WORLD HEALTH ORGANIZATION - · PDF fileWORLD HEALTH ORGANIZATION ... 2001 Evans Road, Carey, NC 27513, ... Vitamins & Fine Chemicals Human Nutrition and Health Viamin Research Group

.WORLD HEALTH ORGANIZATION

INTERNATIONAL AGENCY FOR RESEARCH ON CANCER

IARC Handbooks of Cancer Prevention

Carotenoids

Volume 2

This publication represents the views and expert opinionsof an IARC Working Group on the

Evaluation of Cancer-preventive Agents,which met in Lyon,

10-16 December 1997

1998

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Published by the International Agency for Research on Cancer,150 cours Albert Thomas, F-69372 Lyon cedex 08, France

(Ç International Agency for Research on Cancer, 1998

Distributed by Oxford University Press, Walton Street, Oxford, UK OX2 6DP (Fax: +44 1865267782) and inthe USA by Oxford University Press, 2001 Evans Road, Carey, NC 27513, USA (Fax: + 1 919 677 1303).

AIlIARC publications can also be ordered directly from IARCPress

(Fax: +33 4 72 73 83 02; E-mail: press (Q iarc.fr).

Publications of the World Health Organization enjoy copyright protection inaccordance with the provisions of Protocol 2 of the Universal Copyright Convention.

Ali rights reserved.

The designations used and the presentation of the material in this publication do not imply theexpression of any opinion whatsoever on the part of the Secretariat of the World Health Organization

concerning the legal status of any country, territory, city, or area or of its authorities,or concerning the delimitation of its frontiers or boundaries.

The mention of specifie companies or of certain manufacturers' products does not implythat they are endorsed or recommended by the World Health Organization in preference to others

of a similar nature that are not mentioned. Errors and omissions excepted,the names of proprietary products are distinguished by initial capitalletters.

The authors alone are responsible for the views expressed in this publication.

The International Agency for Research on Cancer welcomes requests for permission toreproduce or translate its publications, in part or in fulL. Applications and enquiries should be addressed

to the Editorial & Publications Service, International Agency for Research on Cancer,which will be glad to provide the latest information on any changes made to the text, plans for new

editions, and reprints and translations already available.

IARC Library Cataloguing in Publication Data

Carotenoids/IARC Working Group on the Evaluation ofCancer Preventive Agents (1997: Lyon,

France)

(IARC handbooks of cancer prevention; 2)

1. Carotenoids - congresses. 1. IARC Working Group on the Evaluation of CancerPreventive Agents Il Series

ISBN 92 832 3002 7ISSN 1027-5622

(NLM Classification: W1)


International Agency For Research On CancerThe International Agency for Research on Cancer (lARC) was established in 1965 by theWorld Health Assembly, as an independently financed organization within the framework ofthe World Health Organization. The headquarters of the Agency are in Lyon, France.

The Agency conducts a programme of research concentrating particularly on the epi-demiology of cancer and the study of potential carcinogens in the human environment. Itsfield studies are supplemented by biological and chemical research carried out in theAgency's laboratories in Lyon and, through collaborative research agreements, in nationalresearch institutions in many countries. The Agency also conducts a programme for theeducation and training of personnel for cancer research.

The publications of the Agency contribute to the dissemination of authoritative infor-mation on different aspects of cancer research. A complete list is printed at the back of thisbook. Information about IARC publications, and how to order them, is also available via theInternet at: http://www.iarc.fr/

Note ta the Reader

Anyone who is aware of published data that may influence any consideration in theseHandbooks is encouraged to make the information available to the Unit of

Chemoprevention, International Agency for Research on Cancer, 150 Cours AlbertThomas, 69372 Lyon Cedex 08, France

Although a11 efforts are made to prepare the Handbooks as accurately as possible, mis-takes may occur. Readers are requested to communicate any errars to the Unit of

Chemoprevention, so that corrections can be reported in future volumes.

Contents

List of Participants .................................................... 1

Preamble ............................................................... 3

General Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Carotenoids1 Chemical and physical characteristics of carotenoids ...................... 23

1.1 Structure and nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231.2 General properties ................................................... .27

1.2.1 Relationships between structure, properties and biological activity .......... .27

1.2.2 Molecular size and shape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .271.2.3 Solubility ..................................................... .27

1.2.4 Properties and molecular interactions of carotenoids in vivo ..... . . . . . . . . . . .271.3 Properties of the conjugated polyene chain ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28

1.3.1 Light absorption and photochemical properties ...... . . . . . . . . . . . . . . . . . . . .281.3.2 Chemical properties .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28

1.4 Isolation and analysis ................................................. .29

1.4.1 General methods ............................................... .29

1.4.2 Identification................................................... .30

1.4.3 Quantitative analysis ............................................ .30

1.5 Data on individual compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .301.5.1 ß-Carotene.................................................... .301.5.2 a-Carotene.................................................... .311.5.3 Lycopene..................................................... .311.5.4 Lutein........................................................ .321.5.5 Canthaxanthin ................................................. .33

1.5.6 Zeaxanthin.................................................... .331.5.7 ß-Cryptoxanthin ................................................ .34

1.5.8 a-Cryptoxanthin ................................................ .34

1.5.9 Zeinoxanthin .................................................. 35

2 Occurrence, commercial sources, use and application, analysis and human exposure 35

2.1 Carotenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .362.1.1 ß-Carotene.................................................... .36

2.1.2 a-Carotene.....................................................412.1.3 Lycopene......................................................43

2.2 Xanthophylls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .442.1.1 Lutein and zeaxanthin ............................................44

2.2.2 Cryptoxanthin...................................................45

2.2.3 Canthaxanthin ..................................................46

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Table of Contents

3 Metabolism, klnetics and genetic variation ............................... 463.1 Humans ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47

3.1.1 Intestinal digestion and absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .473.1.2 Transport in plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .483.1.3 Serum carotenoid concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49

3.1.3.1 Effects of ß-carotene supplements ........................... .50

3.1.3.2 Effects of tobacco smoking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .513.1.3.3 Effects of alcohol drinking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .513.1.3.4 Effects of other modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52

3.1.4 Tissue carotenoid concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .523.1.4.1 Effects of dietary intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .523.1.4.2 Effects of supplemental intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54

3.1.5 Kinetics ...................................................... .54

3.1.6 Metabolism .................................................... .553.1.6.1 ß-Carotene ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .553.1.6.2 ß-Apocarotenoids ...................................... . . .57

3.1.6.3 Other carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .573.1.7 Genetic variation ............................................... .58

3.2 Experimental models ................................................. .583.2.1 Non-human primates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .583.2.2 Preruminant calves and cows ..................................... .59

3.2.3 Ferrets ...................................................... .613.2.4 Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .623.2.5 Other animal species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .633.2.6 Conclusion.................................................... .64

4. Preventive effects ................................................... 644.1 Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64

4.1.1 Studies of cancer occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .644.1.1.1 Observational studies based on blood and other tissue measures of

carotenoids ............................................... .64

4.1.1.2 Observational studies based on dietary questionnaires .......... . . .92

4.1.1.3 Intervention trials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1184.1.2 Studies of intermediate end-points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .130

4.1.2.1 Observational studies of dietary and serum carotenoids .......... .130

4.1.2.2 Experimental studies of colorectal adenoma ................... .131

4.1.2.3 Experimental studies of cellular dysplasia and atypia . . . . . . . . . . . . . .1314.1.2.4 Experimental studies of oral leukoplakia ...................... .132

4.1.2.5 Experimental studies of cell proliferation ...................... .133

4.1.2.6 Experimental studies of chromosomal damage ................. .134

4.1.2.7 Experimental studies of immunological end-points. . . . . . . . . . . . . . . .1354.1.2.8 Summary ............................................. .136

4.2 Experimental models ................................................ .1374.2.1 Experimental animais . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .137

4.2.1.1 ß-Carotene ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1374.2.1.2 ß-Carotene with other potential inhibitors . . . . . . . . . . . . . . . . . . . . . . .1794.2.1.3 Canthaxanthin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1914.2.1.4 Canthaxanthin with other potential inhibitors ................... .205

4.2.1.5 Lycopene ............................................. .207

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4.2.1.6 Lutein................................... . . . . . . . . . . . . .2114.2.1.7 a-Carotene........................................... .2124.2.1.8 Fucoxanthin.......................................... .2154.2.1.9 Astaxanthin........................................... .215

4.2.1.10 Crocetin ............................................. .216

4.2.1.11 Mixtures of carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2164.2.1.12 Other end-points ....................................... 219

