Chapter 4

RESULTS AND DISCUSSIONS

This chapter includes evaluation of mutagenic potential of Nitrite-based

preservatives, showing the results and the discussions of the study. Results of this study

will answer the research objectives during the conduct of the study .

Lethal Dose of Prague Powder 1 to Allium cepa L.

In the different concentrations of Prague Powder 1 used in the FRT, LD50 was

determined at 600 ppm. This was consequently used as the standard concentration to

induced chromosomal aberrations to Allium cepa L.. The concentration was also used as

the positive control in the subsequent study. Figure 4.1 shows the result of Find Ranging test,

where Allium cepa roots were measured metrically getting half of population was compared to

the control set up. The lethal dose50 was found in 400 ppm and 600 ppm but the latter

concentration was used as LD50 because it is nearer to the LD50 obtained during the dry

run of LD50 test which was 700 ppm.. Results revealed that Allium cepa exposed to

different concentrations of curing salt had an effect due to its decreasing root length

compared to control.

Figure 4.1 Find Ranging Test

Figure 4.1 shows the result of Find Ranging test, Lethal Dose 50 was found in 400 ppm and 600 ppm

Morphological Responses of Allium cepa L. to Prague Powder 1

26

control

Results in this test were shown in Figure 4.2 where the variable responses in the average

number of Allium cepa L. roots subjected to different concentration of Prague powder 1. The

findings showed that Prague powder 1 increased the root numbers and root length in low

and medium concentrations unlike to control group. Nonetheless, in high treatment

compared to control, root length decreased due to RNA interference that binds together to

protect roots from Prague powder 1. RNA is a vital part of immune system in viruses and

other foreign bodies, also, in plants wherein it prevents the self-propagation of

transposons medium treatment. In addition, Robinson (2005) stated that RNAi doesn’t

just shut up gene regulation but also used to protect an organism from genes of viruses or

foreign bodies. The medium treatment(300 ppm in Figure 4.2 and Figure 4.3) gained the

greatest number of roots and mean root length compared to other treatment. This result

was explained by Mochizuki (2014) that treatments with Nitrite increased the formation

of roots. Unlike the study of (Olorunfemi and Ehwre, 2011) that maximum root growth

was achieved in control group. However, for other treatments, control, low and high

concentration of curing salt, among the five replicates in each treatment only 4 bulbs

sprouted root. Decrease in the number of roots could be attributed to the inhibition of

prophase which may lead to abnormal mitosis (Shannguan, 2004). Increased in the

number of roots in 300 ppm and 600 ppm of Prague powder 1 was attributed to the

enhancing effect of nitrite present in Prague powder no. 1 compound (De Jesus and

Yllano, 2005).

Values of root length in both control and treated group were subjected to Analysis

of Variance. The p – level of root length was 0.022917 and was found significant to the

chromosomal aberration induced by curing salt in Allium cepa L. where root number p-

27

level shows 0.060692 and recorded as not significant value for chromosomal aberrations.

The outcome of macroscopic analysis was different from the study of Fiskesjo (1985) and

Olorunfemi et. al. (2011) in which macroscopical parameters has a significant correlation

in the aberration of chromosomes brought by food preservatives or chemicals exposed to

onions. However, the remarkable increase in root length observe in Allium cepa at 300

ppm curing salt concentration was due to the presence of growth promoting element like

nitrogen (De Jesus and Yllano, 2005).

control 150ppm 300ppm 600ppm0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

TREATMENT

RO

OT

LEN

GTH

Figure 4.2 Mean root length

Figure 4.2 Average length of Allium cepa L. root under different concentration of

Prague powder 1.

28

control 150ppm 300ppm 600ppm0

2

4

6

8

10

12

14

16

TREATMENT

RO

OT

NU

MB

ER

Figure 4.3 Mean root numberFigure 4.2. Shows the average number of Allium cepa L. under different concentration of

Prague powder 1.

