Toxicology (1) - Ullmann

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Toxicology, 1. Fundamentals

WOLFGANG DEKANT, Institute of Toxicology, University of Wuerzburg, Germany

SPIRIDONVAMVAKAS, Institute of Toxicology, University of Wuerzburg, Germany

1. Introduction. . . . . . . . . . . . . . . . . . . . . . 128

1.1. Definition and Scope . . . . . . . . . . . . . . . 128

1.2. Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

1.3. History. . . . . . . . . . . . . . . . . . . . . . . . . . 131

1.4. Information Resources. . . . . . . . . . . . . . 132

1.5. Terminology of Toxic Effects . . . . . . . . . 134

1.6. Types of Toxic Effects . . . . . . . . . . . . . . 135

1.7. DoseResponse: a Fundamental Issue inToxicology . . . . . . . . . . . . . . . . . . . . . . . 136

1.7.1. Graphics and Calculations . . . . . . . . . . . . 138

1.8. DoseResponse Relationships for

Cumulative Effects. . . . . . . . . . . . . . . . . 140

1.9. Factors Influencing DoseResponse . . . . . 141

1.9.1. Routes of Exposure . . . . . . . . . . . . . . . . . 142

1.9.2. Frequency of Exposure . . . . . . . . . . . . . . 143

1.9.3. Species-Specific Differences in

Toxicokinetics. . . . . . . . . . . . . . . . . . . . . 143

1.9.4. Miscellaneous Factors Influencing the

Magnitude of Toxic Responses. . . . . . . . . 145

1.10. Exposure to Mixtures . . . . . . . . . . . . . . 1452. Absorption, Distribution,

Biotransformation and Elimination of

Xenobiotics . . . . . . . . . . . . . . . . . . . . . . 145

2.1. Disposition of Xenobiotics . . . . . . . . . . . 145

2.2. Absorption . . . . . . . . . . . . . . . . . . . . . . . 147

2.2.1. Membranes . . . . . . . . . . . . . . . . . . . . . . . 147

2.2.2. Penetration of Membranes by Chemicals . 147

2.2.3. Mechanisms of Transport of Xenobiotics

through Membranes. . . . . . . . . . . . . . . . . 148

2.2.4. Absorption . . . . . . . . . . . . . . . . . . . . . . . 1492.2.4.1. Dermal Absorption . . . . . . . . . . . . . . . . . 149

2.2.4.2. Gastrointestinal Absorption . . . . . . . . . . . 1512.2.4.3. Absorption of Xenobiotics by the

Respiratory System . . . . . . . . . . . . . . . . . 153

2.3. Distributionof Xenobiotics byBody Fluids 155

2.5. Storage of Xenobiotics in Organs and

Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . 158

2.6. Biotransformation . . . . . . . . . . . . . . . . . 159

2.6.1. Phase-I and Phase-II Reactions. . . . . . . . . 159

2.6.2. Localization of the Biotransformation

Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . 160

2.6.3. Role of Biotransformation in Detoxication

and Bioactivation . . . . . . . . . . . . . . . . . . 160

2.6.4. Phase-I Enzymes and their Reactions . . . . 1602.6.4.1. Microsomal Monooxygenases: CytochromeP450. . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

2.6.4.2. Microsomal Monooxygenases: Flavin-Dependent Monooxygenases . . . . . . . . . . 163

2.6.4.3. Peroxidative Biotransformation:Prostaglandin-synthase. . . . . . . . . . . . . . . 164

2.6.4.4. Nonmicrosomal Oxidations . . . . . . . . . . . 1652.6.4.5. Hydrolytic Enzymes in Phase-I

Biotransformation Reactions . . . . . . . . . . 166

2.6.5. Phase-II Biotransformation Enzymes and

their Reactions . . . . . . . . . . . . . . . . . . . . 1662.6.5.1. UDP-Glucuronyl Transferases . . . . . . . . . 1672.6.5.2. Sulfate Conjugation . . . . . . . . . . . . . . . . . 1672.6.5.3. Methyl Transferases. . . . . . . . . . . . . . . . . 1682.6.5.4. N-Acetyl Transferases . . . . . . . . . . . . . . . 1692.6.5.5. Amino Acid Conjugation. . . . . . . . . . . . . 1692.6.5.6. Glutathione Conjugation of Xenobiotics and

Mercapturic Acid Excretion . . . . . . . . . . . 169

2.6.6. Bioactivation of Xenobiotics . . . . . . . . . . 1712.6.6.1. Formation of Stable but Toxic Metabolites 1712.6.6.2. Biotransformation to Reactive Electrophiles 1722.6.6.3. Biotransformation of Xenobiotics to

Radicals . . . . . . . . . . . . . . . . . . . . . . . . . 1742.6.6.4. Formation of Reactive Oxygen Metabolites

by Xenobiotics . . . . . . . . . . . . . . . . . . . . 1742.6.6.5. Detoxication and Interactions of Reactive

Metabolites with Cellular Macromolecules 1752.6.6.6. Interaction of Reactive Intermediates with

Cellular Macromolecules . . . . . . . . . . . . . 176

2.6.7. Factors Modifying Biotransformation and

Bioactivation . . . . . . . . . . . . . . . . . . . . . . . . 1792.6.7.1. Host Factors Affecting Biotransformation. 1802.6.7.2. Chemical-Related Factors that Influence

Biotransformation . . . . . . . . . . . . . . . . . . 183

2.6.8. Elimination of Xenobiotics and their

Metabolites . . . . . . . . . . . . . . . . . . . . . . . 1842.6.8.1. Renal Excretion. . . . . . . . . . . . . . . . . . . . 1842.6.8.2. Hepatic Excretion . . . . . . . . . . . . . . . . . . 1852.6.8.3. Xenobiotic Elimination by the Lungs . . . . 186

2.7. Toxicokinetics . . . . . . . . . . . . . . . . . . . . 187

2.7.1. Pharmacokinetic Models . . . . . . . . . . . . . 1872.7.1.1. One-Compartment Model. . . . . . . . . . . . . 1872.7.1.2. Two-Compartment Model . . . . . . . . . . . . 189

2.7.2. Physiologically Based Pharmacokinetic

Models . . . . . . . . . . . . . . . . . . . . . . . . . . 189

3. Mechanisms of Acute and Chronic

Toxicity and Mechanisms of Chemical

Carcinogenesis . . . . . . . . . . . . . . . . . . . . 1903.1. Biochemical Basis of Toxicology . . . . . . 190

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3.2. Receptor-Ligand Interactions . . . . . . . . 191

3.2.1. Basic Interactions . . . . . . . . . . . . . . . . . . 191

3.2.2. Interference with Excitable Membrane

Functions . . . . . . . . . . . . . . . . . . . . . . . . 193

3.2.3. Interference of Xenobiotics with Oxygen

Transport, Cellular Oxygen Utilization, and

Energy Production. . . . . . . . . . . . . . . . . . 194

3.3. Binding of Xenobiotics to Biomolecules . 196

3.3.1. Binding of Xenobiotics or their Metabolites

to Cellular Proteins . . . . . . . . . . . . . . . . . 196

3.3.2. Interaction of Xenobiotics or their

Metabolites with Lipid Constituents . . . . . 197

3.3.3. Interactions of Xenobiotics or their

Metabolites with Nucleic Acids . . . . . . . . 198

3.4. Perturbation of Calcium Homeostasis by

Xenobiotics or their Metabolites . . . . . . 198

3.5. Nonlethal Genetic Alterations in Somatic

Cells and Carcinogenesis . . . . . . . . . . . . 198

3.6. DNA Structure and Function. . . . . . . . . 200

3.6.1. DNA Structure . . . . . . . . . . . . . . . . . . . . 200

3.6.2. Transcription. . . . . . . . . . . . . . . . . . . . . . 201

3.6.3. Translation . . . . . . . . . . . . . . 201

3.6.4. Regulation of Gene Expression . . . . . . . . 202

3.6.5. DNA Repair . . . . . . . . . . . . . . . . . . . . . . 202

3.7. Molecular Mechanisms of Malignant

Transformation and Tumor Formation . 203

3.7.1. Mutations . . . . . . . . . . . . . . . . . . . . . . . . 203

3.7.2. Causal Link between Mutation and Cancer 204

3.7.3. Proto-Oncogenes and Tumor- Suppressor

Genes as Genetic Targets. . . . . . . . . . . . . 205

3.7.4. Genotoxic versus Nongenotoxic

Mechanisms of Carcinogenesis. . . . . . . . . 205

3.8. Mechanisms of Chemically Induced

Reproductive and Developmental Toxicity. 206

3.8.1. Embryotoxicity, Teratogenesis, and

Transplacental Carcinogenesis . . . . . . . . . 206

3.8.2. Patterns of DoseResponsein Teratogenesis,

Embryotoxicity, and Embryolethality . . . . 207

References . . . . . . . . . . . . . . . . . . . . . . . 208

1. Introduction

1.1. Definition and Scope

Chemicals that are used or of potential use incommerce, the home, the environment, andmedical practice may present various types ofharmful effects. The nature of these effects is

determined by the physicochemical character-istics of the agent, its ability to interact withbiological systems (hazard), and its potential to

come into contact with biological systems(exposure).

