Ullmann’s Industrial ToxicologyCopyright c© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-31247-1

Toxicology 3

Toxicology

Wolfgang Dekant, Institute of Toxicology, University of Wuerzburg, Germany

Spiridon Vamvakas, Institute of Toxicology, University of Wuerzburg, Germany

1. Introduction . . . . . . . . . . . . . . 61.1. Definition and Scope . . . . . . . . . 61.2. Fields . . . . . . . . . . . . . . . . . . . 61.3. History . . . . . . . . . . . . . . . . . 81.4. Information Resources . . . . . . . 91.5. Terminology of Toxic Effects . . . 111.6. Types of Toxic Effects . . . . . . . . 131.7. Dose–Response: a Fundamental

Issue in Toxicology . . . . . . . . . . 131.7.1. Graphics and Calculations . . . . . . 151.8. Dose-Response Relationships for

Cumulative Effects . . . . . . . . . . 181.9. Factors Influencing

Dose–Response . . . . . . . . . . . . 191.9.1. Routes of Exposure . . . . . . . . . . 191.9.2. Frequency of Exposure . . . . . . . . 201.9.3. Species-Specific Differences in

Toxicokinetics . . . . . . . . . . . . . 211.9.4. Miscellaneous Factors Influencing

the Magnitude of Toxic Responses . 221.10. Exposure to Mixtures . . . . . . . . 232. Absorption, Distribution,

Biotransformation andElimination of Xenobiotics . . . . 23

2.1. Disposition of Xenobiotics . . . . . 232.2. Absorption . . . . . . . . . . . . . . . 242.2.1. Membranes . . . . . . . . . . . . . . . 242.2.2. Penetration of Membranes by

Chemicals . . . . . . . . . . . . . . . . 252.2.3. Mechanisms of Transport of

Xenobiotics through Membranes . . 262.2.4. Absorption . . . . . . . . . . . . . . . 272.2.4.1. Dermal Absorption . . . . . . . . . . 272.2.4.2. Gastrointestinal Absorption . . . . . 302.2.4.3. Absorption of Xenobiotics by the

Respiratory System . . . . . . . . . . 312.3. Distribution of Xenobiotics by

Body Fluids . . . . . . . . . . . . . . 332.4. Storage of Xenobiotics in Organs

and Tissues . . . . . . . . . . . . . . . 362.5. Biotransformation . . . . . . . . . . 372.5.1. Phase-I and Phase-II Reactions . . . 372.5.2. Localization of the

Biotransformation Enzymes . . . . . 38

2.5.3. Role of Biotransformation inDetoxication and Bioactivation . . . 38

2.5.4. Phase-I Enzymes and theirReactions . . . . . . . . . . . . . . . . 39

2.5.4.1. Microsomal Monooxygenases:Cytochrome P450 . . . . . . . . . . . 39

2.5.4.2. Microsomal Monooxygenases:Flavin-Dependent Monooxygenases 41

2.5.4.3. Peroxidative Biotransformation:Prostaglandin-synthase . . . . . . . . 42

2.5.4.4. Nonmicrosomal Oxidations . . . . . 442.5.4.5. Hydrolytic Enzymes in Phase-I

Biotransformation Reactions . . . . 442.5.5. Phase-II Biotransformation

Enzymes and their Reactions . . . . 452.5.5.1. UDP-Glucuronyl Transferases . . . 452.5.5.2. Sulfate Conjugation . . . . . . . . . . 462.5.5.3. Methyl Transferases . . . . . . . . . . 472.5.5.4. N-Acetyl Transferases . . . . . . . . 472.5.5.5. Amino Acid Conjugation . . . . . . 472.5.5.6. Glutathione Conjugation of

Xenobiotics and Mercapturic AcidExcretion . . . . . . . . . . . . . . . . 48

2.5.6. Bioactivation of Xenobiotics . . . . 492.5.6.1. Formation of Stable but Toxic

Metabolites . . . . . . . . . . . . . . . 502.5.6.2. Biotransformation to Reactive

Electrophiles . . . . . . . . . . . . . . 502.5.6.3. Biotransformation of Xenobiotics to

Radicals . . . . . . . . . . . . . . . . . 522.5.6.4. Formation of Reactive Oxygen

Metabolites by Xenobiotics . . . . . 532.5.6.5. Detoxication and Interactions of

Reactive Metabolites with CellularMacromolecules . . . . . . . . . . . . 53

2.5.6.6. Interaction of ReactiveIntermediates with CellularMacromolecules . . . . . . . . . . . . 55

2.5.7. Factors ModifyingBiotransformation and Bioactivation 58

2.5.7.1. Host Factors AffectingBiotransformation . . . . . . . . . . . 58

2.5.7.2. Chemical-Related Factors thatInfluence Biotransformation . . . . . 62

4 Toxicology

2.5.8. Elimination of Xenobiotics and theirMetabolites . . . . . . . . . . . . . . . 62

2.5.8.1. Renal Excretion . . . . . . . . . . . . 632.5.8.2. Hepatic Excretion . . . . . . . . . . . 642.5.8.3. Xenobiotic Elimination by the

Lungs . . . . . . . . . . . . . . . . . . . 652.6. Toxicokinetics . . . . . . . . . . . . . 652.6.1. Pharmacokinetic Models . . . . . . . 662.6.1.1. One-Compartment Model . . . . . . 662.6.1.2. Two-Compartment Model . . . . . . 672.6.2. Physiologically Based

Pharmacokinetic Models . . . . . . . 683. Mechanisms ofAcute andChronic

Toxicity and Mechanisms ofChemical Carcinogenesis . . . . . . 69

3.1. Biochemical Basis of Toxicology . 693.2. Receptor-Ligand Interactions . . 703.2.1. Basic Interactions . . . . . . . . . . . 703.2.2. Interference with Excitable Mem-

brane Functions . . . . . . . . . . . . 723.2.3. Interference of Xenobiotics with

Oxygen Transport, Cellular OxygenUtilization, and Energy Production 73

3.3. Binding of Xenobiotics toBiomolecules . . . . . . . . . . . . . . 74

3.3.1. Binding of Xenobiotics or theirMetabolites to Cellular Proteins . . 75

3.3.2. Interaction of Xenobiotics or theirMetabolites with Lipid Constituents 76

3.3.3. Interactions of Xenobiotics or theirMetabolites with nucleic Acids . . . 76

3.4. Perturbation of CalciumHomeostasis by Xenobioticsor their Metabolites . . . . . . . . . 77

3.5. Nonlethal Genetic Alterations inSomatic Cells and Carcinogenesis 78

3.6. DNA Structure and Function . . . 793.6.1. DNA Structure . . . . . . . . . . . . . 793.6.2. Transcription . . . . . . . . . . . . . . 803.6.3. Translation . . . . . . . . . . . . . . . 803.6.4. Regulation of Gene Expression . . . 803.6.5. DNA Repair . . . . . . . . . . . . . . . 813.7. Molecular Mechanisms of

Malignant Transformation andTumor Formation . . . . . . . . . . 81

3.7.1. Mutations . . . . . . . . . . . . . . . . 813.7.2. Causal Link between Mutation

and Cancer . . . . . . . . . . . . . . . 833.7.3. Proto-Oncogenes and Tumor-

Suppressor Genes as Genetic Targets 833.7.4. Genotoxic versus Nongenotoxic

Mechanisms of Carcinogenesis . . . 84

3.8. Mechanisms of ChemicallyInduced Reproductive andDevelopmental Toxicity . . . . . . . 84

3.8.1. Embryotoxicity, Teratogenesis, andTransplacental Carcinogenesis . . . 85

3.8.2. Patterns of Dose–Response in Ter-atogenesis, Embryotoxicity, andEmbryolethality . . . . . . . . . . . . 86

4. Methods in Toxicology . . . . . . . 874.1. Toxicological Studies: General

Aspects . . . . . . . . . . . . . . . . . 874.2. Acute Toxicity . . . . . . . . . . . . . 904.2.1. Testing for Acute Toxicity by the

Oral Route: LD50 Test and Fixed-Dose Method . . . . . . . . . . . . . . 90

4.2.2. Testing for Acute Skin Toxicity . . 924.2.3. Testing forAcute Toxicity by Inhala-

tion . . . . . . . . . . . . . . . . . . . . 944.3. Repeated-Dose Toxicity

Studies: Subacute, Subchronicand Chronic Studies . . . . . . . . . 95

4.4. Ophtalmic Toxicity . . . . . . . . . . 964.5. Sensitization Testing . . . . . . . . . 974.6. Phototoxicity and

Photosensitization Testing . . . . . 994.7. Reproductive and Developmental

Toxicity Tests . . . . . . . . . . . . . 994.7.1. Fertility and General Reproductive

Performance . . . . . . . . . . . . . . 1004.7.2. Embryotoxicity and Teratogenicity 1004.7.3. Peri- and Postnatal Toxicity . . . . . 1014.7.4. Multigeneration Studies . . . . . . . 1014.7.5. The Role of Maternal Toxicity in

Teratogenesis . . . . . . . . . . . . . . 1024.7.6. In Vitro Tests for Developmental

Toxicity . . . . . . . . . . . . . . . . . 1024.8. Bioassays to Determine the

Carcinogenicity of Chemicalsin Rodents . . . . . . . . . . . . . . . 103

4.9. In Vitro and In Vivo Short-termTests for Genotoxicity . . . . . . . . 105

4.9.1. Microbial Tests for Mutagenicity . . 1064.9.1.1. The Ames Test for Bacterial Muta-

genicity . . . . . . . . . . . . . . . . . 1064.9.1.2. Mutagenicity Tests in Escherichia

coli . . . . . . . . . . . . . . . . . . . . 1114.9.1.3. Fungal Mutagenicity Tests . . . . . . 1124.9.2. Eukaryotic Tests for Mutagenicity . 1124.9.2.1. Mutation Tests in Drosophila

melanogaster . . . . . . . . . . . . . . 1124.9.2.2. In Vitro Mutagenicity Tests in

Mammalian Cells . . . . . . . . . . . 1124.9.3. In VivoMammalian Mutation Tests 1144.9.3.1. Mouse Somatic Spot Test . . . . . . 114

Toxicology 5

4.9.3.2. Mouse Specific Locus Test . . . . . 1144.9.3.3. Dominant Lethal Test . . . . . . . . . 1144.9.4. Test Systems Providing Indirect

Evidence for DNA Damage . . . . . 1144.9.4.1. Unscheduled DNA Synthesis (UDS)

Assays . . . . . . . . . . . . . . . . . . 1144.9.4.2. Sister-Chromatid Exchange Test . . 1154.9.5. Tests for Chromosome Aberrations

(Cytogenetic Assays) . . . . . . . . . 1164.9.5.1. Cytogenetic Damage and its

Consequences . . . . . . . . . . . . . 1164.9.5.2. In Vitro Cytogenetic Assays . . . . . 1174.9.5.3. In Vivo Cytogenetic Assays . . . . . 1174.9.6. Malignant Transformation of

Mammalian Cells in Culture . . . . 1184.9.7. In Vivo Carcinogenicity Studies of

Limited Duration . . . . . . . . . . . 1194.9.7.1. Induction of Altered Foci in the

Rodent Liver . . . . . . . . . . . . . . 1194.9.7.2. Induction of Lung Tumors in

Specific Sensitive Strains of Mice . 1204.9.7.3. Induction of Skin Tumors in Specific

Sensitive Strains of Mice . . . . . . . 1204.9.8. Methods to Assess Primary DNA

Damage . . . . . . . . . . . . . . . . . 1204.9.8.1. Alkaline Elution Techniques . . . . 1204.9.8.2. Methods to Detect and Quantify

DNA Modifications . . . . . . . . . . 1214.9.9. Interpretation of Results Obtained in

Short-Term Tests . . . . . . . . . . . . 122

4.10. Evaluation of Toxic Effects on theImmune System . . . . . . . . . . . . 123

4.11. Toxicological Evaluation of theNervous System . . . . . . . . . . . . 124

4.11.1. Functional Observational Battery . 1244.11.2. Locomotor Activity . . . . . . . . . . 1254.12. Effects on the Endocrine System . 1265. Evaluation of Toxic Effects . . . . 1265.1. Acceptable risk, Comparison of

Risks, and EstablishingAcceptable Levels of Risk . . . . . 127

5.2. The Risk Assessment Process . . . 1295.2.1. Hazard Identification Techniques . 1295.2.2. Determination of Exposure . . . . . 1315.2.3. Dose-Response Relationships . . . . 1325.2.4. Risk Characterization . . . . . . . . . 1335.2.4.1. The Safety-Factor Methodology . . 1335.2.4.2. Risk Estimation Techniques for

Nonthreshold Effects . . . . . . . . . 1355.2.4.3. Mathematical Models Used in High-

to Low-Dose Risk Extrapolation . . 1365.2.4.4. Interpretation of Data from Chronic

Animal Bioassays . . . . . . . . . . . 1375.2.4.5. Problems and Uncertainties in Risk

Assessment . . . . . . . . . . . . . . . 1375.3. Future Contributions of

Scientifically Based Procedures toRisk Assessment and QualitativeRisk Assessment for Carcinogens 141

5.4. Risk Assessment for Teratogens . 1456. References . . . . . . . . . . . . . . . 146

Abbreviations:

Ah-R arylhydrocarbon receptorAP apurinic/apyrimidinic siteAPS adenosine 5′-phosphosulfateBHK baby hamster kidneyBIBRA British Industrial Biological Re-

search AssociationCoA Coenzym ADDT 1,1′-(2,2,2-trichloro-

ethylidene)bis-(4-chlorobenzene)DHHS U.S. Department of Health and

Human ServicesDHP delayed hypersensitive responseECETOC European Chemical Industry

Ecology and Toxicology CentreED effective doseELISA enzyme-linked immunosorbent

assayFCA Freund’s complete adjuvant

FAD flavine adenine dinucleotideGABA γ-aminobutyrateGC/MS gas chromatography/mass spec-

troscopyGOT glutamic acid oxalacetic transam-

inaseGSH glutathioneGSSG glutathione disulfideGST glutathione S-transferaseGTP guanosine 5′-triophosphateHGPRT hypoxanthine – guanine phospho-

ribosyltransferaseIPCS International Programme on

Chemical SafetyLDH lactate dehydrogenaseLOAEL lowest-observed-adverse-effect

levelLOEL lowest-observed-effect levelMIF migration inhibition factor

6 Toxicology

mRNA messenger RNAMTD maximum tolerated doseNADPH nicotinamide dinucleotide phos-

phate (H)NOEL no-observed-effect-levelNTP National Toxicology ProgramPAPS 3′-phosphoadenosine-5′-phos-

phosulfatePG prostaglandinrRNA ribosomal RNASHE Syrian hamster embryoSMART somatic mutation and recombina-

tion testT, or TCDD 2,3,7,8-tetrachlorodibenzodioxinTD tumor doseTK thymidine kinasetRNA transfer RNAUDP uridine diphosphateUDPG uridine diphosphate glucoseUDPGA uridine diphosphate glucuronic

acidUDS unscheduled DNA synthesis

1. Introduction

1.1. Definition and Scope

Chemicals that are used or of potential use incommerce, the home, the environment, andmed-ical practice may present various types of harm-ful effects. The nature of these effects is deter-mined by the physicochemical characteristics ofthe agent, its ability to interact with biologicalsystems (hazard), and its potential to come intocontact with biological systems (exposure).Toxicology studies the interaction between

chemicals and biological systems to determinethe potential of chemicals to produce adverse ef-fects in living organisms. Toxicology also inves-tigates the nature, incidence,mechanismsof pro-duction, factors influencing their development,and reversibility of such adverse effects. Ad-verse effects are defined as detrimental to thesurvival or the normal functioning of the individ-ual. Inherent in this definition are the followingkey 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 of dif-ferent exposure doses on the magnitude andincidence of the toxic effect should be quan-titated. Such dose-response relationships areof prime importance in confirming a causalrelationship between chemical exposure andtoxic effect (for details, see Section 1.7).