4.2.2 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2204.2.2.1 Mammalian cells in vitro. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2204.2.2.2 Antimutagenicity in short-term tests. . . . . . . . . . . . . . . . . . . . . . . . . .229

4.3 Mechanisms of cancer prevention ....................................... 251

4.3.1 Antioxidant properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2514.3.2 Modulation of carcinogen metabolism ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2544.3.3 Effects on cell transformation and differentiation ....................... 254

4.3.4 Effects on cell-to-cell communication ............................... .254

4.3.5 Inhibition of cell proliferation and oncogene expression ................. .254

4.3.6 Effects on immune function ...................................... .254

4.3.7 Inhibition of endogenous formation of carcinogens . . . . . . . . . . . . . . . . . . . .. 254

5 Other beneficial effects ............................................. .2555.1 Photosensitivity disorders ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2555.2 Cardiovascular disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2555.3 Age-related macular degeneration and cataract ............................ .257

5.4 Other effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .259

6 Carcinogenicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2596.1 Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .259

6.1.1 ATBC study ................................................... .259

6.1.2 CARET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2596.1.3 Interpretation of trials suggesting carcinogenicity ....................... .260

6.2 Experimental animais ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2616.3 Mechanisms of carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .262

7 Other toxic effects ................................................. .2637.1 Toxic and other adverse effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .263

7.1.1 Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2637.1.2 Experimental animais .................................................. 264

7.2 Reproductive and developmental eftects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2647.2.1 Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2647.2.2 Experimental animaIs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..265

7.3 Genetic and related eftects ................................................... .266

7.3.1 Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2657.3.2 Experimental systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .266

7.3.2.1 ln vitro .................................................... 266

7.3.2.2 ln vivo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .270

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Table of Contents

8 Summary of data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2708.1 Chemistry, occurrence and human exposure ............................ .2708.2 Metabolism and kinetics ............................................... 270

8.2.1 Humans .................................................... 2708.2.2 Experimental models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

8.3 Cancer-preventive effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2718.3.1 Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2718.3.2 Experimental systems ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

8.3.2.1 Cancer-preventive activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2728.3.2.2 Genetic and related effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

8.3.3 Mechanisms of cancer prevention .................................. 275

8.4 Other beneficial effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2758.5 Carcinogenic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

8.5.1 Humans .................................................... 2768.5.2 Experimental animais ........................................... 276

8.6 Other toxic effects .................................................... 276

8.6.1 Humans ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2768.6.2 Experimental systems ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

9 Recommendations for research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

10 Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27810.1 Cancer-preventive activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

10.1.1 Humans .................................................... 27810.1.2 Experimental animais ........................................... 278

10.2 Overall evaluation .................................................... 278

11 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

Appendix 1

Appendix 2

The concept of activity profiles of mutagenicity . . . . . . . . . . . . . . . . . 323

Definitions of test codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

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J.A. Baron7927 Rubin BldgOartmouth-Hitchcock Medical Center1 Medical Center Orive

Oartmouth Medical SchoolLebanon, NH 03756USA

J.S. BertramCancer Research Center of HawaiiUniversity of Hawaii1236 Lauhala StHonolulu, HI 96813USA

G. BrittonThe University of LiverpoolLife Sciences BuildingSchool of Biological SciencesCrown StreetLiverpool L69 7ZBUnited Kingdom

E. BuiattiCentro di Oocumentazione per la SaluteVia Triachini 1740100 Bologna

Italy

S. De FloraInstitute of Hygiene and Preventive MedicineUniversity of GenoaVia A. Pastore 1

16132 GenoaItaly

V.J. FeronTNO-Nutrition and Food Research InstituteToxicology OivisionPO Box 3603700 AJ ZeistThe Netherlands

M. GerberCentre de Recherche en CancerologieCentre Val d'AurelleParc Euromedicine34298 Montpeller Cedex 5France

List of participants

Volume 2. Carotenoids

Lyon, 10-16 December 1997

E.R. GreenbergNorris Cotton Cancer CenterOartmouth-Hitchcock Medical Center

1 Medical Center OriveLebanon, NH 03756USA

R.J. KavlockReproductive Toxicology Oivision (MO-71)United States Environmental Protection AgencyNHEERLResearch Triangle Park, NC 27711

USA

P. KnektNational Public Health InstituteMannerheimintie 166

00300 HelsinkiFinland

W. MaloneChemoprevention Branch

National Cancer InstituteExecutive Plaza North

Suite 201Bethesda, MD 20892USA

S.T. MayneOepartment of Epidemiology and Public HealthSchool of MedicineYale University,60 College StreetPO Box 208034New Haven, CT 06520-8034USA

H. NishinoOepartment of BiochemistryKyoto Prefectural University of MedicineKawaramachi-Hi rokojiKamigyo-kuKyoto 602Japan

J.A. OisonIowa State UniversityBiochemistry and Biophysics OepartmentAmes, Iowa 50011

USA

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H. PfanderDepartment of Chemistry and BiochemistryUniversity of BernFreiestrasse 33012 Bern

Switzerland

R.M. Salkeld

F. Hoffmann-La Roche LtdVitamins & Fine ChemicalsHuman Nutrition and HealthViamin Research Group

CH-4070 BaselSwitzerland

W. StahlInstitut für Physiologische Chemie 1Heinrich-Heine UniversitatPostfach 10 37 51

4000 DusseldorfGermany

J.F. SteghensFédération de Biochimie

Höpital Edouard Herriot69437 Lyon Cedex 03France

D.1. ThurnhamHoward Professor of Human NutritionUniversity of UlsterColeraine County, LondonderryBT52 1 SAUnited Kingdom

Secretariat:

J. VirtamoNational Public Health InstituteMannerheimintie 166

00300 HelsinkiFinland

P. BoffettaE. Heseltine (Editor)R. KaaksV. KrutovskikhC. Malaveille

A.B. MillerH. OhshimaB. PignatelliM. PlummerM. RautalahtiE. Riboli

R. SankaranarayananH. Vainio (Responsible Dfficer)J. WilbournM.L. Zaidan Dagli

R.G. Ziegler

Nutitional Epidemiology BranchNational Cancer InstituteExecutive Plaza North 430Bethesda, MD 20892USA

Technical Assistance:Observers:

P. AstorgINRA, Centre de Recherches de DijonUnité de Toxicologie NutritionnelleBP 1540

17 rue Sully21034 Dijon CedexFrance

M. LézèreA. MeneghelD. MiettonS. RuizJ. MitchellS. ReynaudJ. Thévenoux

R. GoralczykF. Hoffmann-La Roche LtdVitamins & Fine ChemicalsHuman Nutrition and HealthCarotenoid Research GroupCH-4070 BaselSwitzerland

Unable ta attend:H.K. Biesalski, Universitat Hohenheim, Institut für Biologische Chemie, Institut 140, Fruwirthstr. 12,70599 Stuttgart, GermanyG.S. Omenn, Executive Vice President for Medical Affairs, 608 Fleming Administration Building, Ann Arbor, Michigan 48109-1340, USA

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Preamble to the IARC Handbooksof Cancer PrevenüonThe prevention of cancer is one of the key objec-tives of the International Agency for Research onCancer (lARe). This may be achieved by avoidingexposures ta known cancer-causing agents, byincreasing host defences through immunization orchemoprevention or by modifying lifestyle. Theaim of the lAC Monographs programme is to evalu-ate carcinogenic risks of human exposure to chem-ical, physical and biological agents, providing ascientific basis for national or international

decisions on avoidance of exposures. The aim ofthe series of IARC Handbooks of Cancer Prevention is

to evaluate scientific information on agents andinterventions that may reduce the incidence of ormortality from cancer. This preamble is dividedinto two parts. The first addresses the generalscope, objectives and structure of the Handbooks.

The second describes the procedures for evaluatingcancer-preventive agents.

Part OneScopePreventive strategies embrace chemical, immuno-logical, dietary and behavioural interventions thatmay retard, block or reverse carcinogenic processesor reduce underlying risk factors. The term 'cancerprevention' is used to refer to interventions withpharmaceuticals, vitamins, mineraIs and otherche mi cals to reduce cancer incidence. The IARCHandbooks address the efficacy, safety and mecha-nisms of cancer-preventive strategies and the ade-quacy of the available data, inc1uding those ontiming, dose, duration and indications for use.

Preventive strategies can be applied across acontinuum of: (1) the general population;

(2) subgroups with particular predisposing hostor environmental risk factors, inc1uding geneticsusceptibilty to cancer; (3) persons with precan-cerous lesions; and (4) cancer patients at risk forsecond primary tumours. Use of the same strategies

or agents in the treatment of cancer patients to

control the growth, metastasis and recurrence of

tumours is considered to be patient management,not prevention, although data from c1inical trialsmay be relevant when making a Handbooks

evaluation.

