Handbook of food toxicology 2002 dekker

These compounds can be of ural origin as products of the metabolic processes of ani-mals, plants, and microorganisms from which the food isderived; as biological and chemical contaminant

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To all those who appreciate my sole existence, especially my wife Usha and daughter Maithili and

To Ratan, Shastry, Suresh, and Prakash, for being such wonderful friends through thick and thin

Food toxicology is deeply rooted in the history of

civiliza-tion In their quest for food, our ancestors must have

at-tempted to eat a variety of plants and animals and soon

recognized that there were harmful as well as beneficial

effects of their consumption The selection by nomadic

hu-mans of only a handful of plant species for domestication

and cultivation from the estimated 350,000 species of

plants documented in the annals of botany and plant

sci-ences is certainly not a chance occurrence Gathering

peo-ples perhaps accidentally found the very species that were

the most predisposed to domestication, as well as the most

well-suited to fulfill human nutritional requirements Our

experience throughout history has taught us much about

how to avoid injury from consuming natural products We

now know which products not to eat under any

circum-stances, which can be eaten under some circumcircum-stances,

and how to process other products to render them safe for

consumption

History has thus taught us how to classify all stances into two classes: those that are safe and those that

sub-are harmful or poisonous Such a classification, however,

is not warranted in a strict scientific sense, primarily

be-cause a strict line of demarcation classifying and

separat-ing beneficial and harmful chemicals cannot be drawn, and

because the degree of harmfulness of any compound is

es-sentially related to the amount consumed In fact, over 400

years ago Paracelsus pointed out that “all substances are

poisons; there is none which is not a poison The right

does differentiates a poison and a remedy.” Indeed, the

en-tire concept of toxicity needs to be evaluated from the

viewpoint of a risk/benefit concept associated with the

consumption of any given material There is no such thing

as absolute safety

Our daily lives are still shaped by our acceptance ofnumerous acceptable risks Nearly everything we con-sume, including salt, sugar, starch, fat, protein, some min-erals and vitamins, and even water, has a harmful effectwhen consumed in high enough concentration Hazardoussubstances associated with food include toxic or antinutri-tive compounds that are naturally present in plants and an-imals; toxins that are produced ruing processing;incidental contaminants such as pesticides, antibiotic drugresidues, and environmental pollutants; and foodbornepathogens However, this does not mean that food is haz-ardous to human beings Toxic components in foods—al-though they should indeed be minimized—are inevitablehazards of living A substance that is considered to betoxic/antinutritive has a more or less pronounced capacity

to induce deleterious effects on an organism when tested

by itself in certain doses This does not always happen der usual dietary conditions We consume many toxic sub-stances in our normal diet every day without showing anysigns of intoxication This is probably because natural tox-icants usually exert their effects only when other potentiat-ing substances are available Also, the concentration ofthese compounds occurring naturally in food is often solow that the item must be consumed in extraordinarilylarge amounts daily over a prolonged period for intoxica-tion to occur

un-Similarly, most toxic effects of potentially hazardouschemicals are not additive In fact, antagonistic reactionsthat make some ingredients interfere and reduce the toxic

effects of other components are not unusual Thus, many

natural products that are common in the human diet have

found wide acceptance not because they are free of toxic

substances, but because they do not contain enough toxins

to be harmful when consumed in reasonable quantities as

part of a balanced diet—or because cooking or another

process eliminates their toxic activity In the vast majority

of instances, our food supply is quite wholesome In the

unfortunate incidents when some link(s) in the food

pro-duction, processing, and distribution scheme fail(s), such

foods, when consumed, have produced adverse toxic

re-sponses that vary in severity from insignificant to fatal

As compared with naturally occurring tritive compounds in the human food chain, the situation is

toxic/antinu-quite different with microbial contaminant of foods In

fact, perhaps the greatest damage, in terms of both

mortal-ity and morbidmortal-ity worldwide, can be directly attributed to

microbial contamination Although changes and

improve-ments in food processing operations as well as in sanitary

practices have contributed to an important increase in the

life span of humans in the last century, these significant

improvements are now challenged by the appearance of

microbes resistant to multiple antibiotics (e.g., Salmonella

sp and the emergence of new bacterial and fungal

patho-gens (e.g., Campylobacter, Listeria, E Coli 0157:H7,

fu-monisins) In the United States alone, between 6.5 million

and 81 million cases of foodborne illness and as many as

10,000 related deaths from seven major foodborne

patho-gens occur each year, costing $6.6 billion to $37.1 billion

in economic losses The situation is grim even in

develop-ing countries where water-borne and food-borne diseases

such as cholera, jaundice, and diarrhea—which impair

hu-man health to a great degree, and therefore the body’s

effi-ciency of food absorption—are perhaps more important

factors affecting human health than many naturally

occur-ring toxic/antinutritive compounds in the food chain

These effects are further magnified by a shortage of such

basic commodities as a clean and safe supply of drinking

water and adequate food for subsistence—this alone was

good enough motivation for me to undertake this project

The primary aims for Handbook of Food Toxicology

are twofold (1) to provide basic coverage of the principles

of toxicology relevant to food science and nutrition, and

(2) to provide the latest information on various toxic and

microbial hazards associated with modern-day foods This

book is divided into two parts that comprise a total of 18

chapters The first part, consisting of Chapters 1–6, deals

with the science and principles of toxicology,

manifesta-tions of toxic effects, biotransformamanifesta-tions of toxicants

rele-vant to food science and human nutrition, and some of the

regulatory and QA/QC issues Chapters 7 through 18 scribe the basic aspects of toxicity associated with com-monly occurring dietary components and substances(naturally occurring, intentionally added, or incidental), aswell as those associated with microbial contamination offoods A basic understanding of the principles behind theoccurrence of microbes in the food chain and their toxicity

de-or toxic mechanisms not only allows us to appreciate thecomplexity of our food supply but is essential for develop-ing newer and safer food production, processing, handling,and distribution technologies

No single food toxicology book can cover all aspects

of the toxicity and safety of the myriad of food used inmany different ways by humans worldwide Indeed, vol-umes and monographs are available on the topic of practi-cally every chapter in this book, and even on those ofmany of the chapter sections Every effort, however, wasmade to cover important toxic hazards associated withfood consumption For some toxins, only historical view-points are described, since research during the past decade

on many of these compounds (e.g., flavonoids, phytates,antioxidants) has shown several positive health benefits as-sociated with their consumption as part of a normal, well-balanced diet In contrast, in-depth coverage is provided

on microbial toxins and food pathogens, since these pear to be the predominant causes of morbidity and mor-tality associated with our food supply Hopefully, thisbook represents a compromise between the historicalviews associated with the traditional, well-known toxiccomponents found in our food supply and the exciting newdevelopments occurring on several other fronts, especially

ap-on foodborne infectiap-ons and intoxicatiap-ons It is my sincerehope that the information presented in this book will serveprofessionals in many disciplines, including agriculture,food science, nutrition, microbiology, toxicology, publichealth, medicine, and other health-related areas Selectedchapters can also be used as college-level teaching material.Finally, it is inevitable in a book of this breadth thatomissions, occasional errors, and lapses in the accuracy ofinterpretation will have escaped the detection of even themost assiduous proofreaders I hope that any such mis-takes are both minor and minimal, and I accept full andexclusive responsibility for them I welcome commentsand suggestions for improvement and for correction of anyerrors

Sincere appreciation is extended to the editorial andproduction staff of Marcel Dekker, Inc., especially to Ms.Maria Allegra, Ms Lila Harris, Ms Katie Stence, Ms.Theresa Stockton, and Ms Susan Thornton Without theircooperation and tremendous patience, this book would

never have been written I gratefully acknowledge the

original treatise in this field: the late Professor Jose M

Concon’s groundbreaking two-volume Food Toxicology,

published in 1988 In fact, the origin of this book can be

traced back to his monumental work in the field I am also

greatly indebted to Professor D K Salunkhe of Utah State

University, who first encouraged me to undertake this task

Finally, no words will ever fully describe the untiring andcontinued support and encouragement provided by mywife Usha and daughter Maithili during the arduous task

of putting together this book

S S Deshpande

Preface

SCIENCE AND PRINCIPLES OF TOXICOLOGY

1 The Science of Toxicology

2 Principles of Toxicology

3 Manifestations of Organ Toxicity

4 Carcinogenesis, Mutagenesis, and Teratogenesis

9 Toxicants Resulting from Food Processing

10 Toxicants and Antinutrients in Plant Foods

11 Fungal Toxins

12 Food-Borne Infections

13 Bacterial Toxins

14 Seafood Toxins and Poisoning

15 Mushroom Toxins

16 Toxic Metals, Radionuclides, and Food Packaging Contaminants

17 Pesticides and Industrial Contaminants

18 Drug Residues

The Science of Toxicology

1.1 INTRODUCTION

The origins of toxicology appear to be deeply rooted in the

history of human civilization Our ancestors in their quest

for food must have attempted to eat a variety of foods of

both plant and animal origin and soon recognized that

there were harmful as well as beneficial consequences

as-sociated with the consumption of such material The rise

of agricultural knowledge has been traced back to ancient

times, when humankind made the transition from nomadic

hunting/gathering tribes to more settled societies

sup-ported by domesticated animal herds and cultivated crops

In terms of archaeological findings, primitive agriculture

may have developed as early as 9000–7000 B.C in the

Near East (Garfield, 1990)

The selection of only a handful of plant species fordomestication and cultivation by the nomadic human is

certainly not a chance occurrence It must predate

agricul-ture by at least some thousands of years Its enormous

complexity is further illustrated by the 3000-plus species

of the estimated 350,000 species of plants documented in

the annals of botany and plant sciences that have been

used historically in some form to feed humans

(Desh-pande, 1992; Borlaug, 1981; Wittwer, 1980) Fewer than

300 are used currently worldwide in organized agriculture

Among these, at least 150 different species are grown in

sufficient quantities to enter the world trade In contrast,

Wittwer (1980) suggests, today some 24 crops essentially

stand between people and starvation In approximate order

of importance these crops are rice, wheat, corn, potato,

barley, sweet potato, cassava, soybean, oat, sorghum,

mil-let, sugarcane, sugar beet, rye, peanut, field bean,

chick-pea, pigeon chick-pea, mung bean, cowchick-pea, broad bean, yam, nana, and coconut Although cereals with nine species andlegumes with eight constitute the preponderance of theglobal food production, together they constitute only0.005% of the available wealth in the plant kingdom.Present-day humans, no doubt, have diversified the uses ofimportant economic plants greatly They, however, haveadded relatively little to this list of basic staples

ba-Domestication of only such a few plant species ashuman food sources is truly one of the most extraordinarystories of our history Beginning only by collecting, thegathering peoples perhaps accidentally(?) chanced uponthe very species that were the most predisposed to domes-tication and were well suited to fulfill human nutritionalrequirements Our experience throughout history has thustaught us much about how to prevent injury from consum-ing natural products as foods We now know which prod-ucts not to eat under any circumstances, which can beeaten under some circumstances, and how to process cer-tain other products to render them safe for consumption.History has thus taught us how to classify all sub-stances in two classes: those that are safe and the others

that are harmful Traditionally, the term food was used for

those materials that were beneficial and essential for thefunctioning of human body Substances that were dis-

tinctly harmful to the body were classified as poisons.

This concept involving the division of chemicalsinto two categories has persisted to the present day Itreadily places certain biological and botanical and, in fact,all distinctly harmful chemicals into a category that is ac-corded due respect Loomis (1978), however, suggestedthat such a classification, in a strictly scientific sense, is

not warranted, primarily because a strict line of

demarca-tion classifying and separating the beneficial and harmful

chemicals cannot be drawn and because the degree of

harmfulness of any compound is essentially related to the

amount consumed

Indeed, the entire concept of toxicity needs to beevaluated from the viewpoint of a risk/benefit concept as-

sociated with the consumption of any given material In

fact, Paracelsus (1493–1541) over 400 years ago pointed

out that “all substances are poisons; there is none which is

not a poison The right dose differentiates a poison and a

remedy.” Since all substances can produce injury or death

under some exposure conditions, it is evident that there is

no such thing as an absolute safe substance or chemical

that will be free of injurious effects under all conditions of

exposure As a corollary, it is also true that there is no

chemical that cannot be used safely by limiting the dose or

exposure Our daily lives are still shaped by our

accep-tance of numerous acceptable risks Nearly everything we

consume, including salt, sugar, starch, fat, protein, some

minerals and vitamins, and even water, has a harmful

ef-fect when consumed in high enough concentration

How-ever, this does not necessarily mean that the substance is

hazardous to human beings

Toxic compounds in our foods, medicines, and ronment—though they should indeed be minimized—are

envi-inevitable hazards of living A substance that is considered

to be toxic/antinutritive has a more or less pronounced

ca-pacity to induce deleterious effects on the organism when

tested by itself in certain doses This does not always

hap-pen under the usual dietary conditions We consume many

toxic substances in our normal diet every day without

showing any signs of intoxification This is probably

be-cause natural toxicants usually exert their effects only

when they are consumed under special conditions or when

there are other potentiating substances present Also, the

concentration of these compounds occurring naturally in

the food is often so low that the item must be consumed in

usually unrealistically large amounts every day for a

pro-longed period for intoxification to occur Furthermore,

hu-mans can handle small amounts of various toxicants

Similarly, most toxic effects of various chemicals that are

potentially hazardous do not have an additive effect In

fact, antagonistic reactions that make some ingredients

in-terfere with and reduce the toxic effects of other

compo-nents are not unusual Thus, many natural products that are

common articles of the diet have found wide acceptance,

not because they are free of toxic substances, but because

they do not contain enough to be harmful when consumed

in reasonable quantities as a part of a balanced diet, or

be-cause cooking or some other process eliminates their toxic

activity

It is thus the “dose” of any given substance that termines its degree of harmfulness If a sufficient dose isingested or is in contact with a biological mechanism, then

de-a hde-armful or toxic effect will be the consequence in thesense that the ability of that biological mechanism to carry

on a function is either seriously impaired or destroyed.Such harmful effects often do not occur suddenly as thedose is increased from minimal to maximal levels Rather

a graded response related to progressive changes in dose isobserved The basic premise of the science of toxicologywith respect to any biological effects of a chemical agent

is to study this relationship that exists between the dose orconcentration and the response that is obtained

Furthermore, toxic chemicals may be selective ornonselective in exerting their harmful effects on biologicalsystems Thus selective chemicals may exert their harmfuleffects only in a few living species, primarily becausesome protective mechanisms may be present in the resis-tant species In contrast, those that act nonselectively exert

an undesirable or harmful effect on all living matter nately, such compounds seldom find their way into ourfood chain under normal conditions

Fortu-The term toxicology is derived from Latin and Greek origins (Latin toxicum meaning “poison”; Greek toxikom meaning “arrow poison”; and Latin logia, meaning “sci-

ence” or “study”) and literally means a study of poisons inrelation to living organisms The science of toxicologytherefore can be best approached as the study of the effects

of chemicals on biological systems, with emphasis on themechanisms of harmful effects of chemicals and the con-ditions under which such harmful effects can occur In itsbroadest sense, it also includes socioeconomic consider-ations as well as legal ramifications

1.2 HISTORY OF TOXICOLOGY

Ironically, toxicology must rank as one of the oldest tical sciences because humans, from the very beginning,needed to avoid the numerous toxic plants and animals intheir environment The Egyptian Ebers Papyrus (circa

prac-1500 B.C.) and the Hindu Vedas (circa 5000 B.C.) rank astwo of the earliest surviving medical records that containinformation on several poisons The surviving medicalworks of Hippocrates, Aristotle, and Theophrastus, pub-lished during the period 400–250 B.C., all included somemention of poisons Dioscorides, a Greek employed by theRoman emperor Nero in about A.D 50, first attempted toclassify plants according to their toxic and therapeuticeffects

