oxi-
dized and reduced forms.

Mn-antioxidants are divalent manganese complexes of small metabolites,

and while the nature of the intracellular Mn-complexes has not been clearly
defined, phosphate and lactate Mn-complexes have been shown to display
the capacity to react efficiently with superoxide in vitro.52,54,55 Complexes of
both iron and copper exhibit superoxide scavenging activity, however these
metal ions also exhibit pro-oxidant activity.56–59 In contrast, manganese ions
react poorly with hydrogen peroxide and do not generate the highly toxic
hydroxyl radical, providing a beneficial antioxidant activity without the pro-
oxidant side effects of other redox active metals.50,60
It appears that Mn-antioxidants can serve to enhance oxidative stress
protection when enzymatic antioxidants are insufficient in various organ-
isms.53,61–63 A critical role for Mn-antioxidants has been demonstrated in
Deinococcus radiodurans, a bacterium that is extremely resistant to radiation
and desiccation. In this organism, survival under extreme exposure to ra-
diation and other oxidative stress conditions is not dependent on anti-
oxidant enzymes but instead relies on the accumulation of millimolar
concentrations of manganese and the subsequent formation of Mn-
antioxidants.50,51,60,63 Interestingly, Lactobacillus plantarum, while resistant
to oxidative stress, does not express the antioxidant enzyme superoxide
dismutase.64,65 Indeed, L. plantarum appears to rely exclusively on Mn-
antioxidants for protection against oxidative stress,54,66,67 highlighting the
power of this alternative ROS detoxification pathway.
4 Chapter 1

The majority of the information on manganese antioxidants has come

from investigation of bacterial and yeast systems; however, it is also likely
that these complexes are present in multicellular organisms. Elevated
manganese accumulation in the nematode Caenorhabditis elegans enhances
thermotolerance and oxidative stress resistance, and extends life span.68,69
The mechanism of the enhanced stress resistance due to manganese sup-
plementation in C. elegans has not been fully elucidated but is suspected to
involve elevated antioxidant activity.

1.2.3 Manganese and Bacterial Virulence

Manganese is either known or proposed to be important for virulence in
bacterial species such as Salmonella enterica, Mycobacterium tuberculosis,
Staphylococcus aureus, Yersinia pestis, and Streptococcus pneumoniae.19,30,31
Invasion and initial survival within host cells is not dependent on manga-
nese; however, extended survival appears to require the element.70,71 The
expression of manganese transporters is required to enhance bacterial sur-
vival when challenged by host defenses.15,18,70,72–74 Whether different classes
of manganese transporter are redundant or involved at different stages of
infection is not known. Models have been proposed in which manganese
transporters, as well as iron transporters, are essential for virulence because
of competition between the infecting bacterium and host cells for metal
ions.31,71 The need for manganese in bacterial virulence appears to go
beyond its role as a cofactor in ROS detoxifying enzymes such as Mn-
superoxide dismutase and catalase. Enterobacteria are capable of rapidly
increasing uptake of manganese in response to stress, and can accumulate
millimolar levels of manganese.17 This concentration of manganese far
exceeds the level needed to supply Mn-superoxide dismutase with its
cofactor. It appears that the formation of non-protein Mn-antioxidant
complexes may also be an important virulence factor in some bacterial
species. The additional protection against reactive oxygen species generated
by the host cells may allow invading bacteria to survive the initial stages of
infection, and thus promote colonization.

1.3 Manganese Transport in Bacteria

1.3.1 Bacterial Manganese Uptake Systems
In prokaryotic cells, which lack internal compartmentalization, metal ion
homeostasis is maintained primarily by tight regulation of metal cation flux
across the cytoplasmic membrane. Manganese uptake in bacteria pre-
dominantly involves members of two transporter families, Nramp (MntH)
and cation-transporting ABC permeases (MntABCD and related), with many
species containing both types of transport system.17,20,70,73,75,76 In addition,
utilization of other transport systems for manganese, such as a P-type
adenosine triphosphatase (ATPase) by Lactobacillus species (MntP) and
Manganese Transport, Trafficking and Function in Invertebrates 5
23,77
Mycobacterium tuberculosis (CtpC), has also been observed. Exposure to
excess manganese leads to repression of these dedicated manganese trans-
port systems.20,74 However, the tight control of manganese influx can be
bypassed via other transporters that are capable of facilitating the uptake of
manganese but escape regulation by this metal. An example of this is PitA,
an inorganic phosphate transporter with high affinity for Mn–HPO4 com-
plexes that appears to be a major source of manganese uptake during con-
ditions of excess.28

