Alcohol: Methods and Protocols (Methods in Molecular Biology)

416 Pages·2008·4.82 MB·English

by  Laura E. Nagy

Alcohol
Methods and Protocols
Methods in Molecular BiologyTM
John Walker, Series Editor
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Alcohol
Methods and Protocols
Laura E. Nagy
Departments of Gastroenterology and Pathobiology, Cleveland Clinic;
Department of Nutrition, Case Western Reserve University, Cleveland, OH
Editor
Laura E. Nagy
Departments of Gastroenterology and Pathobiology
Cleveland Clinic
and
Department of Nutrition
Case Western Reserve University, Cleveland
[email protected]
Series Editor:
John and Jan Walker
28 Selwyn Avenue
Hatfield, Hertfordshire
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ISBN 978-1-58829-906-2 e-ISBN 978-1-54745-242-7
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Preface
Alcohol consumption is often characterized as an environmental stress to the organ-
ism. In response to the stress of alcohol exposure, complex cellular and organismal
adaptations occur to and manage this insult. In most individuals, modest alcohol
consumption over the course of a lifetime does not result in substantive health risks,
and in the pathophysiology of some diseases, such as type 2 diabetes, modest alco-
hol consumption may actually be protective. Yet, chronic and heavy alcohol con-
sumption poses significant health risks. Scientists who study the effects of acute
and chronic alcohol consumption know little about what marks the transition from
a benign or even protective effect of alcohol to its pathophysiological effect in the
development of tissue injury and disease.
Understanding the transition to injury is an important clinical and public health issue,
as excessive alcohol consumption can impact nearly every tissue in the body, contribut-
ing to more than 60 different medical conditions. The effects of excessive alcohol occur
both in the developing organism as well as in the adult. Although the liver, as the site of
ethanol metabolism, is particularly sensitive to chronic alcohol exposure, alcohol con-
sumption also leads to damage of other tissues, including brain, heart, and cardiovascu-
lar system, as well as disruptions in regulation of endocrine and immune systems. The
contribution of alcohol to the development of chronic diseases, such as osteoporosis,
heart disease, and diabetes, is particularly relevant today, given the increased incidence
of these diseases in our aging population.
Recent advances in understanding the pleiotropic effects of ethanol have been
possible because of the development of relevant and rigorously controlled animal
and cell culture models of acute and chronic ethanol exposure. Although each of
the various models for ethanol exposure may not perfectly model exposure of
humans to alcohol, many model systems have now been developed that can mimic
particular conditions of ethanol exposure in target tissues and organs. One of the
primary goals of this volume is to provide detailed procedures for several of the
more common models of acute and chronic ethanol exposure, enabling studies on
the effects of ethanol in both the developing organism and in the adult. Use of these
clearly defined models of ethanol exposure, presented in the first section of this
volume, will allow for comparison of results among different laboratories, as well
as among multiple tissue and organ targets of acute and chronic ethanol exposure.
One of the themes arising in recent studies that investigate the mechanisms of
ethanol action on target tissues is the commonality in the impact of ethanol on regu-
lation of cellular metabolism. Thus, in addition to the effects of acute and chronic
v
vi Preface
alcohol on the complex physiology of the intact organism, alcohol exposure also
has a profound impact on the biology of individual cells. As with studies in whole
animals, investigations to study the impact of ethanol on cellular biology must be
rigorously controlled and designed. Recent advances in the development of specific
methodologies to mimic the impact of ethanol metabolism in cultured cells,
detailed in the second section of this volume, have furthered our understanding of
the molecular mechanisms by which ethanol disrupts cellular function.
Although there are common mechanisms of ethanol action on a variety of cell types,
studies of the effects of ethanol on cellular function must also take into consideration
the complex differentiated function of individual cells and tissues. Thus, expertise in the
use of models of ethanol exposure, as well as in the design and analysis of experiments
to ascertain the effects of ethanol on the highly regulated function of each differentiated
cell and tissue type, must be combined to finely dissect the mechanisms of ethanol
action. Therefore, an additional theme of this volume embraces the methodologies to
investigate a variety of cells and tissues that are known to be disrupted by ethanol, from
intestinal epithelial cells, to cells in the liver, including hepatocytes and Kupffer cells,
to cells in the periphery, including skeletal muscle, adipose and bone. Specific method-
ologies to investigate the effects of ethanol on neuronal function, including the use of
neuronal cell lines and organotypic cultures, are also presented.
It is likely that the effects of ethanol on cell, tissue, and organismal function are
fundamentally based on the impact of ethanol on transcriptional and post-transcrip-
tional regulation of gene expression. Novel methodologies to study the molecular
mechanisms of ethanol action include the use of gene arrays, as well as proteomic
analysis of the post-translational modifications of proteins in organelles and cells
exposed to ethanol. Chapters providing the specific expertise required for the design
and analysis of gene array and proteomic studies are included in this volume to enable
investigators new to these data-rich approaches to successfully “mine” the vast
amount of data that can be obtained by these approaches.
In the final analysis, studies into the molecular mechanisms for ethanol action
