Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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be prepared in diverse recipes that can provide great

satisfaction to human sensorials. Their unique chem-

ical composition encourages the fight against dia-

betes, alimentary ulcers, tumors, blood cholesterol,

and hypertension. As low-calorie foods, they are diet-

ary items ideal for slimming. With the examples of

mushroom species mentioned above, the chemical

constituents described and their biological properties,

it is clear that mushrooms, in addition to being an

item of food delicacy, can claim value as ‘health con-

ditioner.’ Probably within another decade, more re-

search will further define more quantitatively the

biomedicinal values of mushrooms.

See also: Enzymes: Functions and Characteristics;

Mushrooms and Truffles: Classification and

Morphology; Mycotoxins: Classifications; Occurrence

and Determination; Toxicology; Protein: Food Sources;

Vitamins: Overview

Further Reading

Chang H and Butt P (1986) Pharmacology and Application

of Chinese Materia Medica, pp. 642–663. Singapore:

World Scientific Publishing.

Chang ST and Hayes WA (eds) (1978) The Biology and Culti-

vation of Edible Mushrooms. London: Academic Press.

Chang ST and Quimio TH (eds) (1982) Tropical Mush-

rooms – Biological Nature and Cultivation Methods.

Hong Kong: Chinese University Press.

Chang ST, Buswell JA and Miles PG (eds) (1993) Genetics

and Breeding of Edible Mushrooms. USA: Gordon and

Breach.

Chen AW and Miles PG (1996) Biomedical research

and the application of mushroom nutriceuticals from

Gandoderma lucidum. In: Royse DJ (ed.) Mushroom

Biology and Mushroom Products, pp. 161–175. Penn-

sylvania: Penn State University.

Chihara G (1990) Lentinan and its related polysaccharides

as host defence potentiator: their application to infec-

tious diseases and cancer. In: Masihi KN and Lange W

(eds) Immunotherapeutic Prospects of Infectious Dis-

eases, pp. 9–18. Heidelberg: Springer Verlag.

Mizuno T (1995) Bioactive biomolecules of mushrooms:

food function and medicinal effect of mushroom fungi.

In: Teranishi R and Honstein I (eds) Food Reviews Inter-

national, pp. 7–21. USA: Marcel Dekker.

Rajarathnam S and Zakia Bano (1988) Pleurotus mush-

rooms: part II. Chemical composition, nutritional value

post-harvest physiology, preservation and role as human

food. In: Clydesdale FM (ed.) CRC Critical Reviews in

Food Science and Nutrition, Vol 27(2), 87–158. Boca

Raton, FL: CRC Press.

Rajarathnam S and Zakia Bano (1991) Biological utiliza-

tion of edible fruiting fungi. In: Arora DK, Mukerji KG

and Marth EH (eds) Handbook of Applied Mycology:

Foods and Feeds, vol. 3, pp. 241–292. New York:

Marcel Dekker.

Rajarathnam S, Shashirekha MN and Zakia Bano (1992)

Biopotentialities of the Basidiomacromycetes. In:

Advances in Applied Microbiology, Vol. 37. USA:

Academic Press.

Rajarathnam S, Shashirekha MN and Zakia Bano (1998)

Biodegradative and biosynthetic capacities of mush-

rooms: present and future strategies. In: Stewart GF

and Russell I (eds) Critical Reviews in Biotechnology,

vol. 18, pp. 91–236. Boca Rator FL: CRC Press.

Rajarathnam S, Zakia Bano and Shashirekha MN (1999)

Mushrooms: an item of food delicacy or health condi-

tioner. In: Rajak RC (ed.) Microbial Biotechnology

for Sustainable Development and Productivity, pp.

140–151. Jodhpur (India), Scientific Publishers.

tbl0010 Table 10 Mushroom poisoning

Mushroom species Compounds Nature of compound Symptoms

Amanita phalloides Phallotoxin

Cyclopeptide FatalAmanita phalloides Amatoxin

)

Amanita virosa Virotoxin

Gyromitra esculenta Gyromitrin Affects autonomic nervous system

Coprinus atramentarius Coprine Amino acid Drunken sickness

Clitocybe acromelalga Clitidine

Novel amino acids NeurotoxinAcromelic acid

)

Clithioneine

Amanita muscaria Muscarine Amino acid Affects parasympathetic, cholinergic nervous system

Inocybe patouillardii

)

Clitocybe acromelalga

Amanita pantherina Mycoatropine

;

Alkaloids Psychotropic poisoning

Amanita muscaria

)

Amanita regalis

Psilocybe species Psilocybin

Panaeolus species

)

Panaeolina species

Stropharia species

Cortinarius orellanus Orellanin Polypeptide Fatal

Ramaria formosa Emodin Laxative effects

4054 MUSHROOMS AND TRUFFLES/Use of Wild Mushrooms

Mussels See Shellfish: Characteristics of Crustacea; Commercially Important Crustacea; Characteristics of

Molluscs; Commercially Important Molluscs; Contamination and Spoilage of Molluscs and Crustaceans;

Aquaculture of Commercially Important Molluscs and Crustaceans

MUSTARD AND CONDIMENT PRODUCTS

J S Hemingway, Brundall, UK

Introduction

0001 The term ‘mustard’ is believed to be derived from the

use of seeds as condiment, the sweet ‘must’ of old wine

being mixed with crushed seeds into a paste –‘hot

must’ or ‘mustum ardens’. Mustard seed has been

known as a spice since the earliest recorded times; it

is described in Sanksrit and Sumerian texts back to

3000 bc, Egyptian to 2000 bc, and Chinese before

1000 bc; seed fragments exist from the Indus Valley

civilizations around 2000 bc and Egyptian tombs at

Thebes of the same period. Records of mustard being

traded as a manufactured condiment extend back to

the days of the Avignon popes and Shakespeare.

