TECHNICAL METHODS SECTION

Activated Mutatox Assay for Detection

of Genotoxic Substances

B. THOMAS JOHNSON
Environmental Microbiology,
National Fisheries Contaminant Research Center,
Fish and Wildlife Service,
US.Department of the Interior,
Columbia, Missouri 65201

1.o OBJECTIVES
This method is intended as a n initial screening assay to detect
the presence of DNA-damaging substances (genotoxins) in new
products or their metabolites, or in complex mixtures (i.e., wa-
ters, sediments, wastewaters, and leachates), or as an analyti-
cal tool t o evaluate chemicals for structural alerts of DNA-
reactivity.

2.0 SCOPE
This method is applicable to the following types of samples:
(i) a chemical that is soluble in water or soluble in nontoxic
concentrations of organic solvent
(ii) surface water, groundwater, or wastewater; and
(iii) a n aqueous or organic extract
(a) from contaminated surface or groundwater,
(b) from sediment or pore water, or
( c ) from domestic or industrial wastewater.

3.0 PRINCIPLE
3.1 A short-term in vitro procaryotic assay and protocol known as
the activated Mutatox Genotoxicity Assay (MutatoxTh',Micro-

Environmental Toxicology and Water Quality: An International Journal

Vol. 8, 103-113 (1993)
Not subject to copyright in the United States.
Published by John Wiley & Sons, Inc. CCC 1053-4725/93/010103-11
104IJOHNSON

bics Corp., Carlsbad, CA) uses rat hepatic S9 for exogenous

metabolic activation and bioluminescent bacteria Photobacte-
rium phosphoreum for detection of genotoxins.

3.2 A genotoxic response indicates the ability of the test chemical,

metabolite, or sample extract to restore the luminescent state
in a dark strain of luminescent bacteria.

3.3 The intensity of light emission from bacterial cells indicates

the relative genotoxicity of the test sample.

3.4 Tests are conducted using a single tester strain of the lumines-
cent bacteria.

3.5 A rat hepatic postmitochondrial supernatant fraction (S9), a

mammalian surrogate for exogenous enzymes, is used for meta-
bolic activation of progenotoxins to genotoxins.

4.0 REAGENTS, BIOLOGICALS, MATERIALS,

ANDAPPARATUS
4.1 Reagents
4.1.1 All chemicals are of the highest purity commercially available.

4.1.2 List of Chemicals

4.1.3 Solvents:
Acetone
Dimethylsulfoxide (DMSO)
Distilled water

4.1.4 Positive Assay Controls:

2-aminoanthracene (2-AA) (Sigma Chemical Co., St. Louis,
MO)
2-aminofluorene (2-AF)(Aldrich Chemical Co. Inc., Milwaukee,
WI)
Benzo(a)pyrene (BaP) (Aldrich)
Pyrene (PY) (Sigma)
Phenol (Sigma)

4.2 Biologicals
4.2.1 Tester strain: Photobacterium phosphoreum M 169, lyophilized
 MUTATUX ASSAY FOR GENOTOXIC SUBSTANCES/lo5

and stored frozen at -20°C in vials (Microbics Corp., 2232 Ruth-

erford Road, Carlsbad, CA; suggested shelf-life 1 year).

4.2.2 Mutatox Assay Medium: prepackaged dehydrated medium with

essential nutrients, salts, buffer, and S9 cofactor regeneration
system (nicotinamide adenine dinucleotide phosphate and glu-
cose-6-phosphate) (Microbics; suggested shelf-life 1 year).

4.2.3 Exogenous Activation Source: Aroclor 1254-induced rat liver

S9 fraction, stored frozen in liquid nitrogen or a t -8O"C, avail-
able in 1-mL vials a t 35-40 mg protein/mL (Microbiological
Associates Inc., 9900 Blackwell Road, Rockville, MD 20850,
USA, or Molecular Toxicology Inc., 111 Gibraltar Road, Ana-
polis, MD 21401; suggested shelf-life l year).