There appear to have been very few noteworthy vances in either medicine or toxicology during the Middle

ad-Ages The only significant contribution to toxicology

dur-ing this period appears to be that of Moses ben Maimon, or

Maimonides (1135–1204) He published a treatise,

Poi-sons and Their Antidotes, a first-aid guide to the treatment

of accidental or intentional poisonings and insect, snake,

and mad dog bites Maimonides recommended that

suc-tion can be applied to insect stings or animal bites as a

means of extracting the poison and advised application of

a tight bandage above a wound located on a limb He also

noted that the absorption of toxins from the stomach could

be delayed by ingestion of oily substances such as milk,

butter, or cream On the basis of critical and cautious

ob-servations, he also rejected numerous popular remedies of

the day after finding them to be ineffective and mentioned

his doubts concerning the efficacy of others

During the later Middle Ages, Philippus AureolusTheophrastus Bombastus von Hohenheim Paracelsus

(1493–1541) made revolutionary contributions to this

discipline we now call toxicology He first proposed the

toxic agent as a chemical entity and was clearly aware of

the dose-response relationship The following four

princi-ples he first laid out still form the core of toxicological

at the University of Paris, is often cited as the father of

modern toxicology Orfila was the first to attempt a

sys-tematic correlation between the chemical and biological

information of the then well-known poisons He also

sin-gled out toxicology as a discipline distinct from others and

defined it as the study of poisons He wrote in 1815 the

first book of general toxicology that was exclusively

de-voted to adverse effects of chemicals Concerned with

le-gal implications of poisoning, Orfila also pointed out the

importance of determining a chemical analysis to establish

a definitive cause of poisoning Some of the analytical

pro-cedures he developed are still in use today

Since Orfila’s pioneering work, developments intoxicology slowly evolved Although toxicology’s origins

predate those of most other biological sciences and

per-haps even those of medicine, most of the useful

informa-tion related to modern toxicology has only been learned

since the turn of the 20th century In fact, the emergence of

toxicology as a distinct discipline is a much more recentphenomenon There are many reasons for this, includingthe development of new analytical methods since the end

of the Second World War, the emphasis on drug testingthat followed the thalidomide tragedy, the focus on pesti-

cide testing since the publication of Rachel Carson’s Silent

Spring, public concerns about hazardous waste disposal,

and, more recently, the increased incidence of food sonings by microbial pathogens

poi-Detailed descriptions of the historical developments

in the field of toxicology can be found in several excellentreviews (Loomis, 1978; Holmstedt and Liljestrand, 1981;Doull and Bruce, 1986)

1.3 SCOPE/DIVERSITY OF TOXICOLOGY

Modern toxicology is a multidisciplinary field, which hasextracted many of the principles and techniques from sev-eral basic biological and chemical sciences (Figure 1.1) It

is primarily an applied science, dedicated to the ment of the quality of life and the protection of the envi-ronment It draws heavily on tools of chemistry andbiochemistry Those of chemistry provide analytical meth-ods for toxic compounds, particularly for forensic toxicol-ogy and residue analysis, and those of biochemistryprovide the techniques to investigate the metabolism andmode of action of toxic compounds Toxicology may also

enhance-be considered an area of fundamental biology since the aptation of organisms to toxic environments has importantimplications for ecology and evolution Sciences such asimmunology, biomathematics, and ecology are also impor-tant, but to a more limited extent

ad-The science of toxicology contributes heavily in thefield of medicine, especially forensic medicine, clinicaltoxicology, pharmacy and pharmacology, public health,and industrial hygiene It also contributes in an importantway to veterinary medicine and to such aspects of agricul-ture as the development and safe use of agrochemicals Itscontributions to the field of environmental studies areamong the most rapidly expanding areas in the worldtoday

Thus, any attempt to define the scope of toxicologymust take into account the fact that the various subdisci-plines shown in Figure 1.1 are not mutually exclusive andfrequently are heavily interdependent Because of theoverlapping of mechanisms, chemical classes, and useclasses and effects, clear division into distinct branches oftoxicology is often not possible However, for the sake ofconvenience, subdivisions of toxicology can be defined byfollowing any of the several classifications suggested by

Hodgson (1987) These are briefly described in the

follow-ing sections

1.3.1 Applied Toxicology Based

on Disciplines

Various aspects of applied toxicology can be defined as

they occur in or relate to a particular field (Figure 1.1)

These include the following

Clinical and Forensic Toxicology

Clinical toxicology is concerned with the diagnosis and

treatment of human poisoning It encompasses the study

of chemicals originating from any and all sources and is

primarily concerned with all aspects of the interaction

be-tween these chemicals and people Forensic toxicology, in

contrast, combines analytical chemistry with essential

toxicological principles in order to deal with the

medi-colegal aspects of the toxic effects of drugs and chemicals

on humans Its primary role is to aid in establishing

cause-effect relationships between exposure to a drug or

poison and the toxic or lethal effects of the compound It

relies heavily on specific and highly sensitive analytical

methods, which can efficiently isolate, identify, and

quan-titate the toxic compound in question from clinical and

Environmental Toxicology

The broad discipline of environmental toxicology passes the study of chemicals that are the contaminants offood, water, soil, or the atmosphere It is primarily con-cerned with the movement of chemicals and toxicants andtheir metabolites in the environment and in food chain, andthe effect of such toxicants on individuals and populations.Environmental toxicology is also concerned with toxicsubstances that may enter the lakes, streams, rivers, andoceans The most common problems dealt with in this as-pect of toxicology are water-borne viruses and bacteria, ra-dioactive waste, sewage eutrophication, and industrialpollutants

protect the worker from poisonous substances and, in

gen-eral, to make the working environment safe The objective

obviously is to prevent impairment of health of an

individ-ual while on the job In the United States, the Occupational

Safety and Health Act (OSHA) was passed in 1970 to

pro-tect the health of workers Two agencies, the National

In-stitute for Occupational Safety and Health (NIOSH),

responsible for developing safety and health standards,

and the American Conference of Governmental Industrial

Hygienists (ACGIH), devoted to setting safety standards

for chemicals in the working environment, are primarily

responsible for the enactment of OSHA guidelines under

the jurisdiction of the Department of Labor

1.3.2 Classification Based on Measurement

of Toxicants and Toxic Actions

By using a variety of techniques derived from analytical

chemistry, bioassays, and applied mathematics, toxicants

and their toxic effects can be measured and quantitated

This aspect of toxicology includes a number of important

fields (Figure 1.1) as follows:

Analytical Toxicology

Analytical toxicology is a branch of analytical chemistry

that is concerned with methods for the identification and

assay of toxic chemicals and their metabolites in

biologi-cal and environmental samples

Toxicity Testing

The branch of toxicity testing involves the use of living

systems to estimate toxic effects of various chemicals It

covers the entire gamut from short-term tests for

genotox-icity such as the Ames test and cell culture techniques to

the use of live animals for acute toxicity tests and for

life-time or multigeneration chronic toxicity tests The term

bioassay is used to describe the use of living organisms to

quantitate the amount of a particular toxicant present

Biomathematics and Statistics

Mathematical and statistical techniques are used in a

num-ber of areas of toxicology They deal with data analysis, the

determination of significance, and the formulation of risk

estimates and predictive models The latter is particularly

important in epidemiology and environmental toxicology

Toxicological Pathology

Toxicological pathology deals with the effects of toxic

agents as manifested by changes in subcellular, cellular,

tissue, or organ morphological characteristics

Structure-Activity Studies

Structure-activity studies are important to understanding

of the relationship between the chemical and physicalproperties of xenobiotics and toxicity and, particularly, theuse of such relationships for the prediction of toxicity

Epidemiology

Epidemiology, as it applies to toxicology, is closely related

to the biomathematical and statistical models and is ofgreat importance since it deals with the study of toxicity as

it occurs, rather than in an experimental laboratory setting

1.3.3 Classification Based on Mechanisms

of Toxic Action and Effects

Toxicants can also be classified on the basis of all theevents leading to exertion of their toxic effects in vivo.This involves studies at the fundamental level of organ,cell, and molecular functions, including their uptake, dis-tribution, metabolism, mode of action, and excretion Im-portant disciplines include the following:

Biochemical Toxicology

Biochemical toxicology considers events at the cal and molecular level, including enzymes that metabo-lize xenobiotics, generation of reactive intermediates, andinteraction of xenobiotics or their metabolites with macro-molecules

biochemi-Behavioral Toxicology

Behavioral toxicology deals with the effects of toxicants onanimal and human behavior This involves peripheral andcentral nervous systems as well as effects mediated byother organ systems such as the endocrine glands

Teratogenesis

Teratogenesis includes the chemical and biochemicalevents that lead to deleterious effects on the developmental

process; it involves multigenerational studies of animals

to study the long-term toxic effects on the reproductive

processes

Carcinogenesis

Carcinogenesis includes the study of chemical and

bio-chemical events upon exposure to toxic bio-chemicals that

lead to the large number of effects on cell growth

Organ Toxicity

Organ toxicity considers the effects at the level of organ

functions, e.g., neurotoxicity, hepatotoxicity, and

nephro-toxicity

Regulatory Toxicology

Regulatory toxicology is concerned with the formulation

of laws and regulations authorized by laws to minimize the

effect of toxic chemicals on human health and the

environ-ment In the United States, the risk assessment, which is

the definition of risks, potential risks, and the risk-benefit

equations necessary for regulation of toxic substances, is

primarily under the control of several government

agen-cies These include the Food and Drug Administration

(FDA), the Environmental Protection Agency (EPA), and

OSHA

1.3.4 Classification Based on Chemical

Use Classes

Hodgson (1987) also classified toxicants on the basis of

use classes This classification includes the toxicological

aspects of the development of new chemicals for

commer-cial uses In some of these use classes, toxicity, at least to

some organisms, is a desirable trait; in others, it is an

un-desirable side effect This category includes both synthetic

and natural products The following classes can be

identi-fied by the use criteria

Agricultural Chemicals

Agricultural chemicals include herbicides, fungicides,

pes-ticides, and rodenpes-ticides, in which toxicity to the target

or-ganism is a desired quality, whereas toxicity to nontarget

species must be prevented Development of such

selec-tively toxic chemicals is one of the applied roles of

com-parative toxicology

Clinical Drugs

Although the development of clinical drugs is largely the

responsibility of the pharmaceutical industry and

pharma-cology, the toxic side effects and testing for them fallwithin the science of toxicology

Drugs of Abuse

Drugs of abuse are chemicals, often illegal, taken forpsychological or other effects that cause dependence andtoxicity

Naturally Occurring Substances

Naturally occurring substances include many phytotoxins,mycotoxins, and inorganic minerals that occur naturally inthe environment and may find a way into the human foodchain

Combustion Products

Combustion products are generated primarily from fuelsand other industrial chemicals

1.4 SOURCES OF TOXIC COMPOUNDS

Several natural as well as synthetic compounds are potenttoxicants and can enter the human food chain as contami-nants Although toxic, many find important uses at thera-peutic dose levels in clinical medicine Representativesources of toxic compounds are summarized in Table 1.1

1.5 CLASSIFICATION OF TOXICANTS

Toxic agents can be classified in a variety of ways sen, 1986; Sperling, 1984; Manahan, 1992; Gossel andBricker, 1984; Hodgson and Levi, 1987) For example,they can be classified in terms of their target organ (liver,kidney, lungs, skin, nervous system, hematopoietic sys-tem, etc.), their use (pesticides, solvents, food additives,etc.), their source (animal, plant, microbial toxins), andtheir effects (cancer, mutation, liver injury, etc.) Toxicantscan also be grouped in terms of their physical state (gas,dust, liquid), their labeling requirements (explosive, flam-

(Klaas-mable, oxidizer), their chemical properties (aromatic

amines, polycyclic hydrocarbons, halogenated

hydrocar-bons, etc.), or their toxic potential, as shown in Table 1.2

According to Klaassen (1986), classification on thebasis of the biochemical mechanism of action of toxicants

is usually more informative than classification by general

category, such as irritants and corrosives Nonetheless,more general classifications, such as air pollutants, occu-pation-related agents, and acute and chronic poisons, canstill provide a useful focus on a specific problem

It is quite evident that no single classification can beused to cover the entire spectrum of toxic agents In gen-eral, classification systems that take into considerationboth the chemical and the biological properties of the toxi-cant and the exposure characteristics tend to provide use-ful information for legislative or control purposes as well

as for toxicology in general

1.6 FOOD TOXICOLOGY AND THE SCOPE

OF THE BOOK

Food toxicology can be defined as a systematic study of

toxicants found in foods These compounds can be of ural origin as products of the metabolic processes of ani-mals, plants, and microorganisms from which the food isderived; as biological and chemical contaminants from theair, water, and soil; as intentionally introduced food addi-tives; and as those formed during the processing of foods.Food toxicology is thus concerned with the toxic potential

nat-of food, the conditions and factors affecting the presence

of these toxicants in food, their interactions with essentialdietary nutrients, the response of the human body to thesetoxins, and the means of prevention or minimization ofthese toxic effects as they pertain to food safety and hu-man nutrition

As compared to the presence of toxicants that arenaturally present in various foods, biological contamina-tion of our food supply presents grave food safety con-cerns Food-borne diseases caused by bacteria and viruseshave varying degrees of severity ranging from mild indis-position to chronic or life-threatening illness Their impor-tance as a vital public health problem is often overlookedbecause the true incidence is difficult to evaluate and theseverity of the health and economic consequences is often

Table 1.1 Representative Sources and Examples of Toxic

Compounds

Synthetic organic compounds

Air (transportation, industrial processes, electric power

genera-tion, heating processes)

Carbon monoxide, oxides of nitrogen and sulfur,

hydrocar-bons, particulatesWater (runoffs, sewage, waste products discharged from refiner-

ies, swelters, or chemical plants)

Agrochemicals, hydrocarbons, detergents, heavy metals

Food contaminants

Bacterial, fungal, and animal toxins; pesticide residues; plant

alkaloids, residues of animal feed additives (e.g., antibiotics, estrogens); industrial chemicals

Food additives

Nitrates, nitrites

Chemicals in the workplace

Inorganic metals, aliphatic and aromatic hydrocarbons,

halo-genated hydrocarbons, alcohols, esters, organometallics, pesticides

Drugs of abuse

Cocaine, methamphetamines, lysergic acid diethylamide

(LSD), morphine, nicotine, barbituratesTherapeutic drugs

Essentially all therapeutic drugs, which can be toxic at high

dosesAgrochemicals

Pesticides, herbicides, nematicides, rodenticides

Solvents

Aliphatic and aromatic hydrocarbons, halogenated solvents,

alcoholsPolycyclic aromatic hydrocarbons (incomplete combustion of or-

Ricinine, solanine, chaconine, safrole, quinones, estrogens,

en-zyme inhibitors, lectins, cyanogenic glycosides

Inorganic chemicals

Heavy metals, oxides of nitrogen and sulfur

Table 1.2 Toxicity Rating Chart

Source: Gossel and Bricker (1984) and Klaassen (1986).