0002 In the present day, it is by far the largest volume

spice in international trade, totalling over 170 000

kg 10

per year. Other particular features which

make it unusual as a spice crop are that, for condi-

ment purposes, the entire world supply is grown in

temperate regions, as the crop needs long days; hence,

its production is virtually all concentrated in the

Northern Hemisphere, where it has necessarily been

developed by plant breeding to suit full mechaniza-

tion of every operation of its cropping and the bulk

handling of mustard seed into trade (a further un-

usual aspect of a spice material).

Cropping

0003 For use as spice, the predominant production is in

North America, in the Canadian prairie provinces

of Saskatchewan, Alberta, and Manitoba, and just

southwards into Montana and North Dakota,

USA. Annual crops of the order of 200 000 m 10

in those areas produce total quantities in excess of

150 000 kg 10

of export value US$40 10

. Other

important production centers are the Hungarian

plain and the East Anglian counties of the UK, also

Poland, the Czech Republic, and Slovakia. Smaller-

scale cropping occurs in Argentina and Australasia

for local use, but world manufacture effectively

depends on the single annual crop of the Northern

Hemisphere. The main manufacturing usage is in the

USA, Canada, France, Germany, Japan, and the UK.

0004Botanically, two principal species of the Cruciferae

family are involved: ‘White’ (or ‘yellow’) mustard,

Sinapis alba; ‘brown’ and ‘oriental’ mustards, Bras-

sica juncea. In condiments, S. alba contributes a hot

(in terms of mouth feel) nonvolatile principle and

B. juncea a pungent (i.e., olfactory) volatile principle,

each from the hydrolysis of their respective glyco-

sides. Until the 1950s, seed of B. nigra was almost

the sole source of pungency, but with perhaps unpar-

alleled rapidity in any industry, let alone in an area as

traditional as the spice trade, B. juncea replaced

B. nigra over a single decade. This change met the

requirement for full mechanical harvesting, as the

older type needed to be cut by hand.

0005Mustard species are also of great importance as

oilseed crops, particularly B. juncea in the Indian

subcontinent, China, and the Sarepta area of the

southern Ukraine. In recent years, there has de-

veloped an increasing export trade from Canada to

the Indian subcontinent, principally Bangladesh, of

the order of 50 000 kg 10

per year. In all these

countries, the growth is closely similar to that of

rapeseeed. Mustards also find wide usage as salads,

green manure, and fodder crops and as leaf and stem

vegetables in the Far East.

0006The principal centers of plant breeding and agro-

nomic research on mustard crops have long been

Norwich, UK, and Saskatoon, Canada, where succes-

sions of varieties of improved habit, yield, and quality

aspects (e.g., seed size and shape, lower oil contents,

and higher essential principles, kernel and seed-coat

mucilage contents, all being features of manufactur-

ing importance) have been produced.

0007Mustard crops for condiment are all grown as

spring-sown annuals. They show three marked periods

of growth:

0008A rapid vegetative development of leaves and the

branching framework.

MUSTARD AND CONDIMENT PRODUCTS 4055

2.0009 Three to four weeks of characteristic intensive

yellow flowering during June.

0010 Six to eight weeks of seed filling and ripening.

The crops reach a full height of 1.5–2.0 m, with each

plant branching to bear several racemes of pods, all in a

clear ‘podding zone’ that forms the upper part of the

crop canopy. The main differences between the two

principal species are visible during growth: S. alba

foliageandstemarebrightgreenincolor,withsurface

hairs on the hollow stems; B. juncea is more glaucous,

with smooth, waxy, pith-filled stems. The pods of S. alba

are 2–3 cm in length, with a pronounced ‘beak’ at the tip

and with hairy rough surfaces, containing up to eight

seeds, whereas pods of B. juncea areupto5cminlength,

smoothandtubular,withupto20seeds.

0011 Mustard crops are both easy and reliable to grow,

and perform well on land of reasonable fertility, on

which they need only nitrogen as an added fertilizer.

Brassica juncea has considerable resistance to

drought and can crop with limited rainfall but

S. alba flourishes best with more regular precipita-

tion. As neither species is affected by plant diseases to

economic levels, and both suffer to only a limited

degree from insect pests, they do not require repeated

treatments from pesticides. The requirement for clean

seed necessitates adequate weed control practices, for

which well-proven and simple systems are available.

The crops can tolerate reasonable extremes of

weather, such as spring frosts, hail, or strong winds,

without serious losses, standing well on stiff, erect

stems. In cropping systems in which rotational prac-

tice is important, mustards form an excellent ‘break

crop’ between cereals, especially as a preparation for

wheat. As mentioned earlier, modern varieties have

been developed that suit farming machinery used for

other crops, and do not clash with them in the timing

of harvesting, so that there is no need for special

investment or any hand-labor operations.

0012 Mustards are prolific yielders and can increase over

2000-fold in a season in optimum conditions. Current

mean yields in UK cropping are of the order of 900–

1000 kg of seed per acre (2220–2500 kg per m 10

ranging up to 1500 kg (3700 kg); in the North

American prairie, expectations are lower, at some

400–700 kg per acre (990–1730 kg per m 10

) for

B. juncea and 275–550 kg per acre (680–1360 kg

per m 10

) for S. alba, with substantial variation

according to soil moisture status. Yields in eastern

European countries are intermediate.

Seed Handling and Storage

0013 After harvest in Canada, the seed is usually below

10.5% moisture, at which level, it is safe for long-

term storage; indeed, if thoroughly dry and clean,

there is never any risk of infestation of mustard by

stored product pests. In other countries, the seed may

need to be dried gently to reduce it to safe storage

levels; in such situations, it is important to act

quickly, as the seed can deteriorate very rapidly, and

to restrict the temperature of the drying airflow so

that the seed temperature never exceeds 52



C; other-

wise, damage to its enzymes will result.

