CHAPTER 7 Effect of panmasala on pregnant mice and their...

CHAPTER 7 Effect of panmasala on pregnant

mice and their neonates

7. Effect of panmasala on pregnant mice and their

neonates

7.1. Introduction

By virtue of the active cell proliferation, migration and differentiation

processes during development, the entire intrauterine period of mammals is

highly sensitive to any chemical/physical insult. There are three main critical

periods of sensitivity based upon the developmental stage of the conceptus

(Uma Devi eta/. 2000).

1. Preimplantation - Period from fertilization to implantation. An increase in

cell number, cleavage and cavitation (to form the blastocyst) occurs. Only few

cells of this mass give rise to the embryo. Most of the cells go to develop the

placenta and supporting tissues.

2. Organogenesis - Formation of all major organs in their rudimentary form,

intensive cell proliferation, differentiation and cell migration occurs during this

period.

3. Fetal period - A phase of intrauterine growth, characterized by tissue

differentiation, growth and physiological maturation. Exposure during the fetal

period is most likely to affect growth and functional maturation of central

nervous system and reproductive organs, behavioral, mental and motor

defects among possible outcomes.

The corresponding period for mouse development, where the total

gestation is only 19.5 days, are 0-5 days post conception, p.c.- (pre

implantation), 6-13 days p.c.-(organogenesis), 14-1.9.5 days p.c.-(fetal period).

Various chemicals with diverse structures and specific mechanism of

action may act at different stages of pregnancy thereby affecting varied

organs. Thus, it is worth to investigate the most sensitive period for the

potential effect of panmasala in pregnant animals exposed to panmasala at

different days of pregnancy. One of the major ingredient of panmasala i.e.,

Petoto:{jcity of panmasafa 147

areca nut, has been documented to have adverse effects, including

embryotoxicity in mice (Sinha and Rao, 1985a) and teratogenicity in chick

embryo (Paul et a/. 1999). In humans a similar association of betel

quid/arecoline and adverse pregnancy outcome was observed by various

authors (de Costa and Grew, 1982; Yang et a/. 2001; Garcia- Algar et a/.

2005). Intake of areca nut by lactating mother was reported to alter hepatic

drug metabolizing enzymes and LPO in dams as well as their suckling

neonates (Singh and Rao, 1995). Recently, transplacental exposure to areca

nut in human is confirmed by detecting arecoline in placenta (L6pez-Vilchez et

a/. 2006).

Another main component of panmasala gutkha is tobacco, which is

well known for its detrimental effects. Tobacco smoking during pregnancy is

associated with various adverse neonatal and developmental outcomes.

Various studies have reported transplacental exposure of tobacco metabolites

i.e., nicotine, cotinine and their carcinogenic derivatives in newborns of

smokers (Mosier and Jansans, 1972; Lackmann et a/. 1999; Jauniaux et a/.

1999). Nicotine and cotinine have been reported to accumulate in the fetus

(Luck et a/. 1985; Jauniaux et a/. 1999). Intrauterine nicotine exposure

interferes with fetal development of the hematopoietic system resulting in an

imbalance of mature blood and immune cell production after birth (Serobyan

et a/. 2005). Maternal exposure to PAHs through smoking poses a risk for

increased childhood cancers, leukemia and for increased incidence of adult

cancers (John eta/. 1991; Bocskay eta/. 2005). Newborn PAH-DNA adducts

and cotinine levels were inversely associated significantly with birth weight

and length (Perera eta/. 1998). Further, transplacental exposure to PAHs has

been shown to cause DNA damage in newborns (Whyatt eta/. 2001). The

enzymes that detoxicate drugs and chemicals begin to develop very late in

the fetuses of most of the animals, and are present only at low levels in fetal

tissue at full term (Parke, 1984). It is hypothesized that exposure to

panmasala during pregnancy may lead to fetal nicotine/ cotinine/ arecoline

!f'etoto~city of panmasafa 148

uptake along with panmasala induced maternal toxicity which might lead to

toxicological consequences on fetus.

Some studies have suggested potential of prenatal exposure to

tobacco smoke to affect male reproductive system in adults (Ratcliffe eta/.

1992; Jensen et a/. 2005; Ramlau-Hensen et a/. 2007). Benzo(a)pyrene, the

most potent PAH, when exposed to mice from gestational day 7-16 resulted in

dose dependent abrogated reproductive effects on male offspring and

significant increase in both pre- and post-implantation fetal loss (Mackenzie

and Angevine, 1981 ). Similarly, female exposed to second-hand smoke as a

child or in utero had probability of an increased risk of spontaneous abortion

in adulthood (Meeker et a/. 2007). However, authors suggested additional

studies with more refined estimates of childhood and in utero exposure to

tobacco. No studies have been conducted yet to evaluate fetotoxic potential of

a complex mixture like panmasala plain or gutkha containing both areca nut

and/ or tobacco as major ingredients along with other components.

Studies related to prenatal exposure to panmasala and the outcome is

of great relevance as panmasala is chewed by all the sections of society

including pregnant women and children. Thus, the present study was

designed to address the differences in toxicity response to panmasala at

different stages of pregnancy.

7 .2. Study overview

Treatment of females (positive for vaginal plug) with panmasala was

initiated at three different gestation days and continued throughout lactation.

Maternal observation i.e., body weight of female animals was taken regularly

to ensure the pregnancy. At parturition, each litter was checked for total

number of delivered and live pups. Cannibalized pups were excluded in

calculating the litter size as adopted by (Cavieres eta/. 2002). Each pup was

individually weighed. However, head length and width, body and tail length

were measured in representative number of pups selected randomly from

each litter on postnatal day (PND) 0. All the pups were observed daily for

Cf'etotoxjcity of panmasafa 149

mortality and morbidity. Pups were kept with the dam until weaning. At

weaning dams were sacrificed for the observation of number of implantation

sites. One of the organs of dams i.e., liver was also weighed as it is

considered vital organ for embryo survival (Damasceno et a/. 2002). After

weaning, the pups were separated groupwise according to sex and were

maintained on standard mice feed until necropsy at week 1 0. A minimum of

six animals I sex were sacrificed from each group for organ weight. F1 males

were studied for basic spermiogram and testicular oxidative stress. The

experimental overview is depicted in fig. 1

GDO

GDG

GDI4 ...................................................... :-:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:·:

I Pregnancy II Lactation I Post weaning period I .. I I . I 1 Partunhon I I Weanm~ 1

~r ,

I Dams sacrificed at wean I Pups sacrificed at

I Implantation count. liver wt. I week 10

Organ weight.

Male spenniogram.

Testicnlar OS

Figure 1. Experimental overview: Impregnated dams treated with f~~::.)'lpanmasala

and f;;:,.·;.j standard mice feed

7.3. Materials and Methods

7.3.1. Animals and treatment

Virgin female mice, 1 0-12 weeks old were allowed to mate with non

treated male mice (2 females per male). They were cohabitated at

approximately 5:30p.m. The next morning each female was checked before

9:30a.m. for vaginal plug. Female with vaginal plug was presumed mated and

considered as at 0 day of gestation (Gestation day 0, GD 0). A minimum of six


females were randomly assigned to each group i.e., control and experimental

groups. The experimental groups exposed to panmasala covering three

different developmental periods were as follows-

GD 0- GD 0 to weaning (covering all the stages of pregnancy)

GD 6- GD 6 to weaning (covering postimplantation, organogenesis and fetal

period of pregnancy)

GD 14- GD 14 to weaning (covering fetal stage of pregnancy)

Two different doses i.e., 3 and 6% of both PMP and PMT were used

which simulate the human exposure and these doses are about 1 V2 and 3

times higher doses of panmasala if consider the food intake by human (as

described in Chapter 4.3.1)

7 .3.2. Maternal observations

7.3.2.1. Body weight

Mated females were weighed after mating and before initiation of

panmasala treatment and then every 3rd day till parturition and thereafter

weekly till weaning. Mated female mice were excluded from assessment of

dam weight gain of the group if animals were found non pregnant.