Research in toxicology is mainly concernedwith determining the potential for adverse ef-fects caused by chemicals, both natural and syn-thetic, to assess their hazard and risk of humanexposure and thus provide a basis for appropri-ate precautionary, protective and restrictivemea-sures. Toxicological investigations should per-mit evaluation of the following characteristicsof toxicity:

1) The basic structural, functional, or biochem-ical injury produced

2) Dose-response relationships3) The mechanisms of toxicity (fundamentalbiochemical alterations responsible for theinduction and maintenance of the toxic re-sponse) and reversibility of the toxic effect

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 on thepattern of potential exposure or toxicity, may benecessary in order to determine the risk-benefitratio for their use in specific circumstances andto devise protective and precautionarymeasures.Indeed, with drugs, pesticides, food additives,and cosmetic preparations, toxicology testingmust be performed in accordance with govern-ment regulations before use.

1.2. Fields

Toxicology is a recognized scientific disciplineencompassing both basic and applied issues. Al-though only generally accepted as a specific sci-entific field during this century, its principleshave been appreciated for centuries. The harm-ful or lethal effects of certain chemicals, mainlypresent in minerals and plants or transmitted

Toxicology 7

venomous animals, have been known since pre-historic times. In many countries, toxicology asa discipline has developed from pharmacology.Pharmacology and toxicology both study the ef-fect 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 studied ef-fects separated pharmacology and toxicology.Pharmacology focused on chemicals with bene-ficial effects (drugs) at lower doses whereas tox-icology studied the adverse health effects occur-ring with the same chemicals at high doses. To-day, the main interest of research in toxicologyhas shifted to studies on the long-term effectsof chemicals after low-dose exposure, such ascancer or other irreversible diseases; moreover,most chemicals of interest to toxicologists arenot used as drugs.The variety of potential adverse effects and

the diversity of chemicals present in our environ-ment combine to make toxicology a very broadscience. Toxicology uses basic knowledge fromclinical and theoretical medicine and natural sci-ences such as biology and chemistry (Fig. 1).Because of this diversity, toxicologists usuallyspecialize in certain areas.Any attempt to define the scope of toxicology

must 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 not pos-sible.The professional activities of toxicologists

can be divided into three main categories: de-scriptive, mechanistic, and regulatory. The de-scriptive toxicologist is concerned directly withtoxicity testing. Descriptive toxicology still of-ten relies on the tools of pathology and clinicalchemistry, but since the 1970smoremechanism-based test systems have been included in toxic-ity testing [1]. The appropriate toxicity tests inexperimental animals yield information that isextrapolated to evaluate the risk posed by ex-posure to specific chemicals. The concern maybe limited to effects on humans (drugs, indus-trial chemicals in the workplace, or food addi-tives) or may encompass animals, plants, andother factors that might disturb the balance of

the ecosystem (industrial chemicals, pesticides,environmental pollutants).The mechanistic toxicologist is concerned

with elucidating the mechanisms by whichchemicals exert their toxic effects on living or-ganisms. Such studies may result in the develop-ment of sensitive predictive toxicity tests usefulin obtaining information for risk assessment (seeChap. 4).Mechanistic studiesmayhelp in thede-velopment of chemicals that are safer to use orof more rational therapies for intoxications. Inaddition, an understanding of the mechanismsof toxic action also contributes to the knowl-edge of basic mechanisms in physiology, phar-macology, cell biology, and biochemistry. In-deed, toxic chemicals have been used with greatsuccess as mechanistic tools to elucidate mech-anisms of physiological regulation. Mechanis-tic toxicologists are often active in universities;however, industry and government institutionsare now undertaking more and more research inmechanistic toxicology.Regulatory toxicologists have the responsi-

bility of deciding on the basis of data providedby the descriptive toxicologist and the mecha-nistic toxicologist if a drug or chemical poses asufficiently low risk to be used for a stated pur-pose. Regulatory toxicologists are often activein government institutions and are involved inthe establishment 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 onthe classes of chemicals dealt with or applicationof knowledge from toxicology for a specific field(Table 1).Forensic toxicology comprises both analyti-

cal chemistry and fundamental toxicologic prin-ciples. It is concerned with the legal aspectsof the harmful effects of chemicals on humans.The expertise of the forensic toxicologist is in-voked primarily to aid in establishing the causeof death and elucidating its circumstances in apostmortem investigation. The field of clinicaltoxicology recognizes and treats poisoning, bothchronic and acute. Efforts are directed at treatingpatients poisoned by chemicals and at the devel-opment of new techniques to treat these intoxi-cations. Environmental toxicology is a relativelynew area that studies the effects of chemicalsreleased by man on wildlife and the ecosystemand thus indirectly on human health.

8 Toxicology

Figure 1. Scientific fields influencing the science of toxicology

Table 1. Areas of toxicology

Field Tasks and objectives

Forensic toxicology diagnoses poisoning by analyticalprocedures

Pesticide toxicology studies the safety of pesticides,develops new pesticides

Occupational toxicology assesses potential adverse effectsof chemicals used in theworkplace, recommendsprotective procedures

Drug toxicology studies potential effects of drugsafter high doses, elucidatesmechanisms of sideeffects

Regulatory toxicology develops and interprets toxicitytesting programs and is involvedin controlling the use of chemicals

Environmental toxicology studies the effects of chemicals onecosystems and on humans afterlow-dose exposure from theenvironment

Drug toxicology plays amajor role in the pre-clinical safety assessment of chemicals intendedfor use as drugs. Drug toxicology also eluci-dates the mechanisms of side effects observedduring clinical application. Occupational toxi-cology studies the acute and chronic toxicity ofchemicals encountered in the occupational en-vironment. Both acute and chronic occupationalpoisonings have exerted amajor influence on thedevelopment of toxicology in general. Occupa-

tional toxicology also helps in the developmentof safety procedures to prevent intoxications inthe workplace and assists in the definition of ex-posure limits. Pesticide toxicology is involved inthe development of newpesticides and the safetyof pesticide formulations. Pesticide toxicologyalso characterizes potential health risks to thegeneral population caused by pesticide residuesin food and drinking 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 surviv-ing medical works of Hippocrates, Aristotle,and Theophrastus, published during the period400–250 b.c., all included some mention of poi-sons.The Greek and Roman civilizations know-

ingly used certain toxic chemicals and extractsfor hunting, warfare, suicide, and murder. Up

Toxicology 9

to the Middle Ages, toxicology was restrictedto the use of toxic agents for murder. Poisoningwas developed to an art inmedieval Italy and hasremained a problem ever since, and much of theearlier impetus for the development of toxicol-ogywas primarily forensic. There appear to havebeen few advances in either medicine or toxicol-ogy between the time of Galen (131–200 a.d.)and Paracelsus (1493–1541). The latter laid thegroundwork for the later development ofmoderntoxicology. He clearly was aware of the dose–response relationship. His statement that “Allsubstances are poisons; there is none that is nota poison. The right dose differentiates a poisonand a remedy,” is properly regarded as a land-mark in the development of the science of tox-icology. His belief in the value of experimenta-tion also represents a break with much earliertradition. Important developments in the 1700sinclude the publication of Ramazzini’sDiseasesof Workers, which led to his recognition as thefather of occupational medicine. The correlationbetween the occupation of chimney sweepersand scrotal cancer by Pott in 1775 is also note-worthy.

Orfila, a Spaniard working at the Universityof Paris, clearly identified toxicology as a sepa-rate science and wrote the first book devoted ex-clusively to it (1815). Workers of the later 1800swho produced treatises on toxicology includeChristison, Kobert, and Lewin. They increasedour knowledge of the chemistry of poisons, thetreatment of poisoning, the analysis of both xe-nobiotics and toxicity, aswell asmodes of actionand detoxication. A major impetus for toxicol-ogy in the 1900s was the use of chemicals forwarfare. In World War I, a variety of poisonouschemicalswere used in the battlefields ofFrance.This provided stimulus for work onmechanismsof toxicity as well as medical countermeasuresto poisoning. Since the 1960s, toxicology hasentered a phase of rapid development and haschanged from a science that was almost entirelydescriptive to one in which the study of mech-anisms has become the prime task. The manyreasons for this include the development of newanalytical methods since 1945, the emphasis ondrug testing following the thalidomide tragedy,the emphasis on pesticide testing following thepublication ofRachel Carson’s Silent Spring andpublic concern over environmental pollution anddisposal of hazardous waste.

1.4. Information Resources

Because of the complexity of toxicology as a sci-ence and the impact of toxicological investiga-tions on legislation and commerce, a wide rangeof information on the toxic effects of chemi-cals is available. No single, exhaustive sourceof toxicological data exists; several sources arerequired to obtain comprehensive informationon a particular chemical. Printed sources areoften quicker and easier to use than computerdata bases, but interactive online searching canrapidly gather important information from thehuge number of sources present.The information explosion in toxicology has

resulted in a comprehensive volume dedicatedto toxicological information sources:P. Wexler, P. J. Hakkinen, G. Kennedy, Jr.F.W.Stoss, Information Resources in Toxi-cology, 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 booksare listed below:

C.D. Klaasen,Casarett and Doull’s Toxicol-ogy; The Basic Science of Poisons, 6th ed.,McGraw-Hill, New York, 2001.G.D. Clayton, F. E. Clayton (eds): Patty’sIndustrial Hygiene and Toxicology, Wiley,New York, 1993.J. G. Hardman, L. E. Limbird,Goodman andGilman’s, The Pharmacological Basis ofTherapeutics, 10th ed., McGraw-Hill, NewYork, 2001.W.A.Hayes,Principles andMethods of Tox-icology, 3rd ed., Raven Press, New York,2001.E. Hodgson (Ed.): Textbook of Modern Tox-icology, 3rd ed., Wiley Interscience, 2004.T. A. Loomis, A.W. Hayes, Loomis’s Essen-tials of Toxicology, 4th ed., Academic Press,San Diego, 1996.

The huge volume byN. I. Sax andR. J. Lewis,Dangerous Properties of Industrial Materials,7th ed., Wiley, New York, 1999, contains ba-sic toxicological data on a large selection ofchemicals (almost 20 000) and may serve as auseful guide to the literature for compounds notcovered in other publications.

10 Toxicology

Monographs. The best summary informa-tion on toxicology is published in the formof series by governments and international or-ganizations. Most of these series are summa-rizing the results of toxicity studies on spe-cific chemicals. The selection of these chemi-cals is mainly based on the extent of their usein industry (e.g. trichloroethene), their occur-rence as environmental contaminants (mercury)or their extraordinary toxicity (e.g. 2,3,7,8-tetra-chlorodibenzodioxin):

American Conference of Governmental In-dustrial Hygienists, Threshold Limit Valuesand Biological Exposure Indices (Cincin-nati, OH). Published annually.MAK-Begrundungen, VCH Publishers,Weinheim, Federal Republic of Germany.This German series includes detailed infor-mation on the toxicity of chemicals on theGerman MAK list (ca. 150 reports are avail-able; the series 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.TheEnvironmental ProtectionAgency (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 Ecol-

ogy and Toxicology Centre (ECETOC) issues“Monographs” (more than 20 have been pub-lished) and “Joint Assessments of CommodityChemicals.”The monographs of the International Agency

for Research on Cancer are definitive evalua-tions of carcinogenic hazards. The “Environ-mental Health Criteria” documents of the Inter-national Programme on Chemical Safety (IPCS)assess environmental and human health effectsof exposure to chemicals, andbiological or phys-ical agents. A related “Health and Safety Guide”series give guidance on setting exposure limitsfor national chemical safety programs.The National Institute for Occupational

Safety and Health (NIOSH), has published 50“Current Intelligence Bulletins” on health haz-ards of materials and processes at work.The technical report series of the National

Toxicology Program (NTP) reports results oftheir carcinogenicity bioassays, which include

summaries 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 “Annual Re-view of Current DHHS [U.S. Department ofHealth and Human Services], DOE [U.S. De-partment of Energy] and EPA Research” relatedto toxicology.A large number of internet-based resources

are also available to collect information ontoxic effects of chemicals and methods for riskassessment. Some information sites containinglarge amounts of downloadable information arelisted below:

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

US Environmental ProtectionAgency (EPA), ECOTOX Database,http://www.epa.gov/ecotox/

Organisation for Economic Co-operationand Development (OECD), test guidelines,http://www.oecd.org

Agency for Toxic Substances andDisease Registry (ATSDR), toxi-cological profile information sheethttp://www.atsdr.cdc.gov/toxprofiles/

European Chemicals Bureau,http://ecb.jrc.it/

National Toxicology Programm,http://ntp-server.niehs.nih.gov/htdocs/liason/-Factsheets/FactsheetList.html

United Nations Environment Programm,Chemicals http://www.chem.unep.ch/

Journals Results of toxicological researchare published in more than 100 journals. Thoselisted below mainly publish research closely re-lated to toxicology, but articles of relevancemayalso be found in other biomedical journals:

Archives of Environmental Contaminationand ToxicologyArchives of ToxicologyBiochemical PharmacologyChemical Research in ToxicologyCRC Critical Reviews in ToxicologyClinical ToxicologyDrug and Chemical ToxicologyEnvironmental Toxicology and Chemistry

Toxicology 11

Table 2. Toxic effects of different chemicals categorized by time scale and general locus of action

Exposure Site Effect Chemical

Acute local lung edema chlorine gassystemic liver damage carbon tetrachloride

narcosis halothaneSubchronic local sensitization toluene diisocyanate

systemic neurotoxicity hexaneChronic local bronchitis sulfur dioxide

nasal carcinoma formaldehydesystemic bladder carcinoma 4-amino-biphenyl

kidney damage cadmium

Food and Chemical ToxicologyFundamental and Applied ToxicologyJournal of the American College of Toxi-cologyJournal of Analytical ToxicologyJournal of Applied ToxicologyJournal of Biochemical ToxicologyJournal of Toxicology and EnvironmentalHealthNeurotoxicology and TeratologyPharmacology and ToxicologyPractical In Vitro ToxicologyRegulatory Toxicology and PharmacologyReproductive ToxicologyToxicologyToxicology and Applied PharmacologyToxicology and Industrial HealthToxicology In VitroToxicology 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. Sinceon-line searching of commercial data basessuch as STN-International may be expensive,CD-ROM-based systems are increasingly be-ing used. The major advantages are speed, theability to refine searches and format the results,and non-text search options, such as chemicalstructure searching on Beilstein and ChemicalAbstracts.Useful information about actual research on

the toxicology of chemicals may be obtained bysearching Chemical Abstracts or Medline withthe appropriate keywords. Specific data bankscovering toxicology are the Registry of ToxicEffects of Chemical Substances, which givessummary data, statistics, and structures; Toxline

(available in DIMDI) gives access to the litera-ture.