Toxicity Symptoms of Prague Powder 1

Accounted from the result were the morphological deformities observed in tested

concentrations were browning, hooked, short, bent, spiral and crochet-like roots and no

root growth. These values indicate that the samples were toxic comparatively, from 96

hours of exposing the roots under tested concentrations (Olorunfemi, et. al. 2011).

Symptoms observed in the roots of Allium cepa L. subjected to Prague Powder 1

treatments were shown in Figure 4.3. Prominent symptoms of toxicity includes

browning of the root tips because of the toxic effects which causes the death of the cell,

bending of the roots, and abnormal swelling of the roots. Browning of the root tips,

29

abnormal swelling of the roots, hooked and twisted roots were found in 600 ppm were

shown in figure 4.8. In 300 ppm, shown in Figue 4.7 some toxicity recorded were crooked roots

and swelling. ppm. In 300 ppm, crooked roots and swelling in some was also observed. In

150 ppm, twisted, hooked and no growth of roots were also the findings shown in Figure

4.6. In contrast, the Allium cepa bulbs in Figure 4.5 ,exposed to distilled water (control)

manifested no root growth was just the toxicity symptoms. Accounted from the result were the

morphological deformities observed in tested concentrations were browning, hooked, short, bent,

spiral and crochet-like roots and no root growth at a concentration. These values indicates that

the samples were toxic; comparatively, from 96hours of exposing the roots under tested

concentrations. (Daniel I. Olorunfmi and Emanuel O. Ehwra, 2011). Therefore, the higher the

concentration of Prague powder 1, the greater effect of its toxin on its roots.

30

Figure 4.4. Shows the macroscopic analysis of roots in A.treatment 1 (control), B. treatment 2 (150 ppm), C. treatment 3 (300 ppm) and D. treatment 4 (600 ppm).

31

D

C

B

A

Figure 4.5. Shows the normal morphological responses of Allium cepa roots on treatment 1 (control).

32

Figure 4.6. Shows the hooked, crooked and twisted morphological responses of Allium cepa roots under treatment 2 (150 ppm).

33

Figure 4.7. Swelling, browning, twisting and hooked morphological responses of Allium cepa roots under treatment 3 (300 ppm).

34

Figure 4.8. Twisting, browning, hooked, crooked and swelling of roots responses of Allium cepa exposed to treatment 4 (600 ppm).

35

Mitotic Index

Thus mitotic index was measured the actively growing cells. Figure 4.9 shows

the mean mitotic index of Allium cepa root tip following exposure to different Prague

powder no. 1 concentrations. Findings revealed that the lower mean of mitotic index of

samples exposed to different Sodium Nitrite-based preservative treatments. This result

agrees with the previous studies on Allium cepa of Kumar, et. al. (2007) and Gomurgen

(2004) which reduced the mitotic index and chromosomal mutation when exposed to food

preservatives. It is similar with the results of Rencuzogullari, et. al. (2001a) in the

decreased of mitotic index at all concentrations and treatment periods using food

preservative, sodium metabisulfite. It was concluded that reduction of the mitotic activity

seems to be common effect of some food preservatives (Rencuzogullari, et. al. 2001a,

2001b). According to Fernandez, et. al. (2007), the cytotoxicity level of a test compound can be

determined based on the increase or decrease in the mitotic index (MI), which can be used as a

parameter of cytotoxicity in studies of environmental biomonitoring. Significant reduction in

mitotic index, noted in present study may be due to inhibition of DNA synthesis or the blocking

of G2 phase of the cell cycle (Khanna, M., Sharma, S. 2013).In the study presented by Njagi

and Gopalan (1982), reduced mitotic index inhibits the DNA synthesis due to food

additives sodium benzoate and sodium sulphite which induce the formation of anaphase

bridges, premature chromosome condensation and chromatin erosion in root meristems of