Toxicology studies the interaction betweenchemicals and biological systems to determinethe potential of chemicals to produce adverseeffects in living organisms. Toxicology alsoinvestigates the nature, incidence, mechanismsof production, factors influencing their devel-

opment, and reversibility of such adverse ef-fects. Adverse effects are defined as detrimen-tal to the survival or the normal functioning of

Abbreviations

Ah-R: arylhydrocarbon receptor

AP: apurinic/apyrimidinic site

APS: adenosine 50-phosphosulfate

CoA: Coenzym A

DDT: 1,10-(2,2,2-trichloroethylidene)

bis-(4-chlorobenzene)

DHHS: U.S. Department of Health and

Human Services

ECETOC: European Chemical Industry

Ecology and Toxicology Centre

ED: effective dose

FAD: flavine adenine dinucleotide

GABA: g-aminobutyrate

GSH: glutathione

IPCS: International Programme on

Chemical Safety

mRNA: messenger RNA

NADPH: nicotinamide dinucleotide phos-

phate (H)

NTP: National Toxicology ProgramrRNA: ribosomal RNA test

T, or TCDD: 2,3,7,8-tetrachlorodibenzodioxin

TD: tumor dose

tRNA: transfer RNA

UDP: uridine diphosphate

UDPG: uridine diphosphate glucose

UDPGA: uridine diphosphate glucuronic

acid

128 Toxicology, 1. Fundamentals Vol. 37


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the individual. Inherent in this definition arethe following key issues in toxicology:

1. Chemicals must come into close structuraland/or functional contact with tissues or or-gans to cause injury.

2. All adverse effects depend on the amount ofchemical in contact with the biological sys-tem (the dose) and the inherent toxicity ofthe chemical (hazard). When possible, theobserved toxic effect should be related tothe degree of exposure. The influence ofdifferent exposure doses on the magnitudeand incidence of the toxic effect should bequantitated. Such dose-response relation-ships are of prime importance in confirminga causal relationship between chemical ex-posure and toxic effect (for details, seeSection 1.7).

Research in toxicology is mainly concernedwith determining the potential for adverse ef-fects caused by chemicals, both natural andsynthetic, to assess their hazard and risk ofhuman exposure and thus provide a basis forappropriate precautionary, protective and re-strictive measures. Toxicological investigations

should permit evaluation of the following char-acteristics of toxicity:

1. The basic structural, functional, or biochemi-cal injury produced

2. Dose-response relationships3. The mechanisms of toxicity (fundamental

biochemical alterations responsible forthe induction and maintenance of the toxicresponse) and reversibility of the toxiceffect

4. Factors that modify response, e.g., route ofexposure, species, and gender

For chemicals to which humans may poten-tially be exposed, a critical analysis, based onthe pattern of potential exposure or toxicity,may be necessary in order to determine therisk-benefit ratio for their use in specific cir-cumstances and to devise protective andprecautionary measures. Indeed, with drugs,pesticides, food additives, and cosmetic

preparations, toxicology testing must be per-formed in accordance with government regula-tions before use.

1.2. Fields

Toxicology is a recognized scientific disciplineencompassing both basic and applied issues.Although only generally accepted as a specificscientific field during this century, its principles

have been appreciated for centuries. The harmfulor lethal effects of certain chemicals, mainlypresent in minerals and plants or transmittedvenomous animals, have been known since pre-historic times. In many countries, toxicology as adiscipline has developed from pharmacology.Pharmacology and toxicology both study theeffect of chemicals on living organisms and haveoften used identical methods. However, funda-mental differences have developed. Years ago,only the dependence on dose of the studiedeffects separated pharmacology and toxicology.Pharmacology focused on chemicals with bene-ficial effects (drugs) at lower doses whereastoxicology studied the adverse health effectsoccurring with the same chemicals at high doses.Today, the main interest of research in toxicologyhas shifted to studies on the long-term effects ofchemicals after low-dose exposure, such as can-cer or other irreversible diseases; moreover, mostchemicals of interest to toxicologists are not used

as drugs.The variety of potential adverse effects andthe diversity of chemicals present in our envi-ronment combine to make toxicology a verybroad science. Toxicology uses basic knowl-edge from clinical and theoretical medicine andnatural sciences such as biology and chemistry(Fig. 1). Because of this diversity, toxicologistsusually specialize in certain areas.

Any attempt to define the scope of toxicologymust take into account that the various subdisci-

plines are not mutually exclusive and frequentlyare heavily interdependent. Due to the overlap-ping mechanisms of toxicity, chemical classes,and observed toxic effects, clear divisions intosubjects of equal importance are often notpossible.

The professional activities of toxicologistscan be divided into three main categories: des-criptive, mechanistic, and regulatory. The des-criptive toxicologist is concerned directly withtoxicity testing. Descriptive toxicology still often

relies on the tools of pathology and clinicalchemistry, but since the 1970s more mecha-nism-based test systems have been included in

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toxicity testing [1]. The appropriate toxicity testsin experimental animals yield information that isextrapolated to evaluate the risk posed by expo-sure to specific chemicals. The concern may belimited to effects on humans (drugs, industrialchemicals in the workplace, or food additives) ormay encompass animals, plants, and other factorsthat might disturb the balance of the ecosystem(industrial chemicals, pesticides, environmentalpollutants).

The mechanistic toxicologist is concernedwith elucidating the mechanisms by which che-micals exert their toxic effects on living organ-isms. Such studies may result in the development

of sensitive predictive toxicity tests useful inobtaining information for risk assessment (see! Toxicology, 2. Assessment Methods). Mech-anistic studies may help in the development ofchemicals that are safer to use or of more rationaltherapies for intoxications. In addition, an under-standing of the mechanisms of toxic action alsocontributes to the knowledge of basic mechan-isms in physiology, pharmacology, cell biology,and biochemistry. Indeed, toxic chemicals havebeen used with great success as mechanistic tools

to elucidate mechanisms of physiological regu-lation. Mechanistic toxicologists are often activein universities; however, industry and govern-

ment institutions are now undertaking more andmore research in mechanistic toxicology.

Regulatory Toxicologists have the responsi-bility of deciding on the basis of data provided bythe descriptive toxicologist and the mechanistictoxicologist if a drug or chemical poses a suffi-ciently low risk to be used for a stated purpose.Regulatory toxicologists are often active in gov-ernment institutions and are involved in theestablishment of standards for the amount ofchemicals permitted in ambient air in the envi-ronment, in the workplace, or in drinking water.Other divisions of toxicology may be based on

the classes of chemicals dealt with or applicationof knowledge from toxicology for a specific field(Table 1).

Forensic Toxicology comprises both analyt-ical chemistry and fundamental toxicologic prin-ciples. It is concerned with the legal aspects ofthe harmful effects of chemicals on humans. Theexpertise of the forensic toxicologist is invokedprimarily to aid in establishing the cause of deathand elucidating its circumstances in a postmor-

tem investigation. The field ofclinical toxicologyrecognizes and treats poisoning, both chronic andacute. Efforts are directed at treating patients

Figure 1. Scientific fields influencing the science of toxicology



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poisoned by chemicals and at the development ofnew techniques to treat these intoxications. En-vironmental toxicology is a relatively new areathat studies the effects of chemicals released byman on wildlife and the ecosystem and thusindirectly on human health.

Drug Toxicology plays a major role in thepreclinical safety assessment of chemicals in-tended for use as drugs. Drug toxicology alsoelucidates the mechanisms of side effects ob-served during clinical application.Occupation-al toxicology studies the acute and chronictoxicity of chemicals encountered in the occu-pational environment. Both acute and chronicoccupational poisonings have exerted a majorinfluence on the development of toxicology in

general. Occupational toxicology also helps inthe development of safety procedures to preventintoxications in the workplace and assists in thedefinition of exposure limits. Pesticide toxicol-ogy is involved in the development of newpesticides and the safety of pesticide formula-tions. Pesticide toxicology also characterizespotential health risks to the general populationcaused by pesticide residues in food and drink-ing water.

1.3. History

Toxicology must rank as one of the oldest prac-tical sciences because humans, from the verybeginning, needed to avoid the numerous toxicplants and animals in their environment. Thepresence of toxic agents in animals and plantswas known to the Egyptian and Greek civilisa-tions. The papyrus Ebers, an Egyptian papyrusdating from about 1 500 B.C., and the surviving

medical works of HIPPOCRATES, ARISTOTLE, andTHEOPHRASTUS, published during the period 400250 B.C., all included some mention of poisons.

The Greek and Roman civilizations knowing-ly used certain toxic chemicals and extracts forhunting, warfare, suicide, and murder. Up to theMiddle Ages, toxicology was restricted to the useof toxic agents for murder. Poisoning was devel-oped to an art in medieval Italy and has remaineda problem ever since, and much of the earlierimpetus for the development of toxicology wasprimarily forensic. There appear to have beenfew advances in either medicine or toxicologybetween the time of GALEN (131200 A.D.) andPARACELSUS (14931541). The latter laid thegroundwork for the later development of moderntoxicology. He clearly was aware of the doseresponse relationship. His statement that Allsubstances are poisons; there is none that is not apoison. The right dose differentiates a poison and

a remedy, is properly regarded as a landmark inthe development of the science of toxicology. Hisbelief in the value of experimentation also re-presents a break with much earlier tradition.Important developments in the 1700s include thepublication of RAMAZZINIsDiseases of Workers,which led to his recognition as the father ofoccupational medicine. The correlation betweenthe occupation of chimney sweepers and scrotalcancer by POTTin 1775 is also noteworthy.