Rating/class

Probable lethal oral dose for average humanPractically nontoxic > 15 g/kg

not fully appreciated For most food-borne diseases, only a

small proportion of cases reach the notice of health

ser-vices, and even fewer are investigated (Kaferstein et al.,

1999) It is believed that in industrialized countries less

than 10% of the cases are reported, and in developing

countries reported cases probably account for less than 1%

of the total (WHO, 1984) Studies in some industrialized

countries point to an underreporting factor of up to 350 for

certain food-borne diseases (Todd, 1989; Norling, 1994)

Despite these limitations in reporting, available data give

evidence of a tremendous public health problem

Further-more, even in industrialized countries, the data indicate an

increasing trend Although the situation regarding

food-borne diseases is very serious in developing countries,

even the industrialized countries have experienced a

suc-cession of major epidemics, of which the mad cow disease

is the latest entry in a long list of such food-borne

out-breaks The estimated annual incidence of food-borne

dis-eases in the United States ranges from 6.5 million to 80

million cases Surveys in other countries suggest that up to

10% of the general population may annually suffer from a

food-borne disease (Todd, 1989; WHO, 1994; Notermans

and Hooenboom-Verdegaal, 1992; Notermans and van de

Giessen, 1993)

In the United States, typically 400 to 500 food-borneoutbreaks are reported annually to the Centers for Disease

Control (CDC), with an average of about 40 cases per

out-break, for an average of about 18,000 food-borne disease

cases annually (Bean and Griffin, 1990; Bean et al., 1990a,

1990b) However, these data include many fewer cases

than do laboratory surveillance data For example, for

1983 to 1987, 6249 salmonellosis cases per year were

re-ported in the outbreak data compared with 44,000 cases

per year in the laboratory surveillance data (CAST, 1994)

Bennett and coworkers (1987) estimated that 96% of

sal-monellosis cases were food-borne

In the developing countries, diarrheal diseases, cially infant diarrhea, are the dominant problem and in-

espe-deed one of massive proportions Annually, some 1.5

billion episodes of diarrhea occur in children below the

age of 5, and of these over 3 million die as a result (WHO,

1994) Although traditionally contaminated water supplies

were believed to be the main source of pathogens causing

diarrhea, up to 70% of diarrheal episodes may actually be

due to food-borne organisms (Esrey and Feachem, 1989;

Motarjemi et al., 1993)

In addition to the food-borne diseases, food tamination with mycotoxins, pesticide residues, drug resi-

con-dues, and industrial chemicals is a serious issue that

affects human safety and well-being It should, however,

be noted that such contamination occurs on a sporadic

basis Furthermore, it can easily be prevented by using

careful food production, storage, handling, and tion practices

prepara-The primary aim of writing this book is to providecomprehensive information on the chemical and toxico-logical characteristics of various toxicants that occur in thehuman food chain in a single volume Part I presents infor-mation on the basic concepts of toxicology as a science.Here, general information is presented on the basic princi-ples of toxicology, the chemical and biochemical basis oftoxicity of chemicals, manifestations of toxic effects, car-cinogenesis, and detoxification mechanisms

Information on food toxicants from various sources

is presented in Part II Although these food toxicants aredescribed individually and may indeed appear to presentgrave dangers to human health when consumed, the read-ers should be aware of the fact that any treatment of foodtoxicology must be considered from the viewpoint of over-all nutrition This is primarily because, as described ear-lier, there is no such thing as “absolute safety,” andeverything we consume, from water to salt, sugar, pro-teins, and fats, has some associated toxicity Moreover,toxic effects of these compounds are generally not addi-tive We thus consume many toxic substances in our nor-mal diet without showing any signs of intoxification.Furthermore, several compounds thought to be antinutri-tional or toxic in the 1970s and early 1980s have nowshown to have beneficial effects on human nutrition andwell-being In fact, if it were not for the biological contam-ination of foods and food hygiene issues, we probably nowenjoy a safer food supply in a wider variety of forms than

at any other time in the history of human civilization Theauthor would like to bring out this important message inthis book

REFERENCES

Bean, N.H and Griffin, P.M 1990 Foodborne disease outbreaks

in the United States, 1973–1987: Pathogens, vehicles, andtrends J Food Protect 53:804–817

Bean, N.H., Griffin, P.M., Goulding, J.S., and Ivey, C.B 1990a

Fo o db or n e d i seas e o ut b r eak s, 5- y ea r su mma r y,1983–1987 Mor Mortal Wkly Rep CDC Surveill.Summ Morb Mort Weekly Rep (MMWR) 39(SS-1):15–59

Bean, N.H., Griffin, P.M., Goulding, J.S., and Ivey, C.B 1990b

Fo o db or n e d i seas e o ut b r eak s, 5- y ea r su mma r y,1983–1987 J Food Protect 53:711–728

Bennett, J.V., Holmberg, S.D., Rogers, M.F., and Solomon, S.L

1987 Infectious and parasitic diseases In Closing theGap: The Burden of Unnecessary Illness, eds R.W Amlerand H.B Dull, pp 102–114 Oxford University Press,New York

Borlaug, N.E 1981 Using plants to meet world food needs In

Future Dimensions of World Food and Population, ed

R.G Woods, pp 101–153 Westview Press, Boulder, CO

CAST 1994 Foodborne pathogens: Risks and consequences

Task Force Report No l22 Council for Agricultural ence and Technology, Ames, IA

Sci-Deshpande, S.S 1992 Food legumes in human nutrition: A

per-sonal perspective CRC Crit Rev Food Sci Nutr

32:333–363

Doull, J and Bruce, M.C 1986 Origin and scope of toxicology

In Toxicology: The Basic Science of Poisons, 3rd ed., eds

C.D Klaassen, M.O Amdur, and J Doull, pp 3–10 millan, New York

Mac-Esrey, S.A and Feachem, R.G 1989 Interventions for the

Con-trol of Diarrheal Diseases Among Young Children motion of Food Hygiene World Health Organization,Geneva

Pro-Garfield, E 1990 Journal citation studies 53 Agricultural

sci-ences: Most fruitful journals and high yield researchfields Curr Contents 21(51):3–10

Gossel, T.A and Bricker, J.D 1984 Principles of Clinical

Toxi-cology Raven Press, New York

Hodgson, E 1987 Introduction to toxicology In Modern

Toxi-cology, eds E Hodgson and P.E Levi, pp 1–22 Elsevier,New York

Hodgson, E and Levi, P.E 1987 Modern Toxicology Elsevier,

New York

Holmstedt, B and Liljestrand, G 1981 Readings in

Pharmacol-ogy Raven Press, New York

Kaferstein, F.K., Motarjemi, Y., Moy, G.G., and Quevado, F

1999 Food safety: A worldwide public issue In tional Food Safety Handbook, eds K van der Heijden, M

Interna-Younes, L Fishbein, and S Miller, pp 1–20 Marcel ker, New York

Dek-Klaassen, C.D 1986 Principles of toxicology In Toxicology:The Basic Science of Poisons, 3rd ed., eds C.D Klaassen,M.O Amdur, and J Doull, pp 11–32 Macmillan, NewYork

Loomis, T.A 1978 Essentials of Toxicology 3rd ed Lea & biger, Philadelphia

Fe-Manahan, S.E 1992 Toxicological Chemistry Lewis Publishers,Boca Raton, FL

Motarjemi, Y., Kaferstein, F., Moy, G., and Quevado, F 1993.Contaminated weaning food A major risk factor for diar-rhea and associated malnutrition Bull World Health Or-gan 71(1):79–92

Norling, B 1994 Food poisoning in Sweden: Results of a fieldstudy Report No 41/94 National Food Administration,Uppsala, Sweden

Notermans, S and Hooenboom-Verdegaal, A.S 1992 Existingand emerging foodborne diseases Int J Food Microbiol.15:197–205

Notermans, S and van de Giessen, K 1993 Foodborne diseases

in the 1980s and 1990s Food Control 4(3):122–124.Sperling, F 1984 Toxicology: Principles and Practice JohnWiley, New York

Todd, E.C.D 1989 Preliminary estimates of costs of foodbornediseases in Canada and costs to reduce salmonellosis J.Food Protect 52:586–594

WHO 1984 The role of food safety in health and development:

A report of a Joint FAO/WHO Expert Committee on FoodSafety Technical Report Series No 705 World Health Or-ganization, Geneva

WHO 1994 Program for Control of Diarrheal Diseases 9th gram Report 1992–1993 World Health Organization,Geneva

Pro-Wittwer, S.H 1980 The shape of things to come In The Biology

of Crop Productivity, ed P.S Carlson, pp 413–436 demic Press, New York

Principles of Toxicology

2.1 INTRODUCTION

A poison or toxicant is a chemical that is harmful to living

organisms because of its detrimental effects on tissues,

or-gans, or biological processes Any chemical may be a

poi-son at a given dose and route of administration Three

factors primarily influence the toxicity of any chemical to

a given species: the toxic substance itself and the matrix in

which it is present, the circumstances of exposure, and the

organism and its environment

Usually, an experimentally determined acute oraltoxicity expression, such as an LD50 value, which is the

dose required to kill half of test subjects, is used to express

the toxic potential of any given chemical Such estimates,

however, are not an absolute description of the

com-pound’s toxicity in all individuals or across different

spe-cies They neither assess the inherent capacity of the

compound to produce an injury nor reflect the victim’s

ability to respond in a manner other than predicted Hence,

quantitative estimates of toxicity in terms of mortality are

usually not good parameters for toxicity measurements

Much more widespread than fatal poisoning, andcertainly more subtle, are various manifestations of mor-

bidity or unhealthiness Morbidity can be manifested in

several ways Whereas the effects on vital signs are

obvi-ous, it is the subtle effects that are not life threatening per

se but nonetheless are responsible for minor health

ail-ments that ultimately cost millions of dollars in terms of

treatment expenses and loss of productivity In some

in-stances, a toxic response may not be observed for years It

is therefore essential to distinguish acute toxicity, which

has an effect soon after exposure, and chronic toxicity,

which has a long latency period

In practical situations, therefore, the critical factor isnot the intrinsic toxicity of a chemical, but rather the risk

or hazard associated with its use In food science and trition, it is especially important to understand the con-cepts of relative risks and safety, hazard, and toxicityassociated with the consumption of foods Risk is theprobability that a substance will produce harm under spec-ified conditions Absolute safety, in contrast, is the assur-ance that damage or injury from use of a substance isimpossible However, as discussed in Chapter 1, absolutesafety is virtually unattainable Hence, the concept of rela-

nu-tive safety has been proposed (Hall, 1988, 1991) Relanu-tive

food safety then can be defined as the practical certainty

that injury or damage will not result from the consumption

of food or ingredients used in food processing in a able and customary manner and quantity

reason-Food safety, however, does not refer to the food self, but also to the people consuming it For example,foods considered safe for most people when used in a rea-sonable and customary manner and quantity can be ex-tremely toxic, even lethal, to certain sensitive or allergicindividuals

it-The concepts of toxicity and hazard are also relevant

to any discussions of food safety Toxicity, as defined

ear-lier, is the capacity of a chemical to produce harm or injury

of any kind (chronic or acute) under given conditions.Generally, for humans, any deviation from normal is con-sidered as a possible negative effect, even though thechange may seem to be positive, such as increased growth

rates or enhanced nutrient absorption The change is

as-sumed to be negative until proved beneficial Hazard is the

relative probability that such harm or injury will result

when the substance is used in a proposed manner and

quantity Assessments of whether a food or ingredient is

safe should not be based on its inherent toxicity alone, but

on whether or not a hazard is created

The inability to distinguish between toxicity andhazard as associated with the consumption of foods, espe-

cially by the general public, often results in inaccurate

as-sessments of the relative risks and safety of our food

supply Both the regulations and the way that they are

ap-plied by the regulatory agencies often reflect the public

at-titudes toward particular types of chemicals and specific

kinds of risks The way that people generally perceive risk

is quite different from the way scientists analyze risk, and

this dichotomy has led to conflicts in the public policy

arena Generally, people tend to view catastrophic risks,

e.g., airplane crashes, as greater than ordinary ones, e.g.,

automobile accidents Similarly, synthetic chemicals are

often viewed as riskier than natural ones; voluntary risks,

such as smoking, as less significant than involuntary ones,

such as air pollution; and those with immediate effects as

less of a risk than those with delayed effects Some of

these factors are summarized in Table 2.1

The question of what constitutes an acceptable risk

is also a matter of judgment Such decisions are

multifac-eted and complex and involve a balance of risk and

bene-fit High risks may be acceptable in the use of lifesaving

drugs but be unacceptable for food additives Klaassen

(1986a) has suggested the following factors that must be

considered in determining an acceptable risk:

• Benefits gained from use of the substance

• Adequacy and availability of alternative stances to meet the identified use

sub-• Anticipated extent of public use

• Employment considerations

• Economic considerations

• Effects on environmental quality

• Conservation of natural resourcesPublic perception apart, several biological factorsmodify the response of a species to the toxic agent Real-ization of these factors is intrinsic to our fundamental un-derstanding of the principles of toxicology This forms thebasis of this chapter For a more detailed treatment of thesubject, the readers are advised to consult several excellenttextbooks and monographs (Loomis, 1978; Hodgson andGuthrie, 1980; Timbrell, 1982; Gossel and Bricker, 1984;Klaassen et al., 1986; Hodgson and Levi, 1987; Kamrin,1988; Marquis 1989; Manahan, 1992; Stine and Brown,1996; Niesink et al., 1996)

2.2 TYPES AND CIRCUMSTANCES OF EXPOSURE

The exposure of an organism to a toxic substance is ofprime importance in toxicology In this regard, both theduration of exposure per incident as well as the frequency

of exposure need to be considered The rate of exposure,inversely related to the duration per exposure, and the totalperiod over which the organism is exposed are primarilysituational variables Other factors that affect the toxicity

of a substance include the dose, toxicant concentration, theexposure site, and the route of absorption

It is important to understand the differences betweenacute and chronic poisonings or exposures and the acuteand chronic effects or symptoms Acute and chronic poi-sonings differ in the number and duration of exposures tothe toxicant The toxic effects manifested as a result ofpoisoning may be either local, i.e., confined to a specifictissue or organ, or systemic Generally, acute local expo-sure occurs at a specific location as single exposure to thetoxicant It may occur over a period of few seconds to afew hours and may affect the exposure site, particularlyskin, eyes, or mucous membranes Similar tissues or or-gans can also be affected by chronic local exposure How-ever, the time span for the manifestation of toxic effectscould be several months or even years

As compared to local exposures, systemic exposuresare usually manifested in toxic symptoms or effects in tis-sues or organs that are remote from the entry site Thustoxicants may enter the body by inhalation or ingestionand affect organs such as the liver The acute and chronic

Table 2.1 Public Perception of Risk

Source: Kamrin (1988).