4.3 Materials
Glass cuvettes, 11.75 mm x 50 mm (Microbics)
Disposable pipettes (0.1, 1.0, and 10 mL)
Amber glass bottles with Teflon cap liners (10 and 25 mL)
Standard glass beakers (100 and 250 mL)
Aluminum foil (Standard 12-in. roll)

4.4 Apparatus
Model 500 Analyzer (Microbics)
Incubator, thermostatically controlled (20-30°C)
Vortex mixer
Magnetic stirrer with bar
Cuvette holders
Adjustable pipette dispensers (0.1, 1.0, and 10 mL)
Refrigerator (storage of hazardous materials)
Freezer (-80°C)
Vacuum testing coil (Optional)
Water bath thermostatically controlled (37°C) (optional)

5.0 SAFETY MATERIALS

Gloves (disposable)
Tyvek suits (disposable)
Sleeve protectors (disposable)
Safety glasses
Bench covering (plastic-backed paper)
106IJOHNSON

Disposable plastic bags

Closed receptacle(s1 for toxicant disposables
Laminar flow (class I1 B2) vented bacteriological and toxicant
transfer hood with yellow light
Enclosed vented hood
Warning labels (hazard, genotoxic)

6.0 TESTING PROCEDURE

6.1 Sample Preparation
6.1.1 Prepare all samples under a laminar hood with yellow light
to reduce photodegradation of test chemicals and standards.
Disposable gloves, Tyvek suits, sleeve protectors, and safety
glasses are used for handling genotoxic substances.

6.1.2 Prepare each progenotoxin (2-AF, 2-AA, BaP, and PY) control
in DMSO at 1.0 mg/mL in amber bottles with Teflon cap liners.
Label with hazard warnings and store no longer than 1month
in a refrigerator (0-5°C). These positive controls monitor for
environmental polyaromatic hydrocarbons (PAHs). (Note:
other types of controls may be more appropriate for other classes
of genotoxins.)

6.1.3 Prepare working stock of each control in amber bottles with

cap liners at 100-pglmL in acetone (1.0 mL genotoxin stock:
9.0 mL acetone). Label and store for no longer than 1 month
under refrigeration (04°C). Many progentoxins are unstable
and may degrade during storage in carrier solvents, adversely
effecting the validity of the assay.

6.1.4 Prepare test sample o r extract in assay-compatible solvent:

acetone, DMSO, ethanol, methanol, or combinations. If signifi-
cant cytotoxicity is suspected, see Section 7.3 (Cytotoxicity).
Label and store in amber bottles under refrigeration (04°C).

6.2 Assay Preparation

6.2.1 Reconstitute Mutatox Assay Medium (MAM)with distilled wa-
ter (or comparable quality) and prepare a 100 mL aliquot in
250-mL beaker. One hundred milliters of MAM will provide
sufficient medium t o assay 15 test samples, 1 negative, and 4
positive controls. Mix MAM with magnetic stirrer for several
 MUTATUX ASSAY FOR GENOTOXIC SUBSTANCES/10?

minutes until all components are dissolved; adjust to 23 t 1°C.

(If a water-based test sample exceeds 10% of the MAM, check
with Microbics Corp. for concentrated MAM.)

6.2.2 Remove bacteria tester strain from freezer. Check vial for vac-
uum with testing coil (optional) and record lot number. Recon-
stitute freeze-dried bacteria with 1.0 mL of MAM at 23°C; vor-
tex a few seconds to homogenize.

6.2.3 Inoculate the remaining 99 mL of MAM with 1.0 mL of the

hydrated bacteria and stir a few seconds with a vortex mixer.
Immediately preincubate this mixture at 23 t 1°C in a water
bath or incubator for 30 min. (The optimal test temperature
growth range for the tester strain P . phosphoreum is 23 t
1°C; this strain, while in lag phase, appears very sensitive to
temperatures below 20°C; although cell growth does not appear
to be influenced, sensitivity of the cells to genotoxins is signifi-
cantly reduced.) Do not chill i n a n ice bath.

