19
84
TABLE OF CONTENTS
4.1. INTRODUCTION
4.2. MATERIALS AND METHODS
4.2.1. Bacterial Strains
4.2.1.1. Long Term Storage and Propagation of the Tester Strains
4.2.1.2. Preparation of Frozen Working Cultures
4.2.1.3. Growing Overnight Cultures
4.2.2. Genetic Analysis
4.2.3. Preparation of Mutagens
4.2.4. Preparation of Aqueous Extract of Tobacco
4.2.5. Toxicity Determination of MZ to Salmonella Strains
4.2.6. Determination of Anti-mutagenic Potential of MZ
4.2.6.1. Direct Acting Mutagens
4.2.6.2. Mutagens Needing Microsomal Activation
4.2.6.3. Tobacco Extract
4.3. RESULTS
4.3.1. Genotypes Confirmation of the Salmonella Tester Strains
4.3.2. Non Toxicity of MZ to Tester Strains
4.3.2. Antimutagenic Effect of MZ Against Different Mutagens
4.3.2.1. Sodium Azide (NaN3)
4.3.2.2. 4- Nitro-O-Phenylene Diamine (NPD)
4.3.2.3. N-Methyl- N-nitro-N-Nitrosoguanidine (MNNG)
4.3.2.4. Acetaminofluorene (AAF)
4.3.2.5. Tobacco Extract
4.4. DISCUSSION
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4.1. INTRODUCTION
The Ames Salmonella/microsome mutagenicity assay (Salmonella test; Ames
test) is a short-term bacterial reverse mutation assay specifically designed to detect a
wide range of chemical substances that can produce genetic damage that leads to gene
mutations. The identification of substances capable of inducing mutations has become
an important procedure in safety assessment. Ames test is used world-wide as an
initial screen to determine the mutagenic potential of new chemicals and drugs. The
test is also used for submission of data to regulatory agencies for registration or
acceptance of many chemicals, including drugs and biocides (Mortelmans and Zeiger,
2000). Since many of the carcinogens are potent mutagens, research work related to
discovery, characterization and use of anti-mutagenic agent is highly relevant with
respect to identification of anti-carcinogenic substances.
Carotenoids, one of the phytonutrients belonging to the category of
tetraterpenoids, are found abundantly in dark green leafy vegetables and fruits. More
than 700 carotenoids have been discovered thus far. Of these, only about a dozen have
been studied closely. A voluminous body of literature including in vitro studies,
animal studies, human observational studies and clinical trials has suggested a
multiplicity of health effects of carotenoids in human (Krinski, 1993). meso-
Zeaxanthin (MZ) is one of the xanthophyll carotenoid present in the macula lutea of
primate’s retina and reported to have a role in the prevention of age-related macular
degeneration (AMD). MZ is a powerful antioxidant (Firdous et al., 2010) owing to its
extensive conjugated double bonds. The present study was aimed to explore the anti-
mutagenic potential of MZ using a variety of mutagenic substances acting either
directly or after activation by cytochrome P450 enzymes.
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4.2. MATERIALS AND METHODS
4.2.1. Bacterial Strains
Histidine requiring Salmonella typhimurium strains TA 98, TA 100, TA102
and TA 1535 were used.
4.2.1.1. Long Term Storage and Propagation of the Tester Strains (Preparation
of Frozen Permanent Cultures)
The new strain, which came as lyophilized culture, was rehydrated by
aseptically adding 1 ml of nutrient broth and then the rehydrated culture was
transferred to 4 ml of nutrient broth. A drop of the rehydrated culture was transferred
to a nutrient agar plate and streaked the inoculum for individual colonies across the
surface of the plate. After overnight incubation at 370C, the agar plates were observed
for growth. One healthy colony was picked from the plate and re-streaked it for
individual colonies on glucose minimal (GM) agar plates supplemented with an
excess of biotin and histidine. For the strains carrying plasmid pKM101 and pAQ1,
the agar plates were additionally supplemented with ampicillin and tetracycline
respectively. This purification step was repeated two times. Single colonies were
picked from the second purification plate and transferred to a glucose minimal agar
plate supplemented with the appropriate nutrients/antibiotics and were kept as the
master plates. The master plates were then incubated for 2 days at 370C. A small
inoculum from each of the single isolated colonies on the master plates were
inoculated into 5 ml of nutrient broth and incubated overnight at 370C. Tests for the
confirmation of the genotypes (strain check) of the tester strains were carried out.