7.3.2.2. Fetotoxic evaluation

Fertility, Pregnancy, Live birth, Viability and Weaning indices along with

Gestation length and sex ratio were calculated (as described in Chapter

6.3.2.2 - 6.3.2.3).

7.3.2.3.0rgan weight

At weaning dams were sacrificed; liver was dissected out, blotted and

weighed on electronic balance (Mettler Toledo, Germany).

7.3.2.4. Implantation sites

At the time of weaning, dams were sacrificed to observe implantation

sites as dark rings in uterus (as described in Chapter 6.3.2.4).

Postimplantation loss was calculated (as described in Chapter 6.3.2.4).

PetotoJ(jcity of panmasafa 151

7 .3.3. F1 generation evaluation

All the pups were weighed at birth and on PND 4, 7, 14, 21 and week

6, 8 and 10. A minimum of six pups were selected randomly representing

each litter of study group. The measurements of body and tail length, head

length and width were done at PND 0 using a centimeter (cm)/millimeter scale

as per the procedure adopted by Rao eta/. (2006) (Fig 2).

Body length - measured from the base of the skull to the base of the tail.

Head length- measured from the tip of the snout to the base of the skull.

Head width - measured from one side of pinna attachment to the opposite

side.

Tail length -measured from base of the body to the end of tail.

All the values were expressed in em and mentioned as mean± SE.

-~ ••w ........... , Head length

Body length

Figure 2. Procedure of body measurement at PND 0

7.3.3.1. Developmental Markers

Pinna detachment- Pinnae of both ears unfolded to a fully erect position (Fig

3a). Ears were inspected from PND 2 until any one of the pups from the litter

exhibited complete unfolding.

Fur development- First appearance of fur on the body (Fig. 3b), from PND 4.

<FetotoJ(jcity of panmasafa 152

Eye opening - Eye opening is defined as any visible break in the membrane

covering the eye (Fig. 3c). The eyes of each animal were examined from PND

12 until any one of the eyes of any animal among litter was found open.

a b c

Figure 3. Developmental markers (a) Pinna detachment; (b) Fur formation; (c) eye opening (as marked)

7.3.3.2. Post weaning

Weight of animals was taken biweekly. Representative number of male

and female mice from each group was sacrificed at the age of 10 week.

Male -Testis, epididymis, seminal vesicle, liver, spleen and kidneys were

dissected out and weighed. Reproductive health evaluation was done by

employing sperm count and morphology, spermatid count, estimation of

TBARS, Thiol, GSH and SOD (as described in Chapter 4.3.3; 4.3.9).

Female- Weight of ovary, uterus, liver, spleen and kidney were taken.

7.4. Statistical Analysis

Study variables of experimental animals were compared with control

animals for each gestational period. Data are expressed as mean ± SE.

Statistical analysis was performed using SPSS. Parametric ANOVA followed

by Tukey HSD post-hoc test was to determine significance (P< 0.05) between

control and treated groups. Differences in Fertility and Pregnancy indices

were analyzed using Fisher exact test.


7.5. Results

7.5.1. Fertility and Pregnancy indices

The dose of 3 and 6% PMT led to considerable reduction in fertility

index in mated females treated from GD 0 or GD 6 as revealed by lower

number of pregnant females among the mated ones with respect to control.

However, pregnancy index of all the panmasala treated groups was 1 00% as

all the pregnant females delivered live pups (Fig. 4).

~;,;~;;J Fertility index -+-Pregnancy index

100 • • • • • ~ .,

80 " .~ ., .5 ~ 60 a ~ '"' 40 ., a ~

~ 20 ~

0

Control 3PMP 3PMT GPMP GPMT 3PMP 3PMT GPMP GPMT

GDO GDG

Figure 4: Effect of panmasala on mice Fertility and Pregnancy indices

7.5.2. Body weight

Maternal . body weight gain during in utero panmasala exposure is

shown in fig. 5a, b. The data showed that the weight gain was not affected

significantly in any of the panmasala treated groups with respect to non

treated dams but the gain was slightly lower on day 18 of pregnancy in

animals treated from GD 0 with 3 and 6% of PMT while it was more or less

same in PMP treated animals at both the dose levels.

Petoto]\jcity of panmasaCa 154

80

-+- Control -o-- 3PMP -II- 3PMT - 6PMP -II- 6PMT 70

60

.s 50

~ '!:: 40 IP ~

30 *

20

10

0

3 6 9 12 15 18 a Gestation day

80 -+-Control -c-- 3PMP -II-3PMT -6PMP -11--GPMT

70

60

·a 50 b()

'!:: 40 ]J ~ * 30

20

10

0

9 12 Gestation day

15 18

b

Figure 5: Weight gain during in utero panmasala exposure from a) GO 0; b) GO 6

Petotoxjcity of panmasafa 155

The data on weight gain during lactation is depicted in fig. 6. Weight

gain was maximum on LD 14 with respect to weight gain observed at other

intervals i.e., 7 and 21. The weight gain on lactation day (LD) 14, was about

22.3, 11.6 and 14.5% in control, 6% PMP and PMT treated groups

respectively from GD 0 (Fig. 6a). The difference in lactation weight gain was

non significant between treated and non treated groups. On day 21 of

lactation period, the data indicated that percentage weight gain was

considerably lower (statistically non- significant) in all the panmasala treated

dams from GD 0 as compared to treatment from GD 6 and GO 14 and control.

Panmasala treatment from GD 6 did not alter the weight gain during lactation

noticeably with that of control (Fig. 6b). However, 6% PMP and PMT exposure

from GD 14 showed less weight gain than control mice (Fig. 6c).

25 ~Control ~ 3PMP ..,._ 3PMf - GPMP -o- GPMf

20

15

5

Or-------~------r-------r-----~r-----~------~

7 a

Utctatiou day 14 21

Figure 6a. Body weight gain during lactation in GO 0 panmasala treated group

Petoto~ty of panmasafa 156

25 ~Control -Q- 3PMP _,._ 3PMT -e- GPMP ~ GPMT

20

-~ b()

-= 15 ]l ~

* 10

7 Lactation day 14 21 b

Figure 6b. Body weight gain during lactation in GO 6 panmasala treated group

~Control ~ 3PMP _,._ 3PMf - 6PMP -u- 6PMf

7 Lactation Day

14 21 c

Figure 6c. Body weight gain during lactation in GO 14 panrnasala treated group


7.5.3. Liver weight

The maternal liver weight at weaning is shown in fig. 7. The liver weight

did not alter significantly in any of the panmasala treated groups as compared

to non treated group. The weight of the liver in control group was 1.97g while

it was 1.90g and 2.20g in 6% PMP and PMT treated groups respectively from

GOO.

2.5

2.0

1.5 :§

0.5

ND

0.0 ...,............,__r--+---t

Control 3P 3T 6P 6T 3P 3T 6P 6T 3P 3T 6P 6T

GOO GOG GO 14

Figure 7. Maternal liver weight at weaning (NO-Not done)

7.5.4. Pregnancy outcome

Results of pregnancy outcome of in utero treated mice at different

gestational periods/groups are presented in table 1- 3. The data indicated that

gestation length was lower in all the panmasala treated mice as compared to

control and which was significantly smaller at 3 and 6% PMT treated dams

from GO 0 whilst the decline was non significant in PMP treated groups at

both the dose level i.e., 3 and 6%. In addition, gestation length was also lower

in animals treated with panmasala from GO 6 or GO 14 but the decline was

statistically non significant. Litter size as well as number of live pups delivered


were marginally affected among the panmasala treated mice from GD 0 and

to a greater extent when treated from GD 6 or GD 14 as compared to control.