ORFILA, a Spaniard working at the University

of Paris, clearly identified toxicology as a sepa-rate science and wrote the first book devotedexclusively to it (1815). Workers of the later1800s who produced treatises on toxicologyinclude CHRISTISON, KOBERT, and LEWIN. Theyincreased our knowledge of the chemistry ofpoisons, the treatment of poisoning, the analysisof both xenobiotics and toxicity, as well as modesof action and detoxication. A major impetusfor toxicology in the 1900s was the use of che-micals for warfare. In World War I, a variety of

poisonous chemicals were used in the battlefieldsof France. This provided stimulus for workon mechanisms of toxicity as well as medical

Table 1.Areas of toxicology

Field Tasks and objectives

Forensic toxicology diagnoses poisoning by analytical procedures

Pesticide toxicology studies the safety of pesticides, develops new pesticides

Occupational toxicology assesses potential adverse effects of chemicals used in the workplace, recommends protective procedures

Drug toxicology studies potential effects of drugs after high doses, elucidates mechanisms of sideeffects

Regulatory toxicology develops and interprets toxicity testing programs and is involved in controlling the use of chemicals

Environmental toxicology studies the effects of chemicals on ecosystems and on humans after low-dose exposure from the environment



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countermeasures to poisoning. Since the 1960s,toxicology has entered a phase of rapid develop-ment and has changed from a science that wasalmost entirely descriptive to one in which thestudy of mechanisms has become the prime task.The many reasons for this include the develop-

ment of new analytical methods since 1945, theemphasis on drug testing following the thalido-mide tragedy, the emphasis on pesticide testingfollowing the publication of Rachel CarsonsSilent Spring and public concern over environ-mental pollution and disposal of hazardous waste.

1.4. Information Resources

Because of the complexity of toxicology as ascience and the impact of toxicological investi-gations on legislation and commerce, a widerange of information on the toxic effects ofchemicals is available. No single, exhaustivesource of toxicological data exists; severalsources are required to obtain comprehensiveinformation on a particular chemical. Printedsources are often quicker and easier to use thancomputer data bases, but interactive onlinesearching can rapidly gather important informa-

tion from the huge number of sources present.The information explosion in toxicology hasresulted in a comprehensive volume dedicated totoxicological information sources:

P. Wexler, P. J. Hakkinen, G. Kennedy, Jr. F. W.Stoss, Information Resources in Toxicology,3rd ed., Academic Press, 1999.

Textbooks. The easiest way to obtain infor-mation on general topics in toxicology and sec-

ondary references are a range of textbooks avail-able on the market. Only a few selected books arelisted below:

C. D. Klaasen, Casarett and Doulls Toxicology;The Basic Science of Poisons, 6th ed.,McGraw-Hill, New York, 2001.

G. D. Clayton, F. E. Clayton (eds): Pattys In-dustrial Hygiene and Toxicology, Wiley, NewYork, 1993.

J. G. Hardman, L. E. Limbird, Goodman and

Gilmans, The Pharmacological Basis ofTherapeutics, 10th ed., McGraw-Hill, NewYork, 2001.

W. A. Hayes, Principles and Methods of Toxi-cology, 3rd ed., Raven Press, New York, 2001.

E. Hodgson (Ed.):Textbook of Modern Toxicol-ogy, 3rd ed., Wiley Interscience, 2004.

T. A. Loomis, A. W. Hayes,Loomiss Essentialsof Toxicology, 4th ed., Academic Press, San

Diego, 1996.

The huge volume by N. I. Sax and R. J. Lewis,Dangerous Properties of Industrial Materials,7th ed.,Wiley, New York, 1999, contains basictoxicological data on a large selection of chemi-cals (almost 20 000) and may serve as a usefulguide to the literature for compounds not coveredin other publications.

Monographs. The best summary informa-tion on toxicology is published in the form ofseries by governments and international orga-nizations. Most of these series are summarizingthe results of toxicity studies on specific che-micals. The selection of these chemicals ismainly based on the extent of their use inindustry (e.g. trichloroethene), their occurrenceas environmental contaminants (mercury) ortheir extraordinary toxicity (e.g. 2,3,7,8-tetrachlorodibenzodioxin):

American Conference of Governmental Indus-trial Hygienists, Threshold Limit Values andBiological Exposure Indices (Cincinnati,OH). Published annually.

MAK-Begrundungen, VCH Publishers, Wein-heim, Federal Republic of Germany. ThisGerman series includes detailed informationon the toxicity of chemicals on the GermanMAK list (ca. 150 reports are available; theseries is continuously expanded).

The Commission of the European Communi-ties publishes the Reports of the Scientific Com-mittee on Cosmetology and the Reports of theScientific Committee for Food.

The Environmental Protection Agency (EPA)publishes a huge number of reports and toxico-logical profiles. They are indexed in EPA Pub-lications. A Quarterly Guide.

The European Chemical Industry Ecologyand Toxicology Centre (ECETOC) issues

Monographs (more than 20 have been pub-lished) and Joint Assessments of CommodityChemicals.



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The monographs of the International Agen-cy for Research on Cancer are definitive eva-luations of carcinogenic hazards. The Envi-ronmental Health Criteria documents of theInternational Programme on Chemical Safety(IPCS) assess environmental and human health

effects of exposure to chemicals, and biologicalor physical agents. A related Health and Safe-ty Guide series give guidance on setting ex-posure limits for national chemical safetyprograms.

The National Institute for Occupational Safe-ty and Health (NIOSH), has published 50 Cur-rent Intelligence Bulletins on health hazards ofmaterials and processes at work.

The technical report series of the NationalToxicology Program (NTP) reports results oftheir carcinogenicity bioassays, which includesummaries of the toxicology of the chemicalsstudied. A status report indexes both studies thatare under way and those that have been pub-lished. The program also issues an AnnualReview of Current DHHS [U.S. Department ofHealth and Human Services], DOE [U.S. Depart-ment of Energy] and EPA Research related totoxicology.

A large number of internet-based resources

are also available to collect information on toxiceffects of chemicals and methods for risk assess-ment. Some information sites containing largeamounts of downloadable information are listedbelow:

US Environmental Protection Agency (EPA),Integrated Risk Information System (IRIS),http://www.epa.gov/iris/index.html

US Environmental Protection Agency (EPA),ECOTOX Database, http://www.epa.gov/

ecotox/Organisation for Economic Co-operation and

Development (OECD), test guidelines,http://www.oecd.org

Agency for Toxic Substances and Disease Reg-istry (ATSDR), toxicological profile infor-mation sheet http://www.atsdr.cdc.gov/toxprofiles/

European Chemicals Bureau, http://ecb.jrc.it/National Toxicology Programm, http://ntp-server.

niehs.nih.gov/htdocs/liason/Factsheets/Fact-

sheetList.htmlUnited Nations Environment Programm, Chemi-

cals http://www.chem.unep.ch/

Journals Results of toxicological researchare published in more than 100 journals. Thoselisted below mainly publish research closelyrelated to toxicology, but articles of relevancemay also be found in other biomedical journals:

Archives of Environmental Contamination andToxicology

Archives of Toxicology

Biochemical Pharmacology

Chemical Research in Toxicology

CRC Critical Reviews in Toxicology

Clinical Toxicology

Drug and Chemical Toxicology

Environmental Toxicology and Chemistry

Food and Chemical Toxicology

Fundamental and Applied Toxicology

Journal of the American College of Toxicology

Journal of Analytical Toxicology

Journal of Applied Toxicology

Journal of Biochemical Toxicology

Journal of Toxicology and Environmental Health

Neurotoxicology and Teratology

Pharmacology and Toxicology

Practical In Vitro Toxicology

Regulatory Toxicology and Pharmacology

Reproductive Toxicology

ToxicologyToxicology and Applied Pharmacology

Toxicology and Industrial Health

Toxicology In Vitro

Toxicology Letters

Databases and Databanks. Electronicsources, such as computer data bases or CD-ROM are a fast and convenient way to obtainreferences on the toxicity of chemicals. Since on-line searching of commercial data bases such as

STN-International may be expensive, CD-ROM-based systems are increasingly being used. Themajor advantages are speed, the ability to refinesearches and format the results, and non-textsearch options, such as chemical structuresearching on Beilstein and Chemical Abstracts.

Useful information about actual research on thetoxicology of chemicals may be obtained bysearching Chemical Abstracts or Medline with theappropriate keywords. Specific data banks cover-ing toxicology are the Registry of Toxic Effects of

Chemical Substances, which gives summary data,statistics, and structures; Toxline (available inDIMDI) gives access to the literature.



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1.5. Terminology of Toxic Effects

Toxic effects may be divided according to time-scale (acute and delayed), general locus of action(local, systemic, organ specific), or basic mechan-isms of toxicity (reversible versus irreversible).

Acute toxic effects are those that occur after briefexposure to a chemical. Acute toxic effects usu-ally develop rapidly after single or multiple ad-ministrations of a chemical; however, acute ex-posure may also produce delayed toxicity. Forexample, inhalation of a lethal dose of HCNcauses death in less than a minute, whereas lethaldoses of 2,3,7,8-tetrachlorodibenzodioxin willresult in the death of experimental animals aftermore than two weeks. Chronic effects are thosethat appear after repetitive exposure to a sub-stance; many compounds require several monthsof continuous exposure to produce adverse ef-fects. Often, the chronic effects of chemicals aredifferent from those seen after acute exposure(Table 2). For example, inhalation of chloroformfor a short period of time may cause anesthesia;long-term inhalation of much lower chloroformconcentrations causes liver damage. Carcinogen-ic effects of chemicals usually have a long latencyperiod; tumors may be observed years (in rodents)

or even decades (in humans) after exposure.Toxic effects of chemicals may also be clas-sified based on the type of interaction betweenthe chemical and the organism. Toxic effectsmay be caused by reversible and irreversibleinteractions (Table 3). When reversible interac-tions are responsible for toxic effects, the con-centration of the chemical present at the site ofaction is the only determinant of toxic outcome.When the concentration of the xenobiotic isdecreased by excretion or biotransformation, a

parallel decrease of toxic effects is observed.