Upon completion of the strain check, the colony that had given the best overall results
in terms of genotypic characteristics was selected from the master plate. The colony
was then inoculated into 4.5 ml of nutrient broth and incubated overnight at 370C till
the cells reached a density of 1-2x109 Colony Forming Units (CFU)/ml (O.D. at
540nm is between 0.1 and 0.2). 0.5 ml of DMSO (sterile cryopreservative) was added
to the bacterial culture (final concentration, 10%, v/v) and mixed thoroughly. 1 ml
aliquots were dispensed in sterile eppendorf tubes which were labeled as “permanent
cultures” and stored at −700C.
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4.2.1.2. Preparation of Frozen Working Cultures
The working cultures were prepared by inoculating a small inoculum of frozen
permanent culture into 5 ml of nutrient broth. This procedure was done quickly to
prevent the frozen permanent culture from thawing which might result in decreased
viability and loss of plasmid(s). After overnight incubation at 370C, a loopful of the
culture was streaked on glucose minimal agar plates supplemented with an excess of
biotin and histidine. For the strains carrying plasmid pKM101and pAQ1, the agar
plates were additionally supplemented with ampicillin and tetracycline respectively.
One healthy looking colony was purified twice. Single colonies were picked up from
the second purification plate and transferred to glucose minimal agar plates
supplemented with the appropriate nutrients/antibiotics. These plates were kept as the
master plates. All the subsequent steps were identical to those described above for the
preparation of the frozen permanent cultures and the eppendorf tubes were labeled as
“working cultures”.
4.2.1.3. Growing Overnight Cultures
For each experiment, the tester strain cultures were grown overnight in
nutrient broth to a density of 1–2x109 Colony Forming Units (CFU)/ml.
4.2.2. Genetic Analysis
The tester strains were analyzed for their genetic integrity and spontaneous
mutation rate when frozen cultures were prepared. The strain check was performed
again before an experiment was done. The strain check was performed with bacterial
cultures in the following way.
- Histidine Dependence (his)
For histidine dependence study, the tester strain cultures of TA 98, TA 100, TA
102 and TA 1535 were streaked in parallel stripes using sterile swabs across glucose
minimal agar plates supplemented with an excess of biotin. The plates were incubated
overnight at 370C and the growth was examined after 24 hours.
- Biotin Dependence (bio)
For biotin dependence study, the tester strain cultures of TA 98, TA 100, TA 102
and TA 1535 were streaked in parallel stripes using sterile swabs across glucose
minimal agar plates supplemented with an excess of histidine. The plates were
incubated overnight at 370C and the growth was examined after 24 hours.
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- Biotin and Histidine Dependence (bio:his)
For biotin and histidine dependence study, the tester strain cultures of TA 98, TA
100, TA 102 and TA 1535 were streaked in parallel stripes using sterile swabs across
glucose minimal agar plates supplemented with an excess of biotin and histidine. The
plates were incubated overnight at 37oC and the growth was examined after 24 hours.
- rfa Mutation
For rfa mutation study, 0.1 ml of overnight cultures of the tester strains (TA 98,
TA 100, TA 102 and TA 1535) were added to a tube containing 2 ml of molten agar
held at 45oC. The top agar tubes were vortexed for 3 seconds at low speed and poured
on nutrient agar plates without biotin and histidine. The plates were tilted rotated for
even distribution of the top agar on the plates. The plates were placed on a leveled
surface and allowed several minutes for agar to become firm. 10 µl of 1 mg/ml
solution of crystal violet was pipetted into the centre of sterile paper disc and discs
were transferred to each of the plates using sterile forceps. The discs were lightly
pressed with forceps to embed it slightly in the overlay. The plates were incubated
overnight at 37oC and observed for crystal violet sensitivity after 24 hours.
- uvrB Deletion
For uvrB deletion study, the tester strain cultures of TA 98, TA 100, TA 102 and
TA 1535 were streaked in parallel stripes using sterile swabs across nutrient agar
plates. These plates were irradiated with UV lamp at a distance of 35cm for 8
minutes. The plates were incubated overnight at 37oC and the growth was examined
after 24 hours.