However, all these variations in litter size were statistically non significant. The

number of pups in a litter was about 9.1, 9.6 and 8.6 in 6% PMT treated mice

from GD 0, GD 6 and GD 14 respectively with respect to 9.8 in control group.

No definitive pattern of change was noticed with respect to the number of

implants in panmasala treated and non treated animals. The data on

postimplantation loss indicated that it was considerably higher in all the

panmasala treated groups as compared to control. The postimplantation loss

was about 12.1, 12.3 and 13.2% in 6% PMT treated mice from GD 0, GD 6

and GD 14 respectively as compared to about 2.2% in control. Pup mortality

at birth i.e., neonatal death increased in all the panmasala treated groups

dosed from GD 0 while it was more or less similar in GD 6 group in

comparison to control group. Treatment of dams with 6% of both PMP and

PMT from GD 14 resulted in considerable increase in neonatal death as

compared to control.

Pups born to mothers treated from GD 0 or GD 6 with 3 and 6% PMT

had significantly lower birth weight as compared to control while those

exposed from GD 14 had shown significant birth weight reduction only at 3%

PMT dose level. In comparison to control, exposure of PMP to pregnant

animals in all the treated groups resulted in non significant decline in their pup

birth weight. The observation regarding body measurement of pups at PND 0

showed minor alterations in their body and tail length as well as head length,

width and their ratio (Table 4). All these changes were statistically non

significant. Physical developmental features i.e., fur formation and eye

opening occurred more or less on similar days among all the pups of treated

and non treated groups (Table 5). Pinna detachment was delayed non

significantly in PMT treated groups from GD 0, GD 6 and GD 14. High dose of

PMP i.e., 6% from GD 6 and GD 14 to dams also resulted in slightly late

detachment of pinna (Table 5). However, the alterations were statistically non

significant.

Petoto~ty of panmasafa 159

Table 1. Pregnancy outcome of dam treated with panmasala from GD 0

Control 3PMP 3PMT 6PMP 6PMT

Gestation 19.78 ± 0.28 19.00 ± 0.26

period (days) 18.17 ±0.11* 18.83 ± 0.17 18.67 ±0.24•

litter size 9.89 ± 0.42 9.80 ± 0.70 9.67 ± 0.73 9.33 ± 1.05 9.11±1.17

(n)

live pups 9.78 ± 0.43 9.60 ± 0.75 9.33 ± 0.75 9.17 ± 1.17 8.33 ± 1.08

(n)

Implants 10.11 ± 0.39 12.00 ±0.82 10.00 ± 1.47 10.00 ± 0.82 9.89 ± 1.07

(n)

Postimplant

loss(%) 2.22 ± 1.47 6.25 ± 3.99 6.94 ± 2.54 7.94 ± 4.17 12.12 ± 5.47

Neonatal 0.01 ± 0.01 0.03 ± 0.02

death (n) 0.04 ± 0.02 0.03 ± 0.03 0.07 ± 0.05

Birth weight 1.47 ± 0.01 1.43 ± 0.01 1.39 ± 0.01* 1.42 ± 0.02 1.39 ± 0.01*

(&) (n)-average number; -p<0.05. Data are expressed as mean ± SE; (~ untub6f'

Table 2. Pregnancy outcome of dam treated with panmasala from GO 6

3PMP 3PMT 6PMP 6PMT

Gestation

period (days) 19.50 ± 0.5 18.71 ± 0.18 18.86 ± 0.26 18.67 ± 0.33

litter size 8.00 ± 0.82 8.14 ± 1.1 9.14±1.12 9.67 ± 0.56

(n)

live pups 8.00 ± 0.82 8.00 ± 1.09 9.14 ± 1.12 9.67 ± 0.56

(n)

Implants 9.33 ± 0.92 9.86 ± 1.01 10.14 ± 0.88 11.00 ± 0.37

(n)

Postimplant

loss(%) 14.24 ± 3.90 16.04 ± 8.22 11.54 ± 5.41 12.32 ± 3.05

Neonatal

death (n) 0.00 ± 0.00 0.02 ± 0.02 0.00 ± 0.00 0.00 ± 0.00

Birth weight 1.45 ± 0.04 1.38 ± 0.07• 1.39 ± 0.06 1.38 ± 0.06•

(&) (n)-average number; -p<0.05. Data are expressed as mean ± SE; (n)- JIIIRI~

PetotoJ:jcity of panmasafa 160

Table 3. Pregnancy outcome of dam treated with ranmasala from GD 14

3PMP 3PMT 6PMP 6PMT

Gestation 19.00 ± 0.26 18.75 ± 0.25 18.67 ± 0.33 18.80 ± 0.49

period (days)

litter size 8.67 ± 0.71 10.60 ± 0.51 9.33 ± 0.67 8.60 ± 0.51

(n)

live pups 8.67 ± 0.71 10.60 ± 0.51 8.67 ± 0.84 7.80 ± 0.86

(n)

Implants 9.17 ± 0.75 10.83 ± 0.31 9.83 ± 0.65 10.00 ± 0.32

(n)

Postimplant 5.00 ± 3.42 13.18 ± 9.56 5.14 ± 2.36 13.27 ± 6.86

loss(%)

Neonatal 0.00 ± 0.00 0.00 ± 0.00 0.08± 0.05 0.09 ± 0.09

death (n)

Birth weight 1.46 ± 0.02 1.38 ± 0.01• 1.44 ± 0.01 1.45 ± 0.01

(g) (n)-average number; "p<0.05. Data are exp1-essed as mean± SE; jll) nnntber

Table 4. Body measurement of rur treated in utero with ranmasala at PND 0

Dose and Body length Tail length Head length Head width Head

treatment (em) (em) (em) (em) Length/Width

Control 2.09 ± 0.02 1.12 ± 0.02 1.06 ± 0.02 0.74 ± 0.01 1.44 ± 0.02

GDO

3PMP 2.07 ± 0.03 1.08 ± 0.03 0.94 ± 0.03 0.68 ± 0.01 1.39 ± 0.05

3PMT 2.06 ± 0.03 1.02 ± 0.03 1.05 ± 0.02 0.75 ± 0.01 1.42 ± 0.04

6PMP 2.03 ± 0.06 1.00 ± 0.02 0.99 ± 0.02 0.69 ± 0.03 1.45 ± 0.05

6PMT 2.15 ± 0.06 1.08 ± 0.05 1.03 ± 0.05 0.70 ± 0.04 1.47 ± 0.04

GD6

3PMP 2.09 ± 0.03 1.17 ± 0.05 1.06 ± 0.03 0.83 ± 0.04 1.28 ± 0.04

3PMT 2.02 ± 0.05 1.13 ± 0.03 0.98 ± 0.04 0.73 ± 0.02 1.34 ± 0.03

6PMP 2.06 ± 0.09 0.99 ± 0.03 0.94 ± 0.02 0.74 ± 0.03 1.28 ± 0.05

6PMT 2.27 ± 0.15 0.93 ± 0.03 1.03 ± 0.03 0.77 ± 0.03 1.35 ± 0.05

GD14

3PMP NO

3PMT 1.98 ± 0.10 1.11 ±0.12 0.98 ± 0.03 0.70 ± 0.03 1.41 ± 0.05

6PMP 2.20 ± 0.08 0.88 ± 0.04 0.96 ± 0.02 0.74 ± 0.02 1.30 ± 0.06

6PMT 2.08 ± 0.09 1.00 ± 0.04 1.00 ± 0.04 0.75 ± 0.03 1.33 ± 0.04 ND- Not Done

Cf'etoto)(jcity of panmasafa 161

Table 5. Developmental landmarks of pup treated in utero with panmasala

Dose and Pinna detachment Fur fonnation Eye opening

treatment (days) (days) (days)