After complete excretion of the toxic agent,toxic effects are reduced to zero (see below). Aclassical example for reversible toxic effects iscarbon monoxide. Carbon monoxide binds tohemoglobin and, due to the formation of thestable hemoglobincarbon monoxide complex,binding of oxygen is blocked. As a result of theimpaired oxygen transport in blood from thelung, tissue oxygen concentrations are reducedand cells sensitive to oxygen deprivation will die.The toxic effects of carbon monoxide are directlycorrelated with the extent of carboxyhemoglobinin blood, the concentration of which is dependenton the inhaled concentration of carbon monox-

ide. After exhalation of carbon monoxide andsurvival of the acute intoxication, no toxic effectremains (Fig. 2).

Irreversible toxic effects are often caused bythe covalent binding of toxic chemicals to bio-logical macromolecules. Under extreme condi-tions, the modified macromolecule is not re-paired; after excretion of the toxic agent, theeffect persists. Further exposure to the toxicagent will produce additive effects; many che-micals carcinogens are believed to act through

irreversible changes (see Section 2.6.6).

Table 2. Toxic effectsof differentchemicals categorized by timescale

and general locus of action

Exposure Site Effect Chemical

Acute local lung edema chlorine gas

systemic liver damage carbon tetrachloride

narcosis halothane

Subchronic local sensitization toluene diisocyanate

systemic neurotoxicity hexane

Chronic local bronchitis sulfur dioxide

nasal carcinoma formaldehyde

systemic bladder carcinoma 4-amino-biphenyl

kidney damage cadmium

Table 3.Reversible and irreversible interactions of chemicals with

cellular macromolecules as a basis for toxic response

Mechanism Toxic response Example

Irreversible inhibition of

Esterase neurotoxicity tri-o-cresylphosphate

Covalent binding

to DNA

cancer dimethylnitrosamine

Reversible binding to

Hemoglobin oxygen

deprivation

in tissues

carbon monoxide

Cholinesterase neurotoxicity carbamate pesticides

Figure 2. Reversible binding of carbon monoxide to hemo-globin and inhibition of oxygen transport



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Another distinction between types of effectsmay be made according to the general locus ofaction. Local toxicity occurs at the site of firstcontact between the biological system and thetoxic agent. Local effects to the skin, the respira-tory tract, or the alimentary tract may be pro-

duced by skin contact with a corrosive agent, byinhalation of irritant gases, or by ingestion oftissue-damaging materials. This type of toxicresponses is usually restricted to the tissues withdirect contact to the agent. However, life-threat-ening intoxications may occur if vital organs likethe lung are damaged. For example, inhaledphosgene damages the alveoli of the lung andcauses lung edema. The massive damage to thelung results in the substantial mortality observedafter phosgene intoxication.

The opposite to local effects are systemiceffects. They are characterized by the absorp-tion of the chemical and distribution from theport of entry to a distant site where toxic effectsare produced. Except for highly reactive xeno-biotics, which mainly act locally, most chemi-cals act systemically. Many chemicals thatproduce systemic toxicity only cause damageto certain organs, tissues, or cell types withinorgans. Selective damage to certain organs or

tissues by systemically distributed chemicals istermed organ- or tissue-specific toxicity [2]; theorgans damaged are referred to as target organs(Table 4).

Major target organs for toxic effects are thecentral nervous system and the circulatory sys-tem followed by the blood and hematopoieticsystem and visceral organs such as the liver or thekidney. For some chemicals, both local andsystemic effects can be demonstrated; moreover,chemicals producing marked local toxicity may

also cause systemic effects as secondary re-sponses to major disturbances in homeostasis ofthe organism.

1.6. Types of Toxic Effects

The spectrum of toxic effects of chemicals isbroad, and their magnitude and nature depend onmany factors such as the physiocochemical prop-erties of the chemical and its toxicokinetics, theconditions of exposure, and the presence ofadaptive and protective mechanisms. The latterfactors include physiological mechanisms suchas adaptive enzyme induction, DNA repair, andothers. Toxic effects may be transient, reversible,or irrversible; some are deleterious and others arenot. Toxic effects may take the form of tissuepathology, aberrant growth processes, or alteredbiochemical pathways. Some of the more fre-quently encountered types of injury constitutinga toxic response are described in the following.

Immune-mediated hypersensitivity reactionsby antigenic materials are toxic effects ofteninvolved in skin and lung injury by repeatedcontact to chemicals resulting in contact derma-titis and asthma. Inflammation is a frequently

observed local response to the application ofirritant chemicals or may be a component ofsystemic injury. This response may be acute withirritant or tissue damaging materials or chronicwith repetitive exposure to irritants. Necrosis,that is, death of cells or tissues, may be the resultof various pathological processes resulting frombiochemical interactions of xenobiotics, as de-scribed in Chapter 3. The extent and patterns ofnecrosis may be different for different chemicals,even in the same organ. Chemical tumorigenesis

or carcinogenesis (induction of malignant tu-mors) is an effect often observed after chronicapplication of chemicals. Due the long latencyperiod and the poor prognosis for individualsdiagnosed with cancer, studies to predict thepotential tumorigenicity of chemicals have de-veloped into a major area of toxicological re-search. Developmental and reproductive toxicol-ogy are concerned with adverse effects on theability to conceive, and with adverse effects onthe structural and functional integrity of the

fetus. Chemicals may interfere with reproductionthrough direct effects on reproductive organs orindirectly by affecting their neural and endocrine

Table 4. Organ-specific toxic effects induced by chemicals that are

distributed systemically in the organism

Chemical Species Target organ

Benzene humans bone marrow

Hexachlorobutadiene rodents damage to proximal

tubules of the kidney

Paraquat rodents, humans lung

Tri-o-cresylphosphate humans nervous system

Cadmium humans kidney

1,2-Dibromo-

3-chloropropane

humans, rodents testes

Hexane rodents, humans nervous system

Anthracyclines humans heart



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control mechansims. Developmental toxicitydeals with adverse effects on the conceptusthrough all stages of pregnancy. Damage to thefetus may result in embryo reabsorption, fetaldeath, or abortion. Nonlethal fetotoxicity may beexpressed as delayed maturation, decreased birth

weight, or structural malformation. The mostsensitive period for the induction of malforma-tion is during organogenesis; neurobehavioralmalformations may be induced during laterstages of pregnancy.

1.7. DoseResponse: a Fundamental

Issue in Toxicology

In principle, a poison is a chemical that has anadverse effect on a living organism. However,this is not a useful definition since toxic effectsare related to dose. The definition of a poison thusalso involves quantitative biological aspects. Atsufficiently high doses, any chemical may betoxic. The importance of dose is clearly seenwith molecular oxygen or dietary metals. Oxy-gen at a concentration of 21% in the atmosphereis essential for life, but 100% oxygen at atmo-spheric pressure causes massive lung injury in

rodents and often results in death. Some metalssuch as iron, copper, and zinc are essential nu-trients. When they are present in insufficientamounts in the human diet, specific disease pat-terns develop, but in high doses they can causefatal intoxications. Toxic compounds are notrestricted to man-made chemicals, but also in-clude many naturally occurring chemicals. In-deed, the agent with the highest toxicity is anatural poison found in the bacteriumClostridi-um botulinum (LD50 0.01m/kg).

Therefore, all toxic effects are products of theamount of chemical to which the organism isexposed and the inherent toxicity of the chemi-cal; they also depend on the sensitivity of thebiological system.

The term dose is most frequently used tocharacterize the total amount of material to whichan organism is exposed; dose defines the amountof chemical given in relation to body weight.Dose is a more meaningful and comparativeindicator of exposure than the term exposure

itself. Dose usually implies the exposure dose,the total amount of chemical administered to anorganism or incorporated into a test system.

However, dose may not be directly proportionalto the toxic effects since toxicity depends on theamount of chemical absorbed. Usually, dosecorrectly describes only the actual amount ofchemical absorbed when the chemical is admin-istered orally or by injection. Under these cir-

cumstances, the administered dose is identical tothe absorbed dose; other routes of applicationsuch as dermal application or inhalation do notdefine the amount of agent absorbed.

Different chemicals have a wide spectrum ofdoses needed to induce toxic effects or death. Tocharacterize the acute toxicity of different che-micals, LD50 values are frequently used as a basisfor comparisons. Some LD50 values (rat) for arange of chemicals follow:

Ethanol 12 500

Sodium bicarbonate 4 220

Phenobarbital sodium 350

Paraquat 120

Aldrin 46

Sodium cyanide 6.4

Strychnine 5

1,2-Dibromoethane 0.4

Sodium fluoroacetate 0.2

2,3,7,8-Tetrachlorodibenzodioxin 0.01

Certain chemicals are very toxic and producedeath after administration of microgram doses,while others are tolerated without serious toxicityin gram doses. The above data clearly demon-strate that the toxicity of a specific chemical isrelated to dose. The dependence of the toxiceffects of a specific chemical on dose is termeddoseresponse relationship. Before dosere-sponse relationships can be appropriately used,several basic assumptions must be considered.