1.5. Terminology of Toxic Effects

Toxic effects may be divided according totimescale (acute and delayed), general locusof action (local, systemic, organ specific), orbasic mechanisms of toxicity (reversible ver-sus irreversible). Acute toxic effects are thosethat occur after brief exposure to a chemical.Acute toxic effects usually develop rapidly aftersingle or multiple administrations of a chemi-cal; however, acute exposure may also producedelayed toxicity. For example, inhalation of alethal dose of HCN causes death in less thana minute, whereas lethal doses of 2,3,7,8-tetra-chlorodibenzodioxin will result in the deathof experimental animals after more than twoweeks. Chronic effects are those that appearafter repetitive exposure to a substance; manycompounds require several months of continu-ous exposure to produce adverse effects. Often,the chronic effects of chemicals are differentfrom those seen after acute exposure (Table 2).For example, inhalation of chloroform for a shortperiod of time may cause anesthesia; long-terminhalation of much lower chloroform concentra-tions causes liver damage. Carcinogenic effectsof chemicals usually have a long latency period;tumors may be observed years (in rodents) oreven decades (in humans) after exposure.Toxic effects of chemicalsmay also be classi-

fied based on the type of interaction between thechemical and the organism. Toxic effects maybe caused by reversible and irreversible interac-tions (Table 3). When reversible interactions areresponsible for toxic effects, the concentrationof the chemical present at the site of action is

12 Toxicology

the only determinant of toxic outcome. Whenthe concentration of the xenobiotic is decreasedby excretion or biotransformation, a parallel de-crease of toxic effects is observed.

Table 3. Reversible and irreversible interactions of chemicals withcellular macromolecules as a basis for toxic response

Mechanism Toxic response Example

Irreversible inhibitionof Esterase neurotoxicity tri-o-cresylphosphate

Covalent bindingto DNA

cancer dimethylnitrosamine

Reversible binding toHemoglobin oxygen

deprivation intissues

carbon monoxide

Cholinesterase neurotoxicity carbamate pesticides

After complete excretion of the toxic agent,toxic effects are reduced to zero (see below).A classical example for reversible toxic effectsis carbon monoxide. Carbon monoxide binds tohemoglobin and, due to the formation of thestable hemoglobin–carbon monoxide complex,binding of oxygen is blocked. As a result ofthe impaired oxygen transport in blood fromthe lung, tissue oxygen concentrations are re-duced and cells sensitive to oxygen deprivationwill die. The toxic effects of carbon monoxideare directly correlated with the extent of car-boxyhemoglobin in blood, the concentration ofwhich is dependent on the inhaled concentrationof carbon monoxide. After exhalation of carbonmonoxide and survival of the acute intoxication,no toxic effect remains (Fig. 2).

Figure 2. Reversible binding of carbon monoxide tohemoglobin and inhibition of oxygen transport

Irreversible toxic effects are often caused bythe covalent binding of toxic chemicals to bi-ological macromolecules. Under extreme con-

ditions, the modified macromolecule is not re-paired; after excretion of the toxic agent, the ef-fect persists. Further exposure to the toxic agentwill produce additive effects; many chemicalscarcinogens are believed to act through irre-versible changes (see Section 2.5.6).Another distinction between types of effects

may be made according to the general locusof action. Local toxicity occurs at the site offirst contact between the biological system andthe toxic agent. Local effects to the skin, therespiratory tract, or the alimentary tract maybe produced by skin contact with a corrosiveagent, by inhalation of irritant gases, or by in-gestion of tissue-damaging materials. This typeof toxic responses is usually restricted to the tis-sues with direct contact to the agent. However,life-threatening intoxications may occur if vi-tal organs like the lung are damaged. For exam-ple, inhaled phosgene damages the alveoli of thelung and causes lung edema. The massive dam-age to the lung results in the substantialmortalityobserved after phosgene intoxication.The opposite to local effects are systemic ef-

fects. They are characterized by the absorptionof the chemical and distribution from the port ofentry to a distant site where toxic effects are pro-duced. Except for highly reactive xenobiotics,which mainly act locally, most chemicals actsystemically. Many chemicals that produce sys-temic toxicity only cause damage to certain or-gans, tissues, or cell types within organs. Selec-tive damage to certain organs or tissues by sys-temically distributed chemicals is termed organ-or tissue-specific toxicity [2]; the organs dam-aged are referred to as target organs (Table 4).

Table 4. Organ-specific toxic effects induced by chemicals that aredistributed systemically in the organism

Chemical Species Target organ

Benzene humans bone marrowHexachlorobutadiene rodents damage to

proximal tubulesof the kidney

Paraquat rodents,humans

lung

Tri-o-cresylphosphate humans nervous systemCadmium humans kidney1,2-Dibromo-3-chloropropane humans,

rodentstestes

Hexane rodents,humans

nervous system

Anthracyclines humans heart

Toxicology 13

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 liveror the kidney. For some chemicals, both localand systemic effects can be demonstrated;more-over, chemicals producing marked local toxicitymay 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 dependon many factors such as the physiocochemicalproperties of the chemical and its toxicokinetics,the conditions of exposure, and the presence ofadaptive and protective mechanisms. The latterfactors include physiological mechanisms suchas adaptive enzyme induction, DNA repair, andothers. Toxic effectsmaybe transient, reversible,or irrversible; some are deleterious and othersare not. 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 reactions

by antigenic materials are toxic effects often in-volved in skin and lung injury by repeated con-tact to chemicals resulting in contact dermati-tis and asthma. Inflammation is a frequently ob-served local response to the application of irri-tant chemicals or may be a component of sys-temic 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 re-sult of various pathological processes resultingfrom biochemical interactions of xenobiotics, asdescribed in Chapter 3. The extent and patternsof necrosis may be different for different chem-icals, even in the same organ. Chemical tumori-genesis or carcinogenesis (induction of malig-nant tumors) is an effect often observed afterchronic application of chemicals. Due the longlatency period and the poor prognosis for indi-viduals diagnosed with cancer, studies to pre-dict the potential tumorigenicity of chemicalshave developed into a major area of toxicolog-

ical research. Developmental and reproductivetoxicology are concerned with adverse effectson the ability to conceive, and with adverse ef-fects on the structural and functional integrity ofthe fetus. Chemicals may interfere with repro-duction through direct effects on reproductiveorgans or indirectly by affecting their neural andendocrine control mechansims. Developmentaltoxicity deals with adverse effects on the con-ceptus through all stages of pregnancy. Damageto the fetus may result in embryo reabsorption,fetal death, or abortion. Nonlethal fetotoxicitymay be expressed as delayed maturation, de-creased birthweight, or structuralmalformation.The most sensitive period for the induction ofmalformation is during organogenesis; neurobe-havioral malformations may be induced duringlater stages of pregnancy.

1.7. Dose–Response: a FundamentalIssue in Toxicology

In principle, a poison is a chemical that has an ad-verse effect on a living organism. However, thisis not a useful definition since toxic effects arerelated 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 inrodents and often results in death. Some met-als such as iron, copper, and zinc are essentialnutrients. 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 not re-stricted toman-made chemicals, but also includemany naturally occurring chemicals. Indeed, theagentwith the highest toxicity is a natural poisonfound in the bacterium Clostridium botulinum(LD50 0.01 µ/kg).Therefore, all toxic effects are products of the

amount of chemical to which the organism is ex-posed and the inherent toxicity of the chemical;they also depend on the sensitivity of the biolog-ical system.The term “dose” is most frequently used

to characterize the total amount of material to

14 Toxicology

which an organism is exposed; dose defines theamount of chemical given in relation to bodyweight. Dose is a more meaningful and com-parative indicator of exposure than the term ex-posure itself. Dose usually implies the exposuredose, the total amount of chemical administeredto an organism or incorporated into a test sys-tem. However, dose may not be directly propor-tional to the toxic effects since toxicity dependson the amount of chemical absorbed. Usually,dose correctly describes only the actual amountof chemical absorbed when the chemical is ad-ministered orally or by injection. Under thesecircumstances, the administered dose is identi-cal to the absorbed dose; other routes of appli-cation such as dermal application or inhalationdo not define the amount of agent absorbed.Different chemicals have a wide spectrum of

doses needed to induce toxic effects or death. Tocharacterize the acute toxicity of different chem-icals, LD50 values are frequently used as a basisfor comparisons. Some LD50 values (rat) for arange of chemicals follow:

Ethanol 12 500Sodium bicarbonate 4 220Phenobarbital sodium 350Paraquat 120Aldrin 46Sodium cyanide 6.4Strychnine 51,2-Dibromoethane 0.4Sodium fluoroacetate 0.22,3,7,8-Tetrachlorodibenzodioxin 0.01

Certain chemicals are very toxic and producedeath after administration of microgram doses,while others are tolerated without serious toxic-ity in gramdoses. The above data clearly demon-strate that the toxicity of a specific chemical is re-lated to dose. The dependence of the toxic effectsof a specific chemical on dose is termed dose–response relationship. Before dose–response re-lationships can be appropriately used, severalbasic assumptions must be considered. The firstis that the response is due to the chemical ad-ministered. It is usually assumed that the re-sponses observed were a result of the variousdoses of chemical administered. Under exper-imental conditions, the toxic response usuallyis correlated to the chemical administered, sinceboth exposure and effect arewell defined and canbe quantified. However, it is not always apparentthat the response is the result of specific chem-

ical exposure. For example, an epidemiologicstudy might result in discovery of an “associa-tion” between a response (e.g., disease) and oneor more variables including the estimated doseof a chemical. The true doses to which individ-uals have been exposed are often estimates, andthe specificity of the response for that chemicalis doubtful.Further major necessary assumptions in es-

tablishing dose–response relationships are:

– A molecular site (often termed receptor)with which the chemical interacts to producethe response. Receptors are macromolecularcomponents of tissues with which a chemi-cal interacts and produces its characteristiceffect.

– The production of a response and the degreeof the response are related to the concentra-tion of the 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 an in-dicator for its concentration at the molecularsite.

A further prerequisite for using the dose–response relationship is that the toxic responsecan be exactly measured. A great variety of cri-teria or end points of toxicity may be used. Theideal end point should be closely associatedwiththe molecular events resulting from exposureto the toxin and should be readily determined.However, although many end points are quan-titative and precise, they are often only indirectmeasures of toxicity. For example, changes inenzyme levels in the blood can be indicativeof tissue damage. Patterns of alterations mayprovide insight into which organ or system isthe site of toxic effects. These measures usu-ally are not directly related to the mechanismof toxic action. The dose–response relationshipcombines the characteristic of exposure and theinherent toxicity of the chemical. Since toxic re-sponses to a chemical are usually functions ofboth time and dose, in typical dose–response re-lationships, the maximum effect observed dur-ing the time of observation is plotted against thedose to give time-independent curves. The time-independent dose–response relationship may be

Toxicology 15

used to study dose–response for both reversibleand irreversible toxic effects. However, in riskassessments that consider the induction of ir-reversible effects such as cancer, the time fac-tor plays a major role and has important influ-ences on the magnitude or likelihood of toxicresponses. Thus, for this type of mechanism oftoxic action, dose–time–response relationshipsare better descriptors of toxic effects.The dose–response relationship is the most

fundamental concept in toxicology. Indeed, anunderstanding of this relationship is essential forthe study of toxic chemicals.From a practical point of view, there are two

different types of dose–response relationships.Dose–response relationshipsmay 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 individ-ual or in simple biochemical systems. For ex-ample, addition of increasing concentrationsof 2,3,7,8-tetrachlorodibenzodioxin to culturedmammalian cells results in an increase in theconcentration of a specific cytochrome P450 en-zyme in the cells (for details of mechanisms, seeSection 2.5.4.1). The increase is clearly dose re-lated and spans a wide range (Fig. 3). An exam-ple for a graded toxic effect in an individual maybe inflammation caused by skin contact with anirritant material. Low doses cause slight irrita-tion; as the amount increases, irritation turns toinflammation and the severity of inflammationincreases.

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

In dose–response 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 investi-gated. With increasing amount of a chemicalgiven to a group of animals, the magnitude ofthe effect and/or the number of animals affectedincrease. For example, if an irritant chemical isapplied to the skin, as the amount of the materialincreases, the numbers of animals affected andthe severity of inflammation increases. Quantalresponses such as death induced by a potentiallylethal chemicalwill also be dose-dependent. Thedose dependency of a quantal effect in a popula-tion is based on individual differences in the re-sponse to the toxic chemical. A specific amountof the potentially lethal xenobiotic given to agroup of animals may not kill all of them, butas the amount given increases, the proportion ofanimals killed increases.Althought the distinctions between graded

andquantal dose–response 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

Evenwith a genetically homogenous populationof animals of the same species and strain, theproportion of animals showing the effect willincrease with dose (Fig. 4A). When the num-ber of animals responding is plotted versus thelogarithm of the dose, a typical sigmoid curvewith a log-normal distribution that is symmetri-cal about the 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 dose–reponse curve around the 50%value, the midpoint, gives an indication of the

16 Toxicology

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

ranges of doses producing an effect. A steepdose–response curve indicates that the major-ity of the population will respond over a narrowdose range; a shallow dose–response curve in-dicates that a wide range of doses is required toaffect the majority of the population. The curvedepicted in Fig. 4B shows that the majority ofthe individuals respond about the midpoint ofthe curve. This point is a convenient descriptionof the average response, and is referred to as themedian effective dose (ED50). If mortality is theendpoint, then this dose is referred as medianlethal dose (LD50).Death, a quantal response, is simple to quan-

tify and is thus an endpoint incorporated inmanyacute toxicity studies. Lethal toxicity is usually

calculated initially from specificmortality levelsobtained after giving different doses of a chem-ical; the 50% mortality level is used most fre-quently since it represents the midpoint of thedose range at which the majority of deaths oc-cur. This is the dose level that causes death ofhalf of the population dosed. The LD50 valuesare usually given in milligrams of chemical perkilogram of body weight (from the viewpointof chemistry and for comparison of relative po-tencies of different chemicals, giving the LD50in moles of chemical per kilogram body weightwould be desirable). After inhalation, the ref-erence is to LC50 (LC= lethal concentration),which, in contrast to LD50 values, depends onthe time of exposure; thus, it is usually expressed

Toxicology 17

as X-hour LC50 value. The LD50 or LC50 val-ues usually represent the initial information onthe toxicity of a chemical and must be regardedas a first, but not a quantitative, hazard indicatorthat may be useful for comparison of the acutetoxicity of different chemicals [3].Similar dose–effect curves can, however, be

constructed for cancer, liver injury, and othertypes of toxic responses. For the determinationof LD50 values and for obtaining comparativeinformation on dose–response curves, plottinglog dose versus percent response is not practi-cal since large numbers of animals are neededfor obtaining interpretable data.Moreover, otherimportant information on the toxicity of a chem-ical (e.g., LD05 and LD95) cannot be accu-rately determined due to the slope of sigmoidcurve. Therefore, the dose–response curve istransformed to a log-probit (probit = probabilityunits) plot. The data in the Fig. 4B form astraight line when transformed into probit units(Fig. 4C). The EC50 or, if death is the end point,the LD50 is obtained by drawing a horizontalline from the probit unit 5, which is the 50% re-sponse point, to the dose–effect line. At the pointof intersection a vertical line is drawn, and thisline intersects the abscissa at the LD50 point.Information on the lethal dose for 90% or for10% of the population can also be derived by asimilar procedure. The confidence limits are nar-rowest at the midpoint of the line (LD50) and arewidest at the two extremes (LD05 and LD95) ofthe dose–response curve. In addition to permit-ting determination of a numerical value for theLD50 of a chemical with few groups of dosedanimals, the slope of the dose–response curvefor comparison between toxic effects of differ-ent chemicals is obtained by the probit transfor-mation [4].The LD50 by itself, however, is an insuf-

ficient index of lethal toxicity, particulary ifcomparisons between different chemicals are tobe made. For this purpose, all available dose–response information including the slope of thedose–response line should be used. Figure 5demonstrates the dose–response curves for mor-tality for two chemicals.The LD50 of both chemicals is the same

(10 mg/kg). However, the slopes of the dose–response curves are quite different. Chemical Aexhibits a “flat” dose–response curve: a largechange in dose is required before a significant

change in response will be observed. In contrast,chemical B exhibits a “steep” dose–responsecurve, that is, a relatively small change in dosewill cause a large change in response. The chem-icalwith the steep slopemay affect amuch largerproportion of the population by incremental in-creases in dose than chemicals having a shallowslope; thus, acute overdosing may be a prob-lem affecting the majority of a population forchemicals with steeper slopes. Chemicals withshallower slopes may represent a problem forthe hyperreactive groups at the left-hand side ofthe dose–response curve. Effects may occur atsignificantly lower dose levels then for hyperre-active groups exposed to chemicals with a steepdose–response.While the LD50 values characterize the po-

tential hazard of a chemical, the risk of an expo-sure is determined by the hazard multiplied bythe exposure dose. Thus, even very toxic chem-icals like the poison of Clostridium botulinumpose only a low risk; intoxications with thiscompound are rare since exposure is low. More-over, acute intoxications with other highly toxicagents such as mercury salts are rarely seen, de-spite detectable blood levels of mercury salts inthe general population, since the dose is alsolow. On the other hand, compounds with lowtoxicity may pose a definite health risk whendoses are high, for example, constituents of dietor chemicals formed during food preparation byheat treatment.

