The Ames Test in Twenty-first Century

Research & Reviews: A Journal of Toxicology Volume 2, Issue 1, April 2012, Pages 23-37 __________________________________________________________________________________________

The Ames Test in Twenty-first Century Myriam Arriaga-Alba1, Regina Montero_Montoya2, Javier J. Espinosa Aguirre2* 1 Microbiology Research Laboratory, Research Division, Hospital Juárez de México Avenida Instituto Politécnico Nacional No. 5160, México, D. F. 07760 2 Toxicology Genetic Laboratory, Instituto de Investigaciones Biomédicas, Universidad Autónoma de México, Apartado Postal 70228, México, D. F. 04510 ABSTRACT Since the 19th century, industrial development has increased exposure to mutagens and possible carcinogens. In order to avoid these health risks, several techniques have been developed to recognize genotoxins. In the 1970s, Dr. Bruce Ames and colleagues, studying the histidine operon of Salmonella typhimurium, observed that histidine (−) auxotroph mutants were reverted to histidine (+) upon exposure to a mutagen. The researchers further observed that known carcinogens were mutagenic to S. typhimurium strains. The Ames test has since been employed as a first step in evaluating genotoxic risk. The Ames method has been accepted by several international organizations including the IARC (International Agency of Cancer Research), EPA (Environmental Protection Agency), FDA (Food and Drug Administration), IVGT (In Vitro Genetic Toxicity Testing Project), REAC (European Union Legislation, Registration, Evaluation, Authorization and Restriction of Chemicals), and the Japanese Ministry of Health and Welfare (MHW). Occasionally, research of new products is suspended upon a positive Ames test result. The Ames test has also been improved to evaluate DNA repair mechanisms using S. typhimurium uvrB + /uvrB− strains. Additionally, researchers have genetically modified these strains to investigate metabolic mechanisms that convert pre-mutagens to active mutagens. The Ames test has also been useful for studying alternatives to reduce genotoxic risk. It is widely employed for antimutagenic evaluation and elucidation of their mechanisms of action. Knowledge on antimutagens is important to reduce mutagenic risk of genotoxic compounds which cannot be avoided, such as therapeutic drugs. A major pitfall of the Ames test is the correlation of data obtained from prokaryotic cells and eukaryotic cells. The aim of this review is to analyze the advantages of the Ames test as a method for studying several aspects of genetic toxicology. Keywords: Ames test, risk evaluation, mutagenesis, antimutagenesis

*Author for Correspondence: E-mail: [email protected], [email protected] 1. IS THE AMES TEST STILL USEFUL

scientists have validated a large series of tests to evaluate genotoxic risk. The increasing

IN THE 21ST CENTURY?

numbers of substances to be evaluated Since the middle of the 19th century, human

demands the continued development of tests

civilization has developed extremely quickly.

that are easily performed, are reproducible and

As a consequence, quality of human life has

are highly sensitive. Throughout the 1970s to

improved and life expectancy has significantly

the present times, the Ames test has been

increased.

widely employed to assess the mutagenic

Unfortunately,

modern

industrialization has also increased population

potential

exposure to hazardous pollutants, possibly

Unfortunately, this simple test has been shown

escalating the risks of cancers, teratogenesis

to result in a number of false positives and

and other degenerative diseases. In order to

false negatives, especially for comparing

reduce the risks of genotoxic exposure,

mutagenic and carcinogenic potentials.

ISSN: 2231–3834© STM Journals 2012. All Rights Reserved

of

chemical

compounds.

Page 23


In the present review, we assess the current

3. DEVELOPMENT OF THE AMES TEST

status of this test by presenting an analysis of

AS A SCREENING TOOL

the most frequent applications and discuss the international acceptance of this method for

In 1971, Ames et al. observed that the

evaluating possible

of

Salmonella typhimurium auxotrophic strains

chemicals. We conclude by evaluating whether

were reverted by a mutagen with a mechanism

this application is worthy of continued use for

of action similar to that employed for its

scientific studies in the 21st century (Table I).

construction. Since 1972, Dr. Ames has

genotoxic

effects

conducted studies to evaluate the modes of action of several chemical mutagens and

2. BIOGRAPHICAL SKETCH

carcinogens.