- Presence of Plasmid pKM101 (Ampicillin Resistance)
The tester strain cultures of TA 98, TA 100, TA 102 and TA 1535 were streaked
in parallel stripes using sterile swabs across glucose minimal agar plates
supplemented with ampicillin (24 µg/ml) and an excess of biotin and histidine. The
plates were incubated overnight at 37oC and the growth was examined after 24 hours.
- Presence of Plasmid pAQ1 (Tetracycline Resistance)
The tester strain cultures of TA 98, TA 100, TA 102 and TA 1535 were streaked
in parallel stripes using sterile swabs across glucose minimal agar plates
supplemented with tetracycline (2 µg/ml) and an excess of biotin and histidine. The
plates were incubated overnight at 370C and the growth was examined after 24 hours.
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4.2.3. Preparation of Mutagens
All the mutagens, MNNG (1 μg/plate), AAF (20 μg/ plate), NaN3 (2.5
μg/plate) were dissolved in water, except NPD (20 μg /plate) which was dissolved in
DMSO.
4.2.4. Preparation of Aqueous Extract of Tobacco
Tobacco (100g) was cut into small pieces and boiled in 500 ml of distilled
water for 1 hour. This was then evaporated to dryness in vacuum. Earlier studies have
reported that 50 mg of tobacco extract/plate produces maximum mutagenic response
of Salmonella tester strain TA 102 (Sukumaran and Kuttan, 1995). Hence, this
concentration was used for the studies.
4.2.5. Toxicity Determination of MZ to Salmonella Strains
A preliminary toxicity determination of MZ (1000-5000 μg/plate) was done to
determine an appropriate dose range for the mutagenicity assay using the strains TA
98, TA 100, TA 1535 and TA 102. Untreated plates and plates treated with DMSO
were also kept. The plates were incubated for 48 hours and observed for thinning or
absence of bacterial colonies.
4.2.6. Determination of Anti-mutagenic Potential of MZ
4.2.6.1. Direct Acting Mutagens
Anti-mutagenicity of MZ was tested in S.typhimurium strains TA 1535, TA
102, TA 98 and TA 100 with and without microsomal activation by Ames test (Maron
and Ames, 1983). Plate incorporation method was adopted for mutagenicity assay of
direct acting mutagens. Different concentrations of MZ (50, 100 and 250 μg/plate)
were added to 2 ml of top agar containing 0.045 mM histidine/biotin, strains of
S.typhimurium (0.1 ml having 1X109 cells/ml) and different direct acting mutagens
(MNNG, NaN3 and NPD) at concentrations mentioned above. The mixture was
poured onto glucose minimal agar plates and incubated at 370C for 48 hrs. After
incubation, the number of revertant colonies were counted using colony counter. The
plates with mutagen were considered as positive controls and plates without test
sample and mutagen were considered as negative controls or spontaneous revertants.
The samples were tested against sodium azide in strains TA 100, TA 102 and TA
1535, against NPD in TA 100 and TA 98 and against MNNG in TA 100 and TA
90
1535. Each sample was assayed using triplicate plates and values were expressed as
mean ± SD.
4.2.6.2. Mutagens Needing Microsomal Activation
Microsomal activation was done by pre-incubation method for the metabolic
activation of indirect acting mutagens (Matsushima et al., 1980). Microsomal P450
enzymes were induced in rat liver by oral administration of 0.1% phenobarbitone
dissolved in water for 4 days. The animals were sacrificed on the 5th day after
phenobarbitone administration. Livers were excised aseptically and microsomal S9
fractions were prepared by centrifuging the liver homogenates at 9000g for 15
minutes. S9 activation mix was prepared by mixing S9 fraction (1 ml) with 5 ml
sodium phosphate buffer (0.2 M, pH 7.4), 400 µl NADP (0.1 M), 50µl glucose-6-
phosphate (1 M, pH 7.4) and 200 μl MgCl2–KCl. 500 μl of S9 activation mixture was
mixed with an indirect acting mutagen viz AAF (20 μg/plate), different concentrations
of MZ (50, 100 and 250 μg/plate) and 0.1 ml of bacterial strains (TA 98 and TA 100).