Figure 5. Comparison of dose – response relationships fortwo chemicals (log – probit plot)Both chemicals have identical LD50 values, but differentslopes of the dose – response curve

18 Toxicology

Therefore, for characterizing the toxic risk ofa chemical, besides information on the toxicity,information on the conditions of exposure arenecessary. When using LD50 values for toxicitycharacterisation, the limitations of LD50 valuesshould be explicitly noted. These limitations in-clude 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 LD50values

1) Statistical method2) No dose–response curve3) Time to toxic effect not determined4) No information on chronic toxicity

The most serious limitation on the use ofLD50 values for hazard characterization are thelack of information on chronic effects of a chem-ical and the lack of dose–response information.Chemicals with low acute toxicitymay have car-cinogenic or teratogenic effects at doses that donot induce acute toxic responses. Other limita-tions include insufficient information on toxiceffects other than lethality, the cause of death,and the time to toxic effect. Moreover, LD50values are not constant, but are influenced bymany factors and may differ by almost one or-der of magnitude when determined in differentlaboratories.

1.8. Dose-Response Relationships forCumulative 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 ei-ther the toxic effect or of the administered chem-ical. Accumulation of the administered chemi-cal is observed when the rate of elimination ofthe chemical is lower than the rate of adminis-tration. Since the rate of elimination is depen-dent on plasma concentrations, after long-termapplication an equilibrium concentration of thechemical in the blood is reached. Chemicalsmayalso be stored in fat (polychlorinated pesticides

such as DDT) or bone (e.g., lead). Stored chem-icals usually do not cause toxic effects becauseof their low concentrations at the site of toxicaction (receptor). After continuous application,the capacities of the storage tissues may becomesaturated, and xenobiotics may then be presentin higher concentration in plasma and thus at thesite of action; toxic responses result. Besides cu-mulation of the toxic agent, the toxic effect mayalso cumulate (Fig. 6).

Figure 6. Accumulation of toxic chemicals based on theirrate of excretiona) The rate of excretion is equal to the rate of absorption,no accumulation occurs; b) Chemical accumulates due to ahigher rate of uptake and inefficient excretion; the plasmaconcentrations are, however, not sufficient to exert toxiceffects; c) The plasma concentrations reached after accu-mulation are sufficient to exert toxicity

For chemicals which irreversibly bind tomacromolecules, the magnitude of toxic re-sponses may be correlated with the total doseadministered. In contrast to chemicals whichact reversibly, the effect is not dependent onthe frequency of dosing. Effect accumulationis often observed with carcinogens and ion-izing radiation. In Figure 7 accumulation ofeffects is exemplified by the time- and dose-dependent induction of tumors by 4-(dimethyl-amino)azobenzene, a potent chemical carcino-gen [5]. The TD50 values (50% of the treatedanimals carry tumors) are used to characterizethe potency. Identical tumor incidenceswere ob-served after high doses and a short exposure timeor after low doses and long exposure; the tumorincidence was only dependent on the total doseadministered.Reversibility of toxic responses also depends

on the capacity of an organ or tissue to repairinjury. For example, kidney damage by xeno-

Toxicology 19

biotics is often, after survival of the acute phaseof the intoxication, without further consequencedue to the high capacity of the kidney for cellproliferation and thus the capacity to repair or-gan damage [6]. In contrast, injury to the centralnervous system is largely irreversible since thedifferentiated cells of the nervous system cannotdivide and dead cells cannot be replaced.

Figure 7. Time-dependent induction of tumors after differ-ent daily doses of 4-dimethylaminoazobenzene in rats [5]

1.9. Factors Influencing Dose–Response

In animals and humans, the nature, severity, andincidence of toxic responses depend on a largenumber of exogenous and endogenous factors[7]. Important factors are the characteristics ofexposure, the species and strain of animals usedfor the study, and interindividual variability inhumans [8]. Toxic responses are caused by a se-ries of complex interactions of a potentially toxicchemical with an organism. The type and mag-nitude of the toxic response is influenced by theconcentration of the chemical at the receptor andby the type of interaction with the receptor. Theconcentration of a chemical at the site of actionis influenced by the kinetics of uptake and elimi-nation; since these are time-dependent phenom-ena, toxic responses are also time-dependent.Thus, the toxic response can be separated intotwo phases: toxicokinetics and toxicodynamics(Fig. 8).

Toxicokinetics describe the time dependencyof uptake, distribution, biotransformation, andexcretion of a toxic agent (a detailed descriptionof toxicokinetics is given in Section 2.5). Toxi-codynamics describes the interaction of the toxicagent with the receptor and thus specific inter-actions of the agent (see below). Toxicokinet-ics may be heavily influenced by species, strain,and sex and the exposure characteristics [9–13].Differences in toxic response between species,route of exposure, and others factors are oftendependent on influences on toxicokinetics. Sincetoxicodynamics (mechanism of action) are as-sumed to be identical between species, this pro-vides the basis for a rational interspecies ex-trapolation of toxic effects when differences intoxicokinetics are defined.

Figure 8. Toxicokinetics and toxicodynamics as factorsinfluencing the toxic response

1.9.1. Routes of Exposure

The primary tissue or system by which a xeno-biotic comes into contact with the body, andfrom where it may be absorbed in order to exertsystemic toxicity, is the route of exposure. Thefrequent circumstances of environmental expo-sure are ingestion (peroral), inhalation, and skincontact. Also, for investigational and therapeuticpurposes, intramuscular, intravenous, and sub-cutaneous injections may also be routes of ex-posure.Themajor routes bywhich a potentially toxic

chemical can enter the body are – in descendingorder of effectiveness for systemic delivery – in-jection, inhalation, absorption from the intesti-nal tract, and cutaneous absorption.The relation-ship between route and exposure, biotransfor-mation, and potential for toxicity, may be com-plex and is also influenced by the magnitude andduration of dosing (Table 5).The route of exposure has a major influence

on toxicity because of the effect of route of ex-

20 Toxicology

posure on the bioavailability of the toxic agent.The maximum tissue levels achieved, the timeto maximum tissue levels, and thus the durationof the effect are determined by the rate of ab-sorption and the extent of distribution within thesystem.

Table 5. Toxicity of chemicals applied by different routes of expo-sure (data taken from [13])

Chemical Species Route ofapplication

LD50,mg/kg

DDT rat intravenous 68rat oral 113rat skin contact 1931

Atropine sulfate rat intravenous 41rat oral 620

1-Chloro-2,4-dinitro-benzene

rat oral 1070

rat intraperitoneal 280rabbit skin contact 130

Dieldrin rat oral 46rat intravenous 9rat skin contact 10

Direct injection into veins is usually re-stricted to therapeutic applications, but it is im-portant for the toxicology of intravenously in-jected drugs in addicts. Chemicals applied byintravenous injection are rapidly distributed towell-perfused organs in the blood and thus mayresult in the rapid induction of toxic effects. Therapid dilution of a chemical after intravenousinjection by venous blood permits even the in-jection of locally acting or corrosive chemicalswhich are well tolerated. The likelihood of toxi-city from inhaled chemicals depends on a num-ber of factors, of which the physical state andproperties of the agent, concentration, and timeand frequency of exposure are important. Majorinfluences on the absorption and disposition ofxenobiotics are exerted by species peculiaritiessince the anatomy of the respiratory tract andthe physiology of respiration show major differ-ences between rodents and humans. The watersolubility of a gaseous xenobiotic has amajor in-fluence on penetration into the respiratory tract.As water solubility decreases and lipid solubil-ity increases, penetration into deeper regions ofthe lung, the bronchioli, and the alveoli becomesmore effective.Water-soluble molecules such asformaldehyde,are effectively scavenged by theupper respiratory tract and may have toxic ef-fects on the eye and throat. In contrast, gaseswith low water solubility such as phosgene may

penetrate through the bronchii and bronchioli tothe alveoli. Damage to the alveolar surface mayinitiate a series of events that finally results inlung edema. The degree to which inhaled gases,vapors, and particulates are absorbed, and hencetheir potential to produce systemic toxicity, de-pend on their diffusion rate through the alve-olar mebrane, their solubility in blood and tis-sue fluids, the rate of respiration, and blood flowthrough the capillaries.Uptake through the alimentary tract repre-

sents an important route of exposure for xeno-biotics accumulated in the food chain, for natu-ral constituents of human diet, and, drugs. Ab-sorption from the gastrointestinal tract is de-pendent on the lipophilicity of a chemical, themolecular mass of the xenobiotic, and the pres-ence of certain dietary constituents may influ-ence the extent and rate of absorption. Chemi-cals absorbed from the gastrointestinal tract aretransported to the liver via the portal vein; hep-aticmetabolism (“hepatic first-pass effect”)mayefficiently reduce the concentration of the xeno-biotic available in the systemic circulation afteroral uptake. Compounds undergoing bioactiva-tion in the liver usually exhibit greater toxicitywhen given orally than when absorbed acrossthe respiratory tract, due to the high proportionof material passing through the liver. In contrast,chemicals causing toxicity to extrahepatic, well-perfused organs such as the kidney often showa lower degree of toxicity to extrahepatic targetorgans when given orally.Skin contact is an important route of expo-

sure in the occupational and domestic environ-ments. Local effects may include acute inflam-mation and corrosion, chronic inflammatory re-sponses, immune-mediated reactions, and neo-plasia. The percutaneous absorption ofmaterialsmay also be a significant route for the absorptionof systemically toxicmaterials. Factors influenc-ing the percutaneous absorption of substancesinclude skin site, integrity of skin, tempera-ture, formulation, and physicochemical charac-teristics, including charge, molecular mass, andhydro- and lipophilicity.

1.9.2. Frequency of Exposure

The exposure of experimental animals may becategorized as acute, subacute, subchronic, and

Toxicology 21

chronic. Acute exposures usually last less than24 h, and all above-mentioned routes of expo-sure may be applied. With chemicals of low tox-icity, repeated exposures may be used. Acute in-halation exposure is usually less than 24 h; fre-quently 4–8 h is chosen as timescale. Repeatedexposure refers to application of the chemi-cal for less than one month (subacute), one tothree months (subchronic), and more than threemonths (chronic). Chronic exposures to detectspecific toxic effects (carcinogenicity of a chem-ical) may span most of the lifetime of a rodent(up to two years). Repeated exposure may be byany route; the least labor intensive route is oral,by mixing the chemical with the diet; only forspecific chemicals or to simulate likely routesof exposure for humans are application in drink-ing water, by gastric intubation, and by inhala-tion applied. These are more labor-intensive andrequire skilled personnel and/or sophisticatedtechniques and thus are more expensive.The toxic effects observed after single expo-

sure often are different form those seen after re-peated exposure. For example, inhalation of highconcentration of halothane causes anesthesia inanimals and humans. In contrast, long-term ap-plication of halothane in lower doses causes liverdamage in sensitive species The frequency of ex-posure in chronic studies is important for thetemporal characterisation of exposure. Chem-icals with slow rates of excretion may accu-mulate if applied at short dosing intervals, andtoxic effects may result (see Section 1.6). Also,a chemical producing severe effect when givenin a single high dose may have no detectableeffects when given in several smaller doses. In-terspecies and strain differences in susceptibilityto chemical-induced toxicity may be due to het-erogeneity of populations, species specific phys-iology (for example of the respiratory system),basal metabolic rate, size- and species-specifictoxicokinetics and routes of metabolism or ex-cretion (Table 6). In some cases, animal testsmay give an underestimate, in others an overes-timate, of potential toxicity to humans [14].

1.9.3. Species-Specific Differences inToxicokinetics

Species-specific differences in toxic responseare largely due to difference in toxicokinetics

and biotransformation. Distribution and elimi-nation characteristics are quite variable betweenspecies. Both qualitative and quantitative differ-ences in biotransformation may effect the sen-sitivity of a given species to a toxic response(Table 7).

Table 6. Comparative LD50 values for four different chemicals indifferent animal species and estimated LD50 for humans

Chemical Species LD50, mg/kg

Paraquat rat 134mouse 77guinea pig 41human 32 – 48

Ethanol rat 12 500mouse 8000guinea pig 5500human 3500 – 5000

Acetaminophen rat 3763mouse 777guinea pig 2968human 42 800

Aspirin rat 1683mouse 1769guinea pig 1102human 3492

Table 7. Species and sex differences in the acute toxicity of1,1-dichloroethylene after oral administration and inhalation in ratsand mice (data from World Health Organization, Geneva, 1990)

Species Dosing criteria Estimated LD50/LC50

Rat, male inhalation/4 h 7000 – 32 000mg/LRat, female inhalation/4 h 10 300mg/LMouse, male inhalation/4 h 115mg/LMouse, female inhalation/4 h 205mg/LRat, male gavage 1550mg/kgRat, female gavage 1500mg/kgMouse, male gavage 201 – 235mg/kgMouse, female gavage 171 – 221mg/kg

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 differ-ence in the extent of biotransformation as a fac-tor influencing toxic response is the species dif-ferences in the biotransformation of the inhala-tion anesthetic halothane. Both rats and guineapigsmetabolize halothane to trifluoroacetic acid,a reaction catalyzed by a specific cytochromeP450 enzyme [16–18]. As a metabolic interme-diate, trifluoroacetyl chloride is formed, whichmay react with lysine residues in proteins andwith phosphatidyl ethanolamine in phospho-lipids (Fig. 9).