Employing

S.

typhimurium

From 1950 to 1953, Dr. Ames pursued his

histidine mutants, he discovered a novel,

doctorate

alternative

degree

studying

histidine

method

to

detect

possible

biosynthesis [1, 2], which continued during his

carcinogens in the laboratory. The first strains

post-doctoral research [3–6]. Working as the

TA1950

(hisG46),

TA1951

(hisC207),

chief of the Laboratory of Molecular Biology,

TA1952

(hisC307),

TA1534

(hisD3052)

Section of Microbial Genetics of the National

contained a uvrB deletion and histidine operon

Institute of Arthritis and Metabolic Diseases, a

mutations.

division of the National Institutes of Health from 1962 to 1967, Dr. Ames continued with

The Ames system has been continuously

his research on the histidine operon genes and

improved, such as the addition of an rfa

evaluated histidine auxotroph mutant strains of

mutation at the 48´mark of the S. typhimurium

Salmonella typhimurium [7–10].

genomic map (strains TA1535, TA1537 and TA1538)

to

increase

the

membrane

From 1968 to 2000, Dr. Ames was a professor

permeability to chemical compounds. These

of biochemistry and molecular biology at the

strains also contain uvrB−/uvrB + pairs, such

University of California, Berkeley [11–12].

as

Since 1979, he has been the director of the

TA1537/TA1977

National Institute of Environmental Health

TA1538/TA1978

Sciences Center, U. C. Berkeley. Since 2000,

which allow scientists to evaluate the effects

Dr. Ames has been a professor at the graduate

of DNA excision repair through induced

school of the University of California,

mutational lesions.

the

TA1535/TA1975

(uvrB−/uvrB +),

(uvrB−/uvrB +), (uvrB−/uvrB +)

and variants,

Berkeley(http://www.bruceames.org/bnacv.pp).


Page 24


Table I: Examples of Group 1 IARC Recognised Human Carcinogens with a Positive Ames Test. CAS No.

Agent

Volume

Year

000075-21-8

Ethylene oxide*

97, 100F

in prep

000091-59-8

2-Naphthylamine

4, Sup 7, 99,100F

in prep

001402-68-2

Aflatoxins

56, 82, 100F

in prep

000092-67-1

4-Aminobiphenyl

1, Sup 7, 99, 100F

in prep

000071-43-2

Benzene

29, Sup 7. 100F

in prep

000092-87-5

Benzidine

29, Sup 7, 99, 100F

in prep

000050-32-8

Benzo[a]pyrene*

92, 100F

in prep

Betel quid with tobacco

85, 100E

in prep

Betel quid without tobacco

85, 100E

in prep

000055-98-1

Busulfan

4, Sup 7, 100A

2011

000305-03-3

Chlorambucil

26, Sup 7, 100A

2011

000494-03-1

Chlornaphazine

4, Sup 7, 100A

2011

000050-18-0006055-19-2

Cyclophosphamide

26, Sup 7, 100A

2011

059865-13-3079217-60-0

Cyclosporine 1 50

100A

2011

000050-00-0

Formaldehyde

88, 100F

in prep

000148-82-3

Melphalan

9, Sup 7, 100A

2011

298-81-7

Methoxsalen

24, Sup 7, 100A

2011

4,4'-Methylenebis(2-chloroaniline)* (MOCA)

57, 99, 100F

in prep

MOPP and other combined chemotherapy

Sup 7, 100A

2011

4, Sup 7, 99, 100F

in prep

89, 100E

in prep

100F

in prep

(8-methoxypsoralen)

plus

ultraviolet Aradiation 101-14-4

including alkylating agents 000091-59-8 2-

Naphthylamine

016543-55-8064091-91-4

N'-Nitrosonornicotine (NNitrosomethylamino)-

(NNN)

and

4-

1-(3-pyridyl)-1-

butanone (NNK)*

057465-28-8

3,4,5,3’,4’-Pentachlorobiphenyl (PCB-126)* (NB: Overall evaluation upgraded to Group 1 based on mechanistic and other relevant data)