ObjectiveThe objective of the Handbooks programme is thepreparation of critical reviews and evaluations ofevidence for cancer""prevention and other relevantproperties of a wide range of potential cancer-pre-ventive agents and strategies by internationalworking groups of experts. The resultingHandbooks may also indicate where additionalresearch is needed.

The Handbooks may assist national and interna-tional authorities in devising programmes of healthpromotion and cancer prevention and in ma kingbenefit-risk assessments. The evaluations of lAReworking groups are scientific judgements about theavailable evidence for cancer-preventive effcacy

and safety. No recommendation is given withregard to national and international regulation orlegislation, which are the responsibilty of individualgovernments and/or other international authori-ties. No recommendations for specifie research trialsare made.

IARC Working GroupsReviews and evaluations are formulated by inter-national working groups of experts convened bythe lARe. The tasks of each group are: (1) toas certain that a11 appropriate data have beenco11ected; (2) to select the data relevant for the

evaluation on the basis of scientifie merit; (3) toprepare accurate summaries of the data to enablethe reader to fo11ow the reasoning of the WorkingGroup; (4) to evaluate the significanee of the avail-able data from hum an studies and experimental

3


models on cancer-preventive activity, carcino-genicity and other beneficial and adverse effects;and (5) to evaluate data relevant to the und er-standing of mechanisms of action.

Working Group participants who contributedto the considerations and evaluations within a

particular Handbook are listed, with their addresses,at the beginning of each publication. Each partici-pant serves as an individual scientist and not as arepresentative of any organization, government orindustr. ln addition, scientists nominated by

national and international . agencies, industralassociations and consumer and/or envionmental or-ganitions may be invited as observers. IARC staffinvolved in the preparation of the Handbooks are listed.

Working procedures

Approximately 13 months before a working groupmeets, the topics of the Handbook are announced,and participants are selected by IARC staff inconsultation with other experts. Subsequently, rele-

vant c1inical, experimental and human data are col-lected by the IARC from a11 available sources of pub-lished information. Representatives of producer orconsumer associations may assist in the preparationof sections on production and use, as appropriate.

About eight months before the meeting, thematerial co11ected is sent to meeting participants to

prepare sections for the first drafts of the Handbooks.These are th en compiled by IARC staff and sent,before the meeting, to a11 participants of the WorkingGroup for review. There is an opportunity to returnthe compiled specialized sections of the draft tothe experts, inviting preliminary comments, beforethe complete first-draft document is distributed toa11 members of the Working Group.

Data for HandbooksThe Handbooks do not necessarily cite a11 of theliterature on the agent or strategy being evaluated.Only those data collidered by the Working Groupto be relevant to making the evaluation are

inc1uded. ln principle, meeting abstracts and otherreports that do not provide sufficient detail uponwhich to base an assessment of their quality are notconsidered.

4

With regard to data from toxicological, epidemi-ological and experimental studies and from c1ini-cal trials, only reports that have been published oraccepted for publication in the openly available

scientific literature are reviewed by the WorkingGroup. ln certain instances, government agencyreports that have undergone peer review and arewidely available are considered. Exceptions may bemade on an ad-hoc basis to inc1ude unpublishedreports that are in their final form and publiclyavailable, if their inclusion is considered pertinentto making a final evaluation. ln the sections onchemical and physical properties, on production,on use, on analysis and on human exposure,

unpublished sources of information may be used.

Criteria for selection of topics for evaluation

Agents, classes of agents and interventions to beevaluated in the Handbooks are selected on the

basis of one or more of the fo11owing criteria.

· The available evidence suggests potential forsignificantly reducing the incidence of cancers.

· There is a substantial body of human, experi-mental, clinical and/or mechanistic data suit-able for evaluation.

· The agent is in widespread use and of putativeprotective value, but of uncertain efficacy andsafety.

· The agent shows exceptional promise in experi-mental studies but has not been used inhumans.

· The agent is available for further studies ofhuman use.

Part TwoEvaluation of cancer-preventive agentsA wide range of findings must be taken intoaccount before a particular agent can be recog-

nized as preventing cancer. On the basis of experi-ence from the lARC Monographs programme, a

systematized approach to data presentation isadopted for Handbooks evaluations.

Preamble to the IARC Handbooks of Cancer Prevention

Outlne of data presentation scheme for evaluating cancer-preventive agents

1. Chemical and physical characteristics

2. Occurrence, production, use, analysis and

human exposure

2.1 Occurrence

2.2 Production

2.3 Use

2.4 Analysis

2.5 Human exposure

3. Metabolism, kinetics and genetic variation

3.1 Human studies

3.2 Experimental models

3.3 Genetic variation

4. Cancer-preventive effects

4.1 Human studies


4.2.1 Experimental animais

4.2.2 ln-vitro models

4.3 Mechanisms of cancer-prevention

5. Other beneficial effects

6. Carcinogenicity

6.1 Humans

6.2 Experimental animais

7. Other toxic effects

7.1 Adverse effects

7.1.1 Humans7.1.2 Experimental animais

7.2 Genetic and related effects

7.2.1 Humans7.2.2 Experimental models

8. Summary of data

8.1 Chemistry, occurrence and human exposure

8.2 Metabolism and kinetics

8.3 Cancer-preventive effects


8.3.3 Mechanism of action

8.4 Other beneficial effects

8.5 Carcinogenicity


8.6 Toxic effects

8.6.1 Humans


9. Recommendations for research

10. Evaluation

10.1 Cancer-preventive activity

10.1.1 Humans


10.2 Overall evaluation

11. References

1. Chemical and physical

characteristics of the agentThe Chemical Abstracts Servces Registry Number, the

latest Chemical Abstracts Primary Name, theIUPAC Systematic Name and other definitiveinformation (such as genus and species of plants) aregiven as appropria te. Information on chemical andphysical properties and, in particular, datarelevant to identification, occurrence and biologicalactivity are included. A description of technicalproducts of chemicals includes trade names,

relevant specifications and available information oncomposition and impurities. Sorne of the tradenames given may be those of mixtures in which theagent being evaluated is only one of the ingredients.

2. Occurrence, production, use,

analysis and human exposure

2.1 Occurrence

Information on the occurrence of an agent or mix-ture in the environment is obtained from data

derived from the monitoring and surveilance of

5


levels in occupational environments, air, water, soil,foods and animal and human tissues. When avail-able, data on the generation, persistence and bio-

accumulation of the agent are included. For mix-tures, information is given about a11 agents present.

2.2 Production

The dates of first synthe sis and of first commercialproduction of a chemical or mixture are provided;for agents that do not occur natura11y, this infor-mation may a110w a reasonable estimate to bemade of the date before which no human use of,or exposure to, the agent cou Id have occurred. Thedates of first reported occurrence of an exposureare also provided. ln addition, methods of

synthesis used in past and present commercial pro-duction and methods of production that may giverise to different impurities are described.

2.3 UseData on production, international trade and usesand applications are obtained for representative

regions. Sorne identified uses may not becurrent or major applications, and the coverage

is not necessarily comprehensive. ln the case ofdrgs, mention of their therapeutic applications doesnot necessarily represent CUITent practce, nor does it

imply judgement as to their therapeutic effcacy.

2.4 Analysis

An overview of current methods of analysis ordetection is presented. Methods for monitoringhuman exposure are also given, when available.

2.5 Human exposure

Human uses of, or exposure to, the agent aredescribed. If an agent is used as a prescribed orover-the-cOlinter pharmaceutical product, thenthe tye of person receiving the product in terms

of health status, age, sex and medical conditionbeing treated are described. For nonpharmaceuticalagents, particularly those taken because of culturaltraditions, the characteristics of use or exposureand the relevant populations are described. ln a11

cases, quantitative data, such as dose-response

6

relationships, are considered to be of special

importance.

3. Metabolism, kinetics and genetic

variation

ln evaluating the potential utilty of a suspected

cancer-preventive agent or strategy, a number ofdifferent properties, in addition to direct effects

upon cancer incidence, are described and weighed.Furthermore, as many of the data leading to anevaluation are expected to come from studies inexperimental animaIs, information that faciltatesinterspecies extrapolation is particularly impor-

tant; this includes metabolic, kinetic and geneticdata. Whenever possible, quantitative data,including information on dose, duration andpotency, are considered.

Information is given on absorption, distribution

(inc1uding placental transfer), metabolism and ex-cretion in humans and experimental animaIs.