0014Cleaning operations are quite straightforward with

mustards’ round seeds of uniform size. Indented

cylinders, perforated screens, and spiral separators

are all effective, and surface dust can be removed by

aspirated brush-conveyors. The presence of green or

weathered seeds and of specific inseparable weed

seeds is the main cause of downgrading within the

official grades of the Canadian Board of Grain Com-

missioners, under which system the preponderance of

world trading is operated. Storage is normally in bulk

bins, with pneumatic or mechanical conveying, and

carriage in bulk lorries or rail grain-cars, then bulk

shipping holds. Smaller quantities are shipped in

standard freight containers, occasionally in 100-lb

(45-kg) bags if the customer so requires.

Anatomy of the Seed

0015The seed of S. alba is larger than that of B. juncea,

averaging 148 000 seeds per kg, mean diameter

2.22 mm, against 408 000 and 1.63 mm. Features of

the fine structure of the outside of the testa and the

degree and type of surface reticulation and pitting are

characteristic and of importance in differentiating the

seed from that of other brassicas, e.g., rapeseed.

0016The seed coat consists of four or five layers of cells,

including an outer layer of large cells and an inner

palisade layer. Inside this lies the true endosperm,

with an outer layer of cells that possess thickened

walls and contain fat globules and small aleurone

grains, and an inner, very narrow layer of compressed

cells with no evident structure. The center of the seed

is filled with two hemispherical cotyledons with the

embryo lying between. There is only a small hilum

and micropyle. The testa of S. alba contains a large

number of mucilage glands opening to the surface;

B. juncea has many fewer.

0017In B. juncea, the two distinct seed types are ‘brown’

and ‘oriental.’ The former has a darkly colored

endosperm of more substantial cells, but such color

is absent from the latter, leaving a golden-yellow

shade. In this ‘oriental’ type, the testa occupies only

12–15% of the seed by weight, against 19–22% in

brown; hence, the oriental type has a higher kernel

content, of substantial economic value, provided

that the testa is sufficiently robust to stand up to

mechanical handling without rupture.

4056 MUSTARD AND CONDIMENT PRODUCTS

Seed Composition

0018 General specifications for mustard seed and standard

analytical methods are detailed under ISO 1237–

1981 (E) UDS 633.844.004.1. The gross composition

is shown in Table 1, the variation in content of

common fatty acids in the oils of S. alba and

B. juncea is shown in Table 2, and the principal

amino acids in the protein fractions of these two

species are shown in Table 3.(See Amino Acids:

Properties and Occurrence; Fatty Acids: Properties.)

0019 The organoleptic properties of mustards result

from their contents of the essential mustard oils,

which are colorless, irritating liquids; that of S. alba

has little aroma, as it is nonvolatile, but a hot taste,

whereas B. juncea gives a volatile oil with a pungent

aroma. Within the seed, they exist as glycosides

within intact cells of the cotyledons, and it is only

on the rupture of the cells and in the presence of

adequate moisture that the enzyme myrosinase can

catalyze the reactions to produce the isothiocyanate

principles.

0020 In S. alba, the storage glycoside is sinalbin, which

on breakdown gives p-hydroxybenzyl isothiocyanate;

in B. juncea, the glycoside sinigin gives allyl-isothio-

cyanate. (Certain strains of B. juncea, all originating

in the Indian subcontinent, give rise to a mixture of

allyl and 3-butenyl isothiocyanates, the latter being

an undesirable flavor, at least to the Western palate.)

Processing and Food Uses

0021The availability of these distinct flavor effects, heat

and pungency, from the two mustard species allows

the manufacture of a wide range of condiment prod-

ucts and food ingredients. A variety of processing

techniques are used:

0022wet milling of whole seeds;

0023wet milling with husk separation;

0024dry milling with husk separation;

0025dry grinding of whole seeds;

0026dry grinding after oil pressing.

A French (e.g., ‘Dijon’) mustard uses only brown-

colored B. juncea, which, because it is wet-milled,

loses much of its initial pungency. The extent of

removal of the husk fragments by screening after

milling governs the final color of the paste. Such

mustards are commonly blended with wines and

vinegars, and whole or split mustard grains, herbs,

or other spices may be added. (See Milling: Charac-

teristics of Milled Products.)

0027In contrast, an American ‘cream-salad’ mustard is

made by wet milling only S. alba seed with water in a

colloid-type mill. The viscosity of the product is

greatly aided by the presence of mucilage in the seed

coats. Turmeric is usually added to overlay the nat-

ural pale color to a rich yellow. The product has a

sweet, tangy flavor and can be used in large amounts,

almost as a ketchup.

0028Traditional German mustard is similarly made

from S. alba alone, and is thus nonpungent. The

texture of the paste can be varied by the degree of

grinding, and admixtures of herbs, spices, and water

or vinegar are used according to the product.

0029In contrast, traditional English mustard is a blend

of the dry-milled flours of both S. alba and B. juncea

and is both hot and pungent. This product is sold as a

tbl0001 Table 1 Gross composition of mustard seed

Percentage of seed(dry weight)

Neutral oil 24–45 (Sinapis alba 25–30, Brassica juncea 35–45)

Polar lipids 6–12

Protein 20–30

Carbohydrate 12–18

Glycoside 1–3

Phytins 2–3

Water

8–12

Normal commercial mustard seed as traded on an international basis.

Source: Vose JS (1972) A Review of the Chemistry and Enzymology of

Mustard Seeds. Montreal: Reckitt & Colman (Canada) Ltd.

tbl0002 Table 2 Ranges of variation in content of common fatty acids in

the oils within each species

Percentage of oil

Sinapis alba Brassica juncea

Palmitic 2–3 2–4

Oleic 16–28 7–22

Linoleic 7–10 12–24

Linolenic 9–12 10–15

Eicosenoic 6–11 6–14

Erucic 33–51 18–49

Variations under genetic control have been identified but are not presently

utilized as they appear to affect product quality.