057117-31-4

2,3,4,7,8-Pentachlorodibenzofuran*

100F

in prep

000062-44-2

Phenacetin*

24, Sup 7, 100A

2011

013909-09-6

Semustine [1-(2-Chloroethyl)-3-(4-

Sup 7, 100A

2011

methylcyclohexyl)-1-

nitrosourea,

Methyl-

CCNU] 000505-60-2

Sulfur mustard

9, Sup 7,100F

in prep

000052-24-4

Thiotepa

50, 100A

2011

000095-53-4

ortho-Toluidine

77, 99, 100F

in prep

000299-75-2

Treosulfan

26, Sup 7,100A

2011

http://monographs.iarc.fr/ENG/Classification/ClassificationsAlphaOrder.pdf * (NB: Overall evaluation upgraded to Group 1 based on mechanistic and other relevant data).


Page 25


Table II: Some Representative Strains of Salmonella typhimurium Used in the Ames Test. Strain

Genotype

Mutation Detected

HisC207

hisC207

Frameshift mutation

TA1951

hisC207, bio, chl, gal, uvrB

TA1531

HisC207, rfa, bio, chl, gal, uvrB

TA1976

hisC207, rfa

hisG46

hisG46

TA1950

hisG46 gal uvrB

TA1535

hisG46 rfa, bioh, gal, uvrB

TA1975

hisG46, galE, rfa

TA92

hisG46, pKm101

TA2410

hisG46, bio,chl, gal, uvrB, pKm101

TA100

hisG46 rfa,chl, gal, uvrB, pKm101

hisC3076

hisC3076

TA1537

hisC3076 bio, chl, gal, uvrB

TA1977

hisC3076,gale, rfa

hisD3052

hisD3052

TA1534

hisD3052, bio,chl, gal, uvrB

TA1538

hisD3052 rfa, bio,chl, gal, uvrB

TA1978

hisD3052 rfa

TA94

hisD3052, pKm101

TA1978

hisD3052 rfa, , galE

TA98

hisD3052 rfa, bio,chl, gal, uvrB, pKm101

TA88

hisD26610, his01242

TA110

hisD26610, his01242, pKm101

TA97

hisD26610, his01242, rfa, , bio,chl, gal, uvrB, pKm101

Base pair substitution

Frameshift mutation

Frameshift mutation

Frameshift mutation

hisG428

Oxidative damage

TA104

hisG428, rfa, , bio,chl, gal, uvrB

TA102

HisG428, rfa, pKm101, pQ1

Note: Strains in bold letters are those most frequently employed in the Ames test.

The Ames test was further improved by the

improved by the addition of microsomal

introduction of the pKM101 plasmid harboring

preparations of rodent liver enzymes in order

the umuC and mucB and mucC genes (strains

to enhance the level of Cytochrome P450

TA100 and TA98), which improved the SOS

(CYP) activity [13–20].

repair system. The Ames test was further


Page 26


Further work was undertaken to evaluate large

specificity and sensitivity reported for the

series of chemical compounds as well as

Ames test may vary within a chemical group.

known carcinogens that were previously

Brusik et al. (1983) reported that the Ames test

evaluated using animal models. These studies

may have a predictive value of rodent

demonstrated that some strains were suitable

carcinogenesis up to 80%, making this test

to detect frameshift mutations (e.g., TA98).

suitable for screening suspected compounds.

Other strains, such as TA100, are more

Some studies have demonstrated that the Ames

suitable

test may result in false negatives using

to

detect

base-pair

substitution

mutations [21–26]. The Ames test has also

genotoxic

non-electrophilic

compounds.

been used to detect urinary mutagens in

Therefore, the Ames test alone may not be

patients [27]. Several studies have been

conclusive of an actual biological hazard and

conducted to classify mutagens as possible

should be performed along with in vivo

carcinogens and to evaluate genotoxic risk

genotoxic testing [40, 41].

based on the chemical structures of potential candidates [28–30]. The Ames test is also

The Ames test is used by the pharmaceutical

useful to detect genotoxic risks of chemical

industry when developing new therapeutic

pollutants and environmental hazards as

drugs. Purves et al. [40] found a surprising

possible predictors of cancer. This test has

amount of variation in testing for mutagens

become an important tool for genotoxic risk

between companies, which was evident in how

assessment. [31–34]. By the 1980s, the Ames

companies define a genotoxin. Positive results

test was performed worldwide, where it

from in vivo assays, indicative of a genotoxin,

remains

ranged from 30% to 43%. Indeed, many

a

useful

tool

in

microbiology

laboratories. The Ames test protocol is well

companies,

established

terminate development of a drug only on the

and

standardized

for

reproducibility [35–39] (Table II).

particularly

in

Japan,

will

strength of a clear positive result in an Ames test [42, 43].