Kinetic properties within the target species mayaffect the interpretation and extrapolation of dose-response relationships, su ch as blood concentra-

tions, protein binding, tissue concentrations,

plasma half-lives and elimination rates. Compara-tive information on the relationship between useor exposure and the dose that reaches the targetsite may be of particular importance for extrapola-tion between species. Studies that indicate the meta-bolic pathways and fate of the agent in humans andexperimental animaIs are summarized, and data onhum ans and experimental animaIs are comparedwhen possible. Observations are made oninterindividual variations and relevant metabolicpolymorphisms. Data indicating long-term accu-mulation in human tissues are included.Physiologica11y based pharmacokinetic modeIsand their parameter values are relevant and areincluded whenever they are available. Informationon the fate of the compound within tissues andce11s (transport, role of cellular receptors, compart-mentalization, binding to macromolecules) is given.

Genotyping wil be used increasingly, not onlyto identify subpopulations at increased or

decreased risk for cancers but also to characterize


variation in the biotransformation of, and res-ponses to, cancer-preventive and chemotherapeu-tic agents.

This subsection can include effects of the com-pound on gene expression, enzyme induction orinhibition, or pro-oxidant status, when such dataare not described elsewhere. It covers data obtainedin humans and experimental animaIs, with particu-lar attention to effects of long-term use and exposure.

4. Cancer-preventive effects

4.1 Human studies

Types of studies considered. Human data are derivedfrom experimental and non-experimental study

designs and are focused on cancer, precancer orintermediate biological end-points. The experi-

mental designs include randomized contro11ed tri-als and short-term experimental studies; non-

experimental designs include cohort, case-controland cross-sectional studies.

Cohort and case-control studies relate individ-ual use of, or exposure to, the agents under studyto the occurrence or prevention of cancer in indi-viduals and provide an estimate of relative risk(ratio of incidence or mortality in those exposed toincidence or mortality in those not exposed) as themain measure of association. Cohort andcase-control studies follow an observational

approach, in which the use of, or exposure to, theagent is not contro11ed by the investigator.

Intervention studies are experimental in design- that is, the use of, or exposure to, the agent is

assigned by the investigator. The interventionstudy or clinical trial is the design that can providethe strongest and most direct evidence of a protec-tive or preventive effect; however, for practical andethical reasons, such studies are limited toobservation of the effects among specifica11y

defined study subjects of interventions of 10 years

or fewer, which is relatively short wh en comparedwith the overa11lifespan.

Intervention studies may be undertaken in in-dividuals or communities and may or may notinvolve randomization to use or exposure. The

differences between these designs is important inrelation to analytical methods and interpretation offindings.

ln addition, information can be obtained fromreports of correlation (ecological) studies and caseseries; however, limitations inherent in these

approaches usua11y mean that such studies carry lim-ited weight in the evaluation of a preventive effect.

Qua/ity of studies considered. The Handbooks arenot intended to summarize a11 published studies. Itis important that the Working Group consider thefo11owing aspects: (1) the relevance of the study;

(2) the appropriateness of the design and analysisto the question being asked; (3) the adequacy andcompleteness of the presentation of the data; and(4) the degree to which chance, bias and con-

founding may have affected the results.Studies that are judged to be inadequate or

irrelevant to the evaluation are genera11y omitted.

They may be mentioned briefly, particularly whenthe information is considered to be a useful

supplement to that in other reports or when itis the only data available. Their inclusion does

not imply acceptance of the adequacy of the

study design, nor of the analysis and interpretationof the results, and their limitations are outlned.

Assessment of the cancer-preventive effect atdifferent doses and durations. The Working Groupgives special attention to quantitative assessmentof the preventive effect of the agent under study, byassessing data from studies at different doses. TheWorking Group also addresses issues of timing andduration of use or exposure. Such quantitative assess-ment is important to clarify the circumstances underwhich a preventive effect can be achieved, as well asthe dose at which a toxic effect has been shown.

Criteria for a cancer-preventive effect. After summa-rizing and assessing the individual studies, theWorking Group makes a judgement concerningthe evidence that the agent in question prevents

cancer in humans. ln making their judgement, theWorking Group considers several criteria for eachrelevant cancer site.

7


Evidence of protection derived from interventionstudies of good quality is particularly informative.Evidence of a substantial and significant reductionin risk, inc1uding a dose-response relationship, ismore likely to indicate a real effect. Nevertheless, asma11 effect, or an effect without a dose-responserelationship, does not imply lack of real benefitand may be important for public health if the can-cer is common.

Evidence is frequently available from differenttypes of studies and is evaluated as a whole.

Findings that are replicated in several studies ofthe same design or using different approaches

are more likely to provide evidence of a true pro-tective effect than isolated observations from

single studies.

The Working Group evaluates possible explana-tions for inconsistencies across studies, inc1udingdifferences in use of, or exposure to, the agent,

diferences in the underlying risk for cancer andmetabolism and genetic differences in the population.

The results of studies judged to be of high qualityare given more weight. Note is taken of both theapplicabilty of preventive action to several cancers

and of possible differences in activity, inc1udingcontradictory findings, across cancer sites.

Data from human studies (as we11 as from experi-mental models) that suggest plausible mechanismsfor a cancer-preventive effect are important in

assessing the overa11 evidence.

The Working Group may also determinewhether, on aggregate, the evidence from humanstudies is consistent with a lack of preventive effect.


4.2.1 Experimental animaIs

Animal models are an important component ofresearch into cancer prevention. They provide ameans of identifying effective compounds, of car-rying out fundamental investigations into theirmechanisms of action, of determining how theycan be used optimally, of evaluating toxicity and,ultimately, of providing an information basefor developing intervention trials in humans.Models that permit evaluation of the effects of

-8

cancer-preventive agents on the occurrence of

cancer in most major organ sites are available.Major groups of animal models inc1ude: those inwhich cancer is produced by the administration ofchemical or physical carcinogens; those involvinggenetica11y engineered animaIs; and those in

which tumours develop spontaneously. Most can-cer-preventive agents investigated in such studies

can be placed into one of three categories: COff-

pounds that prevent molecules from reaching orreacting with critical target sites (blocking agents);compounds that decrease the sensitivity of targettissues to carcinogenic stimuli; and compoundsthat prevent evolution of the neoplastic process

(suppressing agents). There is increasing interest inthe use of combinations of agents as a means ofimproving efficacy and minimizing toxicity.Animal models are useful in evaluating such COff-binations. The development of optimal strategiesfor human intervention trials can be faciltated bythe use of animal models that mimic the neoplas-tic process in humans.

Specific factors to be considered in such experi-ments are: (1) the temporal requirements of

administration of the cancer-preventive agents;

(2) dose-response effects; (3) the site-specificity ofcancer-preventive activity; and (4) the number andstructural diversity of carcinogens whose activitycan be reduced by the agent being evaluated.

Other types of studies inc1ude experiments in

which the end-point is not cancer but a definedpreneoplastic lesion or tumour-related, inter-mediate biomarker. An important variable in theevaluation of the cancer-preventive response is thetime and the duration of administration of theagent in relation to any carcinogenic treatment, orin transgenic or other experimental models in

which no carcinogen is administered. Further-more, concurrent administration of a cancer-

preventive agent may result in a decreased inci-dence of tumours in a given organ and an increasein another organ of the same animaL. Thus, in

these experiments it is important that multiple

organs be examined.For a11 these studies, the nature and extent of

impurities or contaminants present in the cancer-


preventive agent or agents being evaluated are

given when available. For experimental studies ofmixtures, consideration is given to the possibilty ofchanges in the physicochemical properties of thetest substance during collection, storage, extraction,concentration and delivery. Chemical and toxico-logical interactions of the components of mixturesmay result in nonlinear dose-response relationships.

As certain components of commonly used dietsof experimental animaIs are themselves known tohave cancer-preventive activity, particular consid-eration should be given to the interaction betweenthe diet and the apparent effect of the agent beingstudied. Likewise, restriction of diet may be impor-tant. The appropriateness of the diet given relativeto the composition of human diets may be com-mented on by the Working Group.

Qualiative aspects. An assessment of the experi-mental prevention of cancer involves several con-siderations of qualitative importance, inc1uding:

(1) the experimental conditions under which thetest was performed (route and schedule of expo-sure, species, strain, sex and age of animaIs stud-ied, duration of the exposure, and duration of thestudy); (2) the consistency of the results, for exam-pIe across species and target organ(s); (3) the stageor stages of the neoplastic process, from preneo-plastic lesions and benign tumours to malignantneoplasms, studied and (4) the possible role ofmodifying factors.