Criteria

Characteristics perceived as lower risk

Characteristics perceived as higher risk

Effect manifestation Immediate Delayed

Severity (number of people

affected per incident)

Ordinary CatastrophicControllability Controllable Uncontrollable

systemic poisonings primarily differ in that the exposure

occurs over a prolonged period in the latter case

It is also likely that an acute exposure or poisoningmay result in chronic symptoms Thus, a single exposure

to potent carcinogens, such as aflatoxins or nitrosamines,

may result in the chronic symptom of cancer, whereas a

chronic exposure to cyanide in sufficient dose always

re-sults in acute symptoms Therefore, the terms acute and

chronic, when used to describe symptoms, refer to the

duration and reversibility of the symptoms An acute

symptom is of short duration, usually severe, and

gener-ally reversible after removal of the toxicant Chronic

symptoms, in contrast, are prolonged and persist even after

removal of the toxicant For example, some

organophos-phate pesticides can produce chronic paralysis (Hays,

1972) Liver carcinogens in small doses may result in

hepatic cancer but acute hepatic damage in large doses

Generally, exposures to toxicants between 24 hr and 90

days are usually referred to as prolonged intoxication

(Concon, 1988)

The symptoms of exposure or poisoning may be mediate or delayed They are influenced by factors such as

im-dose, type of compound, and route of contact Toxic

com-pounds, which produce immediate symptoms, may be

eas-ily identified as the cause of intoxication and, therefore,

can be avoided In contrast, those with delayed symptoms,

especially if the effects do not appear until after several

months or years, are not easily identified Many food

poi-sons fall under the highly delayed category These

com-pounds, therefore, are of concern because of their possible

role in modern epidemic diseases whose causes have been

difficult to establish Furthermore, when symptoms are

de-layed, antidotal therapy, assuming that it is known, may be

difficult to administer unless the symptoms have been

cor-related with those of a particular toxicant Even so,

anti-dotes are of little value when the appearance of symptoms,

such as cancer and other effects indicating structural tissue

damage, is markedly delayed

Although chronic symptoms are not necessarily due

to the accumulation of the poison or toxicant in the tissues,

there are toxicants that accumulate in the tissues to toxic

levels, resulting in chronic delayed toxic symptoms Some

well-known examples of such toxicants include heavy

metals, such as lead and mercury, and organochlorine

pes-ticides such as dichlorodiphenyltrichloroethane (DDT)

Similarly, some toxicants do not elicit chronic soning For example, many of the neurotoxins that are ex-

poi-tremely powerful poisons, e.g., the botulinum exotoxin,

may progress from the no effect level to the lethal level

without passing through the chronic range In contrast,

several carcinogens at the lower doses initiate

carcinogen-esis but produce acute hepatic or renal damage when ministered at high doses

ad-2.2.1 Exposure Assessment

Because the problem of chronicity has significant bearing

on public health, it is essential that those compounds ing this property be identified and their other toxicologicalproperties established Therefore, some quantitative mea-sure of the chronicity of a compound is important How-ever, the determination of chronicity has the inherentweakness in that it is based on data obtained from experi-mental animals with short life spans Therefore, a majorproblem of validly extrapolating such data to humans re-mains Nevertheless, such indices of chronicity may be po-tentially useful in serving as a guide in identifying thosecompounds, which may behave similarly in humans.Defining the exposure to a toxic agent is thus essen-tial in assessing the significance of toxicological tests aswell as understanding the risks to both humans and otherorganisms There is no one uniform, well-established pro-cedure for expressing exposure Some commonly used ex-pressions are summarized in Table 2.2 For proper riskassessment, the units in which exposure is expressed must

hav-be compatible with those that toxicologists commonly use

to report the results of their experiments

The toxic pattern of exposure is also critical To terpret the toxicological significance of an exposure, thefollowing factors need to be considered (Brown andBomberger, 1982; Brown, 1987):

in-1 Duration of each exposure, if not instantaneous

2 Frequency with which the exposure is repeated

3 Variation of exposure level within each period

mg Inhaled/kg body weight

mg Ingested, total

mg Ingested/kg body weightRate of intake or exposure mg/kg Body weightConcentration in body tissue mg/ml Serum

Several possible time patterns of exposure of an dividual to a toxicant are shown in Figure 2.1 The short-

in-term averages are generally appropriate for assessing acute

toxicity responses, whereas the long-term annual average

might be more appropriate for assessing chronic toxicity,

such as carcinogenic or mutagenic potential of a given

tox-icant The latter is especially important when a linear

dose-response relationship is seen

Finally, for the control of various sources of sure for risk assessment analysis, one needs to determine

expo-how much exposure is derived from each source In this

regard, the following factors need to be defined for a

com-plete characterization of exposure to any given toxicant:

• Specification of levels of exposure

• Specification of route(s) of exposure

• Distribution of exposures over time

• Distribution of exposures over geographicalregion

• Distribution of exposure over segments of lation at risk

popu-• Distribution of exposures over various sourcesDetailed information on the methodology used forassessing the exposure to toxicants and the accompanying

risk-benefit/safety analysis is supplied in several reviews

(Brown and Bomberger, 1982; Brown and Suta, 1982;

Klaassen, 1986a; Brown, 1987; Kamrin, 1988) An

excel-lent monograph is also available on the principles of data

interpretation including the statistical techniques used for

such purposes (Tardiff and Rodricks, 1987)

2.3 ROUTES OF TOXICANT EXPOSURE AND ABSORPTION

To exert a toxic effect, a compound must have contact withthe biological system under consideration The toxicantmay exert a local effect at the site on initial exposure, but itmust penetrate the organism in order to have a systemic ef-fect In fact, one of the main factors that influence the dose

at the site of action, or the effective dose, is the route of

exposure or the way in which the individual was exposed.Furthermore, the manner by which a potentially toxicchemical is introduced into the body can influence thetime of onset, intensity, and duration of the toxic effects.The route of exposure may also predict the degree of tox-icity and possibly the target systems that are most readilyaffected

Chemicals may be introduced into the complex logical organisms by a variety of routes The major routes

bio-of accidental or intentional exposure to toxicants bio-of mans and other animals include the skin (percutaneousroute), the lungs (inhalation, respiration, pulmonaryroute), and the mouth (ingestion, oral route) (Figure 2.2).Other minor routes of exposure include rectal, vaginal, andparenteral The parenteral route, viz., intraperitoneal, in-tramuscular, intravenous, or subcutaneous) is primarilyconfined to the administration of therapeutic agents.The chemical and physical properties of each com-pound largely determine the route by which intentional oraccidental exposure occurs The pulmonary system is mostlikely to take in toxic gases or very fine, respirable solid orliquid particles In other than a respirable form, a solidusually enters the body orally The percutaneous or dermalroute is important in the absorption of liquids, solutes insolution, and semisolids through the skin

hu-The site of entry of a toxicant is an important factor

in the manifestation of final toxic effects Thus, pounds taken by the oral route may be hydrolyzed to less(or sometimes more) toxic metabolites when exposed tothe acid conditions in the stomach The intestinal micro-flora may also change the nature of the compound by me-tabolism and thereby affect the toxicity outcome

com-The site of entry is also important to the final sition of the compound Thus, absorption through the skinmay be slow and result in initial absorption into the pe-ripheral circulation Absorption from lungs, in contrast, isgenerally rapid and exposes major organs very quickly.Compounds absorbed from the gastrointestinal (GI) tractafter oral exposure first pass through the liver, where theymay be extensively metabolized Irrespective of the route

dispo-of exposure and absorption, there is always a potential forthe toxicant to be absorbed into the bloodstream If this oc-curs, the toxicant is then transported throughout the body,

Figure 2.1 Time patterns of exposure to toxicants: A,

continu-ous; B, intermittent; C, cyclic; D, random; E, concentrated

(Adapted from Brown and Bomberger [1982].)

thereby potentially exposing all the organs and tissues If

one or more of these parts of the body are more sensitive

than the site of entry, more severe toxic effects may occur

to that organ or tissue Furthermore, if the toxicant or its

metabolites remain in the blood circulation for a long

pe-riod, the tissues and organs are exposed to them

repeat-edly Thus a single external exposure may lead to repeated

internal exposures and possibly toxicity to a number of

or-gans and tissues

Once the toxicant is absorbed and enters the stream, it may undergo several metabolic changes For ex-

blood-ample, it may be excreted or stored in body tissues as is, or

it may interact with other body chemicals and be altered in

some way In the latter instance, since the metabolism is

not 100% efficient, one or more metabolites may be

gener-ated from the parent toxic compound All of the

metabo-lites then undergo fates similar to that of the original

absorbed material Thus exposure to one chemical may

re-sult in the excretion or storage of several different

chemi-cals as well as the potential for a variety of toxic effects It

should be emphasized that these are usually not either/or

possibilities—the absorbed substances and their

metabo-lites are often partially excreted, partially stored, and

par-tially available to produce adverse effects (Kamrin, 1988)

As a general rule, a chemical or toxicant injected bythe intravenous parenteral route would be expected to be

the most toxic Administered by other routes, the

approxi-mate descending order of toxicity would be inhalation >

intraperitoneal > subcutaneous > intramuscular >

intrader-mal > oral > topical Of course, not all of these routes areimportant in food toxicology Nonetheless, the salient fea-tures of various routes of toxicant exposure and absorptionare briefly described in the following sections However,because of its obvious importance in food toxicology, amajor emphasis is placed on the oral route of toxicant ab-sorption involving the GI tract

2.3.1 Percutaneous Exposure

The simplest and most common exposure of humans andanimals to exogenous chemicals of all types is exposurethrough accidental or intentional contact of the chemicalwith the skin Toxicants can enter the skin through epider-mal cells, sebaceous gland cells, or hair follicles By farthe greatest area of the skin is composed of the epidermalcell layers, and most toxicants absorbed through the skinenter through epidermal cells Despite their much smallertotal areas, however, the cells in the follicular walls and insebaceous glands are much more permeable than epider-mal cells (Manahan, 1992)

The skin is a complex, multilayered tissue ing approximately 19,000 cm2 of surface in an average hu-man and contributing approximately 10% of the bodyweight (Guthrie and Hodgson, 1987a) It is a membranethat is relative impermeable to most ions as well as aque-ous solutions However, it is permeable to a large number

compris-of toxicants in the solid, liquid, or gaseous phase

Figure 2.2 Major sites of exposure, metabolism and storage, routes of distribution, and elimination of toxic substances in humans

Although skin is the most readily accessible organ toall forms of chemicals, it is also an efficient barrier to most

environmental toxins The major barrier to dermal

absorp-tion of toxicants is the stratum corneum, or the outermost

horny layer composed of highly keratinized cells The

per-meability of the skin is inversely proportional to the

thick-ness of this layer It varies by location in the body in the

order soles and palms > abdomen, back, legs, arms >

geni-tal (perineal) area Disruption of the stratum corneum

essentially removes all but a superficial deterrent to

pene-tration, since the two other main areas of skin, viz., the

dermis and the subcutaneous tissue, offer little resistance

to penetration Therefore, breaks in epidermis due to

lacer-ation, abrasion, or irritation increase the permeability, as

do inflammation and higher degrees of skin hydration

However, in order to reach systemic circulation, the toxic

compound still has to traverse several layers of cells; in

contrast, in the GI tract, only two cell layers separate the

toxicant from the bloodstream

Compounds, which are well absorbed ously, are generally very lipid-soluble In general, gases

percutane-penetrate quite freely through the epidermal tissues,

liq-uids less freely, and solids that are insoluble in water

prob-ably are incapable of penetrating to a significant degree

Penetration of a toxicant via the percutaneous route is also

time dependent and a function of concentration gradient

2.3.2 Pulmonary/Inhalation Route

The pulmonary system is the site of entry for numerous

toxicants Absorption via the lungs is an important route

for toxic gases, volatile solvents, and aerosols and, in some

cases, airborne particles The pulmonary route can greatly

accentuate the expected onset of toxicity for a given

com-pound for two reasons The rich capillary exchange at the

deeper lung recesses causes a toxicant at the lung surface

to be separated by only 1–2 µm from the circulation,

en-abling exchange of gases to occur in seconds or less In

ad-dition, the surface area of lungs (about 50–100 m2) is some

50 times the area of skin

Because of its unpreventable contact with nated air, the respiratory system has also developed nu-

contami-merous mechanisms to avoid many airborne substances

Particles can be trapped in the upper respiratory or

na-sopharyngeal region Those deposited in the

tracheo-bronchiolar region are cleared upward by the mucus

blanket In addition to upper pathway clearance, lung

pha-gocytosis is very active in both upper and lower pathways

of the respiratory tract and may be coupled to the mucus

cilia Phagocytosis may also direct engulfed toxicants into

the lymph

2.3.3 Oral Route

The oral route is a major site of entry into the body formany toxic compounds Food additives, food toxins, lick-ing or rubbing, and airborne particles excluded from pas-sage to the alveoli and returned to the glottis are amongpotential avenues for accidental ingestion

The GI tract can be regarded as a tube through thebody from the mouth to the anus (Figure 2.3) Although it

is within the body, its contents are essentially exterior tothe body fluids Therefore, toxicants in the GI tract canproduce an effect only on the surface of the mucosal cellsthat line the tract Any systemic effect of toxicant ingested

by the oral route, therefore, requires its absorption throughthe mucosal cells that line the inside of the GI tract.Toxicants can be absorbed throughout the GI tract,including the buccal cavity and rectum However, because

of short residence times, most substances are not readilyabsorbed in the mouth or esophagus The stomach is thefirst part of the GI tract where substantial absorption andtranslocation to other parts of the body may take place.Because of the stomach’s low pH (about 1.0), absorptionthrough stomach is dependent on the amount of nonion-ized form available Therefore, substances that are weak

Figure 2.3 Schematic diagram of the alimentary canal and sociated structures in the human A, parotid gland (salivarygland); B, pharynx; C, sublingual gland (salivary gland); D, sub-mandibular gland (salivary gland); E, esophagus; F, cardiacsphincter; G, stomach; H, liver; I, gallbladder; J, pancreas; K,duodenum; L, splenic flexure of transverse colon; M, hepaticflexure; N, transverse colon; O, descending colon; P, ascendingcolon; Q, jejunum; R, caecum; S, appendix; T, ileum; U, sigmoidcolon; V, rectum; W, anus

as-acids (i.e., ionic at pH near 7.0 and above) are neutral in

stomach so that they readily traverse the stomach walls In

some cases, absorption is affected by stomach contents

other than HCl These include food particles, gastric

mu-cin, gastric lipase, and pepsin

Compared to gastric absorption, intestinal tion is extensive because of the presence of microvilli,

absorp-which provide an extremely large surface area (Figure

2.4) The pH of the contents of the small intestine is close

to neutral, so that weak bases that are charged in the acidic

environment of the stomach are uncharged and absorbable

in the intestine Intestinal contents are moved through the

intestinal tract by peristalsis, which has a mixing action on

the contents and enables absorption to occur along the

length of the intestine

There is also significant absorption of compounds inthe colon The epithelium of the colonal lumen behaves

much as the small intestine does The colon is also the

ma-jor site of water absorption Thus, the dragging effect is

probably highly operative in this part of the GI tract

Fur-thermore, the metabolic activity of the colon microflora

may change the absorbability as well as the toxicological

properties of a compound Their effects in transforming

the acidity of this organ’s secretion as well as the acid-base

and lipid solubility properties of the compound itself by

metabolic degradation may transform the toxicological

characteristics of the food altogether

Toxicants ingested by the oral route can also enterthe bloodstream via the enterohepatic circulation system

(Figure 2.2), which comprises the

intestine-blood-liver-bile loop A toxicant absorbed through the intestine goes

either directly to the lymphatic system or to the portal

cir-culatory system The latter carries blood to the portal vein

that goes directly to the liver The liver serves as a ing organ for xenobiotics, subjecting them to metabolicprocesses that usually reduce their toxicities, and secretesthese substances or their metabolites back to the intestine.For some toxicants, there are mechanisms of active excre-tion into the bile in which the substances are concentrated

screen-by one to three orders of magnitude over their levels in theblood Other substances enter the bile from blood simply

by diffusion The importance of enterohepatic circulation

is discussed in greater detail in the section dealing with theexcretion of toxicants in this chapter

The presence and type of food in the stomach canmodify the absorption of a toxicant A meal rich in protein

or fat usually delays absorption Carbonated beverages crease the rate of intestinal absorption by increasing gas-tric emptying time, with evolution of carbon dioxide.Ingestion of a concentrated chemical frequently causes adecrease in absorption as a result of gastric irritation andconstriction of the pyloric sphincter On the positive side,the oral route of intoxication may provide the body with achance to metabolize the ingested toxicant readily

in-2.4 MECHANISMS OF TOXICANT ABSORPTION

The toxicity of a chemical is dependent on the dose ministered However, it refers not to the dose adminis-tered, but rather to the concentration of the toxic chemical

ad-in the target organ To exert its toxic action, a chemicalmust be absorbed in the biological tissue or organ If thefraction of the chemical absorbed is low or the rate of ab-sorption is low, then only a low concentration of the chem-ical in the target organ may be obtained, resulting in little

or no toxicity In this section, various mechanisms of cant absorption are briefly described Again, a specialemphasis is placed on the mechanisms of intestinalabsorption

toxi-A toxicant may pass through a number of barriersbefore achieving a sufficient concentration in the organwhere it produces its characteristic toxic effects These in-clude membranes of a number of cells In all cases, themembranes of tissue, cell, and cell organelle are basicallysimilar They comprise a bimolecular layer of lipid mole-cules coated on each side with a protein layer Certainbranches of the protein layer appear to penetrate the lipidbilayer, and others extend through the membrane

The lipid portion of the membrane consists rily of phosphatidylcholine, phosphatidylethanolamine,and cholesterol The fatty acids of the membranes do nothave a rigid crystalline structure and at physiologicaltemperatures are quasi-fluid in character The fluidity of

prima-Figure 2.4 Schematic drawing of the lining of the small

intes-tine LP, lamina propria

the membranes is largely determined by the structure and

relative proportion of unsaturated fatty acids When the

membranes contain more unsaturated fatty acids, they are

more fluidlike, and active transport (defined later in this

section) is more rapid (Klaassen, 1986a; Guthrie and

Hodgson, 1987a)