Control 4.22 ± 0.15 7.22 ± 0.15 14.56 ± 0.24

GDO

3PMP 4.30 ± 0.15 8.00 ± 0.37 14.30 ± 0.40

3PMT 4.75 ± 0.25 7.00 ± 0.00 14.25 ± 0.25

6PMP 4.00 ± 0.00 7.20 ± 0.20 14.40 ± 0.24

6PMT 4.40 ± 0.40 7.20 ± 0.20 14.40 ± 0.40

GD6

3PMP 4.17 ± 0.17 7.17 ± 0.17 14.67 ±0.21

3PMT 4.80 ± 0.20 7.00 ± 0.00 15.00 ± 0.00

6PMP 4.50 ± 0.22 7.00 ± 0.00 14.00 ± 0.00

6PMT 4.83 ± 0.17 7.00 ± 0.00 14.50 ± 0.34

GD14

3PMP 4.33 ± 0.21 7.00 ± 0.00 14.17±0.17

3PMT 4.40 ± 0.24 7.00 ± 0.00 14.20 ± 0.20

6PMP 4.60 ± 0.24 7.00 ± 0.00 14.20 ± 0.20

6PMT 4.80 ± 0.20 7.00 ± 0.00 14.00 ± 0.00

7.5.5. Postnatal development

The postnatal data of panmasala treated mice are shown in table 6-8.

Live birth index (LBI) was reduced marginally in animals treated from GD 0 or

GD 14 with 6% PMT whereas in animals treated from GD 6, it was more or

less same (between 98.4-1 00%). The lowest LBI i.e., 91.1% was observed in

animals treated from GD 14 with 6% PMT as compared to 98.8% in control

group. Viability of pups on PND 4 was marginally altered in all panmasala

treated groups and Viability index (VI) was lowest i.e., about 88% in animals

treated with 6% of both types of panmasala from GD 14 with respect to about

97.8% in control. The data on Weaning index (WI) showed that animals

Petotoyifcity of panmasafa 162

exposed from GD 14 had maximum decline, which was about 83 and 75% in

6% PMP and PMT respectively as compared to about 93% in control.

However, the WI was also considerably altered in the GD 0 group while

marginally altered among GD 6 group as compared to control. The alterations

in all the indices i.e., LBI, VI and WI were statistically non significant in any of

the panmasala treated groups with respect to control.

Sex ratio of litters declined in both the doses of panmasala treated

dams of GD 0 group which was lowest in 6% PMT i.e., 1.19 as compared to

1.78 in control. However, the sex ratio of pups delivered by the animals

treated with panmasala from GD 6 or GD 14 did not show any pattern of

changes with respect to control.

Table 6. Effect of panmasala exposure from GO 0 on postnatal development


Live Birth 98.89 ± 1.11 97.42 ± 1.81 96.07 ± 2.25 97.22 ± 2.78 93.33 ± 4.57

Index(%)

Viability 97.84 ± 1.45 95.94 ± 2.38 93.88 ± 3.76 92.73 ± 3.4 95.48 ± 2.93

Index(%)

Weaning 93.58 ± 2.33 89.87 ± 4.39 89.34 ± 6.62 89.09 ± 4.45 77.62 ± 16.2

Index(%)

Sex ratio 1.78 ± 0.43 1.32 ± 0.21 1.28 ± 0.15 1.24 ± 0.36 1.19 ± 0.16

(d'/S?)

Table 7. Effect of panmasala exposure from GO 6 on postnatal development

3PMP 3PMT 6PMP 6PMT

Live Birth 100.00 ± 0.00 98.41 ± 1.59 100.00 ± 0.00 100.00 ± 0.00

Index(%)

Viability 96.06 ± 2.50

Index(%) 93.37 ± 4.61 90.28 ± 5.01 94.63 ± 3.75

Weaning

Index(%) 93.98 ± 4.21 91.70 ± 4.42 87.50 ± 4.69 91.57 ± 4.23

Sex ratio 2.37 ± 1.20 1.68 ± 0.37 1.96 ± 0.70 2.38 ± 0.66

(d'/ )


Table 8. Effect of panmasala exposure from GD 14 on postnatal development

3PMP 3PMT 6PMP 6PMT

Live Birth 100.00 ± 0.00

Index(%) 100.00 ± 0.00 92.21±5.11 91.11 ± 8.89

Viability 97.92 ± 2.08

Index(%) 95.00 ± 5.00 88.48 ± 6.55 88.00 ± 12.00

Weaning

Index(%) 97.92 ± 2.08 90.78 ± 4.60 83.05 ± 7.46 75.00 ± 25.00

Sex ratio 1.59 ± 0.59 2.57 ± 1.37 1.60 ± 0.36 1.80 ± 1.10

(~/~)

7.5.6. Postnatal weight changes in pups

The data on postnatal weight of pups are depicted in fig. 8- 10. None of

the panmasala treated groups either from GD 0, GD 6 or GD 14 had shown

significant alteration in pup weight on PND 4 and 7 as compared to control.

However, significantly higher body weight was observed among pups born to

dam treated from GD 0 with 6% PMP on PND 14 and 21 and with 3% PMT on

PND 21, whereas 6% PMT treated group had significantly lower weight of

pups on PND 14 and 21 than that of control (Fig. 8). Further, exposure of

mother to 6% PMP and PMT from GD 6 resulted in a significant decrease in

weight of the offspring on PND 14 and 21 as compared to control pups (Fig.

9). The decline was also observed in pup weight of 3 and 6% PMT treated

dam from GD 14 on PND 14 as well as 21 with respect to control group which

was significantly lower at 6% PMT on PND 21 (Fig. 1 0).

Petoto.{jcity of panmasafa 164

9

8 •Control :l3PMP C3PMT 06PMP •6PMT

7

#

2

0

0 4 Postnatal day 7 14

Figure 8. Body weight of pups born to panmasala treated dam from GO 0 (*decline at p< 0.05; #elevation at p<0.05)

8

7 • Control ;;;i3 PMP ~ 3PMT U 6 PMP • 6 PMT

6

2

0

0 4 Postnatal day 7 14

21

21

Figure 9. Body weight of pups born to panmasala treated dam from GO 6 (*p< 0.05)

8

7 • Control _13 PMP D 3PMT r 6 PMP m 6 PMT

6

0

0 4 Postnatal day 7 14 21

Figure 10. Body weight of pups born to panmasala treated dam from GO 14 (*p< 0.05)

Petotoxjcity of patzmasafa 165

7.5.7. Post weaning weight changes in pups

After weaning, pups were maintained on standard mice feed until

sacrifice. Alteration in body weight of male and female pups during this period

is presented in table 9. At week 6 of postnatal age, weight of pups born to

dam treated from GD 0 with 3 and 6% PMP was significantly higher from

control but the weight change was not significant in female offspring of 3%

PMP treated group. However, the body weight of pups whose dams were

exposed to 6% PMT from GD 0 was lowered (non significant) as compared to

control. The animals treated in utero with 3% PMP, PMT and 6% PMP from

GD 0 showed marginally higher weight on postnatal week 8 and 1 0 whereas it

was significantly low in male offspring with 6% PMT treatment as compared to

corresponding control. In a similar way, at postnatal week 6, 8 and 10, both

male and female pups of dam treated from GD 6 or,GD 14 with 3% PMP and

PMT had higher body weight whilst it was lowered in pups of dam treated with

6% of both types of panmasala as compared to respective control. Female

pups from 6% PMT treat~d dam from GD 14 had a significantly lower weight

at postnatal week 10 than the corresponding control.

The data on postnatal weight of pup indicated higher body weight of

animals treated in utero with 3% PMP, PMT and 6% PMP and lower weight

among 6% PMT exposure from PND 14 till necropsy at week 10 in GD 0

group as compared to control. Three percent PMT as well as 6% PMP and

PMT treatment from GD 6 and GD 14 showed marginal to considerable

alterations in pup weight till weaning. Once the panmasala treatment was

withdrawn, the body weight of offspring of the dam treated with lower dose

i.e., 3% recovered whereas 6% feeding showed recovery to a lesser extent

(Table 9).