4. SPECIFICITY AND SENSITIVITY OF THE AMES TEST

Three separate mathematical analyses of Ames test results for more than 3300 drugs

Generally, the Ames test is highly sensitive

concluded that the assay had a sensitivity of

and able to detect chemicals that are

88%, with a low percentage of false positives

carcinogenic to rodents and possibly to

and

humans. However, the assay is not highly

verifiably positive mutagenic test results, this

specific, which may result in a large number of

value could be increased from 92% to 97%,

false positives and identification of mutagens

based on the mathematical analyses employed

that are not necessarily carcinogens. The

[44].


false

negatives.

Considering

only

Page 27


Nevertheless, making decisions based only on

evaluate metabolic mechanisms of nitro-

a positive Ames test is ill-advised, as this test

groups to produce active mutagens such as 2-

is based only on the detection of point

nitrofluorene and 1-nitropyrene, as well as the

mutations.

nitration mechanisms of genotoxic compounds

Furthermore,

the

biological

significance and mutagenic potential may vary

[48,

with

studies,

constructed with different levels of N-

including the Ames test, must consider

hydroxyarylamine O-acetyltransferase (OAT)

chemical dosages obtained from the slope of a

activity,

dose-response

intracellular

dosage.

Risk

assessment

curve

for

the

evaluated

compound [45].

49].

Other

an

derivate

enzyme

strains

involved

metabolic

were

in

the

activation

of

N-hydroxyarylamines derived from mutagenic nitroarenes and aromatic amines. The oat

At

present,

the

Ames

test

is

widely

overproducing S. typhimurium strains YG7130

recommended by international organizations

and YG7126 were obtained from strains TA98

such as the EPA, FDA, and IARC and it is

and TA100 by introducing oat genes; these

typically the first step of genotoxic evaluation

strains are useful to gain insight on genotoxic

in risk-assessment studies. It is also widely

mechanisms [50].

employed by the pharmaceutical industry for development of active therapeutic compounds,

A set of modified strains derived from S.

as well as to evaluate the possible genotoxic

typhimurium TA1535 was constructed by the

hazards

deletion of ada or ogt genes (YG7100

of

precursor

and

intermediate

compounds, which may be formed during

ada−/ogt +

YG7104

ada +/ogt−,

G7108

commercial preparations [46, 47].

ada−/ogt−) [51]. These strains have been useful to study DNA repair enzymes of alkylated

5. AMES TEST APPLICATIONS

DNA,

and

to

elucidate

the

mechanism of action of an antimutagen able to reduce mutations induced by N-nitroso-

5.1. Development of New Strains The

Ames

test

strains

were

further

guanidines [52]. Inman et al. introduced other

bioengineered to study xenobiotic metabolism

strains to evaluate the effect of the DNA repair

and DNA repair systems. Nitroreductase

excision

deficient cnr null mutant derivatives of strains

UTH8413, which is the Ames strain TA1975,

TA1535 and TA1538 were constructed and

modified with the pKM101 plasmid and the

named YG7131 and YG7127, respectively.