6.2.4 Carefully thaw 1 mL of rat hepatic S9 (35-40.0 mg protein/

mL) by placing the frozen vial in a 50-mL beaker partially filled
with water at 23°C for a few minutes. Do not warm 5'9 enzymes
at temperatures above 37°C.

6.2.5 Add 1.0 mL of S9 to the inoculated MAM aliquot to prepare a

0.35-0.40 mg protein/mL reaction mixture, vortex for 15 s, and
cover the beaker with aluminum foil. Maintain this reaction
*
mixture at 23 1°C in a water bath or incubator; use within
30 min.

6.3 Test Sample Dilution Technique and Exposure

Experiments are performed under a laminar hood with yellow
light and then incubated in the dark (Caution: Many genotoxins
are very photosensitive and can be destroyed under normal
laboratory light conditions.) Disposable gloves, Tyvek suits,
sleeve protectors, and safety glasses are used for handling po-
tentially genotoxic substances. A standard dilution series con-
sists of at least 10 different concentrations of the test sample,
as well as negative and positive controls, and if necessary,
procedural control. The test sanple is serially double diluted
(1: 1v/v) 9 times in the reaction mixture over a 100x range to
elicit a normal dose-response reaction. This dilution series is
 based on work with arylamines and polyaromatic hydrocar-
bons. (Different dilution ranges may be needed for other test
samples.)

6.3.1 Place 10 clean cuvettes numbered from 1 t o 10 in a row in a

cuvette holder for each test sample.

6.3.2 Pipette 1.0 mL of bacteria-S9-MAM reaction mixture into cu-

vette 1 and 0.5 mL into each cuvette 2-10. (Each 10-cuvette
dilution series requires 5.5 mL of the reaction mixture.)

6.3.3 Place 100 p L of the test sample into cuvette 1. (The carrier
organic solvent should not exceed 10% of reaction mixture vol-
ume (v/v) in cuvette 1.)Vortex the test sample and reaction
mixture briefly.

6.3.4 Transfer with a disposable l-mL pipette, 0.5-mL of the sample

from cuvette 1into cuvette 2 (1: 1 volume/volume) and vortex;
using the same procedure sequentially, dilute the entire series.
Discard 0.5 mL from last test cuvette.

6.3.5 Similarly, prepare a negative control dilution series using 10

cuvettes with 5.5 mL reaction mixture, and substitute 100 p L
of carrier solvent for the test sample.

6.3.6 Positive controls consist of four progenotoxins: 2-AA, 2-AF,

BaP, and PY, tested a t 10, 5,2.5, 1.2, and 0.6 pg/cuvette (five-
tube dilution series only because of their high genotoxic sensi-
tivity).

6.3.7 For each positive control dilution series, place 5 clean cuvettes
numbered 1 to 5 in a row in cuvette holder. Pipette reaction
mixture into cuvettes as described in Section 6.3.2; each 5-
cuvette dilution series requires 3.0 mL of the reaction mixture.
Place 100 pL of the positive control working solution into cu-
vette 1. Prepare a dilution series as described in 6.3.4.

6.3.8 Time to perform dilution series for 15 test samples, 1negative,

and 4 positive controls is 60 min.

6.4 Preincubation
Cover treated samples with aluminum foil and preincubate in
 MUTATUX ASSAY FOR GENOTOXIC SUSSTANCES/lo9

a water bath or incubator at 37°C for 30 min under yellow light,

or in the dark (see caution Section 6.3)

6.5 Incubation
*
Incubate treated samples in the dark at 23 lac,the optimal
growth range for the tester strain Photobacterium phos-
phoreum, for 16-24 h.

6.6 Bacterial Bioluminescence

Bioluminescence of the bacterial cells is determined with a
Model 500 Analyzer, which measures the light intensity of each
cuvette.

6.6.1 Turn on the analyzer for several minutes to permit an initial

instrument warmup and stabilization of digital readings. To
adjust the instrument for maximum light sensitivity, place a
clean, empty cuvette in a turret and press the “Set” button.
When the digital reading is “0” press the “Read” button to
assure a reading of less than 1;the instrument is at maximum
sensitivity and ready for the assay. If not, repeat the above step.