Source: Vose JS (1972) A Review of the Chemistry and Enzymology of

Mustard Seeds. Montreal: Reckitt & Colman (Canada) Ltd.

tbl0003Table 3 Principal amino acids in the protein fractions of Sinapis

alba and Brassica juncea seeds

Amino acid content (mg per gram of nitrogen)

Sinapis alba Brassica juncea

Lysine 362 335

Methionine 97 104

Cystine 124 159

Isoleucine 207 236

Leucine 412 395

Phenylalanine 233 240

Tyrosine 206 167

Threonine 171 251

MUSTARD AND CONDIMENT PRODUCTS 4057

dry fine powder, with no reaction until blended with

water for the table, when the characteristic flavors

develop very rapidly. However, as the pungency is

volatile, and the hotness of the essential principle

from S. alba breaks down within a day, the made

condiment loses strength quite quickly. Its strength

can only be maintained in a bottled paste (‘hot Eng-

lish mustard’) by the inclusion of fruit acids in its

manufacture from milled flours.

0030 The dry milling process to produce flour is broadly

similar to the milling of wheat, with the difference

that mustard seeds are very small and have 30–40%

of oil in their cells. This needs great care, as the oil

causes enormous problems if released by excess pres-

sure or temperature. Sinapis alba and B. juncea are

milled separately as they have distinct seed sizes. The

seeds are first thoroughly cleaned, graded for size and

shape, blended, and then dried to a very low level of

moisture content. Their husks are then moistened (so

that the kernel is dry and friable but the husk slightly

elastic and less brittle) and metered steadily on to the

‘break rolls,’ of fluted surface, on which the seed is

nipped to crack open the husk. This is separated from

the kernels by sieving and currents of air in the ‘puri-

fiers’: a remarkably clean separation can be achieved

with little kernel adhering to the husks, and minimal

presence of fine husk particles, which would show as

specks in the flours milled from the kernel fractions.

0031 The kernels then pass on to a series of smooth

‘reduction rolls,’ which progressively reduce them to

a powder. After each pass through a roll, the fine

particles are sieved off to ultrafine by the use of

swirling sieves, their frames covered with silk bolting

cloths of differing meshes; the coarse particles return

to a further reduction roll. The resultant flours – the

S. alba a deep creamy color and B. juncea a rich

golden-yellow shade – are blended in varying propor-

tions according to the product desired. Some products

may be blended with stabilized wheat flour in small

proportions to attain a standard flavor strength, and

coloring may be enriched by the curcumin present in

ground turmeric.

0032 In Japan, the production of mustard powder is

usually by the fine grinding and sifting of whole

seeds, but after the extraction of some of the seed-

oil content by pressing. The Japanese taste, like the

Anglo-Saxon, is for very strongly flavored mustard

products.

0033 The grinding of whole mustard seeds is usually to

obtain a coarser, granular product for use as an ingre-

dient in sauces, pickles, or chutneys. An increasing

usage of ground S. alba is in the North American meat

products industry, where it adds flavor and protein,

and its mucilage helps to absorb moisture. Some

S. alba seed is now being heat-treated to inactivate

its myrosinase, then pin-milled to coarse particles of

no strength of flavor, thus allowing a higher and

wider usage in such meat products.

0034The byproducts of dry milling are principally the

husks. That of S. alba can be ground into a coarse

meal used to impart viscosity to paste mustards or

sauce products, but the husk of B. juncea has little

value.

0035Beyond the ingredient usages of mustard already

mentioned, based on its range of flavors and applica-

tions of the mucilage content of S. alba, the major

usage is the inclusion of mustard flour – predomin-

antly of S. alba – in the formulation of salad creams

and mayonnaises. It provides some flavor and natural

color, has natural bacteriostatic and antioxidant

properties, and, most importantly, acts as an emulsi-

fying agent, helping to bind the oil and fat phases of

the recipe. This was perhaps a fundamental virtue

within the original popularity of mustard as a spice,

technically to ‘enhance flavor’–or disguise the taste

of salted meats or fish – but also to ‘aid digestion’ by

its use with fatty meats or oily fish, attributes praised

by early medical writers or gourmands. (See Antioxi-

dants: Natural Antioxidants; Emulsifiers: Uses in Pro-

cessed Foods.)

See also: Amino Acids: Properties and Occurrence;

Antioxidants: Natural Antioxidants; Emulsifiers: Uses in

Processed Foods; Fatty Acids: Properties; Milling:

Characteristics of Milled Products

Further Reading

Duffus C and Slaughter C (1980) Seeds and their Uses.

Chichester, UK: Wiley.

Hemingway JS (1995) Mustards. In: Smartt J and Sim-

monds NW (eds) Evolution of Crop Plants. London:

Longman.

Hemingway JS (1995) The mustard species: condiments

and food ingredients, and potential as oilseed crops. In:

Kimber DS and McGregor DI (eds) Brassica Oilseeds,

Production and Utilisation. Wallingford, UK: CAB Inter-

national.

Man R and Weir R (1988) The Compleat Mustard. London:

Constable.

Rosengarten F (1969) The Book of Spices. Wynnewood,

PA: Livingston.

Tsunoda S, Hirata K and Gomez-Campo C (1980) Brassica

Crops and Wild Allies. Tokyo: Japan Scientific Societies

Press.

Vaughan JG and Hemingway JS (1959) The utilisation of

mustards. Economic Botany 13(3): 196–204.

Vose JS (1972) A Review of the Chemistry and Enzymology

of Mustard Seeds. Montreal: Reckitt & Colman

(Canada).