The first is that the response is due to the chemicaladministered. It is usually assumed that the re-sponses observed were a result of the variousdoses of chemical administered. Under experi-mental conditions, the toxic response usually iscorrelated to the chemical administered, sinceboth exposure and effect are well defined and canbe quantified. However, it is not always apparentthat the response is the result of specific chemicalexposure. For example, an epidemiologic studymight result in discovery of an association

between a response (e.g., disease) and one ormore variables including the estimated dose of achemical. The true doses to which individuals



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have been exposed are often estimates, and thespecificity of the response for that chemical isdoubtful.

Further major necessary assumptions in estab-lishing doseresponse relationships are:

. A molecular site (often termed receptor) withwhich the chemical interacts to produce theresponse. Receptors are macromolecular com-ponents of tissues with which a chemical inter-acts and produces its characteristic effect.

. The production of a response and the degree ofthe response are related to the concentration ofthe agent at the receptor.

. The concentration of the chemical at the re-ceptor is related to the dose administered.Since in most cases the concentration of anadministered chemical at the receptor cannotbe determined, the administered dose or theblood level of the chemical is used as anindicator for its concentration at the molecularsite.

A further prerequisite for using the doseresponse relationship is that the toxic responsecan be exactly measured. A great variety ofcriteria or end points of toxicity may be used.

The ideal end point should be closely associatedwith the molecular events resulting from expo-sure to the toxin and should be readily deter-mined. However, although many end points arequantitative and precise, they are often onlyindirect measures of toxicity. For example,changes in enzyme levels in the blood can beindicative of tissue damage. Patterns of altera-tions may provide insight into which organ orsystem is the site of toxic effects. These measuresusually are not directly related to the mechanism

of toxic action. The doseresponse relationshipcombines the characteristic of exposure and theinherent toxicity of the chemical. Since toxicresponses to a chemical are usually functions ofboth time and dose, in typical doseresponserelationships, the maximum effect observed dur-ing the time of observation is plotted against thedose to give time-independent curves. The time-independent doseresponse relationship may beused to study doseresponse for both reversibleand irreversible toxic effects. However, in risk

assessments that consider the induction of irre-versible effects such as cancer, the time factorplays a major role and has important influences

on the magnitude or likelihood of toxic re-sponses. Thus, for this type of mechanism oftoxic action, dosetimeresponse relationshipsare better descriptors of toxic effects.

The doseresponse relationship is the mostfundamental concept in toxicology. Indeed, an

understanding of this relationship is essential forthe study of toxic chemicals.

From a practical point of view, there are twodifferent types of doseresponse relationships.Doseresponse relationships may be quantal (allor nothing responses such as death) or graded.The graded or variable response involves a con-tinual change in effect with increasing dose, forexample, enzyme inhibition or changes in phys-iological function such as heart rate. Gradedresponses may be determined in an individualor in simple biochemical systems. For example,addition of increasing concentrations of 2,3,7,8-tetrachlorodibenzodioxin to cultured mammali-an cells results in an increase in the concentrationof a specific cytochrome P450 enzyme in the cells(for details of mechanisms, see Section 2.6.4.1).The increase is clearly dose related and spans awide range (Fig. 3). An example for a gradedtoxic effect in an individual may be inflammationcaused by skin contact with an irritant material.

Low doses cause slight irritation; as the amountincreases, irritation turns to inflammation and theseverity of inflammation increases.

In doseresponse studies in a population, aspecific endpoint is also identified and the doserequired to produce this end point is determinedfor each individual in the population. Both dose-dependent graded effects and quantal responses(death, induction of a tumor) may be investigated.

Figure 3. Dose-dependent induction of cytochrome P4501A 1 protein in cultured liver cells treated with 2,3,7,8-tetra-chlorodibenzodioxin [3]



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With increasing amount of a chemical given to agroup of animals, the magnitude of the effect and/or the number of animals affected increase. Forexample, if an irritant chemical is applied to theskin, as the amount of the material increases, thenumbers of animals affected and the severity of

inflammation increases. Quantal responses suchas death induced by a potentially lethal chemicalwill also be dose-dependent. The dose dependen-cy of a quantal effect in a population is based onindividual differences in the response to the toxicchemical. A specific amount of the potentiallylethal xenobiotic given to a group of animals maynot kill all of them, but as the amount givenincreases, the proportion of animals killedincreases.

Althought the distinctions between gradedand quantal doseresponse relationships are use-ful, the two types of responses are conceptuallyidentical. The ordinate in both cases is simplylabeled response, which may be the degree ofresponse in an individual, or the fraction of apopulation responding, and the abscissa is therange of administered doses.

1.7.1. Graphics and Calculations

Even with a genetically homogenous populationof animals of the same species and strain, theproportion of animals showing the effect willincrease with dose (Fig. 4A). When the numberof animals responding is plotted versus the loga-rithm of the dose, a typical sigmoid curve with alog-normal distribution that is symmetrical aboutthe midpoint, is obtained (Fig. 4B).

When plotted on a log-linear scale, the ob-tained normally distributed sigmoid curve ap-

proaches a response of 0% as the dose is de-creased, and 100% as the dose is increased, buttheoretically never passes through 0 or 100%.Small proportions of the population at the right-and left-hand sides of the curve represent hypo-susceptible and hypersusceptible members. Theslope of the dosereponse curve around the 50%value, the midpoint, gives an indication of theranges of doses producing an effect. A steepdoseresponse curve indicates that the majorityof the population will respond over a narrow dose

range; a shallow doseresponse curve indicatesthat a wide range of doses is required to affect themajority of the population. The curve depicted in

Fig. 4 B shows that the majority of the indivi-duals respond about the midpoint of the curve.This point is a convenient description of theaverage response, and is referred to as the medianeffective dose (ED50). If mortality is the end-point, then this dose is referred as median lethal

dose (LD50).Death, a quantal response, is simple to quan-

tify and is thus an end point incorporated in manyacute toxicity studies. Lethal toxicity is usuallycalculated initially from specific mortality levelsobtained after giving different doses of a chemi-cal; the 50% mortality level is used most fre-quently since it represents the midpoint of thedose range at which the majority of deaths occur.This is the dose level that causes death of half ofthe population dosed. The LD50 values are usu-ally given in milligrams of chemical per kilogramof body weight (from the viewpoint of chemistryand for comparison of relative potencies of dif-ferent chemicals, giving the LD50 in moles ofchemical per kilogram body weight would bedesirable). After inhalation, the reference is toLC50 (LC lethal concentration), which, incontrast to LD50values, depends on the time ofexposure; thus, it is usually expressed asX-hourLC50 value. The LD50 or LC50 values usually

represent the initial information on the toxicity ofa chemical and must be regarded as a first, but nota quantitative, hazard indicator that may beuseful for comparison of the acute toxicity ofdifferent chemicals [3].

Similar doseeffect curves can, however, beconstructed for cancer, liver injury, and othertypes of toxic responses. For the determinationof LD50 values and for obtaining comparativeinformation on doseresponse curves, plottinglog dose versus percent response is not practical

since large numbers of animals are needed forobtaining interpretable data. Moreover, other im-portant information on the toxicity of a chemical(e.g., LD05and LD95) cannot be accurately deter-mined due to the slope of sigmoid curve. There-fore, the doseresponse curve is transformed to alog-probit (probit probability units) plot. Thedata in the Fig. 4B form a straight line whentransformed into probit units (Fig. 4C). The EC50or, if death is the end point, the LD50is obtainedby drawing a horizontal line from theprobit unit 5,

which is the 50% response point, to the doseeffect line. At the point of intersection a verticalline is drawn, and this line intersects the abscissa



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at the LD50point. Information on the lethal dosefor 90% or for 10% of the population can also be

derived by a similar procedure. The confidencelimits are narrowest at the midpoint of the line(LD50) and are widest at the two extremes (LD05and LD95) of the doseresponse curve. In additionto permitting determination of a numerical valuefor the LD50 of a chemical with few groups ofdosed animals, the slope of the doseresponsecurve for comparison between toxic effects ofdifferent chemicals is obtained by the probittransformation [4].

The LD50 by itself, however, is an insufficient

index of lethal toxicity, particulary if compar-isons between different chemicals are to be made.For this purpose, all available doseresponse

information including the slope of the doseresponse line should be used. Figure 5 demon-

strates the doseresponse curves for mortality fortwo chemicals.

The LD50of both chemicals is the same (10 mg/kg). However, the slopes of the doseresponsecurves are quite different. Chemical A exhibits aflat doseresponse curve: a large change in doseis required before a significant change in responsewill be observed. In contrast, chemical B exhibits asteep doseresponse curve, that is, a relativelysmall change in dose will cause a large change inresponse. The chemical with the steep slope may

affect a much larger proportion of the populationby incremental increases in dose than chemicalshaving a shallow slope; thus, acute overdosing may

Figure 4. Typical dose response curves for a toxic effectPlots are linear linear (A); log linear (B); and log probit (C) for an identical set of data



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be a problem affecting the majority of a populationfor chemicals with steeper slopes. Chemicals withshallower slopes may represent a problem for thehyperreactive groups at the left-hand side of thedoseresponse curve. Effects may occur at signifi-cantly lower dose levels then for hyperreactive

groups exposed to chemicals with a steep doseresponse.While the LD50 values characterize the poten-

tial hazard of a chemical, the risk of an exposureis determined by the hazard multiplied by theexposure dose. Thus, even very toxic chemicalslike the poison ofClostridium botulinum poseonly a low risk; intoxications with this compoundare rare since exposure is low. Moreover, acuteintoxications with other highly toxic agents suchas mercury salts are rarely seen, despite detect-

able blood levels of mercury salts in the generalpopulation, since the dose is also low. On theother hand, compounds with low toxicity maypose a definite health risk when doses are high,for example, constituents of diet or chemicalsformed during food preparation by heattreatment.