6.6.2 Read negative control to establish baseline value for test run
(see 7.1.1, Genotoxicity End Point, for application).

6.6.3 To use the analyzer, place cuvette 1 in turret, press the read
button, and record the digital value-0-199-of the light out-
put. Read an entire dilution series noting the sample(s) with
the maximum light output. If the instrument fails to record a
number value and flashes a warning light, the light value from
that sample has exceeded the current sensitivity range of the
instrument. Use that tube to reset the analyzer, note the change
in sensitivity, and continue the sample analysis. (The analyzer
has a safety mechanism to protect the light-sensing device,
hence no reading or a flashing screen.) Continue reading this
dilution series, to find the sample with the maximum light
output. Use this sample to standardize your instrument; re-
member this value may be l o x , ~OOX,or even 1 0 0 0 ~the
readout value due to resetting the analyzer. Re-read this dilu-
tion series and adjust the values. Remember to reset the instru-
ment sensitivity before reading each series of sample. If neces-
sary, repeat the above steps for maximum sensitivity.
6.6.4 Read positive control(s) to establish relative sensitivity and
acceptability of test run (see 7.2, Assay Confirmation).

6.6.5 Reading time for 15 test samples, 1 negative, and 4 positive

controls is approximatley 60 min.

7.0 EXPRESSION OF RESULTS

7.1 Genotoxicity End Point
7.1.1 The negative control identifies the spontaneous light emission
of the dark mutant strain. A light value of 100 or more and at
least three times the light intensity of the negative control
defines a genotoxic response; sensitivity limits are the maxi-
mum peak concentration (MPC) and lowest detected concentra-
tion (LDC) in each dilution series. The dose-response number
is defined as the number of genotoxic responses recorded at
different concentrations per dilution series.

7.1.2 A dilution series with a dose-response number of three or more

is designated genotoxic, with a dose-response number less than
three is designated suspect, or without a genotoxic response is
designated negative.

7.1.3 The test sample is designated GENOTOXIC when at least three

replicate series indicate a mean dose-response number of three
or more (see 7.2.1, Assay Confirmation).

7.1.4 The test sample is designated SUSPECT when at least three

replicate series indicate a mean dose-response number less than
three.

7.1.5 The test sample is designated NEGATIVE when at least three

replicate series contain no genotoxic response.

7.2 Assay Confirmation

72.1 Test sample confirmation is completed in 572 h. Three replicate
dilution series of each test sample are performed. Each replicate
must use a separate preparation of tester strain, MAM, and S9.
Replicates may be performed simultaneously or on different
days.
 MUTATUX ASSAY FOR GENOTOXIC SUBSTANCES/ 111

7.2.2 An LDC of all positive controls (2-AA,2-AF, BaP,and PY) 11.2

pg/cuvette validates the sensitivity and acceptability of the
assay each day. Remember t o replace these chemicals each
month to reduce errors in false positive controls (see Section
6.1.3). When an LDC of any of the four positive controls is B1.2
pg/cuvette, the assay’s sensitivity is considered suspect, the
results are rejected, and this test run is repeated.

7.3 Statistical Analysis

Sample variability is measured by determining the standard
deviation of the mean. Data are tested by analysis of variance
with a Statistical Analysis System computer package (SAS).
Mean differences are measured by one-way analysis of vari-
ance. If the overall F value is significant ( p 5 0.051, the mean
differences are ascertained by using Fisher’s least-significant
difference test.

7.4 (Optional) Cytotoxicity

The relative cellular toxicity of the test chemical (or complex
organic extracts) is determined by a turbidity test. No effort is
made to determine the cytotoxicity of known genotoxins at
concentrations >50 ,ug/test because the ecological relevance
of such a high concentration is questionable. In addition, the
experimental use of high concentrations of known genotoxins
creates unnecessary handling hazards and disposal problems.

7.4.1 Turbidity method. Bacterial cytotoxicity of the extractk) and

test chemicalb) is determined empirically by comparing tur-
bidity of bacterial growth in treatment and control tubes after
16 h. An aqueous 1%phenol solution, freshly prepared, is used
as a positive control.