TA1978 strain with plasmid pKM101, known

Similar nitroreductase-deficient derivates were

as UTH8414 [53] (Table III).

repair

system:

S.

typhimurium

constructed from TA98 and TA100, which were

named

TA98NR

and

TA100NR,

respectively. These strains have been useful to


Page 28


Table III: New Strains Derived from the Salmonella typhimurium Ames Strains. Strain

Genotype

Utility

Reference

UTH8413

hisD3052 rfa, , gale, pkM101

DNA repair

Inman et al.1983

UTH8414

hisG46, galE, rfa, pKm101

DNA repair

Inman et al.1983

YG7100

hisG46 rfa, bioh, gal, uvrB, ada ST.KMr

DNA repair of Yamada et al. alkylated bases

YG7104

YG7108

hisG46 rfa, bioh, gal, uvrB ogt

..Cm r ST

1995

DNA repair of Yamada et al.

ogt ST..Cm r

alkylated bases

hisG46 rfa, bioh, gal, uvrB ada ST..KMr

DNA repair of Yamada et al. alkylated bases

1995

1995

TA1538-

hisD3052 rfa, bio, chl, gal, uvrB , human Metabolism of Yamasaki et al.

CYP1A1

CYP1A1,

TA1538-


CYP1A2,

CYP1A2,

TA1538-


CYP2A6

CYP2A6,

TA1538-


CYP2C8

CYP2C8,

TA1538-


CYP2C9

CYP2C9

TA1538-


CYP2C19,

CYP2C19,

TA1538-


CYP2C19,

CYP2C19,

TA1538-


CYP2E1

CYP2E1

TA1538-


CYP3A4

CYP3A4

TA1538-


CYP3A5

CYP3A5

frameshifts

frameshifts

frameshifts

frameshifts

frameshifts

frameshifts

frameshifts

frameshifts

frameshifts

frameshifts

2004

2004

2004

2004

2004

2004

2004

2004

2004

2004

5.2. Antimutagenesis

to reduce exposure to those compounds.

Increasing awareness of mutagenic chemicals

Nevertheless, many compounds cannot be

that represent a risk of cancer has been useful

avoided. Therefore, parallel to the research on


Page 29


mutagenesis and mutagenic mechanisms, the

Interesting studies have been performed

study of antimutagens has been developed as a

worldwide on the effects of indigenous plants,

viable alternative to reduce genotoxic risks.

which

Antimutagens are chemical compounds that

mechanism

avoid the initiation steps of cancer. These

employed

compounds

as

studied plants had antioxidant properties, but

desmutagens, which prevent active mutagens

many also contained compounds that inhibited

from reaching target molecules, i.e., DNA and

the metabolic activation of polycyclic aromatic

causing mutagenic lesions. True antimutagens

hydrocarbons. Other plant extracts contained

are those compounds that are able to reduce

compounds capable of improving DNA repair

mutagenic

systems [52, 61−63].

have

been

classified

lesions in DNA, mainly

by

have

permitted of

in

action

insight of

alternative

on

plant

the

extracts

medicine. Most

improving the DNA repair systems [54, 55]. These studies have also demonstrated that The Ames test has, in fact, been employed as a

plant constituents are not always innocuous. In

tool to study and classify naturally-occurring

fact, many edible plants have been shown to

antimutagens. Since the early 1980s, Ames et

have

al. initiated systematic studies to evaluate

Coriandrum sativum, a common herb, is able

reductions of genotoxins and cancer risks

to enhance metabolic activation of three

through a diet rich in antimutagens. They

aromatic

observed that populations consuming diets

genotoxic risk [64]. These studies indicated

with high levels of antimutagens had lower

that the Ames test was useful for screening the

risks of developing gastroenteric cancer. The S.

toxic or antimutagenic effects of natural

typhimurium microsomal Ames test has been

compounds frequently employed in alternative

widely employed to study several plant

medicine.

genotoxic

effects.

amines,

thereby

For

example,

increasing

its

extracts known to reduce mutagenicity. The main mechanism of action described for

5.3. The Ames Test as a Useful Tool for

antimutagens

Studies of Genotoxic Metabolism

is

the

same

as

that

of

antioxidants: scavenging oxygen-free radicals

The lack of metabolic activation of genotoxic

[56−58]. This relatively simple test has been

compounds was believed to be a complication

used

of the Ames test. This problem was solved by

to

evaluate

compounds

including

vitamins and to assess several physiological

the

addition

of

CYP-induced

liver

reactions. For example, results showed that

homogenates prepared from rodents, which

ascorbic acid acts more as a pro-oxidant as

added new capabilities to the Ames test. For

opposed to an antioxidant in the presence of

example, several CYP inducers have been

cationic ions in a Fenton reaction [59, 60].

evaluated, which showed that these enzymes are induced by different types of compounds.