Therefore, for characterizing the toxic risk ofa chemical, besides information on the toxicity,information on the conditions of exposure arenecessary. When using LD50values for toxicity

characterisation, the limitations of LD50 valuesshould be explicitly noted. These limitationsinclude methodological pitfalls influenced by

1. Strain of animal used2. Species of animal used3. Route of administration4. Animal housing

and intrinsic factors limiting the use of LD50

values

1. Statistical method2. No doseresponse curve3. Time to toxic effect not determined4. No information on chronic toxicity

The most serious limitation on the use of LD50values for hazard characterization are the lack ofinformation on chronic effects of a chemical andthe lack of doseresponse information. Chemi-cals with low acute toxicity may have carcino-genic or teratogenic effects at doses that do notinduce acute toxic responses. Other limitationsinclude insufficient information on toxic effectsother than lethality, the cause of death, and thetime to toxic effect. Moreover, LD50values arenot constant, but are influenced by many factorsand may differ by almost one order of magnitudewhen determined in different laboratories.

1.8. DoseResponse Relationships for

Cumulative Effects

After chronic exposure to a chemical, toxic re-sponse may be caused by doses not showingeffects after single dosing. Chronic toxic re-sponses are often based on accumulation of eitherthe toxic effect or of the administered chemical.Accumulation of the administered chemical isobserved when the rate of elimination of the

chemical is lower than the rate of administration.Since the rate of elimination is dependent onplasma concentrations, after long-term applica-tion an equilibrium concentration of the chemicalin the blood is reached. Chemicals may also bestored in fat (polychlorinated pesticides such asDDT) or bone (e.g., lead). Stored chemicalsusually do not cause toxic effects because oftheir low concentrations at the site of toxic action(receptor). After continuous application, the ca-pacities of the storage tissues may become satu-

rated, and xenobiotics may then be present inhigher concentration in plasma and thus at thesite of action; toxic responses result. Besides

Figure 5. Comparison of dose response relationships for

two chemicals (log probit plot)Both chemicals have identical LD50 values, but differentslopes of the dose response curve



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cumulation of the toxic agent, the toxic effectmay also cumulate (Fig. 6).

For chemicals which irreversibly bind tomacromolecules, the magnitude of toxic re-sponses may be correlated with the total doseadministered. In contrast to chemicals which

act reversibly, the effect is not dependent on thefrequency of dosing. Effect accumulation isoften observed with carcinogens and ionizingradiation. In Figure 7 accumulation of effects isexemplified by the time- and dose-dependentinduction of tumors by 4-(dimethylamino)azo-benzene, a potent chemical carcinogen [5]. TheTD50 values (50% of the treated animals carrytumors) are used to characterize the potency.Identical tumor incidences were observed afterhigh doses and a short exposure time or after

low doses and long exposure; the tumor inci-dence was only dependent on the total doseadministered.

Reversibility of toxic responses also dependson the capacity of an organ or tissue to repairinjury. For example, kidney damage by xenobio-tics is often, after survival of the acute phase ofthe intoxication, without further consequencedue to the high capacity of the kidney for cellproliferation and thus the capacity to repair organdamage [6]. In contrast, injury to the central

nervous system is largely irreversible since thedifferentiated cells of the nervous system cannotdivide and dead cells cannot be replaced.

1.9. Factors Influencing DoseResponse

In animals and humans, the nature, severity, andincidence of toxic responses depend on a large

number of exogenous and endogenous factors [7].Important factors are the characteristics of expo-sure, the species and strain of animals used for thestudy, and interindividual variability in humans[8]. Toxic responses are caused by a series ofcomplex interactions of a potentially toxic chem-ical with an organism. The type and magnitude ofthe toxic response is influenced by the concentra-tion of the chemical at the receptor and by the typeof interaction with the receptor. The concentra-tion of a chemical at the site of action is influenced

by the kinetics of uptake and elimination; sincethese are time-dependent phenomena, toxic re-sponses are also time-dependent. Thus, the toxicresponse can be separated into two phases: tox-icokinetics and toxicodynamics (Fig. 8).

Toxicokinetics describe the time dependency ofuptake, distribution, biotransformation, and excre-tion of a toxic agent (a detailed description oftoxicokinetics is given in Section 2.6). Toxicody-namics describes the interaction of the toxic agentwith the receptor and thus specific interactions of

the agent (see below). Toxicokinetics may beheavily influenced by species, strain, and sex andthe exposure characteristics [913]. Differences in

Figure 6. Accumulation of toxic chemicals based on theirrate of excretiona) The rate of excretion is equal to the rate of absorption, noaccumulation occurs; b) Chemical accumulates due to ahigher rate of uptake and inefficient excretion; the plasma

concentrations are, however, not sufficient to exert toxiceffects; c) The plasma concentrations reached after accumu-

lation are sufficient to exert toxicityFigure 7. Time-dependent induction of tumors after differ-ent daily doses of 4-dimethylaminoazobenzene in rats [5]



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toxic response between species, route of exposure,and others factors are often dependent on influ-ences on toxicokinetics. Since toxicodynamics(mechanism of action) are assumed to be identicalbetween species, this provides the basis for arational interspecies extrapolation of toxic effectswhen differences in toxicokinetics are defined.

1.9.1. Routes of Exposure

The primary tissue or system by which a xenobi-otic comes into contact with the body, and fromwhere it may be absorbed in order to exert sys-temic toxicity, is the route of exposure. The

frequent circumstances of environmental expo-sure are ingestion (peroral), inhalation, and skincontact. Also, for investigational and therapeuticpurposes, intramuscular, intravenous, and subcu-taneous injections may also be routes of exposure.

The major routes by which a potentially toxicchemical can enter the body are in descendingorder of effectiveness for systemic delivery injection, inhalation, absorption from the intesti-nal tract, and cutaneous absorption. The relation-ship between route and exposure, biotransforma-

tion, and potential for toxicity, may be complexand is also influenced by the magnitude andduration of dosing (Table 5).

The route of exposure has a major influence ontoxicity because of the effect of route of exposureon the bioavailability of the toxic agent. Themaximum tissue levels achieved, the time tomaximum tissue levels, and thus the duration ofthe effect are determined by the rate of absorptionand the extent of distribution within the system.

Direct injection into veins is usually restricted

to therapeutic applications, but it is important forthe toxicology of intravenously injected drugs inaddicts. Chemicals applied by intravenous injec-tion are rapidly distributed to well-perfused organs

in the blood and thus may result in the rapidinduction of toxic effects. The rapid dilution ofa chemical after intravenous injection by venousblood permits even theinjectionof locally actingorcorrosive chemicals which are well tolerated. Thelikelihood of toxicity from inhaled chemicals de-pends on a number of factors, of which the physicalstate andproperties of theagent,concentration,andtime and frequency of exposure are important.Major influences on the absorption and dispositionof xenobiotics are exerted by species peculiaritiessince the anatomy of the respiratory tract and the

physiology of respiration show major differencesbetween rodents and humans. The water solubilityof a gaseous xenobiotic has a major influence onpenetration into the respiratory tract. As watersolubility decreases and lipid solubility increases,penetration into deeper regions of the lung, thebronchioli, and the alveoli becomes more effec-tive. Water-soluble molecules such as formalde-hyde,are effectively scavenged by the upper respi-ratory tract and may have toxic effects on the eyeand throat. In contrast, gases with low water

solubility such as phosgene may penetrate throughthe bronchii and bronchioli to the alveoli. Damageto the alveolar surface may initiate a series ofevents that finally results in lung edema. Thedegree to which inhaled gases, vapors, and parti-culates are absorbed, and hence their potential toproduce systemic toxicity, depend on their diffu-sion rate through the alveolar mebrane, their solu-bility in blood and tissue fluids, the rate of respira-tion, and blood flow through the capillaries.

Uptake through the alimentary tract represents

an important route of exposure for xenobioticsaccumulated in the food chain, for natural con-stituents of human diet, and, drugs. Absorptionfrom the gastrointestinal tract is dependent on the

Figure 8. Toxicokinetics and toxicodynamics as factorsinfluencing the toxic response

Table 5. Toxicity of chemicals applied by different routes of exposure

(data taken from [13])

Chemical Species Route of application LD50, mg/kg

DDT rat intravenous 68

rat oral 113

rat skin contact 1931

Atropine sulfate rat intravenous 41

rat oral 620

1-Chloro-2,4-

dinitrobenzene

rat oral 1070

rat intraperitoneal 280

rabbit skin contact 130

Dieldrin rat oral 46

rat intravenous 9

rat skin contact 10



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lipophilicity of a chemical, the molecular mass ofthe xenobiotic, and the presence of certain dietaryconstituents may influence the extent and rate ofabsorption. Chemicals absorbed from the gastro-intestinal tract are transported to the liver via theportal vein; hepatic metabolism (hepatic first-

pass effect) may efficiently reduce the concen-tration of the xenobiotic available in the systemiccirculation after oral uptake. Compounds under-going bioactivation in the liver usually exhibitgreater toxicity when given orally than whenabsorbed across the respiratory tract, due to thehigh proportion of material passing through theliver. In contrast, chemicals causing toxicity toextrahepatic, well-perfused organs such as thekidney often show a lower degree of toxicity toextrahepatic target organs when given orally.