The activation mixture was incubated at 370C for 45 minutes. After incubation the
activation mixture was added to 2 ml of top agar containing 0.045 mM histidine/biotin
and poured onto minimal agar plates. The plates were then incubated for 48 hours at
37oC. The number of revertant colonies were counted using colony counter. The
plates with mutagen were considered as positive controls and plates without test
sample and mutagen were considered as negative controls or spontaneous revertants.
Each sample was assayed using triplicate plates and values were expressed as mean ±
SD.
4.2.6.3. Tobacco Extract
Anti-mutagenicity of MZ was tested in S.typhimurium strain TA102 against
the aqueous extract of tobacco by the plate incorporation method. Fresh bacterial
culture (0.1 ml, 1-2×109 cells/ml) was mixed with 2 ml of top agar containing
histidine and biotin, different concentrations of MZ (50, 100 and 250 μg/plate), and
tobacco extract (50 mg/plate). Further it was poured on to minimal glucose agar plate
and incubated for 48 hr at 37oC. After incubation, revertant colonies were counted
using colony counter. The plates with mutagen acted as positive control and plates
without test sample and mutagen were considered as negative controls or spontaneous
revertants. Each sample was assayed using triplicate plates and expressed as mean ±
SD.
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The percentage of inhibition of mutagenicity was calculated using the formula
[(R1-SR) – (R2-SR)] × 100/ (R1-SR) where R1 is the number of revertants in the
presence of mutagen alone, R2 is the number of revertants in the presence of drugs
and SR is the spontaneous revertants.
4.3. RESULTS
4.3.1. Genotypes Confirmation of the Salmonella Tester Strains
Result of histidine dependence (his) study showed that all the Salmonella
strains were histidine dependent as there was no growth on glucose minimal agar
plates supplemented with an excess of biotin [Fig 4.1(a)]. Result of biotin dependence
(bio) study showed no growth on glucose minimal agar plates supplemented with an
excess of histidine except for strain TA102 which is biotin independent [Fig 4.1(b)].
This indicated that all the Salmonella strains except TA102 were biotin dependent. A
deletion mutation through the uvrB-bio genes was present in all tester strains, except
in TA102. The uvrB mutation is part of a deletion mutation extending into a gene for
biotin synthesis; therefore, the biotin requirement is a result of the deletion of this
region. In the biotin and histidine dependence (bio:his) study, growth was observed
with all the strains on glucose minimal agar plates supplemented with an excess of
biotin and histidine [Fig 4.1(c)]. In rfa marker study, all the Salmonella strains
showed a zone of growth inhibition around the disc containing crystal violet on a
glucose minimal agar plate supplemented with an excess of biotin and histidine [Fig
4.1(d)]. This indicated that rfa mutation was present in all the strains. rfa mutation led
to a defective lipolysaccharide (LPS) layer making the bacteria more permeable to
bulky chemicals like crystal violet. Result of uvrB deletion study showed that strain
TA 102 did not have uvrB mutation in its genome because of that it was not affected
by the exposure to ultraviolet light. So TA 102 could grow both in presence and
absence of ultraviolet exposure while other strains did not grow when exposed to UV
light [Fig 4.1(e)]. In the study for the presence of plasmid pKM101 (ampicillin
resistance), growth was observed with all the strains except with TA 1535 (since all
strains except TA 1535 carrying plasmid pKM101) on glucose minimal agar plates
supplemented with ampicillin (24 µg/ml) and an excess of histidine and biotin [Fig
4.1(f)]. In the study for the presence of plasmid pAQ1 (tetracycline resistance), there
was growth only with strain TA102 on glucose minimal agar plates supplemented
92
with tetracycline (2 µg/ml) and an excess of histidine and biotin. TA102 is the only
strain that carries plasmid pAQ1 [Fig 4.1(g)].
4.3.2. Non Toxicity of MZ to Tester Strains
MZ was found to be non-toxic up to a concentration of 5000 μg/plate in all the
tester strains used compared to the control and DMSO control. This can be interpreted
from the result that there was no thinning or complete absence of bacterial colonies by
MZ addition.
4.3.2. Antimutagenic Effect of MZ Against Different Mutagens
4.3.2.1. Sodium Azide (NaN3)
MZ significantly reduced the revertants induced by NaN3 in TA100, TA102
and TA1535 strains in a dose dependent manner. The inhibition of mutagenicity was
found to be 46.70, 64.50 and 97% for TA100 strain, 44, 86.6 and 93.80 % for TA102
strain and 46.40, 68.50 and 79.80 % for TA1535 at concentrations 50, 100 and 250 µg
MZ/plate (Table 4.1).