1.3.1.1 Bacterial Nramp Manganese Transporter, MntH

Members of the Nramp (natural resistance-associated macrophage protein)
transporter family were first identified in yeast and mammalian cells and
subsequently found to play a major role in metal ion homeostasis.78–80
Nramp proteins function in general metal ion transport, and members of
this transporter family have been shown to facilitate the movement of di-
valent metal ions including manganese, zinc, copper, iron, cadmium, nickel,
cobalt, and lead.14,81–85 Transport of metal ions through Nramp is energized
by the symport of protons (Figure 1.2).15,86
The majority of bacterial Nramp1 homologues, typically designated as
MntH,15,20,23,80,87 appear to function in manganese homeostasis.15,73,74 The
MntH transporters are commonly found in bacterial species, although
examples of bacteria lacking Nramp transporters have been described.30
Metal accumulation studies revealed that overexpression of Staphylococcus

Figure 1.2 Typical manganese transporters in bacterial cells. (A) During conditions
of manganese deficiency the high affinity transporters MntH, MntABCD,
and MntP facilitate manganese uptake. However, these manganese
transporters are not present in all bacterial species. (B) Manganese
excess inhibits expression of the high affinity transporters and induces
the manganese efflux protein MntE. Uptake of manganese–phosphate
complexes may be a source of manganese when cells are exposed to toxic
concentrations of this metal.
6 Chapter 1

aureus MntH resulted in increased cell-associated manganese but not cal-

cium, copper, iron, magnesium or zinc, indicating that this Nramp1 trans-
porter was selective for the uptake of manganese.20 Consistent with these
observations, mutants of mntH in Bacillus subtilis exhibited impaired growth
in metal-depleted media that could be rescued by the addition of manga-
nese.74 Direct transport assays also indicated a preference for manganese in
MntH from Salmonella enterica serovar Typhimurium and Escherichia coli. The
affinity for manganese far exceeds that for iron in these MntH proteins,
demonstrating the role of Nramp transporters in bacterial manganese
uptake.30
Species differences in MntH metal ion specificity have been observed, with
some MntH homologues appearing to function in the transport of other
metals in addition to manganese. While S. enterica, E. coli, and B. subtilis
MntH exhibit a strong preference for manganese,30,74 the M. tuberculosis
MntH homologue, Mramp, appears to transport not only manganese but
also significant amounts of iron and zinc.88 Roles for Nramp transporters in
the uptake of other metal ions, especially iron, have been documented in
both prokaryotic and eukaryotic organisms.16,73,80,89–91 Multiple Nramp
isoforms can be present in a single species, and these Nramp transporters,
although highly similar, may have divergent metal ion preferences. Pseudo-
monas aeruginosa expresses two distinct Nramp transporters capable
of transporting manganese, and multiple Nramp isoforms are present in
Burkholderia species, although the metal ion preferences of these transpor-
ters have not been determined.30 While the most physiologically relevant
substrate for the majority of bacterial MntH transporters appear to be
manganese, it is clear that these transporters have the capacity to facilitate
the uptake of other metals when they are present in excess. This broad metal
ion selectivity in Nramp transporters also appears to enhance the uptake of
toxic metal ions, such as cadmium and lead.74,85,92