Skin contact is an important route of exposurein the occupational and domestic environments.Local effects may include acute inflammation andcorrosion, chronic inflammatory responses, im-mune-mediated reactions, and neoplasia. The per-cutaneous absorption of materials may also be asignificant route for the absorption of systemicallytoxic materials. Factors influencing the percutane-ous absorption of substances include skin site,integrity of skin, temperature, formulation, and

physicochemicalcharacteristics, includingcharge,molecular mass, and hydro- and lipophilicity.

1.9.2. Frequency of Exposure

The exposure of experimental animals may becategorized as acute, subacute, subchronic, andchronic. Acute exposures usually last less than24 h, and all above-mentioned routes of exposuremay be applied. With chemicals of low toxicity,

repeated exposures may be used. Acute inhalationexposure is usually less than 24 h; frequently48 his chosen as timescale. Repeated exposure refers toapplication of the chemical for less than one month(subacute), one to three months (subchronic), andmore than three months (chronic). Chronic expo-sures to detect specific toxic effects (carcinogenic-ity of a chemical) may spanmost of the lifetime of arodent (up to two years). Repeated exposure maybe by any route; the least labor intensive route isoral, by mixing the chemical with the diet; only for

specific chemicals or to simulate likely routes ofexposure for humans are application in drinkingwater, by gastric intubation, and by inhalationapplied. These are more labor-intensive andrequire

skilled personnel and/or sophisticated techniquesand thus are more expensive.

The toxic effects observed after single expo-sure often are different form those seen afterrepeated exposure. For example, inhalation ofhigh concentration of halothane causes anesthe-sia in animals and humans. In contrast, long-termapplication of halothane in lower doses causes

liver damage in sensitive species The frequencyof exposure in chronic studies is important for thetemporal characterisation of exposure. Chemi-cals with slow rates of excretion may accumulateif applied at short dosing intervals, and toxiceffects may result (see Section 1.6). Also, achemical producing severe effect when given ina single high dose may have no detectable effectswhen given in several smaller doses. Interspeciesand strain differences in susceptibility to chemi-cal-induced toxicity may be due to heterogeneity

of populations, species specific physiology (forexample of the respiratory system), basal meta-bolic rate, size- and species-specific toxicoki-netics and routes of metabolism or excretion(Table 6). In some cases, animal tests may givean underestimate, in others an overestimate, ofpotential toxicity to humans [14].

1.9.3. Species-Specific Differences inToxicokinetics

Species-specific differences in toxic responseare largely due to difference in toxicokine-tics and biotransformation. Distribution and

Table 6. Comparative LD50 values for four different chemicals in

different animal species and estimated LD50for humans

Chemical Species LD50, mg/kg

Paraquat rat 134

mouse 77

guinea pig 41

human 32 48

Ethanol rat 12 500

mouse 8000

guinea pig 5500

human 3500 5000

Acetaminophen rat 3763

mouse 777

guinea pig 2968

human 42 800

Aspirin rat 1683

mouse 1769

guinea pig 1102

human 3492



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elimination characteristics are quite variablebetween species. Both qualitative and quantita-tive differences in biotransformation may effect

the sensitivity of a given species to a toxicresponse (Table 7).

For example, the elimination half-live of2,3,7,8-tetrachlorodibenzodioxin in rats is 20 d,and in humans it is estimated to be up to sevenyears [15]. An example for quantitative differencein the extent of biotransformation as a factorinfluencingtoxic response is thespeciesdifferencesin thebiotransformation of theinhalationanesthetichalothane. Both rats and guinea pigs metabolizehalothane to trifluoroacetic acid, a reaction cata-lyzed by a specific cytochrome P450 enzyme[1618]. As a metabolic intermediate, trifluoroa-cetyl chloride is formed, which may react withlysine residues in proteins and with phosphatidylethanolamine in phospholipids (Fig. 9).

This interaction initiates a cascade of eventsfinally resulting in toxicity. The metabolism ofhalothane in guinea pigs occurs at much higherrates than in rats, so guinea pigs are sensitive tohalothane-induced hepatotoxic effects and rats

are resistant. Qualitative differences in biotrans-formation are responsible for apparent differ-ences in the sensitivity of rats and guinea pigsto the bladder carcinogenicity of 2-acetylamido-fluorene. In rats, 2-acetylamidofluorene is me-tabolized byN-oxidation by certain cytochrome

P450 enzymes. TheN-oxide is further convertedto an electrophilic nitrenium ion which interactswith DNA in the bladder; this biotransformationpathway explains the formation of bladdertumors in rats after long-term exposure to 2-acetylamidofluoren. In guinea pigs, 2-acetylami-dofluorene is metabolized by oxidation at thearomatic ring; since nitrenium ions cannot beformed by this pathway, guinea pigs are resistantto the bladder carcinogenicity of 2-acetylamido-fluorene (Fig. 10).

With some chemicals, age may significantlyaffect toxicity, likely due to age related differ-ences in toxicokinetics. The nutritional statusmay modify toxic response, likely by alteringthe concentration of cofactors needed for bio-transformation and detoxication of toxic chemi-cals. Diet also markedly influences carcinogen-induced tumor incidence in animals [19] and maybe a significant factor contributing to humancancer incidence.

The toxic response is influenced by the mag-nitude, number, and frequency of dosing. Thus,local or systemic toxicity produced by acuteexposure may also occur by a cumulative processwith repeated exposures to lower doses; also,additional toxicity may be seen in repeated-ex-posure situations. The relationships for cumula-

Figure 9. Halothane metabolism by cytochrome P450 inrats, guinea pigs, and humans

Table 7. Species and sex differences in the acute toxicity of 1,1-

dichloroethylene after oral administration and inhalation in rats and

mice (data from World Health Organization, Geneva, 1990)

Species Dosing criteria Estimated LD50/LC50

Rat, male inhalation/4 h 7000 32 000 mg/L

Rat, female inhalation/4 h 10 300 mg/L

Mouse, male inhalation/4 h 115 mg/L

Mouse, female inhalation/4 h 205 mg/L

Rat, male gavage 1550 mg/kg

Rat, female gavage 1500 mg/kg

Mouse, male gavage 201 235 mg/kg

Mouse, female gavage 171 221 mg/kg

Figure 10. Biotransformation pathways of 2-acetylamido-fluorene in rats and guinea pigs



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tive toxicity by repetitive exposure comparedwith acute exposure toxicity may be complex,and the potential for cumulative toxicity fromacute doses may not be quantitatively predict-able. For repeated-exposure toxicity, the preciseprofiling of doses may significantly influence

toxicity.

1.9.4. Miscellaneous Factors Influencing

the Magnitude of Toxic Responses

A variety of other factors may affect the natureand exhibition of toxicity, depending on theconditions of the study, for example, housingconditions, handling, volume of dosing, vehicle,etc. Variability in test conditions and proceduresmay result in significant interlaboratory variabil-ity in results of otherwise standard procedures.For chemicals given orally or applied to the skin,toxicity may be modified by the presence ofmaterials in formulations which facilitate orretard the absorption of the chemicals. Withrespiratory exposure to aerosols, particle sizesignificantly determines the depth of penetrationand deposition in the respiratory tract and thusthe site and extent of the toxic effects.

1.10. Exposure to Mixtures

In experimental animals most data on the toxiceffects of chemicals are collected after exposureto a single chemical; in contrast, human expo-sure normally occurs to mixtures of chemicals atlow doses. Moreover, prior, coincidential, andsucessive exposure of humans to chemicals islikely. Interactions between the toxic effects of

different chemicals are difficult to predict, ef-fects of exposure to different chemicals may beindependent, additive, potentiating (ethanol andcarbon tetrachloride), antagonistic (interferencewith action of other chemical, e.g., as seen withantidotes administered in case of intoxications),and synergistic. Ethanol exerts a potentiatingeffect on the hepatotoxicity of carbon tetrachlo-ride. In rats pretreated with ethanol, the hepato-toxic effects of carbon tetrachloride are muchmore pronounced than in control animals. This

potentiation is due to an increased capacity forbioactivation (see Section 2.5) of carbon tetra-chloride in pretreated rats due to increased

concentrations of a cytochrome P450 enzymein the liver [20]. Thus, an important considera-tions for the assessement of potential toxiceffects of mixtures of chemicals are toxicoki-netics and toxicodynamic interactions. Toxico-kinetic interactions of chemicals may influence

absorption, distribution, and biotransformation,both to active and inactive metabolites. Mix-tures of solvents often show a competitive inhi-bition of biotransformation. Usually, one of thecomponents has high affinity for a specificenzyme involved in its biotransformation,whereas another component has only a lowaffinity for that particular enzyme. Thus, pref-erential biotransformation of the componentwith the high affinity occurs. Different out-comes of enzyme inhibition are possible: if thetoxic effects of the component whose metabo-lism is inhibited is dependent on bioactivation,lower rates of bioactivation will result in de-creased toxicity; if the toxic effects are inde-pendent of biotransformation, the extent oftoxicity will increase due to slower rate ofexcretion. Toxic effects of mixtures may alsonot be due to a major component, but to traceimpurities with high toxicity. For example,many long-term effects seen in animal studies

on the toxicity of chlorophenols are believed tobe due to 2,3,7,8-Tetrachlorodibenzodioxin,which was present as a minor impurity in thesamples of chlorophenols used for these studies.