not only result in a further understanding of the pathophysiology of ethanol-induced
injury, but also contribute to our understanding of the fundamental mechanisms by
which organisms have adapted to subtle changes in their environment. Although
excessive alcohol consumption can result in profound impairments in the ability of
the organism to develop and function, most organisms can readily handle the subtle
insults associated with moderate alcohol consumption. Understanding the genetic,
molecular, cellular, and physiological responses to ethanol that “tip the balance”
from an adaptive response to a maladaptive/pathological response is critical to the
development of therapeutic strategies for the intervention and/or prevention of the
effects of ethanol on development and tissue injury. I hope that the very detailed
and specific methods presented in this volume will further spur investigators to
delve into the complex and fascinating story of the adaptive and maladaptive
responses humans have developed to the consumption of alcohol.
Laura E. Nagy, PhD
Cleveland Clinic
Acknowledgments
I would like to thank Joyce Nolan for her careful and thorough editing and format-
ting of the chapters in this volume. This work was supported in part by US Public
Health Service Grants RO1 AA11975 and RO1 AA11876.
vii
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
Part I Animal Models of Acute and Chronic Ethanol Exposure . . . . . . . 1
1 Acute Models of Ethanol Exposure to Mice . . . . . . . . . . . . . . . . . . . . . . 3
Timothy P. Plackett and Elizabeth J. Kovacs
2 A Voluntary Oral-Feeding Rat Model for Pathological
Alcoholic Liver Injury. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
George L. Tipoe, Emily C. Liong, Tung-Ming Leung, and Amin A. Nanji
3 Intragastric Ethanol Infusion Model in Rodents . . . . . . . . . . . . . . . . . . 33
Hide Tsukamoto, Hasmik Mkrtchyan, and Alla Dynnyk
4 A Practical Method of Chronic Ethanol Administration in Mice. . . . . 49
Ruth A. Coleman, Betty M. Young, Lucas E. Turner, and Robert T. Cook
Part II Animal Models of Ethanol Exposure During Development. . . . . 61
5 Analysis of Ethanol Developmental Toxicity in Zebrafi sh. . . . . . . . . . . 63
Robert L. Tanguay and Mark J. Reimers
6 The Avian Embryo in Fetal Alcohol Research . . . . . . . . . . . . . . . . . . . . 75
Susan M. Smith
7 Artifi cial Rearing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Hector D. Dominguez and Jennifer D. Thomas
8 Intragastric Intubation of Alcohol During the Perinatal Period. . . . . . 101
Sandra J. Kelly and Charles R. Lawrence
ix
x Contents
Part III Cell Culture Approaches to Studying Ethanol Exposure . . . . . . 111
9 Human Monocytes, Macrophages, and Dendritic Cells:
Alcohol Treatment Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Gyongyi Szabo and Pranoti Mandrekar
10 Generation and Use of Primary Rat Cultures
for Studies of the Effects of Ethanol. . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Amanda Lindke, Barbara Tremper-Wells, and Michael W. Miller
11 Development and Properties of HepG2 Cells That
Constitutively Express CYP2E1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Defeng Wu and Arthur I. Cederbaum
12 Modeling the Impact of Alcohol on Cortical Development
in a Dish: Strategies from Mapping Neural Stem Cell Fate. . . . . . . . . . 151
Rajesh C. Miranda, Daniel R. Santillano, Cynthia Camarillo,
and Douglas Dohrman
Part IV Regulation of Specifi c Organ Systems
in Response to Ethanol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
13 Acetaldehyde-Induced Barrier Disruption and Paracellular
Permeability in Caco-2 Cell Monolayer. . . . . . . . . . . . . . . . . . . . . . . . . 171
R. K. Rao
14 Alcohol-Induced Oxidative Stress in the Liver:
In Vivo Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Gavin E. Arteel
15 Isolation of Kupffer Cells From Rats Fed Chronic Ethanol . . . . . . . . 199
Megan R. McMullen, Michele T. Pritchard, and Laura E. Nagy
16 Dendritic Cells in Chronic In Vivo Ethanol Exposure Models . . . . . . 213
Kevin L. Legge and Annette J. Schlueter
17 Formation and Immunological Properties of Aldehyde-Derived
Protein Adducts Following Alcohol Consumption . . . . . . . . . . . . . . . . 235
Geoffrey M. Thiele, Lynell W. Klassen, and Dean J. Tuma
18 Assessment of Natural Killer (NK) and NKT Cells in Murine
Spleens and Livers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Michael R. Shey and Zuhair K. Ballas
Contents xi
19 Polyclonal and Antigen-specifi c Responses of T Cells
and T Cell Subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Betty M. Young, Susan Wiechert, Ruth A. Coleman,
Prajwal Gurung, and Robert T. Cook
20 B-Cell Studies in Chronic Ethanol Mice . . . . . . . . . . . . . . . . . . . . . . . . 295
Shilpi Verma, Carla-Maria A. Alexander, Michael J. Carlson,
Lorraine T. Tygrett, and Thomas J. Waldschmidt
21 Histological Analysis of Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Urszula T. Iwaniec, Thomas J. Wronski, and Russell T. Turner
22 Assessing Effects of Alcohol Consumption on Protein
Synthesis in Striated Muscles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Thomas C. Vary and Charles H. Lang
23 Methods to Investigate the Effects of Chronic
Ethanol on Adipocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Becky M. Sebastian, Li Kang, Xiaocong Chen, and Laura E. Nagy
Part V Proteomic and Genomic Approaches to Study
Response to Ethanol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
24 Proteomic Approaches to Identify and Characterize
Alterations to the Mitochondrial Proteome in Alcoholic Liver Disease . . . 369
Shannon M. Bailey, Kelly K. Andringa, Aimee Landar,
and Victor M. Darley-Usmar
25 Alcoholic Liver Disease and the Mitochondrial Ribosome:
Methods of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
Alan Cahill and Peter Sykora
26 Microarray Analysis of Ethanol-Induced Changes
in Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
Robnet T. Kerns and Michael F. Miles
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411