1.3.1.2 Bacterial ABC-Type Manganese Permeases

The ATP-binding cassette (ABC) transporter superfamily is one of the largest
classes of transporter, and this transporter family utilizes hydrolysis of ATP
to facilitate the import or export of diverse substrates, ranging from ions to
macromolecules.93–95 These transporters are present in the plasma mem-
brane or inner membrane of Gram-negative bacteria,93,95,96 and are well
known for their involvement in multi-drug resistance in both prokaryotic
and eukaryotic cells by enhancing the export of toxins and drugs.97,98
However, ABC transporters functioning as importers have only been de-
scribed in prokaryotic systems.93,95,96 Metal ion transporting ABC permeases
have been identified with important roles in manganese acqui-
sition.19,70,76,99,100 The cation selectivity of manganese ABC-type permeases
extends to other divalent metal ions including iron, zinc, cobalt, nickel,
molybdenum, and cadmium; however, the typical affinities for these metal
ions are 10- to 100-fold lower than for manganese.100–103
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 THE ELEPHANT WANTS TO GO HOME.

If he had known what he ought to do, he would have turned on

those darkies and chased them about half a mile. In that way he
would have managed very soon to relieve himself of his troublesome
pursuers. They would have gone home quite as rapidly as he wanted
to go to his home.
Colonel Myles staid in Africa nearly a year and sometimes had a
good deal of exciting hunting, and at other times weeks would pass
when he was obliged to stay in some native village for want of
transportation and guides.
Sometimes too, he found himself in a part of the country where
there was no large game worth mentioning.
He tried hard to find a gorilla, but never succeeded. He often
heard lions in the night, and once came upon a big fellow who was
lying down by the side of a fallen tree in the very road over which he
and his followers were traveling. The tree was of a yellowish brown
color and the lion was of very much the same color, so the Colonel
did not see him until he came quite near to him.
As quickly as possible he jerked his rifle from his shoulder, but his
horse started and reared, and the lion sprang to his feet, and giving
one hasty look at the advancing party, disappeared in the bushes.
The Colonel was very much disappointed at this mishap, and it
was shortly after he had made up his mind that he never would get a
good shot at a lion, that he concluded to go to India.
A party of traders were on their way to the coast, and the Colonel
joined them.
Reaching the coast he found a vessel nearly ready to sail, and in it
he took passage to Bombay.
He was in India nearly two months before he had an opportunity to
try his favorite rifle on any large game.
He preferred to hunt tigers, for tigers are such scourges to the
localities in which they are found that he felt justified in killing as
many of them as he could. But it turned out that his first hunt was a
buffalo hunt—and not only his first but his second and third, and a
good many after that.
He got in a part of the country in which there were a good many
buffaloes, and as they were needed as food it paid very well to hunt
them, and there were always natives enough who were willing to
help if they might have a large share of the meat.
One day one of these natives had a little more buffalo hunting than
he wanted. The colonel perceived a very large fine bull buffalo
standing in an open space and was just about to take a good aim at
him when the animal began to trot off towards a dense thicket. The
colonel was afraid of losing him and so he fired too quickly. If he hit
the buffalo at all he merely wounded him very slightly, for he dashed
off into the thicket.
The native attendant, however, was quite sure that the buffalo had
been fairly hit and would soon drop, so off he rushed to find him.
 While he was pushing his way through the thicket he heard a
crashing noise, and looking around saw the savage bull charging
down upon him from a little eminence where there was a
comparatively open space.
The man had a gun, but it was not loaded. There was no chance
of his running away, for the bushes and reeds were too close and
strong to allow of that. There was no tree near but one very thick
one, up which he had no time to climb.
The bull stopped for an instant, and then put down his head for
another charge.
The man had no time to do much thinking. Whatever he did in self-
defence must be done quickly, that he knew well. So he darted
behind the tree, and jerking his blanket from his back he put it on his
gun-barrel and waved it about.
The buffalo immediately accepted the challenge, and came at him
full tear. He rushed at the cloth, and as he passed he took it away on
his horns. Then the native hurried off as fast as he could go in the
opposite direction.
The colonel, who had loaded up and was waiting for his attendant
to return, was very much astonished to see a buffalo rush out of the
thicket with a blanket twisted about his horns.
The animal evidently did not notice him, and so he raised his rifle
and shot him dead without the slightest trouble.
 THE NATIVE’S TRICK.
As soon as the buffalo was quite dead, the native appeared from
the thicket and immediately began to boast of the share he had had
in killing the animal.
It is quite certain that had he not succeeded in his very clever trick
the buffalo would either have killed him or would have got away
safely.
A week or two after this the Colonel was invited by an officer in the
English army, named Major Alden, to go wild boar-hunting with him.
The Colonel was quite willing, and so they set out together for the
river, some three miles away, where they expected to find a wild boar
or two. A crowd of natives preceded them, beating up the bushes to
drive out the boars.
Our hunters were both well mounted and armed with long spears
instead of guns. The Major, who wore an undress uniform, carried
also a short sword.
 They had scarcely reached the river-bank when they saw a boar
rush out of some underbrush and make rapidly for the river.
Both horsemen dashed off in pursuit, Major Alden in advance. Just
as he reached the edge of the high bank the Major thought that he
had better rein up, but he did not think soon enough. He stopped
quite near the brink of the bank, and was on the point of turning
back, when the earth caved in beneath his horse, and down into the
water and mud, some ten feet below, went horse and rider!
Fortunately the river was not very deep at that place, but it was
deep enough. The Major went head foremost over his horse’s neck
into the water, and the horse with a tremendous splash, went into the
mud as far as his legs would let him go.
Just in time, our Colonel reined up, and below him he beheld a
doleful sight.
The Major had risen to his feet but was dripping with mud and
water that fell in little cascades from his face, head, and hands and
every part of his body.
The horse was plunging wildly in the river and the poor Major did
not seem to be able to see how to find his way to dry land.
 THE MAJOR’S TUMBLE.