Considerations of importance to the WorkingGroup in the interpretation and evaluation of aparticular study inc1ude: (1) how c1early the agentwas defined and, in the case of mixtures, howadequately the sample composition was reported;(2) the composition of the diet and the stabilty ofthe agent in the diet; (3) whether the source, strainand quality of the animaIs was reported; (4) whetherthe dose and schedule of treatment with theknown carcinogen were appropriate in assays ofcombined treatment; (5) whether the doses of thecancer-preventive agent were adequately moni-tored; (6) whether the agent(s) was absorbed, asshown by blood concentrations; (7) whether thesurvival of treated animaIs was similar to that of

controls; (8) whether the body and organ weights oftreated animaIs were similar to those of con troIs;

(9) whether there were adequate numbers ofanimaIs, of appropriate age, per group;

(10) whether animaIs of each sex were used, ifappropriate; (11) whether animaIs were a110cated

randomly to groups; (12) whether appropriaterespective controls were used; (13) whether theduration of the experiment was adequate;

(14) whether there was adequate statistical analy-sis; and (15) whether the data were adequatelyreported. If available, recent data on the incidenceof specific tumours in historical controls, as well asin concurrent control s, are taken into account inthe evaluation of tumour response. The observa-

tion of effects on the occurrence of lesions

presumed to be preneoplastic or the emergence ofbenign or malignant tumours may in certain in-stances aid in assessing the mode of action of thepresumed cancer-preventive agent. Particular atten-tion is given to assessing the reversibilty of theselesions and their predictive value in relation tocancer development.

Quantitative aspects. The probabilty that tumourswil occur may depend on the species, sex, strainand age of the animaIs, the dose of carcinogen (ifany), the dose of the agent and the route and dura-tion of exposure. A decreased incidence and/or

decreased multiplicity of neoplasms in adequatelydesigned studies provides evidence of a cancer-pre-ventive effect. A dose-related decrease in incidenceand/or multiplidty fuer strengtens fus assodation.

Statistical analysis. Major factors considered in thestatistical analysis by the Working Group inc1udethe adequacy of the data for each treatment group:

(1) the initial and final effective numbers of

animaIs studied and the survival rate;(2) body weights; and (3) tumour incidence andmultiplicity. The statistical methods used shouldbe clearly stated and should be the generally

accepted techniques refined for this purpose. lnparticular, the statistical methods should be appro-priate for the characteristics of the expected datadistribution and should account for interactions in

""9


multifactorial studies. Consideration is given as towhether the appropriate adjustments were madefor differences in survival.

4.2.2 ln-vitro models

Ce11 systems in vitro con tribu te to the early identi-fication of potential cancer-preventive agents andto elucidation of mechanistic aspects. A numberof assays in prokaryotic and eukaryotic systems areused for this purpose. Evaluation of the results ofsu ch assays includes consideration of: (1) thenature of the ce11 type used; (2) whether primaryce11 cultures or ce11 lines (tumorigenic or nontu-morigenic) were studied; (3) the appropriateness ofcontrols; (4) whether toxic effects were consideredin the outcome; (5) whether the data were appro-priately summated and analysed; (6) whetherappropriate quality con troIs were used; (7)whether appropriate concentration ranges wereused; (8) whether adequate numbers of indepen-dent measurements were made per group; and(9) the relevance of the end-points, including inhi-bition of mutagenesis, morphological transforma-

tion, anchorage-independent growth, ce11-ce11 com-

munication, calcium tolerance and differentiation.

4.3 Mechanisms of cancer prevention

Data on mechanisms can be derived from bothhuman and experimental systems. For a rationalimplementation of cancer-preventive measures, itis essential not only to assess protective end-

points but also to understand the mechanisms bywhich the agents exert their anticarcinogenic

action. Information on the mechanisms of cancer-preventive agents can be inferred from relation-ships between chemical structure and biologicalactivity, from analysis of interactions betweenagents and specific molecular targets, from studiesof specific end-points in vitro, from studies of theinhibition of tumorigenesis in vivo and the efficacyof modulating intermediate biomarkers, and fromhuman studies. Therefore, the Working Grouptakes account of mechanistic data in ma king the

final evaluation of cancer-prevention.

10

Several classifications of mechanisms have beenproposed, as have several systems for evaluatingthem. Cancer-preventive agents may act at severaldistict levels. Their action may be: (1) extracellular,for example, inhibiting the uptake or endogenousformation of carcinogens, or forming complexes

with, diluting and/or deactivating carcinogens; (2)

intracellular, for example, trapping carcinogens innon-target ce11s, modifyng transmembrane transport,modulating metabolism, blocking reactive molecules,inbitig cell replication or modulatig gene expression

or DNA metabolism; or (3) at the level of the ce11, tis-sue or organism, for example, affecting ce11 differen-tiation, interce11ular communication, proteases, sig-nal transducton, growth factors, cell adhesion mole-cules, angiogenesis, interactions with the extracellu-lar matrix, hormonal status and the immune system.

Many cancer-preventive agents are known orsuspected to act by several mechanisms, which ffayoperate in a coordinated manner and a110w theff abroader spectrum of anticarcinogenic actvity. There-fore, multiple mechanisms of action are taken intoaccount in the evaluation of cancer-prevention.

BeneficiaI interactions, generally resulting fromexposure to inhibitors that work through comple-mentary mechanisms, are exploited in combiiiedcancer-prevention. Because organisms are natu-ra11y exposed not only to mixtures of carcinogenic

agents but also to mixtures of protective agents, itis also important to understand the mechanisms ofinteractions between inhibitors.

5. Other beneficial effects

Ths secton contains maiy background inormationon preventive activity; use is described in Section 2.3.An expanded description is given, when appropriate,of the effcacy of the agent in the maintenance of anormal healthy state and the treatment of partculardiseases. Information on the mechanisms involvedin these activities is described. Reviews, rather thanindividual studies, may be cited as references.

The physiological functons of agents such as vita-mis and micronutrents can be described briefly, withreference to reviews. Data on the therapeutic effects of

drugs approved for clical use are summaried.


6. Carcinogenicity

Sorne agents may have both carcinogenic and anti-carcinogenic activities. If the agent has been evalu-ated within the IAC Monographs on the Evaluation of

Carcinogenic Risks to Humans, that evaluation is

accepted, unless significant new data have appearedthat may lead the Working Group to reconsider theevidence. When a re-evaluation is necessary orwhen no carcinogenic evaluation has been made,the procedures described in the Preamble to the

lARC Monographs on the Evaluation of CarcinogenicRisks to Humans are adopted as guidelines.

7. Other toxic effects

Toxic effects are of particular importance in thecase of agents that may be used widely over longperiods in healthy populations. Data are given onacute and chronic toxic effects, su ch as organ toxi-city, increased cell proliferation, immunotoxicityand adverse endocrine effects. If the agent occursnatura11y or has been in clinical use previously,the doses and durations used in cancer preventiontrials are compared with intakes from the diet, inthe case of vitamins, and previous clinical expo-sure, in the case of drugs already approved for

humaH use. Wh en extensive data are available,only summaries are presented; if adequate reviewsare available, reference may be made to these. Ifthere are no relevant reviews, the evaluation ismade on the basis of the same criteria as areapplied to epidemiological studies of cancer.

Differences in response as a consequence ofspecies, sex, age and genetic variabilty are pre-sented when the information is available.

Data demonstrating the presence or absence ofadverse effects in humans are included; equa11y,

lack of data on specific adverse effects is stated c1early.Findings in humall and experimental animaIs

are presented sequentia11y under the headings 'Toxicand other adverse effects' and 'Genetic and relatedeffects' .

The section 'Toxic and other adverse effects'includes information on immunotoxicity, neuro-

toxicity, cardiotoxicity, haematological effects and

toxicity to other target organs. Specific case reports

in humans and any previous clinical data are noted.Other biochemical effects thought to be relevantto adverse effects are mentioned. The reproductiveand developmental effects described includeeffects on fertilty, teratogenicity, fetotoxicity andembryotoxicity. Information from nonmam-malian systems and in vitro are presented only ifthey have clear mechanistic significance.

The section 'Genetic and related effects' inc1udesresults from studies in mammalian and nonmam-malian systems in vivo and in vitro. Information onwhether DNA damage occurs via direct interactionwith the agent or via indirect mechanisms (e.g.generation of free radicals) is inc1uded, as is infor-mation on other genetic effects su ch as mutation,recombination, chromosomal damage, aneuploidy,cell immortalization and transformation, and effectson ce11-ce11 communication. The presence and tox-icological significance of cellular receptors for thecancer-preventive agent are described.