'Fetoto:{jcity of panmasafa 166

Table 9. Post weaning body weight (g ± SE) of pups born to panmasala treated dams

Dose and Male Female

treatment

Week6 Week8 Week 10 Week6 Week8 Week 10

Control 17.03 25.56 27.69 15.64 21.45 23.36

± 1.02 ± 0.66 ± 0.36 ± 0.74 ± 0.79 ±0.32

GDO

3PMP 24.07 27.65 28.36 18.54 22.88 24.43

± 0.84• ± 0.65 ± 0.68 ± 1.08 ± 0.53 ± 0.62

3PMT 16.99 25.65 27.53 18.72 22.37 23.75

± 2.08 ± 1.02 ± 0.66 ± 1.06 ± 0.66 ± 0.53

6PMP 23.71 26.25 29.06 19.55 22.32 24.23

± 0.77• ± 0.50 ± 0.47 ± 0.46• ±0.24 ± 0.35

6PMT 15.05 21.29 25.12 13.47 20.19 21.81

± 1.25 ± 2.08• ± 1.14• ± 1.31 ± 0.99 ± 1.97

GD6

3PMP 21.85 26.86 29.15 18.50 21.56 24.36

± 0.99 ± 0.68 ± 0.57 ± 0.80 . ±0.76 ± 0.46

3PMT 20.89 26.43 27.67 18.00 22.74 23.81

± 0.61 ± 0.51 ± 0.48 ± 0.76 ± 0.34 ± 0.36

6PMP 15.38 21.80 24.62 15.71 20.12 21.31

± 0.76 ± 1.05 ± 0.78 ± 0.61 ±0.40 ±0.41

6PMT 16.31 23.58 26.61 16.70 21.25 23.44

± 1.15 ± 0.59 ± 0.50 ±0.78 ±0.53 ± 0.47

GD 14

3PMP 19.48 25.98 27.28 17.83 22.23 22.94

± 1.12 ± 0.52 ± 0.57 ±0.97 ± 0.57 ± 0.58

3PMT 21.76 26.91 27.86 18.95 22.68 23.76

± 0.67 ±0.41 ± 0.46 ±0.53 ±0.48 ±0.33

6PMP 16.86 25.17 27.38 17.35 20.92 23.77

± 1.49 ± 0.60 ± 0.39 ±0.72 ± 0.55 ± 0.47

6PMT 19.31 23.86 25.90 14.71 18.96 21.03

± 1.70 ± 1.62 ± 0.55 ± 1.31 ± 1.17 ± 0.39•

("P< 0.05)

'Fetotoxfcity of panmasafa 167

7.5.8. Reproductive organ weight changes in male pups

The result on male organ weight from each study group at week 1 0 is

presented in table 10. Testicular weight was higher (non significant) in

offsprings born to dam treated from GD 0 with 6% PMP than that of control. In

GD 6 group, testis weight was more or less similar among the PMP treated

groups while slightly declined with PMT treatment as compared to control.

Testis weight was increased at 3% but decreased at 6% of both types of

panmasala treated groups from GD 14 than that of control. The weight of

epididymal tissue was lowered in all the panmasala treated groups with

respect to control. The data indicated that 3 and 6% PMP from GD 0

treatment caused minor alteration in epididymal weight changes while in rest

of the panmasala treated groups; there were a decline as compared to

control. All these changes were statistically non significant. Weight of seminal

vesicle (SV) was increased non significantly in GD 0 treated groups with 3

and 6% PMP whilst it was decreased at 3 and 6% PMT as compared to

control. Within GD 6 group, SV weight increased in all the treated animals

than control except 6% PMP. Panmasala exposure from GD 14 resulted in

increase in SV weight at 3% PMP whereas it decreased in all other treated

groups with respect to control.

7.5.9. Somatic organ weight changes in male pups

Spleen weight was marginally changed in pups born to panmasala

treated mother from GD 0. It declined in 3% PMT and 6% of both PMP and

PMT exposure from GD 6 and with both the doses of PMP from GD 14 as

compared to control. Liver weight was considerably lower with both the doses

of PMT treatment from GD 0 and with 6% of both PMP and PMT exposure

from GD 6. It was more or less similar at 3% PMP of all the three gestation

groups and at 6% PMT of GD 14 group than that of control. In comparison to

control group, weight of kidney was increased in most of the panmasala

treated groups as compared to control. A slight decline was noted in 3% PMT

of GO 14 group and at 6% PMP and PMT of GD 6 and GD 14 group.

However, weight changes of these organs were statistically non significant.

Petotoxjcity of panmasaCa 168

Table 10. Organ weight (g ± SE) of male offspring at week 10.

Dose and Testis Epididymis Seminal vesicle Spleen ,iver Kidney

treatment

Control O.I8I ± 0.005 0.063 ± 0.004 O.I28±0.0I7 0.084 ± 0.006 I l ± 0.045 0.445 ± O.OI

GOO

3PMP O.I81 ± 0.009 0.062 ± 0.004 0.149 ± O.OI3 0.085 ± 0.003 1 ; ± 0.089 0.469 ± 0.037

3PMT 0.184 ± 0.007 0.053 ± 0.005 0.1I8 ± 0.010 0.086 ± 0.006 1 ; ± 0.129 0.421 ±0.037

6PMP 0.205 ± 0.012 0.061 ± 0.004 0.142 ± 0.010 0.074 ± 0.004 1 r ± 0.059 0.471 ±0.029

6PMT 0.1 7 3 ± 0.0 19 0.051 ± 0.008 0.116 ± 0.040 0.087 ± 0.003 1 . ± 0.051 0.435 ± 0.036

GOG

3PMP 0.182±0.012 0.060 ± 0.003 O.I59 ± 0.014 0.080 ± 0.005 1 : ± 0.070 0.494 ± 0.043

3PMT 0.176 ± 0.008 0.05I ± 0.004 0.13I ± 0.018 0.07I ± 0.007 I '±0.110 0.471 ± 0.045

6PMP 0.181 ± 0.007 0.052 ± 0.004 0.125±0.014 0.064 ± 0.003 1 ; ± 0.067 0.385 ± 0.029

6PMT 0.169 ± 0.008 0.052 ± 0.002 0.138 ± 0.021 0.075 ± 0.001 1 : ± 0.034 0.440 ± 0.013

GO 14

3PMP 0.190 ± 0.005 0.058 ± 0.004 0.157 ± O.OI5 0.075 ± 0.003 1 ; ± 0.029 0.465 ± 0.015

3PMT 0.190 ± 0.01I 0.055 ± 0.003 0.111 ± 0.012 0.080 ± 0.008 1 '± 0.089 0.437 ± 0.031

6PMP 0.175 ± 0.007 0.054 ± 0.003 0.124 ± 0.020 0.063 ± 0.003 1 ) ± 0.030 0.434 ± 0.022

6PMT 0.177 ± O.OI5 0.049 ± 0.007 0.092 ± 0.020 0.083 ± 0.003 1 l±0.110 0.437 ± 0.036

'Fetoto*ity of panmasafa 169

7.5.10. Reproductive health evaluation of male pups at sacrifice

Data on spermiogram of male offspring's treated in utero with

panmasala exposure are shown in fig. 11-13. The data indicated that

spermatid count/g testis and ESP were significantly decreased in offspring of

dam exposed to 6% PMT from GD 0 while the decline in sperm count/g cauda

and DSP were non significant (Fig. 11 ). In other panmasala treated gestation

groups i.e., GD 6 and GD 14, a non significant decline in sperm and

spermatid count as well as DSP and ESP were observed (Fig. 12-13).