If the Colonel had been a boy he would have had a good laugh at
this mishap of his companion, but as he was a man he tried not to
add to the discomfiture of the Major by making fun of him. But when
the native beaters came up they set up a shout of laughter, and that
made the Major angry enough, and as he wobbled slowly to shore
he growled out to the “black rascals” a command to stop their noise,
and get his horse out.
The black rascals, although they did see a good deal of fun in this
unfortunate tumble, proved themselves very useful, for they cleaned
the horse and saddle, and while the Major took a bath in the river, (at
a place where it was deeper and with a better bottom) they dried his
clothes and brushed them with bunches of twigs until they looked
quite presentable, and in the afternoon they all set out again on their
hunt.
But the Major seemed doomed to misfortune that day. He had no
spear, for his weapon had been broken in the fall of the morning, and
he had sent one of the men back to the village to get him another.
But before the man returned a boar was started up and Colonel
Myles started off in pursuit. The boar dashed into the underbrush,
the Colonel and a dozen natives after him full tilt, and the farther that
boar ran the madder he got.
He didn’t like being chased, and I suppose no sensible boar would
like it.
Directly he made a sharp turn and rushed out of the bushes to the
river bank where the Major was sitting on his horse waiting for his
spear.
Seeing a man on a horse the boar very naturally thought that he
must be the person who had been after him, and so, full of
vengeance, he dashed at him at full speed, his horrid tusks
glistening in the sunlight.
Instantly the Major pulled his feet out of the stirrups and drew his
sword. There was no time to ride away.
But the sword was short, and the boar was very close to the horse,
who snorted and plunged so that the Major could scarcely keep his
seat, much less get a fair crack at the boar.
 THE MAJOR AND THE BOAR.
If the savage beast had succeeded in getting under the horse he
would have wounded him desperately.
But Major Alden was a cool and a brave man. He kept the horse
away from the boar as well as he could, and at last he got a good
chance, and down came his sword on the boar’s neck.
But this cut did not seem to cool the boar’s courage very much.
The savage animal still charged, the horse plunged and the Major
slashed, and so the fight went on—charge, plunge, slash, until the
Colonel came riding up with his spear, and soon put an end to the
career of the ferocious boar.
The next time the Major and Colonel Myles went out to hunt they
went after tigers.
Tiger hunting is very popular among the white residents of India,
and it is well that it is, for the natives do not often succeed so well in
their hunts after tigers, as the tigers succeed in their hunts after the
natives.
It is astonishing to read, in the government reports, how many
people are annually killed by tigers in some parts of India.
The Colonel’s first tiger hunt was not a very ambitious one. He did
not go out into the jungle on an elephant in company with forty or fifty
natives, but he and the Major, with two or three followers, started off
on foot. They walked a long distance without seeing a sign of tigers,
although they were in a place where two bullocks had been killed by
these animals the previous night.
Towards noon, however, one of the natives discovered the plain
tracks of a tiger, and the party followed the trail until they lost it in a
mass of rocks. In these rocks, however, was a large cave, and the
natives assured them that they would find the animal in this cave.
No one was particularly anxious to go into it to see if the tiger was
there, but, peering carefully in at the entrance of the cave, the
Colonel was sure that he saw something gleaming far back in the
darkness, and he thought that the bright spot must be one of the
tiger’s eyes.
To be sure, unless it was a one-eyed tiger, he ought to see two
bright spots, but he did not stop to consider this point, but took
deliberate aim at the spot and fired.
Nothing happened. No tiger jumped out.
 Then the Major fired, although he was not quite certain that he did
see a shining spot. Still there was no sign of a tiger having been
shot. Even if the beast had been fairly hit by either of the shots, it is
likely that he would have made some disturbance inside the cave, for
tigers are very hard to kill.
Several other shots were fired without effect, and the hunters
came to the conclusion that there was no tiger in the cave.