22 Toxicology

This interaction initiates a cascade of eventsfinally resulting in toxicity. The metabolismof halothane in guinea pigs occurs at muchhigher rates than in rats, so guinea pigs aresensitive to halothane-induced hepatotoxic ef-fects and rats are resistant. Qualitative dif-ferences in biotransformation are responsi-ble for apparent differences in the sensitiv-ity of rats and guinea pigs to the bladdercarcinogenicity of 2-acetylamidofluorene. Inrats, 2-acetylamidofluorene is metabolized byN-oxidation by certain cytochrome P450 en-zymes. The N-oxide is further converted toan electrophilic nitrenium ion which interactswith DNA in the bladder; this biotransforma-tion pathway explains the formation of blad-der tumors in rats after long-term exposureto 2-acetylamidofluoren. In guinea pigs, 2-acetylamidofluorene is metabolized by oxida-tion at the aromatic ring; since nitrenium ionscannot be formed by this pathway, guinea pigsare resistant to the bladder carcinogenicity of 2-acetylamidofluorene (Fig. 10).With some chemicals, age may significantly

affect 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] andmay be a significant factor contributing to hu-man cancer incidence.The toxic response is influenced by the mag-

nitude, number, and frequency of dosing. Thus,local or systemic toxicity produced by acute ex-posure may also occur by a cumulative pro-cess with repeated exposures to lower doses;

also, additional toxicitymay be seen in repeated-exposure situations. The relationships for cu-mulative toxicity by repetitive exposure com-pared with acute exposure toxicity may be com-plex, and the potential for cumulative toxicityfrom acute doses may not be quantitatively pre-dictable. For repeated-exposure toxicity, the pre-cise profiling of doses may significantly influ-ence toxicity.

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

1.9.4. Miscellaneous Factors Influencing theMagnitude of Toxic Responses

A variety of other factors may affect the na-ture and 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,

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

Toxicology 23

toxicity may be modified by the presence of ma-terials in formulations which facilitate or retardthe absorption of the chemicals.With respiratoryexposure to aerosols, particle size significantlydetermines the depth of penetration and deposi-tion in the respiratory tract and thus the site andextent 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 chemicalsat low doses. Moreover, prior, coincidential, andsucessive exposure of humans to chemicals islikely. Interactions between the toxic effects ofdifferent 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 potentiating ef-fect on the hepatotoxicity of carbon tetrachlo-ride. In rats pretreated with ethanol, the hepa-totoxic effects of carbon tetrachloride are muchmore pronounced than in control animals. Thispotentiation is due to an increased capacity forbioactivation (see Section 2.4) of carbon tetra-chloride in pretreated rats due to increased con-centrations of a cytochrome P450 enzyme in theliver [20]. Thus, an important considerations forthe assessement of potential toxic effects ofmix-tures of chemicals are toxicokinetics and toxico-dynamic interactions. Toxicokinetic interactionsof chemicals may influence absorption, distribu-tion, and biotransformation, both to active andinactive metabolites. Mixtures of solvents oftenshow a competitive inhibition of biotransforma-tion. Usually, one of the components has highaffinity for a specific enzyme involved in its bio-transformation, whereas another component hasonly a low affinity for that particular enzyme.Thus, preferential biotransformation of the com-ponent with the high affinity occurs. Differentoutcomes of enzyme inhibition are possible: ifthe toxic effects of the component whose meta-bolism is inhibited is dependent on bioactiva-tion, lower rates of bioactivation will result indecreased toxicity; if the toxic effects are inde-

pendent of biotransformation, the extent of toxi-city will increase due to slower rate of excretion.Toxic effects of mixtures may also not be due toa major component, but to trace impurities withhigh toxicity. For example, many long-term ef-fects seen in animal studies on the toxicity ofchlorophenols are believed to be due to 2,3,7,8-Tetrachlorodibenzodioxin, whichwas present asa minor impurity in the samples of chlorophe-nols used for these studies.

2. Absorption, Distribution,Biotransformation and Eliminationof Xenobiotics

2.1. Disposition of Xenobiotics

The induction of systemic toxicity usually re-sults from a complex interaction between ab-sorbed parent chemical and biotransformationproducts formed in tissues; the distribution ofboth parent chemical and biotransformationproducts in body fluids and tissues; their bindingand storage characteristics; and their excretion.The biological effects initiated by a xenobi-

otic are not related simply to its inherent toxicproperties; the initiation, intensity, and durationof response are a function of numerous factorsintrinsic to the biological system and the admin-istered dose. Each factor influences the ultimateinteraction of the xenobiotic and the active site(Section 1.9). Only when the toxic chemical hasreached the specific site and interacted with itcan the inherent toxicity be realized. The routea xenobiotic follows from the point of adminis-tration or absorption to the site of action usuallyinvolves many steps and is termed toxicokinet-ics. Toxicokinetics influence the concentrationof the xenobiotic or its active metabolite at thereceptor. In the dose–response concept outlinedin Section 1.9 and 1.7, it is generally assumedthat the toxic response is proportional to the con-centration of the xenobiotic at the receptor.How-ever, the same dose of a chemical administeredby different routes may cause different toxic ef-fects. Moreover, the same dose of two differentchemicals may result in vastly different concen-trations of the chemical or its biotransformationproducts in a particular target organ. This differ-ential pattern is due to differences in the dispo-sition of a xenobiotic (Fig. 11).

24 Toxicology

Figure 11. Possible fate of a xenobiotic in the organisms

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 atthe 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 a

xenobiotic 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, an en-zyme, amembrane, or many other possible sites.

2.2. Absorption

The skin, the lungs, and the cells lining the al-imentary tract are major barriers for chemicalspresent in the environment. Except for causticchemicals, which act at the site of first contactwith the organism, xenobiotics must cross thesebarriers to exert toxic effects on one or severaltarget organs. The process whereby a xenobi-otic moves 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 character-istics and the factors that permit transfer offoreign compounds. Membranes are initiallyencountered whether a xenobiotic is absorbed

Toxicology 25

by the dermal, oral, or vapor route. These mem-branes may be associated with several layers ofcells or a single cell. The absorption of a sub-stance from the site of exposure may result frompassive diffusion, facilitated diffusion, activetransport, or the formation of transport vesicles(pinocytosis and phagocytosis). The process ofabsorption may be facilitated or retarded by avariety of factors; for example, elevated tem-perature increases percutaneous absorption bycutaneous vasodilation, and surface-active ma-terials facilitate penetration. Each area of entryfor xenobiotics into the organism may havespecific peculiarities, but a unifying concept ofbiology is the basic similarity of all membranesin tissues, cells, and organelles.

Figure 12. Simplifiedmodel of the structure of a biologicalmembrane

All membranes are lipid bilayers with polarhead groups (phosphatidylethanolamine, phos-phatidylcholine). The polar groups predominateat the outer and inner surfaces of the membrane;the inner space of the membrane consists of per-pendicularly arranged fatty acids [21]. The fattyacids do not have a rigid structure and are fluidunder physiological conditions; the fluid charac-ter of the membrane is largely dominated by thefatty acid composition. Thewidth of a biologicalmembrane is approximately 7–9 nm. Figure 12illustrates the concept of a biological membrane(fluid-mosaic model).Proteins are intimately associated with the

membrane and may be located on the surfaceor inside the membrane structure, or extendcompletely through the membrane. These pro-teins may also form aqueous pores. Hydropho-

bic forces are responsible for maintaining thestructural integrity of both proteins and lipidswithin themembrane structure. The ratio of lipidto protein in differentmembranesmay vary from5:1 (e.g,. myelin) to 1:5 (e.g., the inner mem-brane of mitochondria). Usually, pore diame-ters in membranes are small and permit onlythe passage of low molecular mass chemicals.However, some specialized membranes such asthose found in the glomeruli of the kidney,whichcan have pore sizes of up to 4 nm, also permitthe passage of compounds with molecular massgreater than 10 000.The amphipathic nature of themembrane cre-

ates 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 byChemicals

A chemical can pass through a membrane bytwo general processes: passive diffusion and ac-tive transport. Passive diffusion is described byFick’s law and requires no energy. Active trans-port processes involve the consumption of cellu-lar energy to translocate the chemical across themembrane. Active transportmay 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 diffusion de-pend on their lipid solubility and are often corre-lated with the partition coefficient (solubility inorganic solvents/solubility in water). Lipophilicchemicals may diffuse directly through the lipiddomain of themembrane. However, a certain de-gree of water solubility seems to be required forpassage since many poorly lipid soluble chemi-cals have been shown to penetrate easily. Onceinitial penetration has occurred, the moleculemust necessarily traverse a more polar regionto dissociate from the membrane. Compoundswith extremely high partition coefficients thustend to remain in membranes and to accumulatethere rather than pass through them. Polar com-

26 Toxicology

pounds that are insoluble in the nonpolar, fatty-acid-containing inner space of themembrane of-ten cannot penetrate membranes, although somelowmolecular mass polar chemicals may slowlypenetrate through the aqueous pores of themem-branes.The rates of movement of nonpolar xenobio-

tics through membranes can be predicted basedon the assumptions from Fick’s law of diffusion.Polar compounds and electrolytes of lowmolec-ular mass are believed to behave similarily. Afirst-order equation appears to be applicable tothemajority of xenobiotics. The rate of diffusionof a xenobiotic is related to its concentration gra-dient across the membrane (C1 −C2), the sur-face area available for transfer A, the diameterof the membrane d, and the diffusion constantk. The latter is related to the size and structureof the molecule, the spatial configuration of themolecule, and the degree of ionization and lipidsolubility of the xenobiotic.

Rate of diffusion = kA (C1−C2)

d

As the xenobiotic is rapidly removed afterabsorption, C2 can usually be ignored. and alog/linear plot of the amount of unpenetratedchemicals present over time should be linear.When relatively comparable methods have beenused, calculation of the half-time of penetrationt1/2, is useful. The rate constant of penetrationk is derived from

k =0.693t1/2

When the half-time of penetration after oraland dermal administration of several environ-mental contaminants were compared, rates werefound to vary considerably. Clearly, rates of pen-etration by different routes in mammals showlittle or no correlation.Ionization becomes particularly important

when xenobiotics are introduced into the gas-trointestinal tract, where a variety of pH con-ditions are manifest (see Section 2.2.4.2). Al-though many drugs are acids and bases and thuspotentially ionizable form, most xenobiotics areneither acids nor bases and thus are unaffectedby pH. The amount of a xenobiotic in the ion-ized or unionized form depends upon the pKa of

the xenobiotic and the pH of the medium. Whenthe pH 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 Henderson–Hasselbalch equation:

pKa−pH = log[nonionized]

[ionized]

pKa−pH = log[ionized]

[nonionized]

Since the unionized, lipid-soluble form of aweak acid or base may penetrate membranes,weak organic acids diffuse most readily in anacidic environment, and organic bases in a basicenvironment. There is some degree of penetra-tion even when xenobiotics are not in the mostlipid-soluble form, and a small amount of ab-sorption can produce serious effects if a com-pound is very toxic.

2.2.3. Mechanisms of Transport ofXenobiotics through Membranes

Filtration. Passage of a solution across aporous membrane results in the retention ofsolutes larger than the pores. This process istermed filtration. For example, filtration of so-lutes occurs in the kidney glomeruli, which havelarge pores and retain molecules with molecu-lar masses greater than 10 000. Elsewhere in thebody, filtration by pores may only result in thepassage of relatively small molecules (molecu-lar mass ca. 100), and most larger molecules areexcluded. Thus, uptake of xenobiotics throughthese pores is only a minor mechanism of pene-tration.

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

been implicated in these phenomena. Activetransport may be effected by systems that help

Toxicology 27

Table 8. Special transport processes involved in the passage of xenobiotics through biological membranes

Type of transport Carrier molecule required Examples of substrates Energy required Againstconcentrationgradient

Active transport yes organic acids in the kidney yes yesFacilitated transport yes glucose yes noEndocytosis no proteins yes ?

transport endogenous compounds across mem-branes. Such processes require energy and trans-port xenobiotics against electrochemical or con-centration gradients. Active transport systemsare saturable processes and exhibit a maximumrate of transport; they are usually specific forcertain structural features of chemicals. A car-rier molecule (likely a protein) associates withthe chemical outside the cell, translocates itacross the membrane for ultimate release in-side the cell. This is particularly important forcompounds that lack sufficient lipid solubilityto move rapidly through the membrane by sim-ple diffusion. Active transport plays a major rolein the excretion of xenobiotics from the body,and major excretory organs such as the liver orthe kidney have several transport systems whichmay accept organic acids, organic bases, or evenmetal ions as substrates.In contrast to other special transport pro-

cesses, some carrier-mediated processes do notrequire energy and are unable to move chemi-cals against a concentration gradient. These pro-cesses are termed facilitated transport. Facili-tated transport is particulary beneficial for com-pounds which lack sufficient lipid solubility forrapid diffusion through the membrane. Facili-tated transport is more rapid than simple dif-fusion up to the point at which concentrationson both sides of the membranes are equal. Forexample, the transport of glucose through a vari-ety ofmembranes occurs by facilitated transport.The mechanisms by which facilitated transportoccurs are not well understood.Pinocytosis (liquids) and phagocytosis (so-

lids) are specialized processes in which the cellmembrane invaginates or flows around a xenobi-otic, usually present in particulate form, and thusenables transfer across amembrane.Althoughofimportance once the xenobiotic has gained entryinto the organism, this mechanism does not ap-pear to be of importance in the initial absorptionof a xenobiotic.

2.2.4. Absorption

Absorption is the process whereby xenobioticscross body membranes and are translocated tothe blood stream. The primary sites of absorp-tion of environmental contaminants are the gas-trointestinal tract (gastrointestinal absorption),the skin (dermal absorption), and the lung (res-piratory absorption). Absorption of chemicalsmay also occur from other sites such as muscle,the subcutis, or the peritoneum after administra-tion by special routes. In clinicalmedicine,manydrugs are injected directly into the bloodstreamto circumvent the problems of absorption posedby the peculiarities of the different routes.

2.2.4.1. Dermal Absorption

Human skin can come into contact with manypotentially toxic chemicals. Skin is relativelyimpermeable to aqueous solutions and most xe-nobiotics present as ions. Therefore, it is a rel-atively good barrier separating the human bodyfrom the environment. However, skin is perme-able in varying degrees to a large number of xe-nobiotics, and some chemicals may be absorbedthrough the skin in sufficient amounts to causea toxic response [23]. A striking example of thesignificance of absorption through the skin is thelarge number of agricultural workers who haveexperienced acute poisoning from exposure toparathion (dermal LD50 ≈ 20mg/kg) during ap-plication or from exposure to vegetation previ-ously treated with this pesticide.The human skin is a complex, multilayered

tissue with approximately 18 000 cm2 of sur-face in an average human male. Chemicals tobe absorbed must pass through several cell lay-ers before entering the small blood and lymphcapillaries in the dermis. Transport in blood andlymph then distributes absorbed chemicals in the

28 Toxicology

body. The human skin consists of three distinctlayers (Fig. 13) and a number of associated ap-pendages (sweat and sebaceous glands, hair fol-licles).