4058 MUSTARD AND CONDIMENT PRODUCTS

MUTAGENS

T J Schrader, Sir Frederick Banting Research Centre,

Health Canada, Ottawa, Ontario, Canada

Background

0001 Mutagens are chemical compounds or forms of radi-

ation (such as ultraviolet (UV) light or X-rays) that

cause irreversible and heritable changes (mutations)

in the cellular genetic material, deoxyribonucleic acid

(DNA). Mutagenic lesions persist when they escape

detection by protective cellular DNA repair mechan-

isms, when mistakes occur in the repair process, or

when repair mechanisms are overwhelmed by exten-

sive damage. Upon subsequent cellular replication,

these mutations become fixed in the genome and are

inherited by all daughter cells. In this way, muta-

genesis becomes a cumulative process, stretching

over the lifetime of an organism.

0002 The biological consequences of a mutation depend

upon many critical factors such as the target loci, size

of the mutation, timing during the cell cycle, and

compounding effects of preexisting mutations. Thus,

a mutagenic event occurring in a nonfunctional area

of DNA will have no effect (silent mutation), whereas

a similar change in an actively transcribed region may

profoundly affect gene expression and phenotype or

even lead to cell death (lethal mutation). The influ-

ence of mutations in human health is underscored by

several human disease states caused by mutations that

disrupt regulatory regions or gene coding sequences,

resulting in altered gene expression and protein func-

tion. For example, mutations in genes that promote

or inhibit growth and cellular replication (protoonco-

genes and tumor suppressor genes, respectively) or

code for components of DNA repair pathways are

important contributors to the multistage develop-

ment of cancer. In addition, the accumulation of

mutations over time, leading to gradually less effi-

cient cellular repair capabilities, has been linked to

the aging process and associated degenerative dis-

eases. While the health of a particular individual can

be affected by mutations in somatic cells, mutagenic

events in germ cells (germline mutations) lead to the

transmission of genetic diseases to subsequent gener-

ations. Genetic predispositions to certain breast and

colon cancers, cystic fibrosis, and Huntington’s

disease are included among many examples of such

inheritable diseases.

0003 Although many dietary and environmental agents

have been classified as mutagens, cells are constantly

subjected to a barrage of spontaneous DNA damage.

The spontaneous hydrolysis of DNA bases from the

sugar–phosphate backbone, constant exposure of cel-

lular DNA to oxygen-derived free radicals and low

rates of miscopying during DNA replication all con-

tribute towards the mutational burden. In addition to

these sources, significant exposure to mutagenic com-

pounds can occur through food and water, as well

as through environmental and occupational sources.

Many natural constituents of food are mutagenic and

are produced by plants as defense agents. However,

additional foodborne mutagens can be present as

residues of compounds used during food production

or leached from packaging materials. Mutagenic

compounds can also be produced during food

cooking and preparation. Foods also contain many

compounds that can modulate the activity of muta-

gens. In order to minimize exposure to mutagens, the

majority of developed countries have instituted regu-

latory protocols governing the introduction of new

foods and food-associated chemicals before they are

accepted into the marketplace. With advances in the

understanding of mutagenic processes, existing foods

and food contact items may also be reassessed for

acceptability.

Molecular Mechanisms of Mutagenesis

DNA Damage

0004DNA is a double-helical structure composed of paired

heterocyclic nitrogenous purine and pyrimidine bases

attached to a backbone of deoxyribose and phos-

phoric acid. The purine bases (guanine and adenine)

and pyrimidine bases (cytosine and thymine) form

specific base pairs (guanine/cytosine, adenine/thy-

mine) stabilized by hydrogen bonding. The specificity

of base-pairing and sequence of bases along the DNA

double helix forms the foundation of the genetic code,

and any change can give rise to mutations, expressed

as alterations in gene expression or in protein struc-

ture and function.

0005Spontaneous damage Instability of various parts of

the DNA molecule in aqueous solution leads to spon-

taneous DNA damage and mutations. In addition

to base deamination, the bonds between purines or

pyrimidines and deoxyribose can spontaneously

hydrolyze, creating apurinic/apyrimidinic (AP) sites,

leaving the sugar–phosphate backbone susceptible to

strand breakage. Also, the bases can exist in a number

of slightly different (tautomeric) forms, several of

MUTAGENS 4059

which lead to mispairing because of altered hydrogen

bonding characteristics.

0006 Chemical adducts DNA also comes under assault

from a number of dietary and environmental factors

that interact with several relatively reactive sites

within the structure of DNA, leading to mutagenic

events. Many chemical mutagens are electrophiles

that are reactive, electron deficient species able to

form covalent adducts with nucleophilic sites within

DNA. Binding of chemical adducts to the purine/

pyrimidine bases can stabilize alternative tautomeric

structures or otherwise alter their structural and

hydrogen bonding characteristics. These modifica-

tions not only alter the base pair coding properties

and cause mistakes to be made during replication and

transcription, but also can lead to increased base

hydrolysis and AP site formation. In addition, some

chemical agents produce inter- and intrastrand cross-

linking of DNA, which prevents strand separation

and causes particular difficulties for replication,

transcription, and repair processes.

0007 Oxidative damage Other mutagens can act through

the induction of oxidative stress and cause oxygen or

nitrogen radical formation. Radicals are highly react-

ive compounds that contain unpaired electrons and

include hydroperoxides, hydroxyl, and superoxide

radicals. Transition metal ions such as iron and

copper can catalyze the formation of free radicals

(Fenton reaction). The interaction of free radicals with

DNA results in single- and double-strand breaks,

hydroxylated derivatives of bases and several other

lesions.

0008 DNA intercalation Finally, some mutagens do not

actually react with DNA but modify its structure by

intercalating between the complementary strands of

DNA, disrupting hydrogen bonding between base

pairs. The presence of such compounds causes mis-

reading during replication and transcription, leading

to the generation of mutations.