OUT OF THE CAVE SPRANG AN ENORMOUS TIGER.

However, they consented to let the natives try a plan that they
suggested. This was to smoke the tiger out of his hole. So great
quantities of dried leaves and twigs were collected, and thrust into
the mouth of the cave. While this work was going on, the men were
very enthusiastic about it, and ran up with their arms full of leaves
and sticks, seeming to entirely forget what a predicament they would
be in if a tiger should be within, and if he should make up his mind to
come out before they had finished their job.
When all was ready the dry stuff was fired, and very soon a great
smoke arose, and as the wind blew towards the rocks, most of the
smoke went into the cave.
In about three minutes a horrid growl was heard, and every darkey
took to his heels, one of them making about five jumps towards a
distant tree, up which he climbed like a monkey.
And they were none too quick. Out of the cave sprang an
enormous tiger.
The two white men had their rifles to their shoulders in an instant,
and they fired almost simultaneously. The tiger did not stop,
however, but rushed on, apparently after one of the natives. But
before he reached him the Colonel fired the second barrel of his rifle,
and rolled the beast over. One or two more shots finished him.
This hunt was considered a great success, for the tiger was a very
large one, and was no doubt, the murderer of the bullocks. The
natives were delighted, and went to work to take off the skin, which
was awarded to our Colonel, who had fired the decisive shot.
While in the cave the tiger had probably been lying behind some
rocks, with only part of his head exposed. He had not cared to leave
his entrenchments while they were firing at him, but he evidently did
not like smoke.
The next time the Colonel and Major Alden went out after tigers
they were on an elephant. They rode in a large wooden box on the
elephant’s back, in which they could stand and fire without much fear
of a tiger getting at them. They had wonderful success, for they
came upon no less than five tigers, out in an open space. One of
these was soon killed, and the others ran away in different
directions, like enormous kittens.
But before they got entirely away another was shot dead, and two
more, that ran behind some great rocks, were followed up, and killed
before night.
But one very large one slipped off, growling savagely, into some
reeds by the river bank, and was lost. This tiger was the largest and
most dangerous of the lot, and the man who drove the elephant said
he knew him very well.
 He asserted that this tiger was a man-eater, as a tiger is called
that has once killed a human being. Ever afterward, according to the
native traditions, he has a strong liking for a man for dinner.

THEY CAME UPON NO LESS THAN FIVE TIGERS.