Figure 13. Cross section of human skina) Stratum corneum; b) Sebaceous gland; c) Sweat gland;d) Hair follicle; e) Fat; f ) Muscle

The epidermis is a multilayered tissue vary-ing in thickness from 0.15 (eyelids) to 0.8 mm(palms). This tissue appears to be the greatestdeterrent to the absorption of xenobiotics. Theepithelial tissues of the skin develop andgrowdi-vergently from other tissues. Proliferative layersof the basal cells (stratum germinativum) differ-entiate and gradually replace cells above them assurface cells deteriorate and are sloughed fromthe epidermis. Cells in this layer produce fi-brous, insoluble keratin that fills the cells, anda sulfur-rich amorphous protein that comprisesthe cell matrix and thickened cell membrane.This cell layer, the stratum corneum, providesthe primary barrier to the penetration of for-eign compounds. It consists of several layersof flattened, stratified, highly keratinized cells.These cells are approximately 25–40 µm wideand have lost their nuclei. Although highly wa-ter retarding, the dead, keratinized cells of thestratumcorneumare highlywater absorbent (hy-drophilic), a property that keeps the skin suppleand soft. A natural oil covering the skin, the se-

bum, appears to maintain the water-holding ca-pacity of the epidermis but has no appreciablerole in retarding the penetration of xenobiotics.The rate-determining barrier in the chemical ab-sorption of xenobiotics is the stratum corneum.The dermis and subcutaneous tissue offer lit-

tle resistance to penetration, and once a sub-stance has penetrated the epidermis these tissuesare rapidly traversed. The dermis is a highly vas-cular area that provides ready access to bloodand lymph for distribution once the epithelialbarrier has been passed. The blood supply inthe dermis is subjected to complex, interactingneural and humoral influences whose temper-ature-regulating function can have an effect ondistribution by altering blood supply to this area.Therefore, the extent of absorption of a chemicalthrough the skin may be influenced by tempera-ture, and relative humidity [24].The skin appendages are found in the der-

mis and extend through the epidermis. The pri-mary appendages are the sweat glands (epicrineand apocrine), hair, and sebaceous glands. Thesestructures extend to the outer surface and there-fore may play a role in the penetration of xeno-biotics; however, since they represent only 0.1to 1% of the total surface of the skin, their con-tribution to overall dermal absorption is usuallyminor.Percutaneous absorption can occur by sev-

eral routes, but the majority of unionized, lipid-soluble xenobiotics appear to move by passivediffusion directly through the cells of the stra-tum corneum. Important arguments for the im-portance of transepidermal absorption are thatepidermal damage or removal of the stratumcorneum increases permeability, the epidermalpenetration rate equals whole-skin penetration,epidermal penetration is markedly slower thandermal, and the epidermal surface area is 100–1000 times the surface area of the skin ap-pendages. Very small and/or polarmolecules ap-pear to have more favorable penetration throughappendages or other diffusion shunts, but onlya small fraction of toxic xenobiotics are chemi-cals of this type. Polar substances, in addition tomovement through shunts, may diffuse throughthe outer surface of the protein filaments ofthe hydrated stratum corneum, while nonpolarmolecules dissolve in and diffuse through thenonaqueous lipid matrix between the protein fil-aments.

Toxicology 29

Figure 14. A) Intestinal tract in humans; B) Anatomy of the intestinal wall, the major site of absorption of xenobioticsThe lining of the small intestine is highly folded and has a special surface structure (brush-border membrane) to give a largesurface available for the efficient uptake of nutrients.a) Esophagus (4 – 7.2); b) Stomach (1.0 – 3.0); c) Duodenum (4.8 – 8.2); d) Pancreas; e) Colon (7.9 – 8.0); f ) Jejunum (7.6);g) Ileum (7.6); h) Rectum (7.8); i) Brush-border membraneNumbers in brackets represent pH in different parts of the intestinal tract.

Human stratum corneum displays significantdifferences in structure from one region of thebody to the other, which affect the rate of absorp-tion. Penetration at certain body regions thusvaries according to the polarity and size of themolecule, but it is generally accepted that formost unionized xenobiotics the rate of penetra-tion is in the following order: scrotal> forehead> axilla = scalp > back = abdomen > palm andplantar. The palm and plantar regions are highlydiffuse, but their much greater thickness (100–400 times that of other regions) introduces anoverall lag time in diffusion.The condition of the skin greatly influences

the absorption of xenobiotics. Damage to or re-

moval of the stratum corneum cause a dramaticincrease in the permeability of the epidermisfor xenobiotics. Caustic and corrosive chemi-cals such as acids or alkali or burns will greatlyenhance dermal absorption and thus influencethe toxicity of a xenobiotic applied to the skin.Soaps and detergents are among the damagingsubstances routinely applied to skin. Whereasorganic solventsmust be applied in high concen-trations to damage skin, 1% aqueous solutionsof detergents increase the rate of penetration ofsolutes through human epidermis dramatically.Alteration of the stratum corneum by organicsolvents may also be the cause of increased pen-etration.

30 Toxicology

Organic solvents can be divided into damag-ing and nondamaging categories. Damaging sol-vents include methanol, acetone, diethyl ether,hexane, and some solvent mixtures. These sol-vents and mixtures can extract lipids and prote-olipids from tissues and are thus expected to alterpermeability. Although the mechanical strengthof the stratum corneum is unaltered, delipidiza-tion produces a more porous, nonselective sur-face. Solvents such as higher alcohols, esters,and olive oil do not appear to damage skin ap-preciably. On the contrary, the penetration rateof solutes dissolved in them is often reduced.Surprisingly, lipid-soluble xenobiotics may bemarkedly resistant to washing, even a short timeafter application. For example, 15 min after ap-plication, a substantial portion of parathion can-not be removed from contaminated skin by soapand water.When comparisons across species are made,

human skin appears to be more impermeable, orat least as impermeable, as the skin of the cat,dog, rat, mouse, or guinea pig. The skin of pigsand guinea pigs in particular serves as a use-ful approximation to human skin, but only aftera comparison has been made for each specificchemical.Temperature, surface area of applied dose,

simultaneous application of another xenobiotic,relative humidity, occlusion, age, and hyperther-mia are among a number of chemical, physi-cal, and physiological factors that may alter skinpenetration.

2.2.4.2. Gastrointestinal Absorption

The oral route of entry into the body is speciallyimportant for accidental or purposeful (suicide)ingestion of poisonous materials. Food addi-tives, food toxins, environmental xenobiotics ac-cumulated in the food chain, and airborne par-ticles excluded from passage to to alveoli arealso introduced into the digestive system. Thepenetration of orally administered xenobioticsis primarily confined to the stomach and intes-tine [25].The gastrointestinal tract may be viewed as a

tube traversing the body. It consists of themouth,esophagus, stomach, small and large intestine,colon, and rectum (Fig. 14). The digestive tractis lined by a single layer of columnar cells, usu-

ally protected by mucus, which do not present abarrier to penetration. The circulatory system isclosely associated with the intestinal tract (30–50 µm frommembrane to vasculature), and oncexenobiotics have crossed the epithelium of theintestinal tract, entry into capillaries is rapid. Ve-nous blood flow from the stomach and intestinerapidly removes absorbed xenobiotics and intro-duces them into the hepatic portal vein, whichtransports them to the liver.Absorption of chemicals may take place

along the entire gastrointestinal tract, but mostxenobiotics are absorbed in the stomach and thesmall intestine. A major factor favoring absorp-tion in the intestine is the presence of microvillithat increase the surface area to an estimated 100m2 in the small intestine (see Fig. 14) Becausethe intestinal area thus offers maximal opportu-nity for absorption, it is generally accepted thatabsorption of xenobiotics is greatest in this areaof the gastrointestinal tract. Although the gas-trointestinal tract has some special transport pro-cesses for the absorption of nutrients and elec-trolytes,most xenobiotics seem to enter the bodyfrom the gastrointestinal tract by simple diffu-sion. Exeptions are some heavy metals such asthallium and lead, which mimic the essentialmetals iron and calcium, respectively. They arethus absorbed by active transport systems devel-oped for the uptake of these nutrients.The gastrointestinal tract has areas of highly

variable pH,which canmarkedly change the per-meability characteristics of ionic compounds.For example, passive diffusion is greatly lim-ited except for unionized, lipid-soluble chem-icals. Although variable according to secretoryactivity, the pH of the stomach is ca. 1–3 and thatof the intestine ca. 7. Themeasured pH of the in-testinal contents may not be the same as the pHof the epithelium at the site of absorption, andthis explains the entrance of compounds whosepKa would suggest less favored absorption. Thevariations in pH in the different sections of theintestinal tract may influence the absorption ofacids and bases. Since most xenobiotics are ab-sorbed by diffusion, only the unionized, mem-brane-permeable form may be absorbed. Weakorganic acids are mainly present in the union-ized, lipid-soluble form in the stomach, and pre-dominantly in the ionized form in the intestine.Therefore, organic acids are expected to bemorereadily absorbed from the stomach than from

Toxicology 31

the intestine. In contrast, weak organic basesare ionized in the stomach but present in thelipid-soluble form (unionized) in the intestine.Absorption of such compounds should thereforepredominantly occur in the intestine rather thanin the stomach.However, other factors determining the rate

of membrane penetration such as surface areaavailable for diffusion, blood flow (influencingconcentration gradients), and the law of massaction also influence the site of absorption ofacids or bases from the gastrointestinal tract. Forexample, although only 1% of benzoic acid ispresent in the lipid-soluble, unionized form inthe small intestine, the large surface area andthe rapid removal of absorbed benzoic acid withthe blood result in its efficient absorption fromthe small intestine.Other factors contribute to gastrointestinal

absorption Clearly a xenobiotic must be dis-solved before absorption can take place. Particlesize, organic solvents, emulsifiers, and rate ofdissolution thus also effect absorption. In addi-tion, the presence of microorganisms and hydro-lysis-promoting pH offer opportunities for thebiotransformation of many xenobiotics. Otherfactors affecting gastrointestinal absorption in-clude binding to gut contents, intestinal motility,rate of emptying, temperature of food, effects ofdietary constituents, health status of the individ-ual, and gastrointestinal secretion.

2.2.4.3. Absorption of Xenobiotics by theRespiratory System

The respiratory system is an organ in direct con-tact with environmental air as an unavoidablepart of living. A number of xenobiotics exist ingaseous (carbon monoxide, nitric oxides), va-por (benzene, carbon tetrachloride), and aerosol(lead from automobile exhaust, silica, asbestos)forms and are potential candidates for entry viathe respiratory system. Indeed, the most impor-tant cause of death from acute intoxication (car-bon monoxide) and the most frequent occupa-tional disease (silicosis) are caused by the ab-sorption or deposition of airborne xenobioticsin the lung.The respiratory tract consists of three ma-

jor regions: the nasopharyngeal, the tracheo-bronchial, and the pulmonary (Fig. 15). The na-

sopharynx begins in themouth and extents downto the level of the larynx. The trachea, bronchii,and bronchioli serve as conducting airways be-tween the nasopharynx and the alveoli, the siteof gas exchange between the inhaled air andthe blood. The human respiratory system is acomplex organ containing over 40 different celltypes. These cell types contribute to the pul-monary architecture and function over variouszones of the lung, although to some extent, indi-vidual cell types can be found in several zones.The tracheobronchial system comprises airwayslined with bronchial epithelium with associatedsubmucosal glands and several different tissueswith specific function and the lung vasculature.The absorption of xenobiotics by the respira-

tory route is favored by the short path of diffu-sion, large surface area (50–100 m2), and largeconcentration gradients. At the alveoli (site ofgas exchange), the membranes are very thin (1–2 µm) and are intimately associated with thevascular system. This enables rapid exchange ofgases (ca. 5 ms for CO2 and ca. 200 ms for O2).A thin film of fluid lining the alveolar walls aidsin the initial absorption of xenobiotics from thealveolar air. Simple diffusion accounts for thesomewhat complex series of events in the lungregarding gas absorption. The sequences of res-piration, which involve several interrelated airvolumes, define both the capacity of the lungand factors important to particle deposition andretention. Among the elements important in to-tal lung capacity is the residual volume, that is,the amount of air retained by the lung despitemaximal expiratory effort. Largely due to slowrelease from this volume, gaseous xenobiotics inthe expired air are not cleared immediately, andmany expirations may be necessary to rid theair in the lung of residual xenobiotic. The rateof entry of vapor-phase xenobiotics is controlledby the alveolar ventilation rate, and a xenobioticpresent in alveolar air may come into contactwith the alveoli in an interrupted fashion about20 times per minute. The diffusion coefficientof the gas in the fluids of pulmonary membranesis another important consideration, but doses aremore appropriately discussed in terms of the par-tial pressure of the xenobiotic in the inspired air.On inhalation of a constant tension of a gaseousxenobiotic, arterial plasma tension of the gas ap-proaches the tension of gas in the expired air. Therate of entry is then determined by blood solubil-

32 Toxicology

Figure 15. Anatomy of the human respiratory systema) Trachea; b) Bronchii; c) Bronchioli; d) Alveoli; e) Capillary; f ) Erythrocyte

ity of the xenobiotic and blood flow. For a highblood/gas partition coefficient, a larger amountmust be dissolved in the blood to raise the par-tial pressure. Chemicals with a high blood/gaspartition coefficient require a longer period toapproach the same tension in the blood as in in-spired air than less soluble gases.

Aerosols and Particulates. The entry ofaerosols and particulates is affected by a num-ber of factors. A coal miner inhales ca. 6000 g ofcoal dust particles during his occupational life-time, and only ca. 100 g are found postmortem;therefore, effective protective mechanisms areoperative. The parameters of air velocity and di-rectional changes in air flow favor impaction ofparticles in the upper respiratory systems. Par-ticle characteristics such as size, chemistry ofthe inhaled material, sedimentation and electri-cal charge are important to retention, absorption,

or expulsion of airborne particles. In additionto the other aforementioned lung characteris-tics, amucous blanket propelled by ciliary actionclears the respiratory tract of particles by direct-ing them to the gastrointestinal system (via theglottis) or to the mouth for expectoration. Thissystem is responsible for 80% of lung particu-late clearance. The deposition of various particlesizes in different respiratory regions is summa-rized in Figure 16, which shows that particle sizeis important for disposition and particles largerthan 2 µm do not reach the alveoli [26].The direct penetration of airborne xenobio-

tics at alveolar surfaces or in the upper respira-tory tract is not the only action of toxicolog-ical importance. Both vapors and particulatescan accumulate in upper respiratory passagesto produce irritant effects. Irritant gases may bedeposited in the respiratory tract depending ontheir water solubility and may cause localized

Toxicology 33

damage characterized by edema, swelling, mu-cus production, and increased d vascular perme-ability. If major airways are obstructed by theseprocesses or important anatomical structures ofthe respiratory tract like the alveoli are damaged,life-threatening or deadly intoxications may becaused by the inhalation of irritant gases.

Figure 16. Effect of size on the disposition and sedimen-tation of particulates in the respiratory tractThe site of particle sedimentation is determined largely byparticle size; only very fine particles are deposited in thealveoli; larger particles do not reach the lung but are de-posited in the nasopharynx.

Despite the effectiveness of ciliary move-ment and phagocytosis, the cumulative effectsof silica, asbestos, or coal dust ultimately causechronic fibrosis even though direct absorptionis of minor importance. Thus, phagocytosisprevents acute damage but may contribute tochronic toxicity. There is little evidence for ac-tive transport in the respiratory system, althoughpinocytosis may be of importance for penetra-tion. The lung is an area of extensive metabolicactivity; enzymes present in the lung may cat-alyze both activation and detoxication of xeno-biotics (see Section 2.4).