The adequacy of epidemiological studies of toxiceffects, inc1uding reproductive outcomes andgenetic and related effects in humans, is evaluatedby the same criteria as are applied to epidemiologicalstudies of cancer. For each of these studies, the ade-quacy of the reporting of sample characterizationis considered and, where necessary, commented upon.The available data are interpreted critica11y accord-

ing to the end-points used. The doses and concen-trations used are given, and, for experiments in vitro,mention is made of whether the presence of anexogenous metabolic system affected the observa-tions. For studies in vivo, the route of administrationand the formulation in which the agent was

administered are included. The dosing regimens,inc1uding the duration of treatment, are also given.Genetic data are given as listings of test systems,data and references; bar graphs (actvity profies) andcorresponding summary tables with detailed infor-mation on the preparation of genetic activity pro-files are given in appendices. Genetic and otheractivity in humans and experimental mammals isregarded as being of greater relevance th an that inother organisms. The in-vitro experiments providingthese data must be carefully evaluated, since there

--11


are many trivial reasons why a response toone agent may be modified by the addition ofanother.

Structure-activity relationships that may be rele-vant to the evaluation of the toxicity of an agentare described.

Studies on the interaction of the suspected can-

cer-preventive agent with toxic and subtoxic dosesof other substances are described, the objective

being to determine whether there is inhibitionor enhancement, additivity, synergism or potenti-ation of toxic effects over an extended dose range.

Biochemical investigations that may have a bear-ing on the mechanisms of toxicity and cancer-prevention are described. These are carefully evalu-ated for their relevance and the appropriateness ofthe results.

8. Summary of data

ln this section, the relevant human and experimentaldata are summarized. Inadequate studies are gen-erally not summarized; such studies, if cite d, are

identified in the preceding text.

8.1 Chemistry, occurrence and human exposure

Human exposure to an agent is summarized

on the basis of elements that may include production,use, occurrence in the environment and determina-tions in human tissues and body fluids. Quantitativedata are summarized when available.

8.2 Metabolism and kinetics

Data on metabolism and kinetics in humans andin experimental animaIs are given when these

are considered relevant to the possible mechanismsof cancer -preventive, carcinogenic and toxic

activity.

8.3 Cancer-preventive effects

8.3.1 Humans

The results of relevant studies are summarized,

including case reports and correlation studies whenconsidered important.

..12


Data relevant to an evaluation of cancer-pre-

ventive activity in experimental models are

summarized. For each animal species and routeof administration, it is stated whether a changein the incidence of neoplasms or preneoplastic .lesions was observed, and the tumour sites areindicated. Negative findings are also summa-rized. Dose-response relationships and otherquantitative data may be given when available.

8.3.3 Mechanism of actionData relevant to the mechanisms of cancer-pre-ventive activity are summarized.

8.4 Other beneficial effects

When beneficial effects other th an cancer preven-

tion have been identified, the relevant data aresummarized.

8.5 Carcinogenicity

Norma11y, the agent wil have been reviewed and

evaluated within the lARC Monographs pro-

gramme, and that summary is used with the inclu-sion of more recent data, if appropriate.

8.5.1 Humans

The results of epidemiological studies that are

considered to be pertnent to an assessment of humancarciogenidty are sumared. When relevant, casereport and correlation studies are also summaried.


Data relevant to an evaluation of carcinogenic effectsin animal models are summarized. For each animal

species and route of administration, it is statedwhether a change in the incidence of neoplasms orpreneoplastic lesions was observed, and the tumoursites are indicated. Negative findings are also summa-rized. Dose-response relationships and other quanti-tative data may be mentioned when available.

8.6 Toxic effects

Adverse effects in humans are summarized,

together with data on general toxicological effectsand cytotoxicity, receptor binding and hormonaland immunological effects. The results of investi-


gations on the reproductive, genetic and related

effects are summarized. Toxic effects are summa-rized for whole animaIs, cultured mammalian ce11sand non-mammalian systems. When available,data for humans and for animaIs are compared.

Structure-activity relationships are mentionedwhen relevant to toxicity.

9. Recommendations for research

During the evaluation process, it is likely thatopportunities for further research wil be identi-fied. These are clearly stated, with the understand-ing that the areas are recommended for future

investigation. It is made clear that these researchopportunities are identified in general terms onthe basis of the data currently available.

10. Evaluation

Evaluations of the strength of the evidence for

cancer-preventive activity and carcinogenicity

from studies in humans and experimental modelsare made, using standard terms. These terms mayalso be applied to other beneficial and adverse

effects, when indicated. When appropriate, refer-en ce is made to specific organs and populations.

It is recognized that the criteria for these evalu-ations, described below, cannot encompass a11

factors that may be relevant to an evaluation ofcancer-preventive activity. ln considering a11 the

relevant scientific data, the Working Group mayassign the agent or other intervention to a higheror lower category than a strict interpretation ofthese criteria would indicate.

10.1 Cancer-preventive activityThese categories refer to the strength of the evid-

ence that an agent prevents cancer. The evaluationsmay change as new information becomes available.

Evaluations are inevitably limited to the can-

cer sites, conditions and levels of exposure andlength of observation covered by the availablestudies. An evaluation of degree of evidence,

whether for a single agent or a mixture, is limitedto the materials tested, as defined physica11y,

chemica11y or biologically. When the agents evalu-ated are considered by the Working Group to besufficiently closely relate d, they ilay be grouped

for the purpose of a single evaluation of degree of

evidence.Information on mechanisms of action is taken

into account when evaluating the strength ofevidence in humans and in experimental animaIs,as we11 as in assessing the consistency of resultsbetween studies in humans and experiilentalmodels.

10.1.1 Cancer-preventive activity in humans

The evidence relevant to prevention in hUilans isclassified into one of the fo11owing four categories.

· Suftcíent evidence of cancer-preventive activityThe Workig Group considers that a causalrelationship has been established between use ofthe agent and the prevention of hUilan can-

cer in studies in which chance, bias and confound-ing could be ruled out with reasonable confidence.

· Limited evidence of cancer-preventive activityThe data suggest a reduced risk for cancer withuse of the agent but are limited for making adefinitive evaluation either because chance,

bias or confounding could not be ruled out withreasonable confidence or because the data are

restricted to intermediary biomarkers of uncer-tain validity in the putative pathway to cancer.

· Inadequate evidence of cancer-preventive activity

The available studies are of insufficient quality,consistencyor statistical power to permit a con-clusion regarding a cancer-preventive effect ofthe agent, or no data on the prevention of can-

cer in humans are available.· Evidence suggesting lack of cancer-preventive activity

Several adequate studies of use or exposure

are mutua11y consistent in not showing a

preventive effect.The strength of the evidence for any carcinogenic

activity is assessed in para11el. Both cancer-preven-tive activity and carcinogenicity are identified

and, wh en appropriate, tabulated by organ site.The evaluation also cites the population subgroups

..13


concerne d, specifying age, sex, genetic or environ-mental predisposing risk factors and the presenceof precancerous lesions.

10.1.2 Cancer-preventive activity in experimentalanimaIs

Evidence for prevention in experimental animaIsis classified into one of the fo11owing categories.

· Suffczent evidence of cancer-preventive activityThe Working Group considers that a causalrelationship has been established between theagent and a decreased incidence and/or mult-plicity of neoplasms.

· Limited evidence of cancer-preventive activityThe data suggest a preventive effect but arelimited for making a definitive evaluation be-cause, for example, the evidence of prevention isrestricted to a single experiment, the agent de-creases the incidence and/or multiplicity only ofbenign neoplasms or lesions of un certain neo-plastic potential or there is conflcting evidence.

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· Inadequate evidence of cancer-preventive activityThe studies cannot be interpreted as showingeither the presence or absence of a preventive

effect because of major qualitative or quantita-tive limitations (unresolved questions regardingthe adequacy of the design, conduct or interpre-tation of the study), or no data on prevention inexperimental animaIs are available.

· Evidence suggesting lack of cancer-preventive activityAdequate evidence from conclusive studiesin several models shows that, within the limitsof the tests used, the agent does not preventcancer.

10.2 Overall evaluationFina11y, the body of evidence is considered as a

whole, and summary statements are made thatencompass the effects of the agents in hUffanswith regard to cancer-preventive activity, carcino-genicity and other beneficial and adverse effects,as appropriate.