Page 30


The most widely employed CYP inducer is

genetically

arochor-1254, which is nonspecific. Other

containing human CYP, NADPH-cytochrome

specific inducers have also been evaluated. For

P450 reductase (CPR) and cytochrome b

example, cyclohexanol (CH), an industrial

[68, 69]. Yamasaki et al. [68] cloned genes of

solvent, induces CYP enzymes, including the

human

CYP2E and CYP2B subfamilies. S9 induced

CYP2A6,

by CH enhances mutagenesis of nitrosamines

CYP2C19M CYP2D6, CYP2E1, CYP3A4 or

better than S9 induced by Arochlor-1254.

CYP3A5, together with NADPH CPR. These

Albendazol,

strains have been successfully used to study

an

antiparasitic

drug

at

modified

CYP1A1,

using

CYP1A2,

CYP2C8,

metabolism

a

CYP2C8,

of

induced

plasmid

CYP2A6, CYP2C9,

therapeutic doses, activates the induction of

the

frameshift

CYP1A1, CYP1A2 and CYP2B1 and inhibits

mutations, while elucidating the main CYP

CYP2E1. Employing different S9 inducers, it

family involved [68, 69].

was observed that benzo(a)pyrene and 3methylcholanthrene are activated by CYP2A1,

The YG7108 strain lacks ada and ogt genes,

whereas

N-nitroso

which are involved in DNA repair of alkylated

dimethylamine are activated by CYP2E1 and

DNA bases, and is extremely useful to detect

cyclophosfamide by CYP2B and CYP3A [65,

base pair substitution mutagens, such as

66].

nitrosamines. This strain was modified by

nitroso-pyrolidine

and

cloning different human CYP, such as The Ames test was able to evaluate the drug

CYP1A1,

CYP1A2,

CYP1A2,

CYP1B1,

chlomiphene citrate, widely used to induce

CYP2A6, CYP2A13, CYP2D6, CYP3A4 or

ovulation. It was determined that chlomiphene

CYP3A4 into a plasmid with the CPR human

citrate requires an oxidation-reduction reaction

gene. These strains were used to demonstrate

mediated mainly by CYP1A1 and perhaps

that the human CYP2A family was necessary

CYP2B to induce a frameshift mutagenic

to metabolize carcinogenic nitrosamines found

metabolite [67].

in betel quid, a plant formulation used as a stimulant, an antiseptic and a breath-freshener

5.4. Modified

Ames

Strains

Expressing

in many Asian countries [70].

Human CYP450 and CPR The employment of rodent liver homogenates

5.5. Mutagens in Biological Fluids

raised

human

Mutagenicity testing on body fluids has been

cytochromes would be able to metabolize

widely performed on urine, plasma and serum

genotoxins in the same way as the rat S9

samples. Employing laboratory animals, these

enzymes. This question was solved with the

tests

construction of modified S. typhimurium

pharmacokinetics

strains. The S. typhimurium strain TA1538 was

compound formation from aminated drugs

the

question

of

whether


can

be

useful of

to in

evaluate vivo

the

N-nitroso

Page 31


[71]. This assay can also be useful for

levels of IQ (2-amino-3-methylimidazo-(4,5-f)

epidemiological studies of human populations

quinoline) by modifying P450 expression. The

exposed to occupational mutagens, smoking

employment of plants in alternative medicine

habits

environmental

is increasing. This method has also been useful

contaminants. In fact, it was demonstrated that

to evaluate the in vivo antimutagenic effect of

workers exposed to waste products had higher

Phyllanthus amarus, an important plant of the

levels of urinary mutagens than those working

Indian Ayurvedic system of medicine, against

in water-treatment facilities. The Ames test

urinary mutagens induced by benzo(a)pyrene

also

in rats [77, 78].

and

exposure

to

demonstrated that foundry workers

exposed to aromatic polycyclic compounds had lower urinary levels of mutagens, if they