Metabolic Activation

0009 Some mutagenic compounds (direct-acting) are able

to interact with DNA without any further modifica-

tions; however, many other compounds (indirect-

acting mutagens), become mutagenic as unwanted

byproducts of transformations carried out by cellular

detoxification pathways. Several hydroxylation

(phase I) and conjugation (phase II) pathways are

available for detoxification to increase the water solu-

bility of chemical compounds and aid in their excre-

tion. Although the enzyme systems are generally most

highly expressed in the liver, other tissue types main-

tain these capabilities to varying degrees. The phase I

reactions are carried out by the cytochrome P450

enzyme system, which consists of a large family of

related enzymes in which each isozyme shows specifi-

city for particular chemical structures. During phase

II reactions, more polar groups can be added by

glutathione-S-transferase, glucuronide transferase,

microsomal epoxide hydrase, or acetyltransferase.

Generally, phase II reactions inactivate mutagens,

but instances of the opposite outcome have been

found. Several other enzymes, including flavin mono-

oxygenase and prostaglandin H synthetase, are also

known to activate mutagens.

DNA Repair

0010Several mechanisms have evolved to remove lesions in

DNA and to restore the structural and coding proper-

ties of the base sequence in the damaged region.

0011Direct Reversal Direct reversal of damage is per-

haps the simplest process, whereby a single gene

product can repair a particular form of damage. In

mammalian cells, mutagenic O

-methyguanine resi-

dues are removed by a damage-reversal mechanism in

which the O

-alkyl group is removed by an O

methylguanine transferase in a ‘suicide reaction,’

resulting in inactivation of the enzyme. A second

form of damage reversal is observed in the repair of

simple strand breaks, which can be repaired by liga-

tion in a process catalyzed by DNA ligase. Finally, a

DNA purine insertase activity has been described by

which apurinic sites in DNA are repaired by the direct

insertion of purines.

0012Excision Repair The major and most versatile

mechanism used by mammalian cells to remove

altered bases or nucleotides within DNA is known

as excision repair. Excision repair proceeds in several

successive steps in a multiple enzyme process. Three

major excision repair pathways using different sets of

enzymes have been described: base excision repair,

nucleotide excision repair and mismatch repair.

Classes of Mutations

0013Base excision repair removes misincorporated bases

such as uracil as well as most methylated base damage.

The majority of base damage is repaired by the re-

placement of a single damaged nucleotide with its

normal counterpart, but base excision repair can

also result in the synthesis of two to 10 nucleotide

‘repair patches.’ Initially, the damaged base is recog-

nized and removed by a damage-specific glycosylase

leaving an AP site in the DNA. For single nucleotide

repair, the deoxyribose-phosphate residue in the DNA

backbone is removed and a replacement nucleotide

4060 MUTAGENS

inserted by activities associated with DNA polymer-

ase b. Longer patches result when either polymerase b

or another repair DNA polymerase synthesizes a

stretch of DNA. In this process, a flap region of single

stranded DNA is displaced and is then degraded by a

deoxyribonuclease activity. Both types of base exci-

sion repair are completed when DNA ligase seals the

final nick remaining in the DNA backbone.

0014 The nucleotide excision repair pathway involves

20–30 gene products and is responsible for removing

damage caused by agents that introduce bulky

adducts into DNA. The initial recognition of DNA

damage by a recognition complex recruits the entire

multicomponent repair enzyme complex (exinu-

clease) to the lesion. The exinuclease nicks the

damaged strand on either side of the damage site,

releasing an oligonucleotide of approximately 30

base pairs in length. After the gap is filled in by a

DNA polymerase using the opposite strand as a tem-

plate, the nick at the end of the repair patch is sealed

by ligase. Nucleotide excision repair has also been

shown to be closely linked with transcription (tran-

scription-coupled repair) by which actively tran-

scribed genes are preferentially repaired compared

to silent regions of the genome. A component of the

nucleotide excision repair complex (TFIID) is shared

with the complex involved in gene transcription and

mediates the recruitment of DNA repair machinery to

the damage site when RNA polymerase stalls at a

lesion.

0015 Mismatch repair is a form of excision repair that

corrects mismatched base pairs resulting from repli-

cation errors and heteroduplexes formed during re-

combination. The interaction of various components

involved in mismatch repair is less understood but

results in repair patches of 100–1000 nucleotides.

Mutations in mismatch repair genes have been linked

to an increased risk for inherited forms of colon

cancer.

0016 Mutations can range in size from single base pair

changes to the loss or duplication of entire chromo-

somes. Many factors, such as the mutagenic agent,

particular region of DNA affected, preexisting

damage, and timing during the cell cycle can influence

the magnitude of a mutation. Mutations can be

classified into four broad groups: extragenic, gene,

chromosomal, and genomic mutations.

0017 Extragenic Mutations Extragenic mutations occur

outside of the gene coding sequence and are usually

silent. However, if the mutation occurs in an important

regulatory sequence, gene expression can be affected.

0018 Gene Mutations Gene mutations occur within the

coding sequence of a gene. Point mutations involve

the substitution of bases, causing either transitions

(the substitution of a purine for purine, or pyrimidine

for pyrimidine) or transversions (the substitution of a

purine for a pyrimidine or vice versa). If such a muta-

tion occurs in the wobble position of a codon, the

chance of a change occurring in the translated protein

structure or function will be much reduced. However,

a single base pair change elsewhere in the sequence

can result in proteins with decreased or modified

functions and can also affect protein synthesis and

degradation. Frameshift mutations occur when bases

are either lost or inserted, thus resulting in a shift of

the reading frame by the transcriptional machinery.

Frameshift mutations can result in the production of

modified or inactive proteins and may be lethal to the

cell. Block mutations occur when large sections of a

gene are deleted, inverted, or duplicated, and can lead

to defective proteins being produced.