V. faba. The inhibition of DNA synthesis and decrease in mitotic index might be caused

due to the decrease in ATP level and the pressure from the functioning of energy

36

producing centre (Epel, 1963; Jain and Andsorbhoy, 1988) which blocks the G2-phase of

the cell cycle, preventing the cell from entering mitosis. Mitosis was the process of cell

division that functions as cell replacement, growth and sexual reproduction, thus absence

of mitosis due to reduced mitotic index inhibiting DNA synthesis cannot fulfil its role

(Gupta, 2009; Available online: http://mitosis.wikidot.com/lzimmermann-function-of-

mitosis ). ANOVA shows that p –level 0.003986 was statistically significant to the

reduction of mitotic index induced by Prague powder 1. In relation to the Philippine

Regulation on Food Additives those products made of curing salt or Prague powder 1 is

limited to 200 ppm concentrations or lowers if specified in some regulatory agencies.

Federal food safety only allowed the use of nitrate and nitrite not more than 200mg/kg in

curing meats and other products. Available online: http://foodrecap. net/health/safety/

curing-salt-safety. Control and 150 ppm were subjected to ANOVA and found out that p

– level 0.049543 has a significant value in the decreased of mitotic index as Allium cepa

where exposed to Prague powder 1 (Table 2).

37CONTROL 150 PPM 300 PPM 600 PPM

0

2

4

6

8

10

12

Mitotic index

Figure 4.9 Mitotic Index

As shown in Figure 4.9, mean mitotic Index of Allium cepa root tip subjected to

different concentrations of Prague Powder 1 and control. Mitotic index was reduced by

Prague Powder 1 under liquids treated.

Table 1. ANOVA analysis of Control and 150 ppm Mitotic Index

Effect df Effect MS Effect df Error MS Error F P-levelcontrol and 150 MI

1 1 37.5 4 4.833333 7.758621 0.049543

Mitotix index means were subjected to Duncan test and it was revealed in Table 2

that mean differences marked by the asterisks were significant at p < .05. So, between

treatment 1 and 2, T1 and T3, and T1 and T4 were statistically significant in the reducing

effect of mitotic index induced by Prague powder 1.

Table 2. Duncan Test of Mitotic index

TREATMEN{1}

9.333333{2}

4.333333{3}

2.500000{4}

4.500000

t1{1} .005778* .001104* .005708*

t2{2} .005778* .191854 .900191

t3 {3} .001104* .191854 .174015t4 {4} .005708* .900191 .174015

38

*statistically significant

Chromosomal Aberration

Changes in the morphology and structure of the chromosome have played a very

important role as indicator of genetic damage in both cancer and clinical studies. Levan

(1938) as illustrated in Figure 4.10, the mean chromosomal aberrations of Allium cepa

root tip cells after exposure to different concentrations of Prague Powder 1. It was

observed that Prague Powder concentration is inversely proportional with the number of

chromosomal aberrations.

Chromosomal aberration, clastogenic and physiological aberration as discussed by

(Khanna and Sharma, 2013) was also found in the result of the present study. Clastogenic

aberrations include chromatin bridge/s, chromosomal break/s and ring chromosome/s.

Conversely, the physiological aberrations include c-mitosis, vagrant/s, stickiness, delayed

anaphase and laggard/s. Results of study correlate with the study of (Khanna and Sharma,

2013) and Levan (1938) Figure 4.11 shows the chromosome aberrant cells figured out

from treatment 1, control. Some aberrations were identified as anaphase-bridge, breakage,

chromosomal breakage, and telophase-bridge. Figure 4.12 shows the identified

chromosome aberrant cells observed under 150 ppm. Some aberrant cells identified were

irregular anaphase, chromosomal breakage and anaphasic bridge, chromosomal breakage,

indicates telophase bridge, disturbed spindle fibers, breakage Figure 4.13 illustrated the

aberrant cells of Allium cepa L. roots under 300 ppm . Shown in Figure 4.14 some

identified aberrant cells at 600 ppm. Results of this current study agree with Turkoglu

(2007) which he mentioned that prompting lagging chromosomes were due to treatment

39

of food additives. In this aberration, chromosomes failed to attach to the spindle fiber and

to move either of the two poles.