4.3.2.2. 4- Nitro-O-Phenylene Diamine (NPD)
MZ showed significant anti-mutagenic activity against NPD induced
mutagenicity. Percentage of inhibition increased with an increase in the concentration
of MZ, which is 48, 62.7 and 65 % for TA98 and 49.1, 57.3 and 62.8 % for TA100 at
concentrations of 50, 100 and 250 µg/plate of MZ (Table 4.2).
4.3.2.3. N-Methyl- N-nitro-N-Nitrosoguanidine (MNNG)
MZ inhibited mutagenesity produced by MNNG to S.typhimurium strains
TA100 and TA1535. The inhibition of mutagenesity was 39, 51.94 and 56.8 % for
TA100 and 32.5, 56.23 and 63.9 % for TA1535 at 50, 100 and 250 µg/plate of MZ
(Table 4.3).
4.3.2.4. Acetaminofluorene (AAF)
MZ inhibited mutagenesity induced by AAF, which needs microsomal (S9)
activation. The inhibition of mutagenesity in S.typhimurium strain TA98 was 45.4,
63.7 and 77.4% and in TA100 strain was 47.7, 66.7 and 86.2 % at concentrations 50,
100 and 250µg/plate (Table 4.4).
Table 4.1. Antimutagenic Activity of MZ on Mutagenicity Induced
by NaN3 in S.typhimurium Strains TA100, TA102 and TA1535
Concentration of
drug (µg/ plate)
Average
number of
revertants/ plate
Average
number of
revertants/
plate
Average
number of
revertants/
plate
TA100 TA102 TA1535
NaN3 979 ± 49.6 608± 51.7 847 ± 48.7
NaN3+DMSO 633 ± 37.8 517 ± 31.6 756± 48.7
NaN3+ 50µg MZ
557 ± 37.4***
(46.7%)
368 ± 33.1***
(44%)
461± 42.4***
(46.4%)
NaN3+ 100µg MZ
396 ± 29.6***
(64.5%)
136 ± 27.3***
(86.6%)
277 ± 19.0***
(68.5%)
NaN3+ 250µg MZ
102 ± 19.4***
(97%)
97 ± 17.3***
(93.8%)
168±21.0***
(79.8%)
SR 75 ± 12.6 63 ± 9.6 15 ± 8.0
The values are mean ± SD of 3 different determinations. The mean values
were statistically analyzed by one way analysis of variance (ANOVA) followed by
appropriate post hoc test (Dunnett’s multiple comparison test) using Graph pad Instat
3 Software. ***P < 0.001 significant against mutagen treated positive control groups.
SR is spontaneous reversion. Values in parentheses indicated percentage inhibition.
Percentage inhibition was calculated from NaN3 alone treated group.
Table 4.2. Antimutagenic Activity of MZ on Mutagenicity
Induced by NPD in S.typhimurium Strains TA98 and TA100
Concentration of drug
(µg/ plate)
Average number
of revertants/ plate
Average number
of revertants/
plate
TA98 TA100
NPD 1110 ±43.6 790 ±52.9
NPD+DMSO 1082 ± 34.2 786 ± 61.2
NPD+ 50µg MZ
589 ± 48.7
(48%)
459 ± 32.4***
(49.1%)
NPD+ 100µg MZ
429 ± 26.3
(62.7%)
404 ± 25.8***
(57.3%)
NPD+ 250µg MZ
404 ± 31.3
(65%)
367 ± 26.4***
(62.8%)
SR 22 ± 6.2 116 ± 14.6
The values are mean ± SD of 3 different determinations. The mean values
were statistically analyzed by one way analysis of variance (ANOVA) followed by
appropriate post hoc test (Dunnett’s multiple comparison test) using Graph pad Instat
3 Software. ***P < 0.001 significant against mutagen treated positive control groups.
SR is spontaneous reversion. Values in parentheses indicated percentage inhibition.
Percentage inhibition was calculated from NPD alone treated group.