Exposure to genotoxic compounds might

employed gloves while working [72, 73]. Lupi

reveal information on the metabolism of

et al. recommended the use of urinary analysis

genotoxic compounds. Several mutagens are

to evaluate the effect of chronic exposures to

frequently eliminated as conjugates with

low mutagen levels [74].

glucuronic acid which are liberated by the addition of β-glucuronidase. Some compounds

Urinary mutagenicity studies performed on

are known to be cancer risk factors if they are

human populations must take into account diet

not eliminated in the urine. Varga et al.

and smoking habits. In fact, it is well known

showed

that cigarette smoking is perhaps the most

carcinogen,

important source of urinary mutagens [75, 76].

including sister chromatid exchange test in

Furthermore, meals that potentially contain

bone marrow cells of rats, but could not be

mutagens should also be avoided. For example,

detected

fried beef is an important source of mutagenic

Nevertheless, Choi et al. recommended urinary

compounds, such as mutagenic derivatives

mutagen detection to identify hazardous

from amino-acid pyrolysis. Meals rich in

exposure to chemicals in the workplace and

nitrites should also be avoided, as they might

reported a positive predictive value of 72%

present a source of nitrosamine precursors [71].

[80].

In vivo studies of urinary mutagens are a

6. PERFORMING THE AMES TEST

that

as

asbestos,

gave

a

a

a

positive

urinary

well-known test

mutagen

result

[79].

valuable tool for antimutagenic evaluation. Vitamin C administration inhibits the in vivo

Most laboratories using the Ames test follow

formation of N-nitroso compounds in mice

the method described by Maron and Ames

treated with aminated drugs and nitrite [71].

[39], which has reproducibility up to 89.0%. In

The consumption of green tea for one month

fact, a comparison of the test outcomes among

was shown to reduce the urinary mutagenic

different investigators resulted in an inter-


Page 32


laboratory consistency of 89.5% [81]. The

a TAMix matrix with low spontaneous

Ames test is perhaps one of the most employed

reversion that allows the detection of small

methods

and

mutagen concentrations. Strains are plated in

carcinogenic activities of a large variety of

triplicate in 384-well plates. A positive result

chemicals. A study among several laboratories

is scored by a visible growth or a change of

demonstrated that analysis of experimental

color from purple to yellow. The second

errors and results showed that this test does not

generation Ames 11 test can be performed

fit a Poisson distribution. Snee and Irr in 1984

with an automated analyzer. The kit is

[82] found that analysis of variance, regression

furnished with reagents employing Aroclor or

analysis, and the Student’s t-test were more

phenobarbital

appropriate to analyze Ames test results.

traditional Ames test, nevertheless, remains

for

screening

mutagenic

BNF-induced

S9.

The

the gold standard [39], but is highly correlated Currently,

several

simplified

assays

are

to the Ames 11 test [81].

performed to augment the Ames test. A variation of this test, called the spiral model,

7. CONCLUSIONS

utilizes a dose gradient on the same plate, as opposed to employing different serial dilutions

The Ames test is performed in laboratories

over several plates [83]. Another modification

worldwide to evaluate a large variety of

involves

multi-well

chemicals as the first step in assessing

plaques and observing whether growth is

genotoxic risks. This test also presents a guide

dependent on varying doses and different

to predict interactions between chemical

strains within the same plate. This variation is

compounds and DNA.

the

employment

of

known as the Ames 11 assay [81]. The

Ames

test

is

very

useful

for

The Ames 11 assay was developed as a

antimutagenicity studies to elucidate the

predictive screening assay for genotoxicity.

mechanisms of action and elucidate the role of

This assay can detect both frameshift and base

CYP

pair substitution mutations with or without S9

premutagens. Lastly, the Ames test can be

activation. Frameshift mutations are detected

employed to evaluate the metabolism that

by S. typhimurium TA98, whereas base pair

occurs in in vivo systems. In fact, detecting

mutations are detected using six different

mutagenic metabolites in biological fluids,

strains containing six different base pair

such as urine, has been useful to elucidate the

substitutions developed specifically for this

metabolism of genotoxins, and can be further

assay. Strains are incorporated on the plaque in

employed in epidemiological studies.


enzymes

in

the

metabolism

of

Page 33


11. G. R. Fink, J. R. Roth. Journal of

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