0019Chromosomal Mutations Compared with gene mu-

tations, chromosomal mutations represent a class of

mutations that affect larger regions of DNA within

chromosomes and may involve the deletion, trans-

location, duplication, or further amplification of

whole genes. Chromosomal mutations may lead to

either increased or decreased gene expression and can

also result in the abnormal expression of normally

silent parts of the genome.

0020Genomic Mutations Genomic mutations involve

the duplication or loss of entire chromosomes. Such

mutations can have far-reaching effects on gene ex-

pression and survival. Typically, the progression of a

cancer is marked by cells that have developed pro-

gressively altered complements of chromosomes.

Mutagenicity Assays

0021Many assay systems have been developed to examine

mutagenicity and induction of DNA damage (See

Carcinogens: Carcinogenicity Tests). In many in-

stances, mutagenicity assays are used as shorter

term, more economical surrogates for the 2-year rat

carcinogenesis bioassay. Although a high correlation

exists between mutagenicity and carcinogenicity, nei-

ther this correlation nor that between results obtained

with different mutagenicity tests is perfect. Therefore,

test chemicals are generally subjected to a battery of

three tests or more to gain a more balanced assess-

ment. Widely used mutagenicity assays include both

short-term bacterial, fungal, and mammalian cell-

based (in vitro) and animal-based (in vivo) assay

systems. Mutations within a target gene are detected

as a loss of function (forward mutations)orasa

reversal of the effect of a previous mutation (reverse

MUTAGENS 4061

mutations). These assays generally rely upon a change

in phenotype as an endpoint and therefore may miss

some silent mutations. In vitro systems are generally

conducted in the presence and absence of a source of

metabolic activation, which can include rat liver

homogenate (S9 microsomes) or liver cells. The appli-

cation of molecular biology and biotechnology to

many of these systems has led to further refinement

and increased molecular characterization of the mu-

tations induced.

0022 The Ames Salmonella Test and the E. coli trypto-

phan assay are bacterial systems that measure

reversions induced at genes responsible for the syn-

thesis of histidine and tryptophan, respectively.

Growth of the bacteria on minimal plates requires

back mutations at the indicated loci, and therefore,

test chemical mutagenicity is assessed according to

colony formation. The Ames Salmonella Test has

been widely accepted and validated as a bacterial

assay with different strains available that respond to

base pair and frameshift mutagens as well as oxida-

tive damage. Various refinements to the Ames Sal-

monella Test have been developed, including a series

of bacterial strains that are reverted at specific base

pairs, allowing the spectrum of induced mutations to

be assessed, as well as bacteria that express various

mammalian Phase I and Phase II genes to examine the

effects of particular types of activation. Several

targets can be examined for the induction of forward

mutations in mammalian cells, including gene loci

encoding thymidine kinase in L51278Y mouse

lymphoma cells, hypoxanthine guanine phosphoribo-

syl transferase (HGPRT) in Chinese hamster ovary or

V-79 cells and Na

ATPase in V-79 cells. Muta-

genesis at the HGPRT locus has also been examined

in human lymphocytes and fibroblasts. Plasmid

shuttle vectors have also been developed, which can

be exposed to the test chemical, introduced into mam-

malian cells for repair, and then recovered and re-

introduced into an indicator strain of E. coli for

scoring of mutations. If needed, the plasmids can be

recovered again and sequenced.

0023 Germ-line mutations may be assessed in rodents

using the specific locus test, dominant lethal assay,

and heritable translocation test. Transgenic mice

incorporating l phage LacI or LacZ gene sequences

have also been developed, which provide shorter-

term, in vivo mutagenicity assays. DNA isolated

from these animals is incorporated into l-phage

which is used to infect the proper E. coli host,

forming either clear or colored plaques in the bacter-

ial lawn. The transgenic assays allow mutations to be

examined in all somatic tissues as well as germ-line

tissues, and provide a well-characterized target for

sequence analysis.

0024Chromosomal aberrations can be examined both

in vitro using cell lines or in vivo using animal tissues,

generally bone marrow. Stained metaphase chromo-

some spreads are examined microscopically for struc-

tural chromosomal aberrations. A newer variation of

this technique, fluorescent in situ hybridization, uses

fluorescent labeled DNA probes specific for particular

chromosomal sequences to test for aberrations. Another

chromosomal aberration assay is the micronucleus test

using bone marrow and peripheral blood erythrocytes

or lymphocytes. Micronuclei are small particles contain-

ing acentric fragments of chromosomes or entire

chromosomes that are left behind in the cytoplasm

after mutagen treatment. Micronuclei formation may

be enumerated by microscopy or flow cytometry.

0025Sister chromatid exchange uses differential labeling

techniques to measure the crossing over between each

half of a replicated chromosome. The biological sig-

nificance of sister chromatid exchange is not known,

as the phenomenon does not appear to be related to

the mechanisms involved in chromosomal aberration

formation and is not directly related to mutagenesis.

However, most alkylating agents and compounds that

create adducts and single-strand breakage increase

sister chromatid exchange. Therefore, the assay

tends to be used as a complement to mutagenesis

and chromosomal aberration assays.

Food-associated Mutagens

0026The investigation of mutagens in food is an important

component of food-safety research. Mutagens

found in food can be divided into three groups: nat-

urally occurring compounds, those formed by heating

or processing, and additives and contaminants,

including pesticides.

Naturally Occurring Mutagens

0027Natural components of food account for over 99%

of the carcinogenic and toxic chemicals to which

humans are exposed. Most of these natural mutagens

are produced by plants as natural pesticides. Many of

these intrinsic chemicals are almost impossible to

regulate or remove, although guidelines for accept-

able exposure limits of some natural contaminants

such as mycotoxins are enforced.