2. Absorption, Distribution,

Biotransformation and Elimination of

Xenobiotics

2.1. Disposition of Xenobiotics

The induction of systemic toxicity usually resultsfrom a complex interaction between absorbedparent chemical and biotransformation productsformed in tissues; the distribution of both parentchemical and biotransformation products in bodyfluids and tissues; their binding and storage char-acteristics; and their excretion.

The biological effects initiated by a xenobio-tic are not related simply to its inherent toxicproperties; the initiation, intensity, and duration

of response are a function of numerous factorsintrinsic to the biological system and the admin-istered dose. Each factor influences the ultimate



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interaction of the xenobiotic and the active site(Section 1.9). Only when the toxic chemical hasreached the specific site and interacted with it canthe inherent toxicity be realized. The route axenobiotic follows from the point of administra-tion or absorption to the site of action usually

involves many steps and is termed toxicoki-netics. Toxicokinetics influence the concentra-tion of the xenobiotic or its active metabolite atthe receptor. In the doseresponse concept out-lined in Section 1.7, it is generally assumed thatthe toxic response is proportional to the concen-tration of the xenobiotic at the receptor. Howev-er, the same dose of a chemical administered bydifferent routes may cause different toxic effects.Moreover, the same dose of two different che-micals may result in vastly different concentra-tions of the chemical or its biotransformationproducts in a particular target organ. This differ-ential pattern is due to differences in the disposi-tion of a xenobiotic (Fig. 11).

The disposition of a xenobiotic consists ofabsorption, distribution, biotransformation, andexcretion, which are all interrelated. The com-plicated interactions between the different pro-cesses of distribution are very important deter-minants of the concentration of a chemical at the

receptor and thus of the magnitude of toxicresponse. They may also be major determinantsfor organ-specific toxicity.

For example, in the case of absorption of axenobiotic through the gastrointestinal tract, thechemical proceeds from the intestinal lumen intothe epithelial cells. Following intracellular trans-port, it passes through the basal membrane andlamina propria and enters the blood or lymphcapillaries for transport to the site of action orstorage. At that site, the xenobiotic is releasedfrom the capillaries, into an interstitial area, andfinally through various membranes to its site ofaction, which may be a specific receptor, anenzyme, a membrane, or many other possible sites.

Figure 11. Possible fate of a xenobiotic in the organisms



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2.2. Absorption

The skin, the lungs, and the cells lining thealimentary tract are major barriers for chemicalspresent in the environment. Except for causticchemicals, which act at the site of first contact

with the organism, xenobiotics must cross thesebarriers to exert toxic effects on one or severaltarget organs. The process whereby a xenobioticmoves through these barriers and enters thecirculation is termed absorption.

2.2.1. Membranes

Because xenobiotics must often pass throughmembranes on their way to the receptor, it isimportant to understand membrane characteris-tics and the factors that permit transfer of foreigncompounds. Membranes are initially encoun-tered whether a xenobiotic is absorbed by thedermal, oral, or vapor route. These membranesmay be associated with several layers of cells or asingle cell. The absorption of a substance fromthe site of exposure may result from passivediffusion, facilitated diffusion, active transport,or the formation of transport vesicles (pinocyto-

sis and phagocytosis). The process of absorptionmay be facilitated or retarded by a variety offactors; for example, elevated temperature in-creases percutaneous absorption by cutaneousvasodilation, and surface-active materials facili-tate penetration. Each area of entry for xenobio-tics into the organism may have specific pecu-liarities, but a unifying concept of biology is thebasic similarity of all membranes in tissues, cells,and organelles.

All membranes are lipid bilayers with polar

head groups (phosphatidylethanolamine, phos-phatidylcholine). The polar groups predominateat the outer and inner surfaces of the membrane;the inner space of the membrane consists ofperpendicularly arranged fatty acids [21]. Thefatty acids do not have a rigid structure and arefluid under physiological conditions; the fluidcharacter of the membrane is largely dominatedby the fatty acid composition. The width of abiological membrane is approximately 79 nm.Figure 12 illustrates the concept of a biological

membrane (fluid-mosaic model).Proteins are intimately associated with the

membrane and may be located on the surface or

inside the membrane structure, or extendcompletely through the membrane. These pro-teins may also form aqueous pores. Hydrophobicforces are responsible for maintaining the struc-tural integrity of both proteins and lipids withinthe membrane structure. The ratio of lipid toprotein in different membranes may vary from5:1 (e.g,. myelin) to 1:5 (e.g., the inner membrane

of mitochondria). Usually, pore diameters inmembranes are small and permit only the passageof low molecular mass chemicals. However,some specialized membranes such as those foundin the glomeruli of the kidney, which can havepore sizes of up to 4 nm, also permit the passageof compounds with molecular mass greater than10 000.

The amphipathic nature of the membranecreates a barrier for ionized, highly polar com-pounds; however, changes in lipid composition,

alterations in the shape and size of proteins, andphysical features of bonding may cause changesin the permeability of membranes [22].

2.2.2. Penetration of Membranes by

Chemicals

A chemical can pass through a membrane by twogeneral processes: passive diffusion and activetransport. Passive diffusion is described by

Ficks law and requires no energy. Active trans-port processes involve the consumption of cellu-lar energy to translocate the chemical across the

Figure 12. Simplified model of the structure of a biologicalmembrane



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membrane. Active transport may also act againsta concentration gradient and result in the accu-mulation of a xenobiotic in a specific organ, celltype or organelle.

Diffusion of Chemicals through Mem-

branes. Many toxic chemicals pass membranesby simple diffusion. Their rates of diffusiondepend on their lipid solubility and are oftencorrelated with the partition coefficient (solubil-ity in organic solvents/solubility in water). Lipo-philic chemicals may diffuse directly through thelipid domain of the membrane. However, a cer-tain degree of water solubility seems to be re-quired for passage since many poorly lipid solu-ble chemicals have been shown to penetrateeasily. Once initial penetration has occurred, themolecule must necessarily traverse a more polarregion to dissociate from the membrane. Com-pounds with extremely high partition coefficientsthus tend to remain in membranes and to accu-mulate there rather than pass through them. Polarcompounds that are insoluble in the nonpolar,fatty-acid-containing inner space of the mem-brane often cannot penetrate membranes, al-though some low molecular mass polar chemi-cals may slowly penetrate through the aqueous

pores of the membranes.The rates of movement of nonpolar xenobio-tics through membranes can be predicted basedon the assumptions from Ficks law of diffusion.Polar compounds and electrolytes of low molec-ular mass are believed to behave similarily. Afirst-order equation appears to be applicable tothe majority of xenobiotics. The rate of diffusionof a xenobiotic is related to its concentrationgradient across the membrane (C1 C2), thesurface area available for transferA, the diameter

of the membraned, and the diffusion constant k.The latter is related to the size and structure of themolecule, the spatial configuration of the mole-cule, and the degree of ionization and lipidsolubility of the xenobiotic.

Rate of diffusion k AC1C2

d

As the xenobiotic is rapidly removed after ab-sorption, C2 can usually be ignored. and a log/

linear plot of the amount of unpenetrated che-micals present over time should be linear. Whenrelatively comparable methods have been used,

calculation of the half-time of penetration t1/2, isuseful. The rate constant of penetration k isderived from

k0:693

t1=2

When the half-time of penetration after oral anddermal administration of several environmentalcontaminants were compared, rates were foundto vary considerably. Clearly, rates of penetrationby different routes in mammals show little or nocorrelation.

Ionization becomes particularly importantwhen xenobiotics are introduced into the gastro-intestinal tract, where a variety of pH conditionsare manifest (see Section 2.2.4.2). Although

many drugs are acids and bases and thus poten-tially ionizable form, most xenobiotics are nei-ther acids nor bases and thus are unaffected bypH. The amount of a xenobiotic in the ionized orunionized form depends upon the pK

a of the

xenobiotic and the pH of the medium. When thepH of a solution is equal to the pKa of thedissolved compound, 50% of the acid or baseexists in the ionized and 50% in the unionizedform. The degree of ionization at a specific pH isgiven by the HendersonHasselbalch equation:

pKapH lognonionized

ionized

pKapH log ionized

nonionized

Since the unionized, lipid-soluble form of a weakacid or base may penetrate membranes, weakorganic acids diffuse most readily in an acidic

environment, and organic bases in a basic envi-ronment. There is some degree of penetrationeven when xenobiotics are not in the most lipid-soluble form, and a small amount of absorptioncan produce serious effects if a compound is verytoxic.

2.2.3. Mechanisms of Transport of

Xenobiotics through Membranes

Filtration. Passage of a solution across aporous membrane results in the retention ofsolutes larger than the pores. This process is



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termed filtration. For example, filtration of so-lutes occurs in the kidney glomeruli, which havelarge pores and retain molecules with molecularmasses greater than 10 000. Elsewhere in thebody, filtration by pores may only result in thepassage of relatively small molecules (molecular

mass ca. 100), and most larger molecules areexcluded. Thus, uptake of xenobiotics throughthese pores is only a minor mechanism ofpenetration.

Special Transport Mechanisms. Specialtransport processes include active transport, fa-cilitated transport, and endocytosis (Table 8).Often, the movement of chemicals across mem-branes is not due to simple diffusion or filtration.Even some very large or very polar moleculesmay readily pass through membranes.

Active transport systems have frequently beenimplicated in these phenomena.Active transportmay be effected by systems that help transportendogenous compounds across membranes.Such processes require energy and transportxenobiotics a

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Toxicology (1) - Ullmann