The mechanism of the movement of toxicants acrossmembranes is a poorly researched area A toxicant may

pass through a membrane by one of two general processes

(Klaassen, 1986a): diffusion or passive transport of the

chemical, in which the cell expends no energy in its

trans-fer, and specialized transport, in which the cell takes an

ac-tive part in the transfer of the toxicant through the cell

membranes In spite of these processes, the intestinal

mu-cosae are relatively impermeable tissues to many

sub-stances, including various electrolytes, many organic

compounds, and water-soluble macromolecules such as

starches, pectins, and other heteropolysaccharides and

hy-drocarbons (Crane, 1979; Henning, 1979; Concon, 1988)

2.4.1 Passive Transport

Most toxicants cross membranes by simple diffusion

Sim-ple diffusion of compounds with appropriate water/lipid

partition coefficients largely determines the rate of

toxi-cant movement However, the nature of the mucosal

mem-brane is such that even passive or simple diffusion of

compounds is selective Six factors primarily govern the

passive diffusion of substances: (a) Fick’s law, (b)

molecu-lar size, (c) lipid solubility, (d) degree of ionization, (e)

“drag” effect or bulk flow of the absorption of water, and

(f) the Donnan distribution effect Their relationship to the

movement of toxicants across the membrane barriers is

briefly described in the following sections

Fick’s Law

Many small hydrophilic compounds diffuse across cell

membranes through aqueous channels For molecules that

can easily pass through these pores, Fick’s law can predict

their rate of diffusion

J = KA (C e – C i )/h

= PA (C e – C i)Where

J = rate of diffusion

k = diffusion coefficient

A = area of surface diffusion

C e , C i= concentrations of the solute outside and

inside the cell, respectively

P = K/h, permeability coefficient

Fick’s law holds quite well for nonelectrolytes such

as urea However, an additional mechanism is also tive with substances such as fructose, mannose, and xy-lose, even though their absorption also appears to followFick’s law These sugars appear to be absorbed by facili-tated diffusion Simple diffusion through the cell mem-brane obeying Fick’s law generally cannot take placeagainst a concentration gradient and is not inhibited bymetabolic inhibitors Furthermore, there is no competitiveabsorption with other substances (Schanker, 1961; Levine,1970; Concon, 1988)

opera-Molecular Size

The molecular size of a chemical also influences the rate

of intestinal absorption Generally, an inverse relationship

is observed as the rate of absorption decreases with creasing molecular size However, several factors influ-ence the effect of molecular size on the absorption of thesecompounds In this regard, lipid solubility and ionizationeffects are more important in regulating the passive ab-sorption of compounds through the intestinal tract

in-Lipid Solubility

Some substances seem to be absorbed through the nal tract by passive diffusion even though their molecularsize greatly exceeds the postulated average pore size of theintestinal epithelium Thus a route of entry through the cellmembrane other than through the aqueous channels must

intesti-be available to these molecules These substances enter thecell by “dissolving” into the cellular membrane material,which is highly lipophilic in nature Thus, their rate of ab-sorption can be correlated to their solubility in oil or lipidsolvents Often, a linear relationship between lipid solubil-ity and absorbability is seen for compounds with similarchemical properties However, such correlation is oftenpoor for compounds that, in addition to their lipid solubil-ity characteristics, also behave as weak electrolytes inaqueous medium The rate of absorption of such com-pounds through the cell membranes can be markedly af-fected by a change in pH as described later

Degree of Ionization

Many toxic chemicals exist in solution in both ionized andnonionized forms The ionized form is often unable topenetrate the cell membrane because of its low lipid solu-bility, whereas the nonionized form may be sufficientlylipid-soluble to diffuse across the cell membrane Diffu-sion is thus primarily dependent on the lipid solubility ofthe nonionized form of the compound This phenomenon

is observed with a wide range of chemicals, including

weak acids and ammonium salts, and dyes This is

particu-larly true of chemicals for which absorption is not also

mediated by mechanisms other than simple diffusion

The degree of ionization of a compound, i.e., the tio of ionized and nonionized species (A–/HA), can be cal-

ra-culated from the Henderson-Hasselbalch equation

pH = pKa + log (A–/HA)where A– and HA are the ionized and nonionized species,

respectively The amount of weak organic acid or base in

the nonionized form is dependent on its dissociation

con-stant The pH at which an acid is 50% dissociated (ionized

= nonionized) is called its pK a It is defined as the negative

logarithm of the acid dissociation constant

Convention-ally, the dissociation constant for both acids and bases can

be expressed as a pKa

The degree of dissociation and ionization of a weakacid or base is dependent on the pH of the medium This

relationship for a weak acid, benzoic acid, and a base,

aniline, is shown in Figure 2.5 As the pH decreases, more

of the acid becomes nonionized The converse is true as

the pH is increased For an organic base like aniline, the

opposite occurs Thus, weak acids with pKa values of

around 2 are ionized in the small intestine at pH 6 to the

extent of approximately 10,000:1, and with weak bases

with pKa of around 9, the degree of dissociation is around

1000:1 Since the effective mucosal cell pH is around 5,

Schanker and coworkers (1958) postulated that for weak

acids with a pKa of 2, the degree of dissociation in the cell

is around 1000:1, whereas that of weak bases with pKavalues around 9 is about 10,000:1 These values appear to

be the minimal ratios that must be present for effective sorption of these compounds in the GI tract

ab-Since the lipid soluble form (nonionized) of a weakelectrolyte is the species that crosses cell membranes, or-ganic acids are more likely to diffuse across membraneswhen they are in an acidic environment, whereas a basicenvironment favors diffusion of bases across membranes.Thus, on the basis of the effect of pH in the absorption ofcompounds, the degree of toxicity of a weak acid or basemay be predicted from the pH of the GI tract

However, when the total area of absorptive surfaceand the rate of passage are considered, the pH effect maynot have a significant influence on the total amount ab-sorbed For example, the absorption of a weakly acidicsubstance may be highly favored in the stomach as the pH

of its contents decreases, but more of the substance may beabsorbed in the intestines, assuming a normal rate of emp-tying occurs This is because even though greater dissocia-tion occurs in the intestines, the pH here permits theexistence of a significant proportion of nonionized species,and the large surface area compared to that of stomach re-sults in greater total absorption Thus, even though the rate

of absorption in the intestines per unit area may be lessthan that in the stomach, the greater total surface area ofthe former results in greater total absorption Therefore,the large surface area of the intestines may obviate any ad-vantage or disadvantage that the ionization effect mayhave in the absorption of chemicals

The ionizing effects also explain the poor ability by passive diffusion in the small intestines of salts,strong acids, bases, and organic and essential cations such

absorb-as Fe2+, Fe3+, Mg2+, Ca2+, Zn2+, Mn2+, Co2+, and othertrace metallic ions, and the anions, such as PO43– and Cl–.Examples of organic anions that are poorly absorbed arecitrates, lactates, and tartrates The poor absorbability ofthese substances, in fact, forms the physiological basis fortheir cathartic effects Similarly, nonabsorbable sulfatesand phosphates are also cathartics

Therefore, for nutritionally essential minerals, cial mechanisms are necessary for absorption in order toprovide adequate amounts for the animal’s nutritional re-quirements However, even with these special mecha-nisms, absorption of these minerals is limited and iscontrolled by the body’s physiological requirements.These limitations afford a measure of protection for the or-ganism because above a certain level, these substances arequite toxic (Ulmer, 1977; Concon, 1988) The mechanismsinvolving active or carrier-mediated transport are de-scribed later in this section

spe-Figure 2.5 Effect of pH on the ionization of a weak organic

acid, benzoic acid (pKa = 4, curve A), and a weak organic base,

aniline (pKa = 5, curve B)

“Drag” Effect and Bulk Flow of the Absorption of Water

When water flows in bulk across a porous membrane, any

solute that is small enough to pass through the pores

flows with it Passage through these channels is called

fil-tration, since it involves bulk flow of water due to a

hy-drostatic or osmotic force Because such aqueous

channels in most cells are relatively small (4–10 nm),

only chemicals with molecular weight of 100 to 200

dal-tons can pass through (Schanker, 1961, 1964) Larger

molecules are excluded except in more highly porous

tis-sues (approximately 70-nm pore size) such as kidneys

and liver These allow molecules smaller than albumin

(molecular weight 68,000) to pass through Because

many toxicants are relatively large molecules, this

path-way is often of limited importance

The bulk flow of absorption of water follows seuille’s law:

Poi-V = 8 πr4Phl

where

V = rate of flow

r = radius of pore

P = hydrostatic pressure or osmotic pressure

h = viscosity of solution flowing through pore

l = length of pore

The extent of hydrodynamic flow across the brane of intestinal epithelium differs from what can result

mem-from the osmotic gradients across the membrane of the

small intestines This difference is explained on the basis

of the concept of bulk flow (Concon, 1988) The bulk flow

can increase transport of solutes through the pore by

“dragging” the molecules in the moving stream The

drag-ging effect may be expected to be greater at the time when

absorption by the various mechanisms is at full capacity,

so much so that the total osmolar concentration in the

plasma is much greater than that in the intestinal lumen In

this case, the greater the total rate of absorption, the higher

the total osmolar concentration of the plasma

Conse-quently, the dragging effect is also greater The dragging

effect will obviously be expected to be greatly increased

when the amount of fluid ingested is large, since, in

addi-tion to osmotic effects, the hydrostatic pressure is also

greater

However, because of the very nature of dragging andbecause of the relatively large molecules of many toxi-

cants the contribution of the dragging phenomenon to the

total absorption of toxicants is probably much less than

that from other mechanisms, such as the active transport

processes

Donnan Distribution Effect

A pH differential on each side of a membrane allows onlyundissociated particles to diffuse out The process, termed

the Donnan distribution effect, causes a net transfer of a

chemical to another compartment Thus, an ionizablechemical that is free to diffuse through a membrane disso-ciates in the compartment where such dissociation is fa-vored This ionization reduces the concentration of thediffusible chemical so that a concentration differential isestablished More of the diffusible chemical then migrates

to the compartment where it can be dissociated more vorably until equilibrium between the two compartmentswith respect to this chemical is attained

fa-The Donnan distribution effect may be a significantfactor governing the flow of a weak acid from the stomach(pH 2 to 4) or the small intestine (pH 4.5 to 6.5) to theblood (pH 7.35 to 7.45) The degree to which the Donnaneffect enhances the net transfer of a substance across the

GI mucosa is highly dependent on the pH differential tween the intestinal medium and the blood and the pKa ofthe substance An example of this type of distribution isshown by the study of Shore and associates (1957), whoinjected acidic and basic drugs intravenously into dogs Inthe gastric juice, where their ionization is favored, the con-centration of basic drugs at equilibrium increased to asmuch as 40 times that in the plasma In contrast, acidicdrugs, whose ionization is not favored, either were absent

be-or increased in concentrations to no mbe-ore than six-tenths

of those in the plasma Zawoiski and colleagues (1958)also observed that the concentration in the gastric juice of

an organic base injected into the dog increased as the pHdecreased

The Donnan distribution effect is apparently less fective in the transfer of weak bases, such as some alka-loids, from the GI tract to the blood This is becauseionization in the blood is less favorable and, hence, there is

ef-a compef-aref-atively smef-all, if ef-any, concentref-ation differentief-al.The Donnan effect also occurs if one of the chargedcomponents in one compartment is a macromolecule toolarge to diffuse through the membrane When this mole-cule dissociates, and one of the particles so formed isfreely diffusible, the movement of this particle to the op-posite side must be accompanied by a movement in the op-posite direction of a particle of the same charge tomaintain electrical neutrality Thus, this process may causethe transfer of a different substance to the compartmentcontaining the macromolecule If, on the other hand, thecompartment opposite that containing the macromoleculehas freely diffusible positive and negative ions and the ionsare the same as those associated with the macromolecule,then at equilibrium a net increase of these ions is found inthe macromolecule compartment This type of transfer of

charged particles, to a large extent, may not be applicable

in the case of the GI tract because charged particles are not

freely diffusible across the GI membrane Thus, it is

essen-tial that the charged substances that are important to life

are transported by special mechanisms

A net transfer of a substance can also result from theDonnan distribution effect, if in one compartment, a non-

diffusible substance, such as a protein, can bind the

diffus-ible molecule The movement of the diffusdiffus-ible substance

bound by the protein follows Fick’s law, since in effect, a

large concentration gradient is present as long as the

bind-ing capacity of the protein is not exhausted

2.4.2 Special Transport

There are a number of instances in which the movement of

a compound across a membrane cannot be explained by

simple diffusion or filtration because the compound is too

water-soluble to dissolve in the cell membranes and too

large to flow through the channels Thus, if the GI

absorp-tion were to rely solely on passive transport, the

limita-tions imposed on this process would exclude many

compounds, even those essential to the survival of the

organism Those compounds that meet the conditions

nec-essary for passive absorption in many cases cannot be

ab-sorbed at a rate commensurate with the needs of the body

Furthermore, the requirement of these substances by the

body may be such that their concentration in the lumen of

the GI tract may be lower than that in the blood Therefore,

even though the GI epithelium is an “open” membrane,

ab-sorption under these conditions is thermodynamically

im-possible Instead of absorption, leakage into the GI lumen

results Thus, for the transport of many essential

sub-stances, such as sugars, amino acids, and nucleic acids, as

well as some foreign molecules, the GI epithelium is

equipped with a number of special transport systems

The special transport mechanisms thus are mostmanifest in GI absorption These mechanisms attain much

greater importance in the elimination of toxicants, however,

in which special transport is important to the removal of

xe-nobiotics and their metabolites An important characteristic

of the special transport systems, when operable, is that they

allow movement of compounds with lesser lipid solubility,

compounds that would ordinarily be expected to move very

slowly through the highly lipophilic cell membranes These

processes are briefly described in the following

Active Transport

Active transport requires energy and permits the

absorp-tion of the compound even against a concentraabsorp-tion

gradi-ent The following features characterize an active transport

trans-3 The transport system is selective Therefore,certain basic structural requirements exist forchemicals to be transported by the same mech-anism with the potential for competitiveinhibition

4 The system requires the expenditure of energy

so that metabolic inhibitors block the transportprocess

Compounds that are actively transported across acell membrane are presumed to pass into the cell interior

by forming a complex with a macromolecular carrier, erally proteins, on one side of the membrane The complexsubsequently traverses to the other side of the membrane,where the compound is released The carrier moleculethen returns again to the original surface to repeat thetransport cycle

gen-Carrier proteins involved in the active transport cesses generally have specificities for certain kinds ofchemical groups and configurations (Crane, 1979) Thistype of active transport system can be inhibited by a vari-ety of compounds For example, the transport of sugarsand amino acids can be inhibited by cyanide, dinitrophe-nol, malonate, fluoroacetate, arsenate, and copper (Therand Winne, 1971)

pro-The carrier system can also be saturated This anism imposes a limit on the rate of absorption, eventhough the amount absorbed generally is a function of thedose Many exogenous compounds can also compete withthe carrier systems of essential nutrients, especially sub-stances that structurally resemble the nutrients and otherendogenous physiologically essential compounds

mech-The active transport of organic chemicals appears to

be closely associated with the sodium transport Thus,compounds that inhibit sodium transport also inhibit thetransport of the organic compounds (Ther and Winne,1971; Concon, 1988) The energy-requiring active trans-port mechanism can also be inhibited by interference inthe metabolic sources of energy Thus, compounds that areinhibitors of oxidative phosphorylation also inhibit activetransport of a variety of nutrients