General Remarks

Experiments with animal models conducted in

the 1960s and 1970s demonstrated that highdoses of retinoids could inhibit carcinogenesis in

several organs, including the respiratory tract.Many epidemiological studies carried out duringthe late 1970s and early 1980s showed negative

associations between estimated intakes of vitaminA or ß-carotene (provitamin A) and the risk fordeveloping cancer at various sites. ln we11-nour-

ished populations, however, no consistent rela-tionships were found between cancer risk andplasma retinol concentrations. Fo11owing these

observations, it was postulated that ß-carotene

itself, without transformation into retinol, mightprotect against cancer by its antioxidant capacityor by direct action or conversion to retinoid-likemolecules. These suggestions captured the imagi-

nations of many workers and stimulated mu chresearch (Peto et al., 1981). This monograph sum-marizes a great deal of that work and attempts todraw conclusions about the present and futuretrends in research.

CarotenoidsOf the various classes of pigments in nature,the carotenoids are among the most wide-spread and important and have attracted inter-est for a considerable time. ln 1831,Wackenroder isolated carotene from carrots(Daucus carota), from which the class of com-pounds derives its name. ln 1837, Berzelius

gave the name 'xanthophy11s' to the yeHow pig-ments from autumn leaves. These reports markthe beginning of carotenoid research. Because

of their ubiquitous occurrence, different func-tions and interesting properties, carotenoids arethe subject of interdisciplinary research in

many branches of science. The industrial pro-duction of carotenoids has also contributed

mu ch to knowledge in this field.Early work on carotenoid chemistry is sum-

marized in the book Carotenoide (Karrer &

Jucker, 1948), which was fo11owed by another,edited by Isler et al. (1971), who devised an eco-nomica11y feasible method for the industrial

synthesis of carotenoids. Biochemical aspects

have been covered in The Biochemistr of the

Carotenoids (Goodwin, 1980, 1984), in TheBiochemistr of Natural Pigments (Britton, 1983)and, more recently, in Plant Pigments,

(Goodwin, 1988). The technical and nutrition-al applications of carotenoids have been treatedin a book edited by Bauernfeind (1981).Recently, a new series, Carotenoids (Britton etaL., 1995a,b), was started which wil cover theentire field. Advances in a11 areas of research oncarotenoids are covered in the published pro-

ceedings of the International Symposia on

Carotenoids (Weedon, 1976; Goodwin, 1979;Britton & Goodwin, 1982; Davies & Rau, 1985;

Krinsky et aL., 1989; Britton, 1991, 1994, 1997).ß-Carotene is one of the agents being evalu-

ated in the frarrework of the US National

Cancer Institute chemoprevention plan. Thisprogramme includes a number of in-vitro andin-vivo preclinical studies and phase-l, phase-IIand phase-II clinical chemoprevention trials(Ke11off & Boone, 1994).

Carotenoids occur in aH three domains oflife, Le. eubacteria, archeobacteria and eukary-otes. More than 600 naturally occurringcarotenoids are known today (Pfander et al.,1987; Ku11 & Pfander, 1995). Algae are a ri chsource of carotenoids, and more than 100 suchcompounds have been isolated from theseorganisms and characterized (Haugan et aL.,1995). The most important source ofcarotenoids for humans is plants, in which thebrillant colours of the carotenoids are often

masked by chlorophy11, e.g. in green leaves. Thecarotenoids are responsible for the beautifulcolours of many fruits (citrus fruits, tomatoes,paprika, rose hips) and flowers (Eschscholtzia,

Narcissus), as we11 as the colours of many birds(flamingo, cock of the rock, ibis, canary), insects(ladybird) and marine animaIs (crustaceans,salmon). Norma11y, carotenoids occur in low

concentrations; nevertheless, total carotenoidproduction in nature has been estimated to be

about 108 tonnes per year (Isler et aL., 1967).Analysis of serum and human breast milk hasshown that about 20 carotenoids derived fromfruits and vegetables may be absorbed andmetabolized by humans (Khachik et al., 1997).

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Carotenoids produced industria11y by synthesisor from natural extracts are widely used in feed tocol our egg yolk, chickens, shrimp and farm-raisedsalmon or to colour food products such as mar-garine and cheese. Various methods have beendeveloped for incorporation of carotenoids intofoods: a microcrystallne dispersion in an edible

fat is used to colour margarine, and ß-carotene inthe form of a microdispersion in a hydrophilc

protective co11oid is used in fruit juices.Besides the ß-carotenes, which are of commer-

cial interest, many other natura11y occurring

carotenoids have been synthesized. The mainemphasis has been on the synthesis of carotenoidsin optica11y active forms, as it is these that are

found in nature and are useful as reference stan-dards for analytical work.

Given the large number of naturalcarotenoids, a detailed systematic nomenclaturehas been developed (Section 1). This handbookdeals specifica11y with those compounds foundpredominantly in hum an blood and tissues andwith which the most extensive studies of cancerprevention have been undertaken. The term'carotenoid covers a11 of these compounds; theterm 'carotene' is restricted to the hydrocarbons,that is, compounds containing only hydrogenand carbon, and carotenoids containing oxygenfunctions are known as 'xanthophy11s'. The provi-tamin A carotenoids are ß-carotene and othercompounds that con tain one unsubstituted ß

ring, e.g. a-carotene and ß-cryptoxanthin.Because of their long system of conjugated

double bondsJ any carotenoid can theoretica11yexist not only in the a11-trans (all-E) form but also

in alternative forms with different geometrical

arrangements of one or more of the double bonds,Le. as cis (Z) isomers. Interconversion between thegeometrical isomers occurs readily. The geometri-cal isomers have different physical and chemicalproperties, which must lead to differences in theirbioavailabilty and biological activity. For a11 pur-poses of this handbook, E = trans and Z = cis, andthe terms trans and cis are used consistently.

The form in which a carotenoid is ingested isalso important. Supplements can consist of for-mulations of pure, often crystallne material as asuspension in oil or in a stabilzed, water-dis-

persible form. ln natural foods, the structuralmatrix and molecular environment of a

..16

carotenoid are major determinants of its bioavail-abilty. The isomeric composition of samples of aparticular carotenoid from different sources mayalso vary. A11 ß-carotene samples, for example,

consist of mixtures of geometrical isomers, but theisomeric compositions are not always the same.

Thus, ß-carotene obtained from the alga Dunaliellacan contain up to 50% cis isomers, whereas iiimost natural sources the level of cis isomers pre-sent is sma11 (usually no more than 5-10%).

The many biological properties and functionsof the carotenoids establish the importance of thisc1ass of compounds (Krinsky, 1994). During pho-tosynthesis, direct excitation of carotenoids bylight results in an excited singlet state; subsequenttransfer of this excitation energy to chlorophyllinitia tes photosynthesis. The involvement ofcarotenoids can effectively extend the wavelengthof light availableto an organism for photosynthe-sis. Carotenoids are also important for photoprotection of ce11s and tissues (Section 1).

ln humans and in animaIs that require vita-min A for normal growth and development, themost important source is the ingestion andmetabolism of carotenoids that can be convertedto vitamin A. An adequate supply of vitamin A isnot critical in affuent societies but may stil be asevere problem in countries of the Third World.

According to an estimate of WHO (Underwood &Arthur, 1996), 250000-500000 children go blindevery year due to xerophthalmia, a disease caused

by a deficiency of vitamin A, or die as a conse-

quence of infectious diseases.Oxidative processes may damage macromole-

cules such as proteins, lipids and DNA bases. Suchdamage may affect the proper functioning of ce11sand con tribu te to the development of cancer andof cardiovascular and other degenerative disease.Oxidative pro cesses are also a necessary part of

essential biological functions in ce11s, however,

including those involved in intrace11ular signaltransduction and control of ce11 proliferation orapoptosis. Thus, the health of an organism

depends on a balance between oxidants andantioxidants.

The abilty of carotenoids to act as antioxidantsin vitro is we11 established; what is currently ofgreat interest is whether they also behave

as antioxidants in vivo, e.g. in low-density

lipoproteins and in the membranes of ce11s sus-

General Remarks

ceptible to carcinogenesis. Other possible anti-carcinogenic effects of carotenoids could derivefrom their influence on immune functioning orinduction/suppression of enzymes involved inxenobiotie detoxification, su ch as cytochrome

P450, and the postulated growth regulationmediated by gap-junctional communication.Some carotenoids are metabolized to retinol orfurther to other retinoids, and may also inducebiological effects mediated by retinoic acidreceptors.