Analysis of variance result of p-level of chromosomal aberrations was recorded as

0.132431 and statistically identified not significant value in mutagenic effect of Nitrite-

based preservative to Allium cepa root cells. The study of National Toxicology Program

(2001) shows that under the conditions of this 2-year drinking water study, there was no

evidence of carcinogenic activity of sodium nitrite in male or female F344/N rats exposed

to 750, 1500, or 3000 ppm. There was also no evidence of carcinogenic activity of

sodium nitrite in male B6C3F1 mice exposed to 750, 1500, or 3000 ppm. Therefore,

there was no detrimental effect of applied concentrations of Prague powder 1 in this

study in human health. However, as stated in the Philippine Regulation on Food

Additives those products made of curing salt or Prague powder 1 is limited to 200 ppm

concentrations or lowers if specified in some regulatory agencies. Federal food safety

only allowed the use nitrate and nitrite not more than 200mg/kg in curing meats and other

products. Available online: http://foodrecap.net/health/safety/curing-salt-safety/. Thus,

chromosomal aberration nearest to 200 ppm in the present study which is 150 ppm was

compared to control. As shown in Table 3, ANOVA p – level 0.015719 revealed that 150

ppm was statistically significant to chromosomal aberration induced by Prague powder 1.

40

CONTROL 150 PPM 300 PPM 600 PPM0

5

10

15

20

25

30

35

40

45

50

Treatments

Chro

mos

omal

Abe

rrati

on

Figure 4.10 Chromosomal Aberration

As illustrated in Figure 4.10 the mean chromosomal aberrations of Allium cepa root

tip cells after exposure to different concentrations of Prague Powder 1.

Table 3. ANOVA analysis of Control and 150 ppm Chromosomal Aberration

Effect df Effect MS Effect df Error MS Error F P-levelCA 1 1 678.1763 4 41.73451 16.24977 0.015719

41

CONTROL 150 PPM 300 PPM 600 PPM0

5

10

15

20

25

30

35

40

45

50

Treatments

Chro

mos

omal

Abe

rrati

on

Figure 4.11. Photomicrograph of Allium cepa root tip cells in control group (Treatment 1). A –irregular anaphase, arrow indicates chromosomal breakage and anaphasic bridge, B -chromosomal breakage, C – Normal telophase, D – Normal Metaphase, E – arrow indicates telophase bridge, F –disturbed spindle fibers , G - prophase, H – telophase. Magnification 1000x.

42

Figure 4.12. Photomicrograph of Allium cepa root tip cells in low concentration (Treatment 2, 150 ppm). A – Normal anaphase, B –irregular anaphase , C – sticky chromosomes, D –sticky metaphase E –anaphase fragment, F – normal Metaphase, G –normal telophase , H – Chromosomal breakage I- telophase breakage. Magnification at 1000x.

43

Figure 4.13 Photomicrograph of Allium cepa root tip cells in medium concentration (Treatment 3, 300 ppm). A –broken chromosomes , B –Laggard chromosomes , C – normal metaphase, D –spindle fibers disturbed at prophase , E –normal telophase, F-anaphase double bridge G- anaphase small fragment, H- normal metaphase, I- multiple bridge, J-sticky prophase. Magnification 1000x.

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Figure 4.14. Photomicrograph of Allium cepa root tip cells in high concentration (Treatment 4, 600 ppm). A – Normal Telophase, B – Irregular metaphase, chromosomes not aligned in the equatorial line, C –irregular anaphase, arrow indicates bridge, D –irregular anaphase, arrow indicates chromosomal breakage, E- sticky metaphase, G- disturbed spindle fibers in prophase. Magnification 1000x.

45