Various sections of the GI tract seem to have specificpreference for the active absorption of a compound orgroups of compounds For example, sugars as well as theneutral amino acids are largely absorbed in the middle por-tions of the small intestine, whereas the basic amino acidsare absorbed equally in all parts of the small intestine(Concon, 1988) Bile salts and vitamin B12 are absorbed

mostly in the ileum; Ca , Fe , and Cl are absorbed

mostly in the upper small intestine In contrast, Na+

ap-pears to be equally absorbed in all parts of the small

intes-tine and colon; H+ is absorbed most in the ileum and

colon

The active transport process is of fundamental portance in toxicology It is involved in the elimination of

im-foreign compounds from the organism To transport

sub-stances out of the cerebrospinal fluid, the central nervous

system has two transport systems at the choroid plexus,

one each for organic acids and organic bases (Klaassen,

1986a) Likewise, the kidney has two active transport

sys-tems that eliminate foreign compounds from the body, and

the liver has at least four active transport systems, two for

organic acids, one for organic bases, and one for neutral

organic compounds

The active transport system itself is also potentially

a target of many toxic compounds, which inhibit the

pro-cess in one way or another From the nutritional

stand-point, active transport’s being subject to competitive

inhibition even among the nutrients is also relevant to the

toxic effects of nutrient excesses

Facilitated Transport

The facilitated transport mechanism is similar to active

transport except that in this carrier-mediated transport

sys-tem, the substrate is not moved against a concentration

gradient Also, this process does not require any energy

expenditure Hence, the term facilitated diffusion was

coined by Danielli (1954) The transport of glucose from

the GI tract into blood, from plasma into red blood cells,

and from blood into the central nervous system is thought

to occur through facilitated diffusion There is evidence

that facilitated transport may also apply to exogenous

chemicals (Fiese and Perrin 1969)

Similarly to active transport, facilitated diffusion isalso subject to saturation phenomena, competitive inhibi-

tion of similar compounds, stimulation by sodium ions,

and a temperature effect

Endocytosis

Pinocytosis (for liquids) and phagocytosis (for solids) are

specialized transport processes in which the membrane

in-vaginates or flows around a toxicant, allowing more ready

transfer across cell membranes This type of transfer

across cell membranes is important to removal of

particu-late matter from the alveoli by alveolar phagocytes and for

removal of some toxic substances from the blood by the

reticuloendothelial system of liver and spleen

A defect in the intestinal epithelium may enhancepinocytosis of macromolecules (Concon, 1988) This pro-

cess therefore explains the absorption of protein toxins andother toxic materials that otherwise would be excludedfrom the intestinal epithelium for reasons of molecularsize alone Since many toxic substances in foods are mac-romolecules, their toxicity is therefore related to the ability

of the small intestine to absorb them

2.5 FACTORS AFFECTING INTESTINAL ABSORPTION

The toxicity of compounds absorbed through the GI tract

is generally much less than that of compounds that gainentry through other routes This is because the GI tract im-poses certain limitations on their rates of absorption Mostsubstances absorbed in the GI tract must pass through theliver, where they can be metabolized to derivatives oflesser or greater toxicity In addition, several other factorscan influence the absorption of toxicant through the GItract Their importance in the manifestations of toxic ef-fects is briefly described

2.5.1 Effect of Blood Flow

Compounds that influence the blood flow generally alsoinfluence the rate of absorption Thus, vasoconstrictivedrugs such as serotonin, norepinephrine, and vasopressindiminish blood flow and consequently absorption of water

In contrast, ethanol, which increases the blood flow rate, isabsorbed at a rapid rate in the stomach (Magnussen, 1968).Blood flow can also influence absorption by its ef-fect on the supply of oxygen and other nutrients Thisproperty follows from the fact that active transport re-quires oxygen Indeed, there appears to be a critical bloodflow rate through the splanchnic area below which activetransport ceases (Robinson et al., 1964, 1966) The loss ofactive transport capacity of the intestinal epithelium is alsoobserved in intestinal ischemia Such loss of active trans-port capacity can be prevented by perfusion of the is-chemic tissues with solution saturated with oxygen.The draining effect of blood increases the rate of ab-sorption simply from a consideration of Fick’s law, sincethe removal of absorbed substances in the serosal side ofthe GI epithelium maintains a large concentration gradi-ent Normally, the rate of blood flow in the portal vein(Figure 2.2) is approximately 1.2 L/hr/kg, with a 30% in-crease in blood flow through the splanchnic area after ameal (Concon, 1988) Therefore, an increased absorption

of toxicant may result if it is ingested during a meal, suming that the pH is favorable and the toxicant is notbound to other components in the food As a corollary, adecrease in blood flow rate lowers the intestinal absorption

as-of toxicants Thus, a normal blood flow rate in the

intesti-nal epithelium is essential to maintenance of a normal rate

of absorption of chemical; compounds that increase the

blood flow generally displaying an enhanced toxicity

2.5.2 Effect of Lymph Flow Rate

The lymphatic flow rate, only about one-six hundredth to

one-thousandth of that of the blood, is important for highly

toxic substances, such as the botulinum toxin, that are

transported by the drag effect and bulk flow mechanism

However, very little is known about the effect of lymph

flow rate on the absorption of exogenous toxicants

2.5.3 Gut Motility and Emptying Time

Gut motility and the rate of passage and elimination of

food from the GI tract also influence the rate of absorption

of chemicals Higher absorption rates are observed with

increased residence times of food in the GI tract

There-fore, any conditions that decrease or increase gut motility

and emptying or passage time have a corresponding effect

on the toxicity of compounds

If smaller amounts of compounds are presentedslowly for absorption in the GI tract, there may be suffi-

cient time and several ways to dispose of the undesirable

compounds Consequently, a toxicologically insignificant

amount, if any, ultimately reaches its target organ or tissue

The emptying or passage time in the GI tract canalso influence the toxicity of compounds The intestinal

microflora play an important role in this regard For

exam-ple, a compound that is made toxic by action of the

intesti-nal microflora may show no toxic effects if it moves

slowly through the intestinal tract The reason is that only

small amounts unabsorbed by the intestine reach the

co-lon, where microflora are most abundant In contrast, a

substance may move rapidly in the GI tract for various

rea-sons (e.g., stimulation by cathartics), so that a fraction of

the toxic dose is absorbed The same reasoning applies to

stomach emptying for those compounds absorbed in the

il-eum or colon The rate of appearance of substances in the

colon also regulates their metabolism by the microflora,

influencing both their absorption and their toxicity

The gastric emptying time is influenced by the typeand volume of the meal, the acidity of the gastric content,

the neutralization process in the duodenum, and drugs

such as the pressor amines, norepinephrine, histamine, and

tyramine (Levine and Walsh, 1975; Holz, 1968; Concon,

1988) These drugs are found in many types of food

prod-ucts even though their concentrations are often too low to

affect normal intestinal function They also have potent

ef-fects on the motility of the intestines and colon (Holz,1968)

Microbial infections of the intestinal wall and otherdisease symptoms can also affect the intestinal motility.Such diseases produce a rapid transit time of intestinalcontents, as in diarrhea, or the opposite effect, as in consti-pation These conditions are extreme examples of contrast-ing effects of gut motility and transit time

Pathological conditions in the GI tract may also fect the integrity of intestinal mucosa and the toxicantabsorption For example, because many toxic substancesare lipid soluble and are absorbed and transported in asso-ciation with lipids via the lymphatic system, any mal-absorption of fats may have far-reaching toxicologicalimplications other than those involving these substancesdirectly

af-2.5.4 Chemical Factors Affecting Absorption

Chemicals may affect the absorption of compounds by(a) formation of insoluble precipitates, or complexes withspecific substances, or formation of chelates that facili-tate or inhibit absorption and solubilization; (b) competi-

ti on for bi ndi ng or c arrie r prot eins involved i nabsorption; and (c) modification of the motility or ab-sorptive capacity of the GI mucosa

Compounds such as phytic acid, oxalates, and sypol can form insoluble complexes with bivalent metalions, amino acids, and proteins Insoluble precipitates canalso be formed by phosphates, fatty acids, and alkalis (e.g.,antacids) Sometimes, chelation can also improve the ab-sorption of toxicants For example, citric acid can increasethe absorption of lead (Graber and Wei, 1974), and magne-sium improves the absorption of dicoumarol (Ambre andFischer, 1973)

gos-Competition for carrier proteins also influences theabsorption of toxicants In this regard, similarity in gen-eral structure may be sufficient to influence the absorp-tion of compounds Toxicants can also modify theabsorptive capacity of the intestinal mucosa by inter-action with its structural constituents Lectins, for ex-ample, bind strongly with specific receptors in cellmembranes The absorptive capacity of the intestinal mu-cosa may be affected by changes in the acidity of the in-testinal mucosa Thus, compounds that inhibit carbonicanhydrase cause a decrease in intestinal pH (Concon,1988) The general metabolic integrity of the GI mucosaltissues is thus essential to their structural and functionalstatus Any substance or condition that destroys the meta-bolic integrity of these tissues has an adverse effect on

their function and structure, thereby affecting the

absorp-tion of compounds

2.6 BIOLOGICAL TARGETS OF TOXIC

COMPOUNDS

A knowledge of the interactions of a toxicant with

spe-cific molecular targets, the role that molecular target

plays in the chemical dynamics of the cell, and the

re-sponse of the cell to either the presence of the toxicant or

the perturbations the toxicant elicits is fundamental to our

understanding of the manifestations of toxic effects In

this section, the potential molecular, subcellular, and

cel-lular targets available to a toxicant for interaction are

briefly described

2.6.1 Molecular Targets

The four basic macromolecules, viz., proteins, lipids,

car-bohydrates, and nucleic acids, involved in the dynamic

ex-ecution of living processes in the biological systems are

frequently the target of toxic compounds The small

me-tabolites of the cell are quickly and easily replaced after

modifications, since they are part of the flux of material

throughout the metabolic pathways In contrast, the

mac-romolecules are of complex biosynthetic origin Their

re-placement in the cellular system is energy intensive and

requires a dietary supply of precursors, such as essential

amino acids, fatty acids, and vitamins

Generally, interactions of toxicant with cules, especially cellular enzymes involved in important

macromole-metabolic pathways, often result in improper levels

(ex-cessive or deficient) of a cellular component This effect,

in turn, may produce a range of subtle but pervasive

ef-fects, varying from a disturbance of the osmotic strength

of the cell’s cytoplasm to the interruption of energy

metabolism In the cell, one perturbation may trigger

an-other, in a cascading series of reactions that may intensify

the potential for harm and, spatially and temporally,

ob-scure the initial triggering reaction At some point in the

series of reactions, the system is irreversibly altered, even

dies At present, our knowledge of such interactions at

cel-lular level is confined to those of a relatively few

well-characterized toxicants, especially those that are very

specific in action and are potent, and in which the

expo-sure produces an acute effect The toxicity of compounds

that either act chronically by a mechanism different from

that of their acute action or produce latent symptoms (e.g.,

the mutagens, carcinogens, and teratogens) is extremely

1 Structural proteins, e.g., collagen

2 Catalytic proteins, e.g., various enzymes

3 Carrier or storage proteins, e.g., hemoglobin,transferrin, ceruloplasmin

4 Informational or regulatory proteins, e.g., peptidehormones such as insulin or repressor proteins

5 Immunological proteins, e.g., immunoglobulinsinvolved in defense mechanisms

Specific proteins that may fill several roles simultaneouslyare not unusual

Toxicants primarily interact with the side chains ofamino acids that constitute the primary backbone of theproteins Because these side chains are also involved inand primarily determine the secondary and tertiary struc-tures of the protein, their interactions with toxicants candisturb protein structure Usually, a disturbance at anylevel of protein structure, especially enzymes, can alter theprotein’s catalytic or biological function

The relative order of nucleophilicity (i.e., capacity ofany atom containing an unshared pair of electrons or anexcess of electrons to participate in covalent bond forma-tion) relative to the major groups in biological moleculescan be summarized as follows:

R-S– > R-SHR-NH2 > R-NH3+R-COO– > R-COOHR-O– > R-OHR-OH = H-OHand finally

R-S– > R-NH2 > R-COO– = R-O–

Amino acids whose side chains are capable of ing with toxic chemicals are listed in Table 2.3 From thepreceding relationships it is obvious that the strongest nu-cleophile in protein molecules is the sulfhydryl group ofcysteine, particularly in the ionized, thiolate form Next inline are the amine groups in their uncharged, unprotonatedforms, including the α-amines at the N terminals, theε-amines of lysyl side chains, the secondary amines of his-tidine imidazolyl groups and tryptophan indole rings, and

react-the guanidino amines of arginine residues Finally, react-the

least potent nucleophiles are the oxygen-containing

ioniz-able groups, including the α-carboxylate at the C

termi-nals, the β-carboxyl of aspartic acid, the γ-carboxyl of

glutamic acid, and the phenolate of tyrosine residues These

side chains of amino acids in a protein molecule are

pre-dominantly involved in interactions with various toxicants

Coenzymes

Coenzymes are a class of biomolecules that participate in

several enzymatic reactions and are present in limited

con-centrations within the cell Their synthesis is complex, and

their replacement is in part dependent on the dietary

sup-ply of vitamins

The coenzymes themselves may be subject to directattack by toxicants For example, some symptoms of

heavy metal poisoning are similar to those of vitamin B1

deficiency Metalloproteins, especially the heme proteins

possessing the iron-containing protoporphyrin ring, are

also quite vulnerable to attack Cyanide poisoning of

cyto-chrome c oxidase, a heme-containing protein involved in

the terminal electron transport process during respiration,

is well known

Nucleic Acids

Nucleic acids are the building blocks of a cell’s genetic

ma-terial, viz., the deoxyribonucleic acid (DNA) and

ribonu-cleic acid (RNA) The DNA molecule offers a target of

considerable size for interaction with toxic chemicals The

introduction of an error in the DNA results in the loss of

quality or quantity of biological information The error

produces a faulty protein molecule or results in a level of

an RNA and/or protein species that is inappropriate to the

cell’s state of differentiation A permanent modification of

DNA is a mutation, which, if expressed, may lead to a

car-cinogenic and teratogenic event

Aside from the inhibition of enzymes involved intheir synthesis, toxic compounds may also affect DNA andRNA formation and function by reacting with the macro-molecules themselves For example, DNA replication,RNA translation, and consequently protein synthesis may

be interfered with by alkylation of the DNA or RNA

pu-rine or pyrimidine bases by N-nitroso compounds, such as

the nitrosamines

Lipids

Lipids primarily serve three cellular functions: storage,structural, and informational Triglyceride stores in thecell and the adipose tissue are mobilized in times of stress

or food deprivation to yield fatty acids and glycerol for ergy production This process is not regarded as an essen-tial function However, lipids are an integral part ofmembranes Their length, their degree of unsaturation, andthe nonlipid moieties attached to them essentially governthe permeability, excitability, and fusion properties of themembrane, as well as influence the activity of membrane-bound enzymes complexes Lipids, in the form of steroids,also serve as hormones

en-The most susceptible function to interactions withexogenous toxicants seems to be related to the lipid’s role

in membrane structure and function Free radicals ated by exogenous agents can cause oxidative changes inthe unsaturated fatty acid constituents of the membranes.Such oxidative changes may in turn lead to carcinogenesis,mutagenesis, and cellular aging mechanisms Disturbances

gener-in steroid metabolism may also lead to cancer

Carbohydrates

Carbohydrate polymers serve structural, recognition, andstorage functions in the cell Carbohydrate moieties on thesurface of a cell are also involved in regulation by thebody’s immune system Cells that have been transformed

to tumor cells display an altered carbohydrate surface terminant Generally, any substantial modification of car-bohydrate structure or function occurs by modification ofthe enzymes involved in carbohydrate metabolism Themolecular weight and the redundancy of their structure inthe cell simply render them too diffuse and inert to sufferattack directly by an exogenous chemical Thus, as com-pared to other macromolecules, carbohydrates are not sen-sitive or frequent targets of exogenous chemicals

de-2.6.2 Subcellular Targets

Many toxicants show little discrimination in their attack

on molecular targets in vitro but do elicit specific

patho-Table 2.3 Amino Acids with Side Chain Functionalities and

Their pKa Values

logical effects in vivo Thus the accessibility of a

parti-cular cellular structural component, as well as the

component’s influence on the integrity of one or another

organelle, determine the specificity of attack Organelles

possess enzyme systems specific to their purpose This

compartmentalization and specialization of function

explain in part the susceptibility of one organelle to a

toxicant to which another is impervious The availability

of such targets also explains why a toxicant elicits one

type of damage in one cell population but has a totally

different effect in another Some specific subcellular

targets available for the exogenous toxicants are

de-scribed next

Nuclei

Nuclei are the site of DNA and RNA synthesis Toxicants

can interfere with the synthesis of both DNA and RNA,

generally by altering the activity of the individual enzymes

involved

Mitochondria

The mitochondria of the cell are the sites of oxidative

phosphorylation and, therefore, are primarily responsible

for adenosinetriphosphate (ATP) synthesis A number of

toxicants, some ancient and notorious, attack various parts

of the mitochondrial oxidation system, producing low ATP

levels and disturbance of the redox state of the cell

Lysosomes

Lysosomes are intracellular vesicles of hydrolytic

en-zymes, including nucleases, phosphatases, and peptidases

The vesicle normally sequesters these potent hydrolases

from the cytoplasm A disruption of the lysosomal

mem-branes by interaction with toxicant releases the hydrolytic

enzymes to attack adjacent cell material

Endoplasmic Reticulum

The endoplasmic reticulum (ER) is the fine filigree of

in-tracellular membrane sheets that, upon cell disruption,

yield the microsomes The ER is divided into two basic

units, rough and smooth, the former masked by the

attach-ment of ribosomes The ER also contains electron transfer

enzymes responsible for oxidation of various lipophilic

compounds, including the steroids, long-chain fatty acids,

and exogenous compounds

Perturbation of the ER upon exposure to and action with a toxicant may result in a disturbance of the

inter-membrane and constituent enzyme activities, or a

pro-liferation of the ER structure and/or specific enzymes

at-tached to it The ability of the ER to metabolize foreign

compounds makes this subcellular system a frequent target

of toxicants The activity is particularly high in the liver,although the system exists to a smaller extent in mostcells In some instances, the products formed by the en-zyme system of the ER are more toxic to the cell than theparent compound Furthermore, since the enzymes of the