8.0 PERSONAL REMARKS

8.1 The activated Mutatox Genotoxicity Assay is sensitive, rapid,
economical, and technically simple, a unique test.

8.1.I Mutatox shows a broad spectra of sensitivity; that is, six classes
of progenotoxins-arylamines, polyaromatic hydrocarbons,
112iJOHNSON

mycotoxins, nitrosoamines, phosphamides, and azo-aromatic

dyes-are detected in the low pg/cuvette range (<lOpg).

8.1.2 The availability of the assay is on demand, and the incubation

time is short; results are obtained in <24 h. Confirmation of a
suspected genotoxic substance can be made in 5 7 2 h.

8.1.3 The assay is technician friendly, requiring minimal technical

or microbiological training. Prepackaged dehydrated media,
freezed-dried bacteria, and standard disposable reaction tubes
require only a few hydration, mixing, and dilution steps to
initiate the test-all without the rigors, tedium, and cost of
sterile technique. Bacteria are clonal and require no reisolation
or reculturing and are completely disposable.

8.1.4 The volume of the reactants is low, reducing the quantity of

toxic wastes and cost of their disposal.

8.1.5 A positive control in the Mutatox Assay is used to ascertain

the acceptable sensitivity of the Photobacterium phosphoreum
strain. The negative control is used to determine viability and
spontaneous light emission of the dark mutant bacteria.

8.1.6 Although all genotoxins produce DNA damage, they do not

represent a homogenous group of chemicals. Some experimen-
tation may be needed that elicits a normal dose response.

8.1.7 Hepatic S9 has been obtained from Aroclor-1254 induced rats,

and used as a surrogate metabolic activation system in both
procaryotic and eucaryotic genotoxicity tests for many years.
Although rat S9 is readily available, commercially the protein
concentrations between batches and sources may vary signifi-
cantly. Protein concentrations should be determined for each
batch on a milligram per milliliter basis. Mixed-function oxi-
dase activity, concentrations of S9, and incubation temperature
are three important factors influencing Mutatox sensitivity.

8.2 The genotoxicity end point of this assay is conservative because

the effect of DNA damage on somatic and germinal cells is
potentially devastating. The final designation of negative (i.e.,
no detectable evidence of genotoxic activity) is made only after
the mean values of at least 30 data points (i.e., three replicate
tests, Section 7.1) over a 1OOX concentration is determined.
 MUTATUX ASSAY FOR GENOTOXIC SUBSTANCES/ 113

8.3 The Mutatox Assay is quality assurance and quality control

friendly because archival tester strains and test media are eas-
ily stored for inspection or audit.

8.4 The activated Mutatox Genotoxicity Assay compares favorably

with the well-known and validated Ames Salmonella Mutage-
nicity Test. The spectra of sensitivity with seven known or
suspected arylamines and polycyclic aromatic hydrocarbon pro-
genotoxins show both qualitative and quantitative similarities;
the lowest genotoxic concentrations are in the low microgram
range (51)for both bacterial assays. In the Ames test, toxic
chemicals frequently induce bacterial cytotoxicity necessitat-
ing additional tests to demonstrate reversion from auxotroph
to prototroph of the histidine-deficient tester strain Salmonella.
The Mutatox tester strain Photobacterium is not a nutritional
auxotroph; therefore, it does not require a tedious and time-
consuming confirmation test.

8.5 The two Photobacterium bioassays marketed by Microbics

Corp., Mutatox and Microtox, should not be confused. In the
Mutatox Genotoxicity Assay, nonglowing luminescent bacteria
(dark mutants) are exposed to a test substance, and the amount
of light increase indicates the degree of genotoxicity in the
sample. Conversely, in the Microtox Acute Toxicity Assay,
glowing luminescent bacteria are exposed t o a test substance,
and the amount of light decrease indicates the degree of toxicity
of the sample.

8.6 The three most common indices of performance-sensitivity,

specificity, and predictability-suggest that the activated Mu-
tatox Genotoxicity Assay is a valuable monitoring tool to detect
genotoxins.