Issues in research on carotenoids andhuman cancer

A variety of investigative approaches has beenused in the extensive body of research on the

effect of carotenoids on carcinogenesis. Findingsfrom in-vitro systems, animal models, human epi-demiologieal studies and human c1inieal trials arediscussed in Sections 4 and 6. Application of a11 of

the disciplines involved in such studies wil beneeded to understand the possible effects ofcarotenoids in the prevention of cancer. Whenfindings from various research fields point in acommon direction, inference is relatively straight-forward. Associations seen in observational epi-demiological studies may be c1arified by c1inicaltrials. The underlying mechanisms may be betterunderstood from studies in animal models and invitro.

The diverse data may, however, be difficult tointegrate, as each research domain suggests

different mechanisms or even different effects.Su ch discrepancies emphasize the limitations ofeach of the research methods, whieh may notprovide relevant information for cancer preven-

tion as currently conducted. Studies in vitro and inanimal models rely on biological models of car-cinogenesis, which may not correspond to thehuman situation with regard to cancer. Forexample, in-vitro systems isolated from tissue andblood proteins and protected from counter-regu-

latory physiologieal responses may suggest mech-anisms that do not occur in humans. Animal

models of cancer often involve specifie carcino-gens and a dosage schedule that is much morerapid than those to whieh humans are usua11y

exposed. ln addition, the physiology of organs

can differ between humans and animaIs, further

complicating extrapolation of results.Many species have been used to study the rela-

tionship between exposure to carotenoids and

cancer. Each species has advantages and limita-tions with regard to the human situation. Forexample, the absorption and transport ofcarotenoids in rodents differ markedly from thosein humans, although the metabolism and func-tions of carotenoids in rodents and humans aresimilar. These issues must be considered carefullywhen extrapolating the results obtained in anygiven species to humans.

Epidemiology is also subject to several poten-tial limitations. Estimates of dietary intake ofcarotenoids derived from questionnaires, foodcomposition tables or biomarkers of nutritionalexposure may be of only limited validity becausequestionnaires tend to reflect recent intake ratherthan intake at the time of cancer induction.

Further, unti recently, dietary databases did not

contain data on the content of specifie carotenesin food items. Confounding is a second major

issue in nutrition al epidemiology. Specific food

constituents tend to be c1ustered by food group;

e.g. vegetables and fruits are the main sources notonly of carotenoids but also of other substances

that have been postulated to protect against can-cer. Sin ce other nutrients ffay have cancer-protec-tive properties, they themselves may underlie anapparent inverse association between carotenoidintake and cancer risk. Conceivably, an apparentprotective effect of a high intake of carotenoidscould be due to associated dietary factors, such aslow fat intake and non-dietary lifestyle factorssu ch as exercise, avoidance of cigarette smokingand leanness. Associations with intermediate end-points, increasingly used in nutritional epidemiol-ogy, may not para11el those with cancer itself.Indeed, it has been diffieult even to define the cri-teria whereby an end-point is a valid intermediatemarker of cancer.

Randomized intervention trials avoid theproblems of measuring exposure and of con-

founding which bedevil observational studies;however, the evidence from clinical trials isrestricted to the doses given, the duration and thestage of the natural history of the cancer understudy. When possible chemopreventive agentssuch as the carotenoids are being tested, the agentbeing administered and the way in which it is

17


administered may also differ in important waysfrom the situation in whieh carotenoids are

obtained from foods. Thus, observational studieson nutrient-disease relationships and interven-tion studies in which an isolated nutrient is givendo not necessarily examine the same questions.

Objectives of this handbook

The Working Group has critically evaluated workrelevant to the potential role of carotenoids andcancer prevention carried out during the last twodecades. An enormous volume of experimentalresearch on the anti-cancer effects of ß-caroteneand other dietary and non-dietary carotenoids

resulted from the enthusiasm that fo11owed the

original observation of an inverse association

between dietary ß-carotene and cancer risk. Thehypothesis did not only affect experimental stud-ies but resulted in expansion of interest in thewhole field of carotenoids. It stimulated improve-ments in analytical techniques to measure thesma11 concentrations of carotenoids in serum, and

this in turn facilitated more detailed research onmetabolism, genetie variation and the kineties ofcarotenoid turnover in tissues. This handbookalso describes and evaluates the outcomes of thelarge intervention trials that were conducted inthe wake of the enthusiasm for the hypothesis

and were made possible by the availabilty ofß-carotene supplements. The outcome of thatwork is evaluated, and the direction that researchis currently ta king and areas where more researchis stil needed are defined.

ReferencesBauernfeind, ie., ed. (1981) Carotenoids as

Colorants and Vitamin A Precursors. Technologicaland Nutritional Applications, New York,Academie Press

Britton, G., ed. (1983) The Biochemistr of NaturalPigments, Cambridge, Cambridge UniversityPress

Britton, G., ed. (1991) Proceedings of the 9thInternational Symposium on Carotenoids,Kyoto, Japan, 1990. Pure AppL. Chem., 63,

1-176

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Britton, G., ed. (1994) Proceedings of the 10thInternational Symposium on Carotenoids,Trondheim, Norway, 1993. Pure Appl. Chem.,66, 931-1076

Britton, G., ed. (1997) Proceedings of the 11thInternational Symposium on Carotenoids,Leiden, . The Netherlands, 1996. Pure AppL.Chem., 69, 2027-2173

Britton, G. & Goodwin, T.W., eds (1982)

Carotenoid Chemistr and Biochemistr, Oxford,Pergamon Press

Britton, G., Liaaen-Jensen, S. & Pfander, H., eds(1995a) Carotenoids, VoL. lA, Isolation andAnalysis, Basel, Birkhäuser Verlag

Britton, G., Liaaen-Jensen, S. & Pfander, H., eds(1995b) Carotenoids, VoL. 1B, Spectroscopy, Basel,

Birkhäuser Verlag

Davies, B.H. & Rau, W., eds (1985) Proceedings of

the 7th International Symposium on Caro-tenoids, Munich, Germany, 1984. Pure AppL.

Chem., 57, 639-821Goodwin, T.W., ed. (1979) Proeeedings of the 5th

International Symposium on Carotenoids, Madison,USA, 1978. Pure AppL. Chem., 51, 435-657

Goodwin, T.W. (1980) Carotenoids in higherplants. ln: Goodwin, T.W., ed., The Biochemistrof the Carotenoids, 2nd Ed, VoL. 1, Plants,London, Chapman & Hall, pp. 143-203

Goodwin, T.W., ed. (1984) The Biochemistr of theCarotenoids, 2nd Ed., VoL. 2, AnimaIs, London,Chapman & Hall

Goodwin, T.W., ed. (1988) Plant Pigments,

London, Academie PressIsler, O., Ruegg, R. & Schwieter, U. (1967) Caro-

tenoids as food colourants. Pure AppL. Chem.,

14, 245-263Isler, O., Gutmann, H. & Solms U., eds (1971)

Carotenoids, Basel, Birkhäuser VerlagKarrer, P. &Jucker, E., eds (1948) Carotinoide, Basel,

Birkhäuser Verlag

Kelloff, G.j. & Boone, e.W., eds (1994) Cancerchemopreventive agents: Drug developmentstatus and future prospects. J. CeU. Biochem.,

Suppl. 20, 110-140Khachik, E, Spangler, e.j. Smith, J.c., Canfield,

L.M., Steck, A. & Pfander, H. (1997)Identification, quantification, and relative con-centrations of earotenoids and their metabo-

lites in hum an milk and serum. Anal. Chem.,

69, 1873-1881

General Remarks

Krinsky, N.l. (1994) The biological properties ofcarotenoids. Pure AppL. Chem.i 66, 1003-1010

Krinsky, N.l., Mathews-Roth, M.M. & Taylor, R.E,eds (1989) Carotenoids: Chemistr and BiologyiNew Yorki Plenum Press

Kun, D. & Pfander, H. (1995) Appendix: List ofnew carotenoids. ln: Britton, G., Liaaen-Jensen,S. & Pfander, H., eds, Carotenoids, Vol. lA,

Isolation and Analysis, Basel, Birkhäuser Verlag,Basel, pp. 295-317

Peto, R., Don, R., Buckley, J.D. & Sporn, M.B.(1981) Can dietary beta-carotene materianyreduce human cancer rates? Naturei 290,201-208

Pfander, H., Gerspacher, M., Rychener, M. &Schwabe, R. (1987) Key to Carotenoids by OttoStraub, 2nd Ed., Basel, Birkhäuser Verlag

Underwood, B.A. & Arthur, P. (1996) The contri-bution of vitamin A to public health. FASEB J.

10, 1040-1048Weedon, B.C.L., ed. (1976) Proceedings of the 4th

International Symposium on Carotenoids,Bern, Switzerland, 1975. Pure AppL. Chem., 47,

97-243

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