ER metabolize primarily endogenous substrates, unnaturalproliferation of this system may result in abnormal levels

of hormones, bile salts, and other normal metabolites.Thus an excess of enzyme activity can be as dangerous tothe biological system as a deficiency

mes-an influx of sodium mes-and calcium ions mes-and mes-an efflux of tassium ions A loss of soluble proteins from the cell mayfinally occur Although it is not possible to decide whether

po-to attribute the change in permeability po-to a primary attack

of the toxicant on the membrane directly, a loss of tive permeability by the plasma membrane is unquestion-ably one of the characteristics of a dying cell

selec-2.6.3 Cellular Targets

The susceptibility of a cell to a toxin primarily depends on

at least the following three factors:

1 The specialization of the cell, i.e., which ceptible organelles are preeminent in the cell’seconomy

sus-2 The distribution of the toxin within the body

3 The cell’s reaction to the presence of the toxin

An example of the first type, i.e., selective toxicitybased on cell specialization, is the sensitivity of the cells

of the myocardium to anoxia These cells depend rily on ATP generated by mitochondrial oxidative pro-cesses and, hence, are critically aerobic An interruption ofthe blood supply (ischemia) quickly produces cell death(an infarction) Another example of selective toxicitybased on specialization is the destruction of rapidly divid-ing intestinal crypt cells by DNA synthesis inhibitors, such

prima-as the nitrogen mustards

The distribution of a toxin in the body and its route

of entry and elimination are also important factors Since a

toxin rarely has homogeneous distribution, it is inevitable

that some cell populations suffer high exposure For

exam-ple, the red blood cells, because of their thorough exposure

to dissolved gases in the lungs and their high level of

he-moglobin, are primary targets of carbon monoxide

poison-ing The liver, in contrast, is infused with the blood

directly from the stomach and small intestine The

nutri-ents, as well as all ingested toxins, therefore, impact

ini-tially on this organ

Finally, what the cell can or cannot do with the toxinalso determines its relative sensitivity to exogenous toxi-

cants The liver has a large complement of enzymes in the

microsomes that can metabolize a wide range of

exoge-nous chemicals In many cases, the liver is successful in

eliminating or decreasing toxicity, but in some instances, it

may create a more toxic metabolite to its own detriment

Other cells, with less active microsomal enzymes, either

are less effective in dealing with a toxicant or are more

re-sistant to it, depending on whether metabolism deactivates

or activates that compound

It should also be emphasized here that the cell is markably resistant and can survive temporary disturbances

re-in its environment Similarly, durre-ing the course of tion, it has also developed several defensive mechanisms

evolu-to counterattack the effects of exogenous chemicals in logical systems Some of these mechanisms are described

bio-in Chapter 5 They are the emergency measures that ate with varying degrees of success and efficiency

oper-2.7 BIOCHEMICAL EFFECTS RESULTING IN TOXIC INJURY

Because the cell’s components are reactive chemicals, icants can react with its components and interfere in its op-eration Exogenous substances that interfere in the cell’s

tox-activity are called intrinsic toxicants Those native or

fa-miliar to the cell and toxic only when present in excess are

called relative toxicants (Concon, 1988) Many of the

compounds of the latter group are in fact essential to thenormal operation of the cell; others are metabolic by-products

As shown in Figure 2.6, a toxicant may be detoxified

by metabolic processes and eliminated from the body,made more toxic (toxified) by metabolic processes anddistributed to receptors, or passed on to receptors as a met-

Figure 2.6 Major steps involved in the overall process leading to toxic effects of chemicals

abolically unmodified toxicant In every biological

sys-tem, there are thus safeguards against the intrusion of

unwanted chemicals However, these safeguards are

im-perfect When they fail, toxic effects result As discussed

in the preceding section, toxic effects eventually indicated

by gross functional disturbances must necessarily initiate

injury at the molecular level Although the biochemical

mechanisms of toxicity of a large number of compounds

are still unknown, the biochemical bases of their

toxici-ties may include one or more of the following general

types

2.7.1 Deficiency of Essential Compounds

Nutrients that cannot be synthesized by higher living

forms necessarily must be supplied from external sources

The urgency of delivering these compounds to the cell

de-pends on their rate of usage and degradation, the extent of

cell storage, and the magnitude of their involvement in

en-ergy production, the cell’s primary need Nutrient

defi-ciencies in cell can occur either through the absence of

nutrients in foods, a primary deficiency, or failure of or

in-terference in their delivery or metabolism Such an internal

obstruction is termed a secondary deficiency One of the

following mechanisms may be responsible for such failure

Inhibition of Digestive Enzymes or Other Digestive Factors

Toxicants can inhibit a variety of enzymes involved in the

digestive processes Well-known examples include

inhibi-tors of the proteolytic and amylolytic enzymes present in

several legumes

Absence of Digestive Enzymes or Other Digestive Factors

Lactose intolerance in certain segments of populations is

well known because of the inherent genetic deficiency of

β-galactosidase enzyme Similarly, certain

oligosaccha-rides present in legumes cannot be metabolized because of

the absence of corresponding digestive enzymes

Interference in the Absorption of Essential Compounds

Interference in the absorption of essential compounds may

arise as a result of the following factors:

1 Chemical or physical combination of one pound with another, resulting in the formation

com-of a nonabsorbable complex: chelation com-of etary essential minerals by phytate falls in thiscategory

di-2 Absence of the compound necessary for the sorption of the compound: for example, vitamin

ab-B12 is not effectively absorbed in the absence ofthe intrinsic factor

3 Interaction or modification of the GI mucosa:

An example is the group of compounds known

as lectins, which bind on the absorptive surfaces

of the intestinal mucosa

4 Inhibition of enzymes involved in absorption:for example, the antibiotic actinomycin D inhib-its RNA and protein synthesis, including pre-sumably enzymes necessary for the absorption

of amino acids (Yamada et al., 1967; Concon,1988)

5 Solubilization of essential compounds in vents that are nonabsorbable For example, min-eral oil, which may dissolve lipid-solublevitamins, may prevent their absorption

sol-6 Increased motility of the GI tract: many factorscan affect this motility Certain factors in food-stuffs may cause diarrhea and similar rapidevacuation of intestinal contents, resulting inpoor absorption of essential compounds

Interference of the Transport of Essential Compounds to the Cells

Nitrite ions, for example, interfere in the transport of gen to hemoglobin

oxy-Degradation of Essential Compounds

Nutrients may be destroyed even before they are absorbed.For example, there are factors, such as thiaminases, thatdestroy thiamine; retinol and carotenoids may be de-stroyed by oxidizing agents; and ascorbic acid, by ascorbicacid oxidase

Inactivation of Essential Compounds

Certain substances may react with some of the essentialnutrients without causing degradation but rendering thembiologically inactive Cyanide, for example, may interactwith cobalamin, vitamin B12, to form cyanocobalamin,which is biologically inactive It is believed that chroniccyanide intoxication may be the cause of tropical amblyo-pia as a result of cyanide inactivation of cobalamin

Interference of the Uptake of Essential Compounds in the Cells or Tissues

Thiocyanate inhibits the iodine uptake of thyroid cells,whereas glucose and amino acid uptake of muscle cellsdoes not occur in the absence of insulin (Concon, 1988;Langer and Stolc, 1964)

Antagonism Between Essential Compounds

Antagonism exists among leucine, isoleucine, and valine

when one of these is present in relatively large excess over

another (Harper et al., 1970); β-carotene and

cholecalcif-erol are also antagonistic (Weits, 1964)

2.7.2 Inhibition of Metabolic and Other

Nondigestive Enzymes

Several toxicants inhibit the activity of metabolic and

other nondigestive enzymes Carbamates and

organo-phosphate pesticides, for example, are potent inhibitors of

acetylcholine esterase, an enzyme necessary for

neu-rotransmission (Aldridge and Reiner, 1969; Concon, 1988,

O’Brien, 1969a, 1969b) The inhibition of monoamine

ox-idase by antidepressant drugs has been shown to increase

the biological activity of pressor amines found in several

foods (Vettorazzi, 1974)

2.7.3 Interference with Neurotransmission

Several aspects of the neurotransmission process are

sus-ceptible to the action of various toxicants The propagation

of the nervous impulse is rapid and is obviously an

energy-requiring process The complexity and rapidity of

neu-rotransmission may also lay bases for its vulnerability The

interference of several toxicants with this process is

usu-ally quite serious and often fatal For example, the deadly

poisons tetrodotoxins, from the puffer fish, and saxitoxin,

from shellfish and clams, derive their lethality from their

capacity to block specifically the sodium gates of the axon

(Kao, 1972; Narahashi, 1972) In contrast, DDT acts in an

opposite way, by keeping the sodium channel open but

partially blocking the potassium channel (Narahashi and

Haas, 1967)

The synaptic mechanism by which the nerve pulse propagates appears to be the specific target of many

im-potent toxicants The release of the neurotransmitter

ace-tylcholine can be blocked by botulinum toxin (Brooks,

1956) Acetylcholine esterase, involved in the regeneration

of acetylcholine after the transmittal of the nervous

impulse, is also a target of many poisons, such as

or-ganophosphates (O’Brien, 1969a, 1969b) and the

cholin-esterase inhibitors in the potato, eggplant, tomato, and

sugar beet (Orgell, 1963)

Many exogenous substances can also mimic the fects of neurotransmitters Muscarine, the toxic alkaloid

ef-from the mushroom Amanita muscaria, behaves in the

same way as acetylcholine (Bradley et al., 1966) Its

ex-treme toxicity is attributed to its resistance to degradation

in the tissues

Toxic reactions directly involving the nervous tem are generally more severe, with an almost immediateappearance of symptoms Furthermore, other toxicitymechanisms, such as interference in the transport of nutri-ents, protein synthesis, energy metabolism, and respira-tion, indirectly affect the nervous system

sys-2.7.4 Phototoxic Reactions

Certain exogenous and endogenous compounds in the skincells, when sufficiently illuminated, may become highlyreactive with cellular components This process by whichlight damages tissues, in the presence of a photosensitive

substance, is called phototoxic reaction and is also known

as photosensitization or photodynamic action.

Ippen (1969) classified two types of phototoxic tions: photoautoreaction and photoheteroreaction In thefirst type, the photosensitive substance merely induces thenormal photochemical reaction of the cell as in sunburnformation In other words, in the presence of the photo-toxic compound, skin may become more readily suscepti-ble to the deleterious effects of sunlight Most phototoxicreactions, such as those caused by furocoumarins, are ofthis type In the second type, a toxic product is formedfrom the photochemical reaction of the photoactive sub-stance This toxic derivative may be formed independently

reac-of the tissues Chlorpromazine and sulfanilamide are amples of compounds that are involved in phototoxic reac-tions of the second type Photoallergic reaction is a form

ex-of photoheteroreaction In this case, the photochemical action produces an allergen

re-The classes of phototoxic compounds include bothnatural and synthetic compounds The natural compoundsinclude the hypericins (Blum, 1964), the furocoumarins orpsoralens (Pathak, 1969), porphyrins (Clare, 1956; Rim-ington et al., 1967), steroids, essential oils (Spikes, 1968),riboflavin (Spikes and Glad, 1964), and flavin mononucle-otide (FMN) (Frisell et al., 1959) The synthetic photo-toxic compounds include many drugs prescribed routinely,such as anesthetics, antibiotics, antihistamines, diuretics,barbiturates, sulfonamides, phenothiazines, dyes and othercoal tar and petroleum products, and perfumes and co-lognes (Spikes, 1968)

2.7.5 Interference with Genetic Material and Function

Interactions of toxicants with DNA and RNA, the buildingblocks of a cell’s genetic material, not only affect cellularreactions such as protein synthesis, but may also lead tomutagenesis and carcinogenesis These processes are de-scribed in greater detail in Chapter 4 Mutagenic effects in-

volving the germinal cells have the potential for hereditary

transmission Therefore, the damage of these effects can

extend beyond one generation and across pedigrees and

population groups

2.8 DISTRIBUTION OF TOXICANTS

Subsequently to absorption, the toxicant is capable of

dis-tribution (translocation) throughout the biological system

The transport processes discussed earlier are the major

factors operative in the distribution of the toxicant from

cell to cell and organ to organ and for movement into total

body water

Body fluids are distributed among three primarycomponents: plasma water, interstitial water, and intracel-

lular water Vascular fluid has the important role in the

dis-tribution of absorbed toxicants Human plasma accounts

for approximately 4% of body weight but 53% of total

blood volume, whereas the interstitial tissue fluids account

for 13% of body weight and intracellular fluids for 41%

(Guthrie and Hodgson, 1987b; Klaassen, 1986b)

Al-though only a small amount of toxicant in the body may be

in contact with the receptor or target site, it is the

distribu-tion of the bulk of the toxicant that governs the

concentra-tion and disposiconcentra-tion of that critical proporconcentra-tion If the

toxicant is distributed only in plasma, a high concentration

is achieved in the vascular tissue On the contrary, if the

same quantity of toxicant is also distributed in the

intersti-tial and intracellular water, concentrations are much lower

in the vascular system The rate of distribution of the

toxi-cant to the tissues of each organ is thus primarily

deter-mined by the blood flow through the organ and the ease

with which the chemical crosses the capillary bed and

pen-etrates the cells of the particular tissue Its eventual

dispo-sition is largely dependent on the ability of the toxic

chemical to pass through the cell membranes and its

affin-ity for the various tissues

The following are some of the factors that affect thedistribution of the toxicant in biological systems