2.3. Distribution of Xenobiotics by BodyFluids

After entering the blood by absorption or by in-travenous administration, xenobiotics are avail-able for distribution throughout the body. Theinitial rate of distribution to organs and tissuesis determined by the blood flow to that organ andthe rate of diffusion of the chemical into the spe-cific organ or tissue. Uptake of xenobiotics intoorgans or tissuesmay occur by either passive dif-fusion or by special transport processes. Withintissues binding, storage, and/or biotransforma-

tion may occur. After reaching equilibrium, thedistribution of a chemical among organs and tis-sues is largely determined by affinity; blood flowdetermines distribution only during the initialphase shortly after uptake.Body fluids are distributed between three dis-

tinct compartments: vascular water, interstitialwater and intracellular water. Plasma water andinterstitial water are extracellular water. Plasmawater plays an important role in the distribu-tion of xenobiotics. Human plasma accounts forabout 4% of the total body weight and 53% ofthe total volume of blood. By comparison, theinterstitial tissue fluids account for 13% of bodyweight, and intracellular fluids account for 41%.The concentration of a xenobiotic in blood fol-lowing exposure will depend largely on its ap-parent volumeof distribution. If the xenobiotic isdistributed only in the plasma, a high concentra-tion will be achieved within the vascular tissue.In contrast, the concentration will be markedlylower if the same quantity of xenobiotic weredistributed in a larger pool including the inter-stitial water and/or intracellular water.Among the factors that affect distribution,

apart frombinding to bloodmacromolecules, arethe route of administration, rate of biotransfor-mation, polarity of the parent xenobiotic or bio-transformation products, and rate of excretionby the liver or kidneys. Gastrointestinal absorp-tion and intraperitoneal administration providefor immediate passage of a compound to theliver, whereas dermal or respiratory routes in-volve at least one passage through the systemiccirculation prior to reaching the liver. The meta-bolism of most xenobiotics results in productsthat are more polar and thus more readily ex-creted than the parent molecules. Therefore, therate ofmetabolism is a critical determinant in thedistribution of a compound, since compoundsthat are readily metabolized are usually readilyexcreted, and thus are proportionally less proneto accumulate in certain tissues. The same prin-ciple applies to polarity, since very polar xeno-biotics will be readily excreted. Chemicals maycirculate either free or bound to plasma proteinor blood cells; the degree of binding and fac-tors influencing the equilibrium with the freeform may influence availability for biotransfor-mation, storage, and/or excretion [27].Patterns of xenobiotic distribution reflect cer-

tain physiological properties of the organism

34 Toxicology

Figure 17. Uptake and redistribution with blood of lipophilic xenobioticsLipophilic xenobiotics in the blood are first distributed to well-perfused organs (A); after some time, they are redistributed toorgans with lower blood flow representing a larger fraction of the body weight (B, C)

and the physicochemical properties of the xe-nobiotics. An initial phase of distribution maybe distinguished that reflects cardiac output andblood flow to organs. Heart, liver, kidney, brain,and other well-perfused organ- receive most of alipophilic xenobioticwithin the first fewminutesafter absorption. Delivery to the smooth mus-cles, most viscera, and skin is slower, and thetime to reach a steady-state concentration of axenobiotic in these organs may be several hours.A second phase of xenobiotic distribution maytherefore be distinguished; it is limited by bloodflow to an organ or tissue and involves a far largerfraction of body mass than the first phase of dis-tribution (Fig. 17).Only a limited number of xenobiotics have

sufficient solubility in blood to account for sim-ple dissolution as a route of distribution; the dis-tribution of many xenobiotics occurs in associa-tion with plasma proteins. The binding of drugsto plasma proteins is of key importance in trans-port. Many organic and inorganic compounds oflow molecular mass appear to bind to lipopro-teins, albumins, and other proteins in plasma and

are transported as protein conjugates. This bind-ing is reversible. Cellular components may alsobe responsible for transport of xenobiotics, butsuch transport is rarely a major route. The trans-port of xenobiotics by lymph is usually quanti-tatively of little importance since the intestinalblood flow is 500–700 times greater than the in-testinal lymph flow.A large number of studies on binding of drugs

by plasma protein have demonstrated that bind-ing to serum albumin is particularly importantfor these chemicals. Only few studies on the re-versible binding of toxic xenobiotics have beenperformed, but available evidence suggests a sig-nificant role of lipoproteins in plasma. Theseplasma proteins may bind xenobiotics as wellas some physiological constituents of the body.Examples for plasma proteins which may bindxenobiotics are albumin, α- and β-lipoproteins,and metal-binding proteins such as transferrin.Lipoproteins are important for the transport oflipid-soluble endogenous chemicals such as vi-tamins, steroid hormones, and cholesterol, butthey may also bind lipophilic xenobiotics. If a

Toxicology 35

xenobiotic is bound to a protein, it is immobi-lized remote from the site of action. The extentof binding to plasma proteins varies consider-ably among xenobiotics. While some are not atall bound, for others more than 90% of admin-istered dose may be bound to plasma proteins.These ligand–protein interactions are reversibleand provide a remarkably efficient means fortransport of xenobiotics to various tissues.Thexenobiotic–protein interaction may be simplydescribed according to the law of mass actionas:

where [T]F and [T]B are the concentrations offree and bound xenobiotic molecules, respec-tively, and k1 and k2 are the rate constants forassociation and dissociation; k2, which governsthe rate of binding to the protein, dictates the rateof xenobiotic release at a site of action or storage.The ratio k1/k2 is identical with the dissociationconstant Kdiss. Among a group of binding siteson proteins, those with the smallest Kdiss for agiven xenobiotic will bind it most tightly.In contrast to the covalent binding to proteins

seenwithmany xenobiotics or their electrophilicmetabolites (see Section 2.5.6.6), the interac-tion of xenobiotics with plasma proteins is mostoften noncovalent and reversible. Noncovalentbinding is of primary importance with respectto distribution because of the opportunities todissociate after transport. Binding of xenobio-tics to plasma proteins may be due to severaltypes of interactions which are summarized inthe following.

Ionic Binding. Electrostatic attraction oc-curs between two oppositely charged ions. Thedegree of binding varies with the chemical na-ture of each compound and the net charge. Dis-sociation of ionic bonds usually occurs readily,but some transition metals exhibit high associa-tion constants (low Kdiss values), and exchangeis slow. Ionic interactions may also contributeto binding of alkaloids with ionizable nitrogengroups and other ionizable xenobiotics.

Hydrogen Bonding. Generally, only themost electronegative atoms form stable hydro-gen bonds. Protein side chains containing hy-droxyl, amino, carboxyl, imidazole, and carb-

amyl groups may form hydrogen bonds, as canthe nitrogen and oxygen atoms of peptide bonds.Hydrogen bonding plays an important role in thestructural configuration of proteins and nucleicacids.

Van der Waals forces are very weak inter-actions between the nucleus of one atom andthe electrons of another atom, i.e., betweendipoles and induced dipoles. The attractiveforces are based on slight distortions inducedin the electron clouds surrounding each nucleusas two atoms come close together. The bindingforce is critically dependent upon the proxim-ity of interacting atoms and diminishes rapidlywith distance. However, when these forces aresummed over a large number of interactingatoms that “fit” together spatially, they can playa significant role in determining specificity ofxenobiotic–protein interactions.

Hydrophobic Interactions. When two non-polar groups come together they exclude thewater between them, and this mutual repulsionof water results in a hydrophobic interaction.In the aggregate they represent the least possi-ble disruption of interactions among polar watermolecules and thus can lead to stable complexes.Some consider this a special case of van derWaals forces. The minimization of thermody-namically unfavorable contact of a polar groupwith water molecules provides the major stabi-lizing effect in hydrophobic interactions.Consequences of the binding to plasma pro-

teins are reduced availability of the free xeno-biotic in the cells and a delayed excretion. Thexenobiotic bound to plasma protein cannot crosscapillary walls due to its high molecular mass.The fraction of dose bound is thus not availablefor delivery to the extravascular space or for fil-tration by the kidney. It is generally accepted thatthe fraction of xenobiotic that is bound may notexert toxic effects; however, many xenobioticsand endogenous compounds appear to competefor the same binding site, and thus one com-poundmay alter the unbound fraction of anotherby displacement, thereby potentially increasingtoxic effects. Plasma proteins that can bind en-dogenous chemicals and xenobiotics are listedbelow, together with examples of bonded xeno-biotics:

Toxicology 147

47. B. J. Smith, J. F. Curtis, T. E. Eling, Chem.Biol. Interact. 79 (1991) 245 – 264.

48. C. Vogel, Curr. Drug Metab. 1 (2000)391 – 404.

49. W. L. Smith, L. J. Marnett, Biochim. Biophys.Acta. 1083 (1991) 1 – 17.

50. B. H. Bach, J. W. Bridges, CRC Crit. Rev.Toxicol. 15 (1985) 217 – 329.

51. R. J. Duescher, A. A. Elfarra, J. Biol. Chem.267 (1992) 19859 – 19865.

52. P. D. Josephy, T. E. Eling, R. P. Mason,Mol.Pharmacol. 23 (1983) 461 – 466.

53. A. J. Kettle, C. C. Winterbourn, J. Biol. Chem.267 (1992) 8319 – 8324.

54. H. Thomas, F. Oesch, ISI Atlas Sci.:Biochemistry 1 (1988) 287 – 291.

55. S. A. Sheweita, A. K. Tilmisany, Curr. DrugMetab. 4 (2003) 45 – 58.

56. B. Burchell, W. H. Coughtrie, Pharmacol.Ther. 43 (1989) 261 – 289.

57. T. R. Tephly, B. Burchell, Trends Pharmacol.Sci. 11 (1990) 276 – 279.

58. R. H. Tukey, C. P. Strassburg, Annu. Rev.Pharmacol. Toxicol. 40 (2000) 581 – 616.

59. J. K. Ritter, Chem. Biol. Interact. 129 (2000)171 – 193.

60. K. W. Bock, B. S. Bock-Hennig, G. Fischer, L.W. D. Ullrich in H. Greim et al. (eds.):Biochemical Basis of ChemicalCarcinogenesis, vol. 1, Raven Press, NewYork 1984, pp. 13 – 22.

61. G. J. Mulder: Sulfation of Drugs and RelatedCompounds, CRC Press, Boca Raton, Fla.,1981.

62. M. W. Duffel, A. D. Marshal, P. McPhie, V.Sharma, W. B. Jakoby, Drug Metab. Rev. 33(2001) 369 – 395.

63. E. Y. Krynetski, W. E. Evans, Pharm Res. 16(1999) 342 – 349.

64. D. A. Evans, Pharmacol. Ther. 42 (1989)157 – 234.

65. P. Meisel, Pharmacogenomics 3 (2002)349 – 366.

66. I. M. Arias, W. B. Jakoby: Glutathione:Metabolism and Function, Raven Press, NewYork 1976.

67. B. Ketterer, D. J. Meyer, A. G. Clark in H. Sies,B. Ketterer (eds.): Glutathione Conjugation:Mechanisms and Biological Significance,Academic Press, London 1988, pp. 73 – 135.

68. R. Morgenstern, G. Lundqvist, G. Andersson,L. Balk, J. W. DePierre, Biochem. Pharmacol.33 (1984) 3609 – 3614.

69. J. L. Cmarik, P. B. Inskeep, M. J. Meredith, D.J. Meyer, B. Ketterer, F. P. Guengerich, CancerRes. 50 (1990) 2747 – 2752.

70. J. Caldwell, W. B. Jakoby: Biological Basis ofDetoxification, Academic Press, New York1983

71. M. W. Anders: Bioactivation of ForeignCompounds, Academic Press, Orlando 1985.

72. M. W. Anders in I. M. Arias, W. B. Jakoby, H.Popper, D. Schachter, D. A. Shafritz (eds.):The Liver: Biology and Pathology, 2nd ed.,Raven Press, New York, 1988, pp. 389 – 400.

73. J. G. Bessems, N. P. Vermeulen, CRC Crit.Rev. Toxicol. 31 (2001) 55 – 138.

74. G. M. B. StClair, V. Amarnath, M. A. Moody,D. C. Anthony, C. W. Anderson, D. G.Graham, Chem. Res. Toxicol. 1 (1988)179 – 185.

75. F. P. Guengerich, D. C. Liebler, CRC Crit. Rev.Toxicol. 14 (1985) 259 – 307.

76. D. C. Liebler, F. P. Guengerich, Biochemistry22 (1983) 5482 – 5489.

77. L. R. Pohl, R. V. Branchflower, R. J. Highet, J.L. Martin, D. S. Nunn, T. J. Monks, J. W.George, J. A. Hinson, Drug Metab. Dispos. 9(1981) 334 – 339.

78. M. W. Anders, W. Dekant:Conjugation-dependent Carcinogenicity andToxicity of Foreign Compounds, AcademicPress, San Diego, 1994.

79. T. J. Monks, M. W. Anders, W. Dekant, J. L.Stevens, S. S. Lau, P. J. van Bladeren, Toxicol.Appl. Pharmacol. 106 (1990) 1 – 19.

80. M. W. Anders, W. Dekant, S. Vamvakas,Xenobiotica 22 (1992) 1135 – 1145.

81. W. Dekant, S. Vamvakas, M. W. Anders, in W.Dekant, H.-G. Neumann (eds.): Tissue SpecificToxicity: Biochemical Mechanisms, AcademicPress, London, 1992, pp. 163 – 194.

82. S. D. Aust, C. F. Chignell, T. M. Bray, B.Kalyanaraman, R. P. Mason, Toxicol. Appl.Pharmacol. 120 (1993) 168 – 178.

83. B. D. Goldstein, B. Czerniecki, G. Witz, EHPEnviron. Health Perspect. 81 (1989) 55 – 57.

84. B. D. Goldstein, G. Witz, Free Radicals Res.Commun. 11 (1990) 3 – 10.

85. J. P. Kehrer, B. T. Mossman, A. Sevanian, M.A. Trush, M. T. Smith, Toxicol. Appl.Pharmacol. 95 (1988) 349 – 362.

86. P. A. Cerutti, B. F. Trump, Cancer Cells 3(1991) 1 – 7.

87. E. Chacon, D. Acosta, Toxicol. Appl.Pharmacol. 107 (1991) 117 – 128.

88. R. H. Burdon, V. Gill, C. Rice-Evans, FreeRadicals Res. Commun. 11 (1990) 65 – 76.

148 Toxicology

89. R. W. Pero, G. C. Roush, M. M. Markowitz, D.G. Miller, Cancer Detect. Prev. 14 (1990)551 – 561.

90. W. B. Jakoby, D. M. Ziegler, J. Biol. Chem.265 (1990) 20715 – 20719.

91. H. Sies, Naturwissenschaften 76 (1989)57 – 64.

92. D. M. Ziegler in W. B. Jacoby (ed.): EnzymicBasis of Detoxification, vol. I, AcademicPress, New York 1980, pp. 201 – 227.

93. E. Boyland, L. F. Chasseaud, Adv. Enzymol.Relat. Areas Mol. Biol. 32 (1969) 173.

94. T. A. Fjellstedt, R. H. Allen, B. K. Duncan, W.B. Jakoby, J. Biol. Chem. 248 (1973)3702 – 3707.

95. R. C. James, R. D. Harbison, Biochem.Pharmacol. 31 (1982) 1829 – 1835.

96. L. I. McLellan, C. R. Wolf, J. D. Hayes,Biochem. J. 258 (1989) 87 – 93.

97. J. A. Hinson, D. W. Roberts, Annu. Rev.Pharmacol. Toxicol. 32 (1992) 471 – 510.

98. S. D. Nelson, P. G. Pearson, Annu. Rev.Pharmacol. Toxicol. 30 (1990) 169 – 195.

99. L. R. Pohl, Chem. Res. Toxicol. 6 (1993)786 – 793.

100. E. R. Stadtman, C. N. Oliver, J. Biol. Chem.266 (1991) 2005 – 2008.

101. E. R. Stadtman, Science 257 (1992)1220 – 1224.

102. H. Esterbauer, H. Zollner, R. J. Schaur, ISIAtlas Sci.: Biochemistry 1 (1988) 311 – 317.

103. H. Kappus, Biochem. Pharmacol. 35 (1986)1 – 6.

104. J. L. Farber, Chem. Res. Toxicol. 3 (1990)503 – 508.

105. J. P. Kehrer, D. P. Jones, J. J. Lemasters, J. L.Farber, H. Jaeschke, Toxicol. Appl. Pharmacol.106 (1990) 165 – 178.