The driver said he had seen him kill a man, and that he knew him
by his peculiar markings. Whether this story was true or not, it was
evident that this was a very large and dangerous beast, and ought to
be killed, if such a thing should prove possible.
So, a few days afterwards, a large hunt was organized, having for
its object the destruction of this particular tiger. The party went out
mounted on three elephants—two men in each howdah or box, and
a driver on each elephant’s neck. Besides the riders, there were
about fifty natives on foot, who went along to beat up the bushes and
make themselves generally useful.
The Colonel and Major Alden were not together this time, but were
on different elephants. Colonel Myles’ companion was a military man
who was a very good shot and quite a noted tiger hunter. His name
was Captain Harrison, and our friend was very glad to go with him,
because he had the best elephants and was likely to see the best
sport.
There is a very great difference in the elephants that are taken on
tiger hunts. Some of them will get frightened and run away the
moment they see or hear a tiger, and then the hunters on their backs
have not much of a chance to get a shot at the beasts. But others
will stand their ground bravely, and the elephant that carried the
Colonel was said to be one of these.
They rode on, close to the river bank for many a mile under a
dreadfully hot sun, and, a little after noon one of the men who had
mounted a tall tree shouted out that he had seen the tiger among the
reeds on the river bank not very far from the spot where our hunting
party sat quietly on their elephants.
Stones were now thrown into the bushes and several shots were
fired by the native hunters. But no tiger made his appearance. The
thicket was full of thorns and was very dense, and there were other
reasons for not entering it—one very good one.
So the shouting and the stone-throwing were continued, and that
was about all that was done for fifteen or twenty minutes.
Then one of the followers, who had a gun, crept on his hands and
knees to the edge of the thicket and peeped in under the bushes.
He looked all about and could see nothing, and then he cast his
eyes to one side, and there lay the tiger not ten feet from him!
It is amazing how quickly he drew back, jumped up, and ran off at
the top of his speed. As soon as he reached what he thought was a
safe distance he turned and fired at the spot where he had seen the
tiger.
And what was very astonishing indeed, he hit it.
Up jumped the beast in a rage, and in an instant he bounded out
of the thicket into the open field.
He was all ready for a big fight, and he growled and gnashed his
teeth in a way that would have made your blood run cold.
 Every body leveled their guns at him, but he did not give them time
to take a good aim, for he charged at headlong speed right for the
foremost elephant. The animal on which the Colonel and his friend
were mounted was a brave one, but he did not fancy such a tiger as
this, and he turned to run away.
But he was not quick enough. The tiger bounded at him like a flash
and had him by the trunk before he could lift it out of harm’s way.
The driver on the elephant’s neck drew up his legs in a hurry, and
the hunters leaned over their box to try and get a shot. But the
elephant’s head was in the way, and they could not get a fair sight at
the tiger.
As for the poor elephant, he did not at all fancy having a tiger
chewing away at his trunk. So he bellowed and floundered about at a
great rate, but that did not seem to inconvenience the tiger, who held
on like a good fellow.
The other elephants and hunters were coming up, but they did not
come fast enough. The elephants seemed to be a little particular
about their trunks, and were in no haste to get near the beast that
was hanging so grimly to their big brother.
 THE TIGER SEIZES THE ELEPHANT BY THE TRUNK.
Then our elephant got tired of this sport. He gave his trunk a swing
under him at the same time that he made a step forward.
This brought the tiger just in front of his right foreleg.
Down knelt the elephant with one great knee directly upon the
tiger’s body.
The elephant weighed tons, and there was a dead tiger under his
knee in less than twenty seconds.
So here was a dangerous and noted tiger killed without a shot
from the brave hunters who went out after him. But they were none
the less brave for that.
The only man who did hit him was a coward, and the elephant that
killed him, would have run away if he could. Things turn out this way
sometimes.
 I can only tell of one more of Colonel Myles’ hunts. He spent many
months in India and killed a good many tigers, for which he had the
thanks of the people and the approval of his own conscience—two
things that hunters do not always have, I can assure you.
His last hunt, as far as we are concerned, was a bear hunt. He
heard that a large bear had been seen a short distance from the
place where he was then encamped, and early the next day after
receiving the news, he went out with one native follower to see if he
could find it. They followed the tracks of the beast until they reached
a place where there were some very high rocks.
Mounting to the top of these they peeped over and saw, at the
bottom of a ravine beneath, the mouth of a cave that appeared to
extend under the rocks a short distance.
In this cave, lying with his head on his paws, they distinctly saw a
large bear, fast asleep. He was, however, in such a position that it
would be very hard to get a good shot at him.
The Colonel then thought of a plan to make him come out. To be
sure they might have hurled stones at him or shouted, but in either
case the bear might have been frightened and drawn himself into his
cave, entirely out of sight, or he might have rushed up the rocks
faster than they would like to have him come.
The Colonel wanted him to come out of his cave, but to stay down
at the bottom of the ravine.
So he whispered to his man to unroll his long turban and to get out
on the branches of a tree that overhung the mouth of the cave. Then
he was to lower the turban down and tickle the bear’s nose.
The man did as he was told, and, as the turban was just long
enough to reach the bear’s nose, he was able to tickle him nicely.
At first the bear just fidgeted a little and then he made a dab with
his paw at the supposed fly that was worrying him.
But the turban continued to tickle him, and at last he woke up with
a start. When he saw the turban hanging before him he made a snap
at it, and then the man jerked it away.
 THE BEAR GAVE THE TURBAN A VIGOROUS PULL.
Up jumped the bear, just like a cat after a handkerchief. He made
a bound after the turban and seized it with his paws and teeth.
He jumped so suddenly and gave the turban such a vigorous pull
that the man came very near being jerked out of the tree, which
might have been bad for him, but our Colonel, who was ready with
his rifle, fired and killed the bear instantly. He was a big fellow and
had a splendid skin.
When the Colonel sailed for home he carried with him half a dozen
bear and tiger skins. They were all fine ones, but the best of them, a
magnificent skin that had once belonged to a very large and savage
Bengal tiger, was a particular favorite with him, and he now has it on
his library floor, just before the big grate where he sits and reads on
winter evenings.
And yet he did not kill the tiger to which the skin belonged. He
cannot point to it as an evidence of his bravery and skill in the
jungles of India.
It is the skin of the tiger that the elephant killed with his knee.
 A SUGAR CAMP.