2.8.1 Binding to Plasma Proteins

Binding by the plasma proteins has an important bearing

upon the distribution of toxicants Serum albumin is the

most important protein in this regard Because many

toxi-cants are very lipophilic, the plasma lipoproteins also play

an important role in toxicant binding The binding is

non-covalent, involving hydrogen, Van der Waals, and ionic

bonds, and the proportion of the toxicant bound depends

on various physicochemical factors

The nonbound (free) portion of the toxicant in theplasma is in equilibrium with the bound portion, but onlythe former passes through capillary membranes There-fore, excessively protein-bound compounds (>90%) arerestricted in terms of equilibrium (distribution) within theorganism Under steady-state conditions, the concentration

in the extravascular water equilibrates with the free centration in the plasma

con-Plasma protein binding sites may be saturated, orone bound compound may be displaced by another Thus,

a dose threshold for toxicity is often seen as a result of uration of plasma protein binding sites, which results in adramatic increase in the plasma concentration of the freecompound Such competitive binding for the same sites on

sat-a protein csat-an hsat-ave sat-an importsat-ant toxicologicsat-al significsat-ance.This is especially true for highly toxic compounds pos-sessing a very high affinity for protein binding sites.Although extensive plasma protein binding affectspassive diffusion, which is concentration dependent, it haslittle effect on active transport processes such as secretion

at the kidney Plasma protein binding of toxicants fore influences the distribution and the half-life of thetoxicant in the body and is responsible for toxic dosethresholds

there-A number of methods are used to study (ligand)-protein interactions, including ultrafiltration,electrophoresis, equilibrium dialysis, solvent extraction,solvent partition, ultracentrifugation, spectrophotometry,and gel filtration or equilibrium Such methods yield datathat are often expressed in terms of the percentage ofligand bound However, it must be noted that as ligandconcentration is lowered, percentage of bound ligand in-creases Thus, if a protein has a high affinity for a ligand,

toxicant-as often occurs with albumin, the percentage bound fallssharply when the total ligand concentration exceeds a crit-ical value

2.8.2 Plasma Level

The plasma level of a toxicant is an important parameter indistribution as it relates more readily to the effect than thedose itself In general, the plasma concentration morenearly reflects the concentration at the site of action, al-though the relationship may not be a simple one if the tox-icant is sequestered in a particular tissue or organ

2.8.3 Tissue Localization

The passage of exogenous chemicals into cells and acrossmembranes, as discussed earlier, is generally restricted tothe nonionized, lipid-soluble form of the chemical Thus,compounds that meet these criteria pass out of the blood,

diffuse through tissues, and are distributed through the

body Lipid-soluble compounds may dissolve in tissues

with a high fat content and may remain sequestered there

for some time

Some compounds may accumulate in a specifictissue because of their affinity for a particular macro-

molecule Such is the case with the binding of carbon

monoxide with hemoglobin These sites may be the target

sites for toxicity Accumulation may also occur in tissues

other than the site of action Such storage depots for

toxi-cants in the biological systems are described later in this

chapter

Toxic compounds thus may be distributed out all the tissues of the body, or they may be restricted

through-to certain tissues Two areas for special consideration

are the brain and the fetus The blood-brain barrier does

not completely prevent the passage of toxicants into the

central nervous system (CNS) but rather represents a

site that is less permeable than most other areas of the

body Therefore, many poisons do not enter the brain in

appreciable quantities

The following are the major anatomical and logical reasons why some toxicants have reduced capacity

physio-for entering the CNS (Klaassen, 1986b):

1 The capillary endothelial cells of the CNS aretightly joined, leaving few or no pores betweenthe cells

2 The capillaries of the CNS are largely rounded by glial cell processes (astrocytes)

sur-3 The protein concentration in the interstitial fluid

of the CNS is much lower than elsewhere in thebody

Thus, in contrast to other tissues, the toxicant hasdifficulty moving between capillaries and has to traverse

not only the capillary endothelium itself, but also the

membranes of the glial cells in order to gain access to the

interstitial fluid Furthermore, the low protein

concentra-tion of the interstitial fluid of the CNS also decreases the

distribution of chemicals to the CNS These features

to-gether act as a protective mechanism to decrease the

distri-bution of toxicants to the CNS and thus the toxicity

In contrast, passage of compounds across the centa occurs generally by passive diffusion Lipid-soluble

pla-compounds are thus readily transported However, if

me-tabolism in utero converts the compound into a more polar

metabolite, accumulation may occur in the fetus

Exoge-nous compounds generally achieve the same concentration

in fetal plasma as in the maternal plasma water In addition

to chemicals, viruses (e.g., rubella, human

immunodefi-ciency virus [HIV]), cellular pathogens (e.g., syphilis

spirochete), antibody globulins, and even erythrocytestraverse the placenta (Goldstein et al., 1974)

2.8.4 Volume of Distribution

As mentioned earlier, body fluids are distributed amongplasma and interstitial and intracellular water The distri-bution of the toxicant into each of these three fluidsprofoundly affects the plasma concentration If a toxicant

is distributed only in the plasma water (approximately 3liters in the average human), the plasma concentration isobviously much higher than if it is distributed in all extra-cellular fluid (approximately 14 liters) or the total bodywater (approximately 40 liters) The volume of distribu-

tion (VD) may be calculated from

VD = dose (mg)/plasma concentration (mg/L)and is expressed in liters A more rigorous determination

of the volume of distribution utilizes the area under theplasma concentration/time curve (area under the curve[AUC]) (Figure 2.7) for the calculation:

VD = dose/(k × area) where k is the elimination rate constant

or

VD = dose/C0where C0 is the plasma concentration at time zero gained

by extrapolation of the log plasma concentration versustime plot (Figure 2.8) Ideally, the compound should beadministered intravenously, unless the degree of absorp-tion is known

The parameter VD yields useful information For

in-stance, a very high apparent VD may indicate sequestration

in a particular tissue or localization in fat Similarly, the tal amount of a toxicant in the body, i.e., the total body

to-Figure 2.7 Plasma level profile for a foreign chemical AUC,area under the curve (From Timbrell [1982])

burden, may be estimated from a knowledge of the plasma

concentration and VD:

Total body burden (mg) = plasma concentration (mg/L) × VD (L)

Only the free rather than the total amount in the

plasma should be used for the calculation of VD, as only

the former is available for distribution

2.8.5 Plasma Half-Life

The plasma half-life is also an important parameter It can

be calculated from measurements of the plasma level at

the various time points The half-life is the time required

for the plasma concentration of the toxicant to decrease by

half from a given point Measurement of the plasma level

of a toxicant at various times after dosing gives a curve

that decays exponentially as shown in Figure 2.7 Plotting

the data semilogarithmically (Figure 2.8) gives a linear

re-lationship from which the half-life can be readily

calcu-lated as follows:

log C = log C0 – (kt/2.303) Slope = –k/2.303

Half-life (t1/2) = 0.693/k where C = plasma concentration, C0 = plasma concentra-

tion at time zero, t = time after dosing, and k = elimination

rate constant

A simple linear relationship is seen if the tion of the compound fits a single compartment model,i.e., the toxicant being distributed in plasma water alone Ifthe toxicant first undergoes distribution and the plasmaconcentration declines more slowly, governed by the pro-cess of elimination and metabolism, then a two-phase de-cay is seen For detailed mathematical treatment of thetopic, the readers are referred to several excellent reviews(Goldstein et al., 1974; Tuey, 1980; Klaassen, 1986b;Gibaldi and Perrier, 1982)

distribu-The half-life of a toxicant reflects the various cesses taking place in vivo after the administration of acompound Thus, following the initial absorptive phase,the toxicant is distributed, metabolized, and excreted, andthese processes, acting in conjunction, determine the rate

pro-of removal pro-of the toxicant from the plasma Changes in thehalf-life of the toxicant may therefore yield valuable infor-mation about changes in these processes For example, thehalf-life indicates the ability of the body to metabolize andexcrete the compound When this ability is impaired, ei-ther through saturation of enzymatic or active transportprocesses or if the liver or kidneys are damaged, the half-life may well be prolonged An indication of the ability ofthe body to metabolize and eliminate the compound may

be gained from the total body clearance This may be culated from the parameters described earlier

cal-Total body clearance = VD × k

or alternatively,Total body clearance = dose/AUCwhere the dose is administered intravenously

The plasma level and half-life are also important rameters when the exposure to a toxicant is chronic Thus,

pa-if the exposure is shorter than the half-lpa-ife, the toxicant cumulates in the body, whereas if the half-life is very shortcompared to the exposure, the toxicant does not accumu-late in the body It is therefore important to measure theplasma concentration of the toxicant for an assessment ofchronic toxicity

ac-2.9 METABOLISM/BIOTRANSFORMATION

OF TOXICANTS

Metabolism is important in a number of body processes,one of which is the detoxification of foreign or exogenouscompounds The biotransformation of a foreign, toxiccompound is thus an important aspect of its disposition invivo A metabolically unmodified toxicant is often re-

ferred to as an active parent compound, and a substance modified by metabolic processes as an active metabolite.

Figure 2.8 A semilog plot of the plasma level of a foreign

chemical against time C0, plasma concentration at time zero

(From Timbrell [1982].)

Both types of species may be involved in the manifestation

of toxic responses

Almost any reactive chemical that is administered to

or ingested by the organism is almost immediately

sub-jected to mechanisms that may confine its translocation

within the organism or terminate its existence as a free

chemical (Figure 2.9) Upon absorption, a toxicant thus

begins changing location, concentration, or chemical

den-sity It may be transported independently by several

com-ponents of the circulatory system, be absorbed by various

tissues or stored, effect an action, be detoxified, or be

acti-vated; the parent compound or its metabolite(s) may react

with body constituents, be stored, or be eliminated The

study of kinetics, known as pharmacokinetics or

toxicoki-netics, involved in these processes is a highly specialized

branch of toxicology

During the metabolic phase, an active parent pound can be present in blood, liver, or extrahepatic tis-

com-sues (nonliver tissue), and in the latter two, it may be

converted to an inactive metabolite or metabolites An

in-active parent metabolite may produce a toxic metabolite or

metabolites in the liver or in extrahepatic tissue; in both

these locations, a toxic metabolite may be changed to an

inactive form Therefore, metabolism of a toxicant

in-volves a number of pathways by which the compound is

converted to a toxicant or to a substance that is eliminated

from the biological system

One of the main results of such metabolic mation thus is the facilitation of the removal from the body

transfor-of toxic compounds, that, unless excreted, would late to toxic levels The types of biotransformations aremany and varied, and the metabolic systems involved arenecessarily very flexible and nonspecific These are de-scribed in detail in Chapter 5 The major factor determin-ing the route(s) of biotransformation is the structure of thecompound itself

accumu-The elimination of the toxicant from the body is theend point for biotransformation Kidneys are the main ex-cretory organ of the body for foreign chemicals, and theyare most efficient at eliminating polar molecules Thus,metabolic processes that are detoxifying in nature gener-ally involve reactions that convert nonpolar molecules intomore polar ones In most cases, these changes are advanta-geous to the body, but in some instances, the process con-verts basically nontoxic nonpolar compounds into moretoxic polar (or more polar) metabolites The nonpolarlipid-soluble compounds are generally reabsorbed fromthe kidney tubules or simply equilibrate between plasmaand bile by passive diffusion, to no effect

Thus, metabolism may not necessarily be a cation process Its primary purpose is often to facilitateelimination of the compound and alter its biological activ-ity In some cases, the effect of metabolism might just be

detoxifi-to alter elimination from the urinary detoxifi-to the biliary route,for example, by increasing the molecular weight

Metabolism or biotransformation is therefore an portant determinant of the activity of a compound, the du-ration of this activity, and the half-life of the compound inthe body Some very lipid-soluble compounds, such aschlorinated hydrocarbons, polychlorinated and polybromi-nated biphenyls, and aflatoxins, which are poorly ab-sorbed, may have whole-body half-lives measured inmonths or even years

im-The chemical alterations that are the basis ofbiotransformation of foreign compounds are catalyzed by

a number of enzymes, depending on the chemical structure

of the compound in question The most important is thecytochrome P-450 monooxygenase system, but numerousother enzymes may be utilized, both those involved in theintermediary metabolism and those whose main function

is the metabolism of xenobiotics

Specific enzymes that recognize particular types ofmolecules are normally present in small quantities, and thebody produces more of them when the need arises, e.g.,after a significant exposure to the appropriate foreignmolecule Unfortunately, this process is not easily revers-ible, and the body does not revert to its preexposure statusvery rapidly Thus exposure to one chemical of a particu-lar type may lead to the presence of a large number ofspecific enzymes when a subsequent exposure to the same

or a similar chemical occurs This process of increasing

Figure 2.9 Schematic representation of the pathways through

which a toxicant or exogenous chemical may pass during its

presence in humans

the enzyme levels is called enzyme induction In general,

this is beneficial since it helps the body to respond rapidly

to repeated exposures Of course, if the metabolic

pro-cesses resulting from the response lead to more toxic

rather than less toxic metabolites, this induction is

coun-terproductive

Although the major organ involved in the formation of exogenous compounds is the liver, other tis-

biotrans-sues and organs may be involved to a greater or lesser

extent The importance of liver in this respect relates to its

position as a portal to the tissues of the body By

metabo-lizing and hence removing toxic substances ingested orally

and absorbed via the hepatic-portal circulation, the liver

protects the organism In some cases, this metabolic

con-version during the absorption phase is almost complete,

resulting in a first-pass effect The gut wall may also carry

out biotransformation and, hence, be responsible for a

first-pass effect, as may the lung for compounds absorbed

by inhalation

2.10 STORAGE OF TOXICANTS

Toxicants are often concentrated in specific tissues in the

body Whereas some achieve the highest concentration at

their site of action (e.g., carbon monoxide in hemoglobin),

others are concentrated at sites other than the site of toxic

action For example, lead is stored in bone, whereas the

symptoms of lead poisoning are due to lead in the soft

tis-sues The compartment where the toxicant is concentrated

but not involved in the toxicological response can be

thought of as a storage depot These can be considered as

protective mechanisms, preventing the accumulation of

high concentrations of toxicants at the site of toxic action

These toxicants in these depots are always in equilibrium

with free toxicant in plasma, and as they are

biotrans-formed or excreted from the body, more is released from

the storage site (Klaassen 1986b) The biological half-life

of stored toxicants thus can be very long

The following are the major sites of storage fortoxicants

2.10.1 Plasma Proteins

Several proteins in the plasma have the ability to bind to

exogenous chemicals as well as some normal

physiologi-cal constituents (Table 2.4) The majority of foreign

chem-icals that are bound to plasma proteins are bound to serum

albumin It is the most abundant protein in plasma and

serves as a depot protein and transport protein for several

endogenous and exogenous compounds Transferrin, a

β1-globulin, is important for transport of iron in the body

Ceruloplasmin, which carries most of the copper in theserum, is the other metal-binding protein in plasma Theα- and β-lipoproteins are very important in the transport oflipid-soluble compounds, such as vitamins, cholesterol,and steroid hormones The γ-globulins are antibodies thatinteract specifically with antigens Compounds possessingbasic characteristics often bind to α1-acid glycoprotein(Klaassen, 1986b; Wilkinson, 1983)

Several relevant aspects of binding of toxicants toplasma proteins were described in conjunction with thedistribution of toxicants in a preceding section in thischapter

2.10.2 Liver and Kidney

Liver and kidney have a high capacity to bind chemicals.These two organs also concentrate more toxicants thanother organs, primarily because both are important to theelimination of toxicants from the body

2.10.3 Intracellular Binding Proteins

Within the liver and kidney, several intracellular proteinsare important in concentrating the toxicants Examples in-clude Y protein or ligandin, which has a high affinity formany organic acids; azo dye carcinogens and cortico-steroids, in the cytoplasm of the liver; and the cadmium-binding protein metallothionein, found in the kidney andliver (Levi et al., 1969; Litwalk et al., 1971; Klaassen andShoeman, 1974)

Table 2.4 Examples of Plasma Proteins That Serve as Storage Depots for Physiological Constituents and Toxicants

Protein Physiological constituent/toxicantAlbumin Calcium, copper and zinc ions, biliru-

bin, uric acid, vitamin C, adenosine, tetracyclines, chloramphenicol, digitonin, fatty acids, suramin, quinocrine, penicillin, salicylate, sulfonamides, streptomycin, acid dyes, phenol red, histamine, triiodo-thyronine, thyroxine, barbiturates

α- and β-Lipoproteins Lipid-soluble vitamins, cholesterol,

steroid hormones, vitamin B12, sialic acid, thyroxine

Immunoglobulins (γ-globulins)

Specific for individual antigens

α1-Acid glycoprotein Basic compounds