Table 4.3. Antimutagenic Activity of MZ on Mutagenicity
Induced by MNNG in S.typhimurium Strains TA100 and TA1535
Concentration of
drug
(µg/ plate)
Average number of
revertants/ plate
Average
number of
revertants/ plate
TA100 TA1535
MNNG 742 ± 18.9 520 ± 13.6
MNNG +DMSO 713 ± 27.0 509 ± 19.7
MNNG + 50µg MZ
500 ± 24.0***
(39%)
358 ± 20.0***
(32.5%)
MNNG + 100µg MZ
420 ± 26.6***
(51.94%)
240±17.5***
(56.23%)
MNNG + 250µg MZ
396 ±26.8***
(56.8%)
202 ±16.9 ***
(63.9%)
SR 122 ± 7.8 22 ± 5.4
The values are mean ± SD of 3 different determinations. The mean values
were statistically analyzed by one way analysis of variance (ANOVA) followed by
appropriate post hoc test (Dunnett’s multiple comparison test) using Graph pad Instat
3 Software. ***P < 0.001 significant against mutagen treated positive control groups.
SR is spontaneous reversion. Values in parentheses indicated percentage inhibition.
Percentage inhibition calculated from MNNG alone treated group.
Table 4.4. Antimutagenic Activity of MZ on Mutagenicity Induced by
AAF in S.typhimurium strains TA98 and TA100 in the Presence of S9 Mix
Concentration of drug
(µg/ plate)
Average number of
revertants/ plate
Average number of
revertants/ plate
TA 98 TA100
AAF 515 ± 18.9 603± 21.3
AAF+ DMSO 500±12.4 588±17.4
AAF+ 50µg MZ 300 ± 6.7***
(45.4%)
415 ± 11.0***
(47.7%)
AAF+ 100µg MZ 213± 12.4***
(63.7%)
341± 11.9***
(66.7%)
AAF+ 250µg MZ 148 ± 13.0***
(77.4%)
265 ± 9.9***
(86.2%)
SR 41 ± 3.0 211 ± 6.1
The values are mean ± SD of 3 different determinations. The mean
values were statistically analyzed by one way analysis of variance (ANOVA)
followed by appropriate post hoc test (Dunnett’s multiple comparison test) using
Graph pad Instat 3 Software. ***P < 0.001 significant against mutagen treated
positive control groups.
SR is spontaneous reversion. Values in parentheses indicated percentage
inhibition. Percentage inhibition calculated from AAF alone treated group.
93
4.3.2.5. Tobacco Extract
Earlier studies have reported that 50 mg of tobacco extract/plate produced
maximum mutagenic response to Salmonella tester strain TA102. MZ showed
significant anti-mutagenic activity against tobacco induced mutagenicity. The
inhibition of mutagenicity by MZ in S.typhimurium strain TA102 was found to be
19.8, 51.56 and 79.4 % at concentrations 50, 100 and 250 µg/plate (Table 4.5).
4.4. DISCUSSION
Ames test serves as a quick way to analyze the mutagenic potential of a
compound. The test uses a number of Salmonella strains with pre-existing mutations
in the histidine operon that make the bacteria unable to synthesize the required amino
acid-histidine. So these bacteria could not grow and form colonies in a histidine
deficient medium. New mutations at the site of these pre-existing mutations or nearby
in the genes can restore the gene’s function and allow the cells to synthesize histidine.
These newly mutated cells can grow in the absence of histidine and form colonies. For
this reason, the test is often referred to as a “reversion assay” (Ames et al., 1975).
When the Salmonella tester strains were grown on a glucose minimal agar plate
containing a trace of histidine, only those bacteria that could revert to histidine
independence i.e., spontaneous revertants were able to form colonies. The number of
spontaneously induced revertant colonies per plate is relatively constant. However,
when a mutagen was added to the plate, bacteria reverse back to histidine independent
and form colonies in histidine deficient medium. Addition of anti-mutagenic agents
considerably reduces reverse mutation capability of mutagens.