Abnormality of sperm head shape was noticeably increased (statistically non

significant) among all the three in utero panmasala treated groups i.e., GD 0,

GD 6 and GD 14 as compared to control (Table 11). The sperm head shape

abnormality was higher in the offspring of dams treated from GD 0 and GD 6

as compared to GD 14. The data on sperm and spermatid count, DSP, ESP

and sperm head morphology indicated the adverse effect of prenatal

exposure to panmasala on sperm quality even though some of the data

obtained were statistically non significant.

1m sperm/g cauda IB spermatid/g testis ITIDSP i9ESP "'o 18

""' "' 16 ..., l'"b

;;:; 14 "' D

~0 12 *

i !l 10 ~ ]

8 "' E ~ 6

'b '<i' 4 "9 ;; u ~ 2 E ~ 0


Figure 11. Spermiogram of offspring of in utero treated dam from GD 0. (*p< 0.05)


18 1m sperm/g cauda m spermatid/& testis !mDSP OESP ~0

;:;;-16 "' <-)

~ 14 ;:;;-"' 0

'b 12 .....

·~ 10

~ 8 . .., "' E ~ 6

'b ..... 4 ';i' "0

~-0 2

f ~ 0


Figure 12. Sperrniogram of offspring of in utero treated dam from GD 6

'"o .....

18

~ 16 <-)

~g_ 14

e; 0 ;.::. 12 ''b .....

Control

m sperm/g cauda &1 spermatid/& testis mosP

3PMP 3PMT 6PMP

Figure 13. Sperrniogram of offspring of in utero treated dam from GD 14

OESP

6PMT


Table 11. Sperm head abnormality in pups of in utero panrnasala treated darns

Dose a11d treatme11t

Control

GDO

3PMP

3PMT

6PMP

6PMT

GD6

3PMP

3PMT

6PMP

6PMT

GD14

3PMP

3PMT

6PMP

6PMT

% Spenn head shape abnormality

2.85±0.74

3.88±0.55

3.80±0.73

5.04 ± 0.75

5.00± 0.51

3.63 ± 0.40

4.94±0.63

5.29 ± 0.73

5.75 ± 0.93

3.25 ±0.35

3.35± 0.65

3.54 ± 0.41

3.75 ± 0.61

7.5.11. Biochemical analysis of testicular tissue

The result of testicular biochemical analysis is presented in table 12.

The data on TSARS production/g testis indicated elevation in LPO among

offspring of all the panmasala treated dams as compared to control. The

values were 7.82 and 8.46 nM/mg testis in 6% PMP and PMT treated mice

respectively from GD 14 group which were about 44% and 55% higher than

control but the induction of TSARS production was non significant. The

concentration of thiol/g testis was significantly higher in 6% PMP and PMT of

both GD 6 and GD 14 groups as well as 3% PMT of GD 6 group with respect

to control. In contrast, activity of SOD (as revealed by % inhibition of

pyrogallol) and concentration of glutathione were non significantly lowered in

all the panmasala treated groups as compared to control. The data on

testicular biochemical analysis revealed that prenatal exposure to panmasala

has the potential to induce oxidative stress in the male offspring.

Petotox:fcity of panmasafa 172

Table 12. Biochemical analysis of testis of pups treated in utero with panmasala

GOO GD6 GD 14

TBARS Control 5.42 ± 0.56

(nM/mz testis)

3PMP 5.71 ± 0.54 5.72 ± 0.64 7.11 ± 1.31

3PMT 8.17±1.76 5.16 ± 0.61 5.76 ± 0.50

6PMP 6.44 ± 0.50 6.44 ± 1.24 7.82 ± 1.04

6PMT 8.34 ± 1.90 6.80 ± 1.42 8.46 ± 1.67

Thiol Control 2.03 ± 0.65

(JlM/mz testis)

3PMP NO 3.17 ± 0.13 2.67 ± 0.08

3PMT 3.30 ± 0.19 4.03 ± 0.41• 2.85 ± 0.08

6PMP 3.07 ± 0.23 3.77 ± 0.14• 3.78 ± 0.23•

6PMT 3.42 ± 0.35 3.78 ± 0.33• 3.58 ± 0.40•

Glutathione Control 37.92 ± 2.73

(Jlz/Z testis)

3PMP ND 27.08 ± 2.20 22.09 ± 1.90

3PMT 30.00 ± 2.62 25.01 ± 2.37 24.27 ±1.67

6PMP 32.33 ± 2.80 30.65 ± 1.76 27.92 ± 2.78

6PMT 25.19 ± 3.18 22.32 ± 3.25 28.38 ± 3.86

SOD Control 75.00 ± 11.27

(% inhibition)

3PMP NO 68.46 ± 9.89 64.42 ± 4.04

3PMT 57.00 ± 8.60 50.64 ± 12.28 61.85 ± 10.09

6PMP 59.44 ± 5.61 57.79 ± 6.97 71.43 ± 5.99

6PMT 58.81 ± 9.98 50.13 ± 5.26 53.75 ± 9.45

ND-Not Done j * y><O·Ot:)

Petotoy;jcity of panmasafa 173

7 .5.12. Reproductive organ weight changes in female pups

The organ weight of female is presented in table 13. The ovary weight

was declined in all the panmasala treated groups of all the three gestation

days' exposure groups. The weight of uterus was higher at 6% of both PMP

and PMT treated groups with respect to control in offspring of GD 0 and GD

14 gestation groups while it was marginally altered at low dose i.e., 3%

panmasala treatment at three gestation groups. However, these changes

were statistically non significant.

7.5.13. Somatic organ weight changes in female pups

A definite pattern of change in liver weight was observed in all the

panmasala treated groups. The liver weight was high as compared to control

but these changes were statistically non significant. Spleen weight was non

significantly higher at both PMT treated groups from GD 0 while it was more

or less similar in GD 6 treated groups with respect to non treated group.

Exposure to 3% PMP and 6% of both PMP and PMT from GD 14 led to an

increase in the weight of spleen (non significant). The weight of kidney was

higher in all the panmasala treated groups at all the three gestation days i.e.,

GD 0, GD 6 and GD 14. The increase was significant at 3 and 6% PMT

treated groups from GD 0 and 6% of both PMP and PMT treatment from GD

14 with respect to control.

Petoto~ty of panmasafa 17 4

Table 13. Organ weight (g ± SE) of female pups treated in utero with panmasala.

Organ Dose GDO GD6 GD14

Ovary Control 0.019 ± 0.003

3PMP 0.014 ± 0.001 0.009 ± 0.001 0.008 ± 0.000

3PMT 0.010 ± 0.002 0.010 ± 0.001 0.006 ± 0.002

6PMP 0.011 ± 0.001 0.009 ± 0.001 0.011 ± 0.001

6PMT 0.012 ± 0.001 0.009 ± 0.001 0.011 ± 0.002

Uterus Control 0.107±0.01

3PMP 0.113 ± 0.02 0.093 ± 0.01 0.106 ± 0.01

3PMT 0.112 ± 0.01 0.089 ±0.02 0.084 ± 0.01

6PMP 0.114 ± 0.02 0.123 ± 0.02 0.156 ± 0.01

6PMT 0.150±0.05 0.103±0.01 0.131 ±0.16

tiver Control 0.954 ± 0.14

3PMP 1.196±0.05 1.292 ± 0.06 1.298 ± 0.08

3PMT 1.070 ± 0.12 1.272 ± 0.06 0.972 ± 0.01

6PMP 1.190 ± 0.05 1.094 ±0.12 1.344 ± 0.14

6PMT 1.307 ± 0.12 1.144 ± 0.09 1.266 ±0.06

Spleen Control 0.081 ± 0.00

3PMP 0.086±0.00 0.089±0.00 0.117 ± 0.03

3PMT 0.095 ±0.02 0.075 ± 0.01 0.073 ±0.00

6PMP 0.079 ±0.00 0.075 ± 0.01 0.094 ±0.01

6PMT 0.101 ±0.01 0.090 ±0.00 0.086±0.00

Kidney Control 0.246±0.00

3PMP 0.263 ±0.01 0.298±0.02 0.304 ±0.02

3PMT 0.316 ± 0.02. 0.283 ±0.02 0.256 ± 0.00

6PMP 0.296 ± 0.01 0.267 ±0.03 o.304 ± o.o1•

6PMT 0.337 ± 0.03. 0.304 ±0.03 0.322 ± 0.03•

<p< 0.05

IFetoto:(jcity of panmasafa 17 5

7 .6. Discussion

The present study suggests that in utero PMT exposure can cause

pregnancy failure however, no considerable change in body weight gain

during pregnancy or lactation was observed. Pregnancy outcome of in utero

panmasala treatment from different gestational periods varied but preterm

birth and low birth weight of newborn among PMT treated animals were

consistent. Neonatal death also increased among some of the PMT treated

animals. The novel finding of the study is that both male and female offspring

treated in utero with panmasala were negatively affected.