106. K. Hemminki, Carcinogenesis 14 (1993)2007 – 2012.

107. B. N. Ames, Mutat. Res. 214 (1989) 41 – 46.108. R. Adelman, R. L. Saul, B. N. Ames, Proc.

Natl. Acad. Sci. USA 85 (1988) 2706 – 2708.109. K. W. Bock, H.-P. Lipp, B. S. Bock-Hennig,

Xenobiotica 20 (1990) 1101 – 1111.110. D. R. Koop, D. J. Tierney, Bioassays. 12

(1990) 429 – 435.111. A. H. Conney, Annu. Rev. Pharmacol. Toxicol.

43 (2003) 1 – 30.112. L. Wu, J. P. J. Whitlock, Proc. Natl. Acad. Sci.

USA 89 (1992) 4811 – 4815.113. J. P. J. Whitlock, Chem. Res. Toxicol. 6 (1993)

754 – 763.114. B. Testa, P. Jenner, Drug Metab. Rev. 12

(1981) 1 – 117.

115. B. Testa, Xenobiotica 20 (1990) 1129 – 1137.116. J. R. Halpert, F. P. Guengerich, J. R. Bend, M.

A. Correia, Toxicol. Appl. Pharmacol. 125(1994) 163 – 175.

117. M. Blum, D. M. Grant, W. McBride, M. Heim,U. A. Meyer, DNA Cell Biol. 9 (1990)193 – 203.

118. M. E. Andersen, CRC Crit. Rev. Toxicol. 11(1981) 105 – 150.

119. M. W. Anders, W. Dekant in M. W. Anders, W.Dekant, D. Henschler, H. Oberleithner, S.Silbernagl (eds.): Renal Disposition andNephrotoxicity of Xenobiotics, AcademicPress, San Diego 1993, p. 155 – 183.

120. R. M. Brenner, F. C. J. Rector: The Kidney, W.B. Saunders Co., Philadelphia 1981.

121. E. C. Foulkes, Proc. Soc. Exp. Biol. Med. 195(1990) 1.

122. B. A. Saville, M. R. Gray, Y. K. Tam, DrugMetab. Rev. 24 (1992) 49 – 88.

123. C. D. Klaassen, J. B. Watkins, Pharmacol.Rev. 36 (1984) 1 – 67.

124. O. Fardel, L. Payen, A. Courtois, L. Vernhet,V. Lecureur, Toxicology 167 (2001) 37 – 46.

125. Y. Kato, H. Suzuki, Y. Sugiyama, Toxicology181–182 (2002) 287 – 290.

126. J. G. Filser, Arch. Toxicol. 66 (1992) 1 – 10.127. M. E. Andersen, Toxicol. Lett. 138 (2003)

9 – 27.128. R. Dixit, J. Riviere, K. Krishnan, M. E.

Andersen, J. Toxicol. Environ. Health. B Crit.Rev. 6 (2003) 1 – 40.

129. M. E. Andersen, M. L. Gargas, H. J. ClewellIII, K. M. Seceryn, Toxicol. Appl. Pharmacol.89 (1987) 149 – 157.

130. M. E. Andersen, D. Krewski, J. R. Withey,Cancer Lett. 69 (1993) 1 – 14.

131. R. B. Conolly, M. E. Andersen, Annu. Rev.Pharmacol. Toxicol. 31 (1991) 503 – 523.

132. S. M. Cohen, L. B. Ellwein, Science 249(1990) 1007 – 1011.

133. B. Butterworth,Mutation Res. 239 (1990)117 – 132.

134. Q. Chen, J. Marsh, B. Ames, B. Mossman,Carcinogenesis 17 (1996) 2525 – 2527.

135. R. A. Roberts, S. Chevalier, S. C. Hasmall, N.H. James, S. C. Cosulich, N. Macdonald,Toxicology 181–182 (2002) 167 – 170.

136. M. S. Denison, S. R. Nagy, Annu. Rev.Pharmacol. Toxicol. 43 (2003) 309 – 34.

137. J. Abel, D. Hohr, H. J. Schurek, Arch. Toxicol.60 (1987) 370 – 375.

138. S. Orrenius, P. Nicotera, Arch. Toxicol. Suppl.11 (1987) 11 – 19.

Toxicology 149

139. S. Orrenius, D. J. McConkey, G. Bellomo, P.Nicotera, Trends Pharmacol. Sci. 10 (1989)281 – 285.

140. E. C. Miller, J. A. Miller, Pharmacol. Rev. 18(1966) 805 – 838.

141. E. C. Miller, J. A. Miller, Cancer 47 (1981)2327 – 2345.

142. P. A. Cerutti, Science 227 (1985) 375 – 381.143. International Agency for Research on Cancer:

Models, Mechanisms and Etiology of TumourPromotion, IARC Sci. Publ., Lyon, 1984.

144. J. M. Bishop, Science 235 (1987) 305 – 311.145. J. M. Bishop, Cell 64 (1991) 235 – 48.146. J. M. Bishop, Cell 42 (1985) 23 – 38.147. J. Ashby, R. W. Tennant,Mutation Res. 257

(1991) 229 – 306.148. R. L. Brinster in T. H. Shepard, J. R. Miller,

M. Marois (eds.):Methods for Detection ofEnvironmental Agents that ProduceCongenital Defects, Elsevier New York, 1975,pp. 113 – 124.

149. J. M. Manson, L. D. Wise, in M. O. Amdur, J.Doull, C. D. Klaassen (eds.): Casarett andDoull’s Toxicology. The Basic Science ofPoisons, Pergamon Press New York 1991, pp.226 – 254.

150. R. P. Bolande in J. G. Wilson, F. C. Fraser(eds.): Handbook of Teratology, vol. 2, PlenumPress New York 1977, pp. 293 – 328.

151. S. Ivankovic: “Perinatal Carcinogenesis,DHEW Publication no. (NIH) 7a–1063”, Nat.Cancer Inst. Monogr. 51 (1979) 103 – 116.

152. E. Schlede, U. Mischke, R. Roll, D. Kayser,Arch. Toxicol. 66 (1992) 455 – 470.

153. M. J. Van den Heuvel, Human Exptl. Toxicol. 9(1990) 369.

154. B. Berner, E. R. Cooper in A. F. Kydonieu, B.Berner (eds.): Transdermal Delivery of Drugs,CRC Press, Boca Raton 1987, pp. 107 – 130.

155. J. A. Shatkin, H. S. Brown, Environ. Res. 56(1991) 90 – 108.

156. B. Ballantyne, T. Marrs, P. Turner: Generaland Applied Toxicology, Macmillan Press,New York 1993.

157. A. P. Worth, M. Balls: Alternative(non-animal) Methods for Chemical Testing:Current Status and Future Prospects,FRAME, Nottingham, UK, 2002.

158. P. A. Botham, D. A. Basketter, T. Maurer, D.Mueller, M. Potokar, W. J. Bontinck, FoodChem Toxicol. 29 (1991) 275 – 86.

159. G. M. Henningen in D. W. Hobson (ed.):Dermal and Ocular Toxicology. Fundamentalsand Methods, CRC Press, Boca Raton 1991,pp. 153 – 192.

160. W. E. Ecvam in European Center for theValidation of alternative methods Alternative(non-animal) Methods for Chemical Testing:Current Status and Future Prospects, vol. 30,Suppl. 1, FRAME, Nottingham, UK 2002, pp.95 – 102.

161. D. B. Clayson, F. Iverson, R. Mueller, Teratog.Carcinog. Mutagen 11 (1991) 279 – 296.

162. J. K. Haseman, A. Lockhart, Fund. Appl.Toxicol. 22 (1994) 382 – 391.

163. D. G. Hoel, J. K. Haseman, M. D. Hogan, J.Huff, E. E. McConnell, Carcinogenesis 9(1988) 2045 – 2052.

164. B. N. Ames, R. Magaw, L. S. Gold, Science236 (1987) 271 – 80.

165. B. N. Ames, L. S. Gold in T. Slaga, B.Butterworth (eds.): Chemically Induced CellProliferation: Implications for RiskAssessment, Wiley-Liss, New York 1991, pp.1 – 20.

166. L. S. Gold, T. H. Slone, B. R. Stern, N. B.Manley, B. N. Ames, Science 258 (1992)261 – 265.

167. D. J. Waxmann, L. Azaroff, Biochem. J. 281(1992) 577 – 592.

168. B. N. Ames, J. McCann, E. Yamasaki,Mutation Res. 31 (1975) 347 – 64.

169. D. M. Maron, B. N. Ames,Mutat. Res. 113(1983) 173 – 215.

170. M. Watanabe, M. J. Ishidate, T. Nohmi,Mutat.Res. 234 (1990) 337 – 348.

171. T. P. Simula, M. J. Glancey, C. R. Wolf,Carcinogenesis 14 (1993) 1371 – 1376.

172. M. Richold, A. Chaudley, J. Ashby, D. G.Gatehouse, J. Bootman, L. Henderson in D. J.Kirkland (eds.): UKEMS Sub-Committee onGuidelines for Mutagenicity Testing, reportpart Revised Basic Mutagenicity Tests,UKEMS Recommended Procedures,Cambridge University Press, Cambridge 1990,pp. 115 – 141.

173. J. J. McCormick, V. M. Mahler, Environ.Molec. Mutag. 14(S 15) (1989) 105 – 113.

174. G. M. Williams, J. H. Weisburger,Mutat. Res.205 (1988) 79 – 90.

175. N. Ozawa, F. P. Guengerich, Proc. Natl. Acad.Sci. USA 80 (1983) 5266 – 5270.

176. H. Bartsch, A. Barbin, M.-J. Marion, J. Nair,Y. Guichard, Drug Metab. Rev. 26 (1994)349 – 372.

177. C. Richter, J.-W. Park, B. N. Ames, Proc. Natl.Acad. Sci. USA 85 (1988) 6465 – 6467.

178. P. B. Farmer, H.-G. Neumann, D. Henschler,Arch. Toxicol. 60 (1987) 251 – 260.

150 Toxicology

179. J. A. Swenberg, N. Fedtke, F. Ciroussel, A.Barbin, H. Bartsch, Carcinogenesis 13 (1992)727 – 729.

180. I. A. Blair, Chem. Res. Toxicol. 6 (1993)741 – 747.

181. E. Randerath, T. F. Danna, K. Randerath,Mutat. Res. 268 (1992) 139 – 153.

182. M. D. Shelby, E. Zeiger,Mutat. Res. 234(1990) 257 – 261.

183. J. G. Vos, Arch. Toxicol Suppl. 4 (1980)95 – 108.

184. A. D. Dayau, R. F. Hertel, E. Hesseltine, G.Kazautzis, E. Smith, M. T. Van der Venne:Immunotoxicity of Metals andImmunotoxicology, Plenum Press, New York1990.

185. D. M. Maloney, Environ. Health Perspect. 101(1993) 396 – 401.

186. C. C. Harris, Science 262 (1993) 1980 – 1981.187. J. V. Rodricks, Environ. Health Perspect. 102

(1994) 258 – 264.188. F. R. Johannsen, CRC Crit. Rev. Toxicol. 20

(1990) 341 – 367.189. A. M. Jarabek, W. H. Farland, Toxicol. Ind.

Health 6 (1990) 199-.190. D. W. Nebert, Mutat. Res. 247 (1991)

267 – 281.191. R. T. H. van Welie, R. G. J. M. van Dijck, N. P.

E. Vermeulen, N. J. van Sittert, CRC Crit. Rev.Toxicol. 22 (1992) 271 – 306.

192. R. W. Tennant, J. Ashby,Mutat. Res. 257(1991) 209 – 227.

193. A. C. Beach, R. C. Gupta, Carcinogenesis 13(1992) 1053 – 1074.

194. K. Crump, CRC Crit. Rev. Toxicol. 32 (2002)133 – 53.

195. C. C. Travis, Toxicology 47 (1987) 3 – 13.196. H. Vainio, M. Sorsa, A. J. McMichael:

Complex Mixtures and Cancer Risk,International Agency for Research on Cancer(WHO), Lyon 1990.

197. J. A. Swenberg, R. R. Maronpot in (ed.):Chemically Induced Cell Proliferation:Implications for Risk Assessment, Wiley-Liss,New York 1991, pp. 245 – 251.

198. S. M. Cohen, L. B. Ellwein, Cancer Res. 51(1991) 6493 – 6505.

199. B. N. Ames, L. S. Gold, Science 249 (1990)970 – 1.

200. B. N. Ames, L. S. Gold,Mutat. Res. 250(1991) 3 – 16.

201. G. M. Gray, J. T. Cohen, J. D. Graham,Environ. Health Perspect. 101 (1993)203 – 208.

202. J. C. Barrett, Environ. Health Perspect. 100(1993) 9 – 20.

203. M. E. Andersen, H. J. Clewell, 3rd, M. L.Gargas, F. A. Smith, R. H. Reitz, Toxicol.Appl. Pharmacol. 87 (1987) 185 – 205.

204. T. Green, Xenobiotica 20 (1990) 1233 – 1240.205. D. G. Hoel, N. L. Kaplan, M. W. Anderson,

Science 219 (1983) 1032 – 1037.206. W. K. Lutz, Carcinogenesis 11 (1990)

1243 – 1247.207. H. Vainio, P. N. Magee, D. B. McGregor, A. J.

McMichael: Mechanisms of Carcinogenesis inRisk Identification, IARC ScientificPublications No. 116, Lyon 1992.

208. C. Shaw, H. B. Jones, Trends Pharmacol. Sci.15 (1994) 89 – 93.

209. P. Grasso, M. Sharratt, A. J. Cohen, Annu. Rev.Pharmacol. Toxicol. 31 (1991) 253 – 87.

210. J. A. Swenberg, Environ. Health Perspect. 101(1993) 39 – 44.

211. L. Poellinger, M. Gottlicher, J.-A. Gustafsson,Trends Pharmacol. Sci. 13 (1992) 241 – 245.

212. S. J. Borghoff, B. G. Short, J. A. Swenberg,Annu. Rev. Pharmacol. Toxicol. 30 (1990)349 – 67.

213. R. M. McClain, Toxicol. Lett. 64/65 (1992)397 – 408.

214. A. M. Tritscher, J. A. Goldstein, C. J. Portier,Z. McCoy, G. C. Clark, G. W. Lucier, CancerRes. 52 (1992) 3436 – 3442.

215. C. Portier, A. Tritscher, M. Kohn, C. Sewall,G. Clark, L. Edler, D. Hoel, G. Lucier, Fund.Appl. Toxicol. 20 (1993) 48 – 56.

216. J. P. V. Heuvel, G. C. Clark, M. C. Kohn, A.M. Tritscher, W. F. Greenle, G. W. Lucier, D.A. Bell, Cancer Res. 54 (1994) 62 – 68.

217. V. H. Frankos, Fund. Appl. Toxicol. 5 (1985)615 – 622.