The Salmonella strains used in the test have different mutations in various
genes in the histidine operon; each of these mutations is designed to be responsive to
mutagens that act via different mechanisms. TA1535 and TA100 detect mutagens
causing base pair substitutions, TA 98 detects frame shift mutagens and TA102
detects mutagen causing oxidative damage (Levin et al., 1982). Mutagens may be
either direct acting or requiring microsomal activation. Direct acting mutagens
interact directly with DNA to produce mutation. In this study when direct acting
mutagens like NaN3, NPD and MNNG were added to histidine deficient glucose
minimal agar plates, there were substantial increase in bacterial colonies. In NaN3
alone treated positive control plates, the increase in bacterial colony formations were
Table 4.5. Antimutagenic Activity of MZ on Mutagenicity
Induced by Tobacco Extract in S.typhimurium Strain TA102
The values are mean ± SD of 3 different determinations. The mean values
were statistically analyzed by one way analysis of variance (ANOVA) followed by
appropriate post hoc test (Dunnett’s multiple comparison test) using Graph pad Instat
3 Software. ***P < 0.001 significant against mutagen treated positive control groups.
SR is spontaneous reversion. Values in parentheses indicated percentage inhibition.
Percentage inhibition calculated from tobacco extract alone treated group.
Concentration of drug
(µg/ plate)
Average number of
revertants/ plate
Tobacco extract 712 ± 34.6
Tobacco extract + DMSO 699 ± 37.5
Tobacco extract + 50µg MZ 585 ± 26.3***
(19.8%)
Tobacco extract+100µg MZ 382 ± 17.3***
(51.56%)
Tobacco extract + 250µg MZ 204 ± 10.8***
(79.4%)
SR 72 ± 17.5
94
13 fold for TA 100, 10 fold for TA 102 and 57 fold for TA 1535 when compared to
those of spontaneous revertants. In NPD alone treated positive control plates, there
were 51 fold increase in the bacterial colony formations for TA 98 and 7 fold increase
for TA 100 when compared to those of spontaneous revertants. In MNNG alone
treated positive control plates, the bacterial colony formations were increased to 6 fold
for TA 100 and 24 fold for TA 1535 when compared to those of spontaneous
revertants. This indicated that all the direct acting agents used were able to reverse the
pre-existing mutations in the bacteria. Addition of MZ at different concentrations (50,
100 and 250 μg/plate) significantly reduced the number of revertant colonies by
inhibiting the reversion mutation capabilities of these mutagens in a concentration
dependent manner (IC50<100 μg/plate).
In tobacco alone treated positive control plates, there was 10 fold increase in
the bacterial colony formation for strain TA 102 when compared to that of
spontaneous revertants. But the addition of MZ at different concentrations (50, 100
and 250 μg/plate) significantly reduced the number of revertant colonies produced by
tobacco in a dose dependent manner (IC50<100 μg/plate). In plates treated with the
highest dose of MZ, the number of revertant colonies per plate was reduced to 79.4%.
Indirect acting mutagens like AAF require microsomal activation to induce
mutation. In human and rodents, the cytochrome-based P450 metabolic oxidation
system is present mainly in liver and to a lesser extent in lungs and kidneys. These
cytochrome P450 enzymes are capable of metabolizing a large number of indirect
acting chemicals to DNA-reactive electrophilic forms. Since bacteria do not have this
metabolic capability, an exogenous mammalian organ activation system needs to be
added to the petri plate together with the test chemical and the bacteria. For this
purpose, a rodent (rat) metabolic activation system was introduced into the test system
(Garner et al., 1972). To increase the level of metabolizing enzymes, the rats were
pre-treated with the mixed-function oxidase inducer viz phenobarbitone. In the
present study we can see that in positive control plates, there was an induction of
reverse mutations in bacteria by the addition of AAF in the presence of a rat liver
metabolic activation system. This was evident from the increase in number of
revertant colonies per plate. The increase in the number of bacterial colonies was 3
fold for TA 100 and 13 fold for TA98 when compared to that of spontaneous
revertants. But the addition of MZ at different concentrations (50, 100 and 250
95
μg/plate) markedly reduced the number of revertant colonies produced by AAF in a
dose dependent manner (IC50<100 μg/plate). In plates treated with the highest dose of
MZ i.e. 250 μg/plate, there was 77.4% reduction in number of revertant colonies per
plate for strain TA 98 and 86.2% reduction for strain TA 100.
Present study clearly indicates that MZ has significant anti-mutagenic activity
against direct acting mutagens and mutagens needing microsomal activation. Since
most of the carcinogens are mutagens, this work reveals the relevance of carotenoid
MZ in the anticancer studies.