0028Flavonoids form a major group of natural plant

compounds, and in a survey of mutagenesis using

the Ames Salmonella Test, 33 out of 70 natural and

synthetic flavonoids proved to be mutagenic, produc-

ing both base pair and frameshift mutations. Studies

indicate that only the flavonol (3-hydroxyflavone)

members of this group are mutagenic. Myricetin and

quercetin are direct-acting mutagenic flavonoids,

whereas kaempferol is indirect-acting. Norwogonin

4062 MUTAGENS

and sexangularetin are two other indirect-acting

flavonoids. The pyrrolizidine alkaloids represent

another large group of widespread mutagenic plant

compounds found in some herbal teas and medicines,

and include compounds such as monocrotaline, dehy-

domonocrotaline, retrorsine, and isatidine. Pyrrolizi-

dine alkaloids are indirect-acting mutagens, with

some compounds inducing DNA-strand breakage,

whereas others form DNA and DNA–protein cross-

links. Another class of mutagenic compounds is the

glucosinolates, which are found in cruciferous vege-

tables. Glucosinolates are broken down into isothio-

cyanates or nitriles, including the mutagenic allyl

isothiocyanate glucoside (sinigrin). The mutagen

aquilide A (ptaquiloside) from bracken fern (fiddle-

heads) is a related glycoside and can also be passed

through cows’ milk. Catechol-type phenolics, such as

tannins and caffeic acid and its esters (chlorogenic

and neochlorogenic acids), are more widespread

than other natural pesticides and can induce chromo-

somal damage. Caffeic acid conjugates can be present

in higher plants, with coffee, apple, and lettuce as

major sources in the human diet. A class of chemicals

known as linear furanocoumarins, found in parsley

and celery, has several mutagenic members, includ-

ing psoralen, 5-methoxypsoralen (bergapten), and

8-methoxypsoralen (xanthotoxin). Linear furanocou-

marins intercalate into DNA and form mono- and di-

adducts in the presence of long-wave UV light. A

number of mutagenic hydrazine compounds such as

hydrazinobenzoate are found in 30 species of mush-

rooms. Estragole and safrole are mutagenic alkenyl-

benzenes that are found in a number of spices such as

black pepper, basil, fennel, and nutmeg. Other food-

related mutagens, include ethyl acrylate from pine-

apple, sesamol from sesame seeds, and benzyl acetate

from basil, jasmine tea, and honey.

0029 Mycotoxins are an important source of mutagenic

contamination resulting from mold growth in food-

stuffs. Human exposure can occur through grain

crops, meat products, milk and eggs, nuts and

peanuts, fruits, and juice. Aspergillus spp. produce a

family of aflatoxin mycotoxins (aflatoxins B1, B2,

G1, G2), of which aflatoxin B1 is the most potent.

Food products produced from livestock fed aflatoxin-

contaminated feeds can also serve as a potential

source of human exposure. Other mutagenic myco-

toxins include vomitoxin and fusarin C produced by

Fusarium sp., luteoskyrin (Penicillium sp.), sterigma-

tocystin (Aspergillus sp.), ochratoxin A, and patulin

(Aspergillus and Penicillium spp.). Alternaria sp., of

which A. alternata is primarily responsible for black

spot on tomatoes and black head on grain, produce a

number of mutagenic toxins such as altertoxins I, II,

and III, and stemphyltoxin III (Figure 1).

Mutagens Formed During Processing, Preparation

or Storage

0030Many very significant mutagens are formed during

the processing and preparation of food. Mutagens

that are formed as a result of heating and preparation

are extremely difficult to avoid or regulate.

0031Grilling or charring of protein rich foods has been

shown to produce some of the most potent mutagens

described to date. Polycyclic heterocyclic amines are

indirect mutagens formed during frying or grilling of

meat and fish. This group includes several related

amino imidazo-azaarene structures belonging to 3

major families: (1) imidazoquinolines (4-MeIQ), (2)

imidazoquinoxalines (8-MeIQx, 4,8-DiMeIQx), and

(3) imidazopyridines (PhIP). Grilling also produces a

number of amino acid pyrolysis products (Trp-P-1,

Trp-P-2, Lys-P-1, Phe-P-1, IQx) and oxygen contain-

ing heterocycles. Generally, PhIP is the predominant

heterocyclic amine found and can be transferred to

infants through breast milk.

0032Heterocyclic amines appear to be formed by a

Maillard reaction between creatine or creatinine, an

amino acid and a sugar such as glucose or fructose.

The Maillard reaction is responsible for browning,

which provides the improved appearance, flavoring,

and aroma of heated foods. The reaction starts as a

condensation between reducing sugars and the amino

groups of amino acids, peptides, or proteins. The

initial products may proceed through a very complex

series of transformations resulting in the formation of

thousands of compounds, including both mutagens

and nonmutagens.

0033Polycyclic aromatic hydrocarbons are a second

class of mutagenic compounds formed during the

grilling or smoking of meat and fish. Polycyclic aro-

matic hydrocarbons are common pyrolysis products

of most organic material, and include benzo[a]pyr-

ene, 1-nitropyrene and 1, 6-dinitropyrene. The furfur-

als, another class of compounds, are produced during

the heating of sugars. Hydroxymethylfurfural is a

heat-induced decomposition product of hexoses that

becomes mutagenic after sulfate conjugation.

0034Other processes can give rise to mutagens. Ureth-

ane (ethyl carbamate) is found in fermented foods and

in beverages treated with the fungicide pyrocarbo-

nate. N-Nitrosodimethylamine is a naturally occur-

ring mutagen present in cheese, soybean oil, canned

fruit, meat products, and alcoholic beverages, but is

also formed from the use of elastic rubber nettings in

cured pork products. Most nitrosamines cross the

placental barrier or pass into the mother’s milk,

thereby leading to possible in utero and infant

exposure.

0035Lipid peroxidation occurs with the storage of fats

and oils, owing to the reaction of oxygen with sites of

MUTAGENS 4063