When I was a boy I knew no more about a “sugar camp” than I

knew of a molasses-candy fort.

THE SUGAR CAMP.

In fact I would probably have thought one as ridiculous as the
other, if it had been mentioned to me.
This was because I did not live in a maple-sugar country. I had
eaten maple-sugar, but I had no idea how it was made, and when I
first saw a sugar-camp, out in the woods, I was both surprised and
interested.
In the first place it was not a camp at all—according to my idea—
for what the people at the farm-house where I was visiting, called the
camp was a house—a very rough one, but still a house. I expected
to find tents and big camp-fires under the trees. I found a fire, but it
was in the house.
It was in February that I went out to the camp, and although there
was still snow on the ground, the day was mild and pleasant.
The men were all at work when I arrived, and I wandered about,
looking at everything and asking questions.
The camp was in the middle of a large grove of sugar-maple trees,
and in each of the large trees a hole had been bored, and a little
spout, made of a piece of elder wood, with the pith scooped out, had
been inserted in each hole. Through these spouts the sap was
dripping into pans and wooden troughs, placed at the foot of each
tree.
As fast as these pans and troughs were filled they were taken to
the house and emptied into boilers that were suspended over the
fire. Here the sap boiled away at a great rate, and the men took turns
in stirring it so that it should not burn.
I found that this sugar-making was quite a tedious operation, as
the sap had to be boiled twice, and a great deal of care and time was
spent upon it before it cooled down into the hard, light-brown maple
sugar of which most boys and girls are so fond.
But I saw enough to make me understand the principles of the
business.
I found that when the sap began to rise in the trees, in the early
spring, there was always enough of it to supply the needs of the tree,
and a good deal besides to supply the needs of the sugar-makers.
I watched all the processes, and tasted the sap when it first flowed
from the tree and in all its different stages. And when I went back to
the farm-house, early in the afternoon, I thought that it would be a
great thing to have a grove of sugar-maples, and to be able to make
one’s own sugar, and to be independent, in that respect at least, of
the grocery-store.