Panmasala gutkha induced higher loss of pregnancy as indicated by

lower fertility index in PMT treated groups than in control or PMP treated

groups. However, all the pregnant mice that completed term gave birth to live

pups, which in turn resulted in 1 00% pregnancy index in all the groups. The

early loss of pregnancy may be due to failed fertilization, or pre-implantation/

implantation failure and early resorption. Arecoline, major alkaloid of areca

nut, had shown to inhibit the growth of implanting mouse embryos in vitro

indicating its potential to exert poisonous effects on implanting embryos in

vivo (Liu and Young, 2005). Areca catechu (areca nut) had also been

described as antifertility agent (Garg and Garg, 1971; Kapoor eta/. 1974). In

addition Acacia catechu (catechu) was also found to inhibit fertility (Azad

Chowdhary eta/. 1984). Recently, it has been demonstrated by Soares eta/.

(2007) that cigarette smoking negatively affects uterine receptiveness

independently from its effect on ovarian function. In rodents, uterine

receptivity to embryos is modulated by ovarian estrogen and progesterone

(Psychyos, 1995). Leutinizing hormone controls progesterone release, and

estrogen not only prepares the uterine endometrium but also activates

blastocysts for implantation (Yoshinaga, 1995). Additionally, copulation in

rodents produces surges of prolactin from the pituitary gland, which stimulate

the production of uterotrophic progesterone (Cross and Rossant, 2001 ).

Incubation of the human granulosa cells with cotinine, combination of nicotine,

cotinine and anabasine, or an aqueous extract of cigarette smoke resulted in

inhibition of progesterone synthesis and slightly influenced estradiol


production (Gocze et a/. 1999). They suggested these concomitant actions

behind the higher incidence of early abortion in pregnant women who smoke.

Gocze and Freeman (2000) further mentioned that cigarette smoke alkaloids

inhibited the progesterone production and cell growth of cultured MA-1 0

Leydig tumor cells and could reduce the fertilization, implantation, and early

human development. Common carcinogens reported to be present in

smokeless tobacco include TSNAs, PAHs (especially benzo(a)pyrene and

polonium -210 (Stratton et a/. 2001). PAHs are known to be capable of

inducing fetal toxaemia, retard fetal growth and development and disrupt the

endocrine system. Hence, the observed effect on decline in fertility might be

through endocrine dysfunction caused by both types of panmasala. The

relatively higher negative effects in PMT than PMP may be due to the

additional effects of tobacco.

Preimplantation embryos comprise undifferentiated pluripotent

populations of cells which depend largely on the maternal supply for essential

hormones, growth factors and nutrients that are taken into embryonic cells by

a number of mechanisms in the absence of a functional placenta (Koshy et a/.

1975; Caro and Trounson, 1984; Kaye and Gardner, 1999). Alterations in the

maternal milieu resulting from changes in environmental, metabolic, hormonal

and nutritional factors may influence the proliferation rate, differentiation and

survival of these pluripotent cells and thus contribute to implantation failure,

embryonic resorption, growth restriction and or malformations (Rehak eta/.

1996). Dams dosed from GD 0 had lower fertility index as compared to

corresponding dams dosed from GD 6, which suggests higher losses before

implantation i.e., preimplantation loss by both types of panmasala treated

groups. The fertility index was more or less equal in PMP while it was slightly

lowered in PMT treated dams exposed from GD 6 as compared to control

indicating that after implantation treatment of PMP and PMT was unable to

induce the same effect as before implantation i.e., GD 0. It is known that

chemicals pass readily from general circulation into uterine fluid and penetrate

the embryo during the preimplantation period. After exposure to exogenous

insults during the preimplantation period, the embryo either dies or survives

PetotoY\fcity of panmasafa. 177

unharmed to term because this phase is all or none period (Giavini et a/.

1990). In this period, stem cells have the capacity to substitute damaged cells,

resulting in a possible embryonic development (Carlson, 1996).

In the present study all the panmasala treated pregnant mice gained

more or less similar body weight during pregnancy but the weight gain was

slightly lower among the PMT treated groups as that of control indicating toxic

effect of PMT. No significant alterations in liver weight of treated dams were

observed at the time of weaning. The weight gain during lactation was

different in dams even though they were treated with similar doses of

panmasala indicating the role of time of initiation of panmasala treatment i.e.,

GD 0, GD 6 or GD 14 in the weight gain. This suggests that time and duration

of exposure during pregnancy might affects health status of dams while

lactating. Knight and Peaker (1982) reported that retained milk markedly

influences the gross weight of mother and by day 20 of lactation the weight

decreases. Dams treated with 6% PMP and PMT and to some extent 3%

PMT had shown decrease in weight gain during lactation that might in turn

affect offspring weight and development.

The most significant impact of maternal consumption of panmasala on

the developing fetus observed in the study is decrease in gestational length

and fetal birth weight with both the doses of gutkha. There are several reports

on humans that smoking mother had shorter gestation period. Paulson et a/.

(1994) suggested that premature delivery in human might, at least in part, be

caused by the increased uterine contractility associated with increased level

of catecholamines stimulated by nicotine. Cigarette smoke (or its individual

constituents) may cause preterm delivery through impairment of placental

function. For example, nicotine has been shown to reduce maternal blood

supply to the placenta and thus, to reduce oxygen supply to the fetus. Another

mechanism is via the alteration of hormonal secretion patterns that are

important for gestation, parturition, or both. The mechanism behind the

shorter gestation length in PMT treated animals might also be due to reasons

stated above.


Finding on fetal birth weight corroborates with previous studies in mice,

rats, hamsters and human with cigarette smoke. Effect of consumption of

tobacco in other than smoking form is also reported to cause low birth weight

in human (Verma et a/. 1983). Since nicotine stimulates the release of

epinephrine, which thereby causes vasoconstriction, chronic reduction in

placental blood flow has been postulated as a potential mechanism for low

fetal birth weight. However, some investigators have not supported this

mechanism (Barr and Brent, 1970; Bruce, 1976; Trend and Bruce, 1989) and

also showed that nicotine is not directly responsible for reduction in fetal

weight (Younoszai et a/. 1969). Recently, Wang et a/. (2002) showed

interaction of metabolic genes and smoking which in turn is associated with

infant birth weight. Hence, the role of tobacco irrespective of its mode of

intake seems the most plausible cause of low birth weight. Earlier Sinha and

Rao (1985a) found an adverse effect on birth weight of pups born to areca nut

fed mice. Recently Garcia-algar et a/. (2005) have hypothesized based on six

newborns to mothers who chewed areca nut during gestation that chronic

exposure of the fetus to arecoline can lead to adverse birth outcomes and

suggested for further study on this aspect. In the present study, PMP having

areca nut as major ingredient did not show significant alterations in pup birth

weight. This might be due to difference in dose, composition and route of

exposure of panmasala plain.

Ishikawa et a/. (2006) mentioned that fetal weight may be principally

regulated by placental growth function during mid gestation i.e., GD 11.5-

13.5. Maternal to fetal exchange of tobacco smoke constituents through the

placenta during this time period had reported to play a role in generation of

low birth weight (Esposito eta/. 2008). In present study, mice exposed to 6%

PMT from GD 14 did not significantly decreased the newborn weight as

compared to the treatment from other gestational periods i.e., GD 0 and GD 6

with the same dose of PMT. The observed lower decline in weight of fetus

might be due to high postimplantation loss leading to lower litter size. Thus, it

is speculated that animals with small litter size might have more weight as

Cf'etoto~ity of panmasafa 179

compared to dams with large litter size. It also might be possible that the

fetus weight declined at 6% PMT of GD 14 group to such an extent that leads

to its in utero death. The increase in postimplantation loss further supports

this proposition.

Pup weight might be affected due to uterine or endometrial toxicity or

uterine crowding or increased litter size which in turn creates competition for

nutrients within a litter. The effects i.e., low birth weight of pups, high

postimplantation loss observed in the present study indicated the fetotoxic

potential of panmasala that is more evident in gutkha treated animals. Three

reasons have been hypothesized for the low birth weight - 1) preterm/

premature births, 2) intra uterine growth retardation and 3) nutritional

deficiencies to dams. In the present study first two causes are possible, as

intake of food was not affected in any of the treated groups as compared to

control. In view of the fact that nicotine has been found to delay embryonic

implantation (Card and Mitchell, 1979), it is possible that PMT induced

decrease in fetal weight may be due to delayed implantation and attendant

generalized developmental delays along with preterm birth.

During lactation weight of pups born to 6% PMT treated dams was

lower than that of control. Recovery in weight after PND 4 was observed more

commonly in 3% PMP and PMT while to a lesser extent with 6% dose level in

GD 6 and GO 14 group indicating the stable damage at cellular and sub

cellular level. A trend toward excess weight in pups above control group was

noted after PND 14 with both the doses of PMP and 3% PMT treatment from

GD 0. This finding corroborates with the observation of Paulson eta/. (1994).

They reported that maternal smokeless tobacco administration (aqueous

extract) resulted in heavier pups than control. They hypothesized interactive

effects, when all constituents are present at their specific concentration, may

show synergism at one concentration but a subtractive or antagonistic effect

at a different dose. This rationale can also be taken into consideration.

Duration of exposure may also play a role as 3% PMT and 6% PMP exposure

to dams from GO 6 and GO 14 had shown decrease in pup weight. It might

Cf'etotoxjcity of panmasafa 180

also be possible that dams treated with different in utero exposures i.e., GD 0,

GD 6 and GD 14 with same lactation treatment respond differently during

lactation.

Increased body weight, which can also be due to obesity, is a symptom

of metabolic syndrome as described in Chapter 6.5. Both paternal betel quid

use and tobacco smoking early in pregnancy have reported to cause

metabolic syndrome. Recently maternal smoking during pregnancy is

suggested to cause future obesity and metabolic syndrome in child (Oken et

a/. 2008; I no, 2009). Hence, the observed higher body weight of offspring from

some of the treated groups from GD 0, GD 6 and GD 14 might be a symptom

of metabolic syndrome caused by panmasala. However this required further

confirmation. On the contrary, offspring born to dam treated with high dose of

panmasala i.e., 6% PMT had remarkable weight reduction and effects were

consistent with pup mortality.

Pup mortality at birth i.e., neonatal death was considerably increased

with maternal panmasala use during GD 0 and GD 14 with high dose of both

PMP and PMT i.e., 6%. There are reports indicating that still births/ perinatal

mortality increases with maternal tobacco use (Krishna, 1978; Paulson et a/.

1994; Krishanamurty, 1997) and the risk was greater in earlier gestational

periods (Gupta and Subramoney, 2006). Perinatal mortality is one of the most

sensitive indices of maternal and child health (Gupta and Subramoney, 2006).

Pup mortality continued during lactation as indicated by reduced viability and

weaning indices of treated mothers in GD 0 and GD 14 groups as compared

to control. The effect was more at 6% dose used in the study. In group GD 6,

pup viability was comparable to control however litter size was smaller than

other groups. It could be due to the panmasala induced postimplantation

failure as the uterine was under standard diet during preimplantation period.

Hence, it might be possible that just after implantation under normal diet,

developing vulnerable fetus could not suddenly tolerate chemical insult and

unable to repair damage leading to in utero death. This is supported by the

higher postimplantation losses along with decreased litter size observed in the

'FetotoJ;jcity of panmasafa 181

study. Hence, lesser mortality at birth was observed among pups of dam

treated with panmasala from GD 6 as compared to GD 0 and GD 14. Viability

and weaning indices were marginally lowered in GD 6 group and this can be

attributed to continuous dosing till weaning. Embryonic loss after implantation

is used as a measure of abortifacient effect. The data on GD 6 points towards

the potential of panmasala as an abortifacient. Earlier Damascene and

Lemonica (1999) reported that anti-implantation effect/ high postimplantation

loss may be due to several reasons, such as morphological alterations in the

uterine epithelium that interfere with embryonic implantation.

Exposure to panmasala during GD 6 and GD 14 did not alter the

sexual differentiation. However, sex ratio was weakly affected only in GD 0

group. Krishnamurty and Joshi (1993) showed decrease male:female ratio of

live newborns among maternal smokeless tobacco use. Further Krishnamurty

(1997) showed maternal tobacco use in any form was associated with male

fetal damage. These factors may attribute to the minor variations on sex ratio

observed in the present study. Once the sexual differentiation occurs, the

subsequent kinetics of development is dramatically different in the male and

female (Pyror eta/. 2000).

Panmasala in utero treatment led to alterations in pups' testis,

epididymis and seminal vesicle weight and the effect was more with PMT

exposure. Weight of somatic organs viz., liver, spleen and kidney were also

marginally affected by 3% PMT and 6% of both PMP and PMT in most of the

treated groups as compared to control. Similar effects on reproductive as well

as somatic organ weight were observed in female offsprings. Organ weight

variation even though non significant point towards the toxic potential of

panmasala in F1 generation.

Reproductive toxic potential of panmasala was also revealed by F1

male spermiogram. The changes in spermatid count/ g testis were significant

at 6% PMT while sperm count/ g cauda was decreased non significantly. An

effect of a substance on spermatid number may indicate that it interferes in

•Fetotoxjcity of panmasafa 182

the process of spermatogenesis, while an effect on sperm number in the

cauda epididymis may not be specific to the alterations of the

spermatogenesis (Dalsenter et a/. 1999). For any given species, there is a

fixed ratio of Sertoli to germ cells; thus agents that affect Sertoli cell

proliferation during the first two weeks of postnatal life will set the sperm

production rate per testis (Pyror et a/. 2000). Efficiency of sperm production

and daily sperm production in pups' testis were hampered after panmasala

treatment to dams. Panmasala dosing in the present study was continued till

weaning (covering first three weeks of postnatal life) and both arecoline and

nicotine are reported to pass to offspring via lactation. Hence, observed effect

might be due to alterations in Sertoli cells functioning and direct or indirect

effect on testicular tissue with 6% dose level. Simultaneously an increase in

sperm head shape abnormality was observed in the treated groups as

compared to control.

Previous studies reported reproductive toxic potential of chewing

tobacco in the form of decrease in human semen quality (Said et a/. 2005)

and panmasala induced sperm head shape abnormality in mice (Kumar et at.

2003). The present study revealed a considerable decline in sperm quality of

offspring prenatally exposed to panmasala. Jensen et a/. (2005) showed non

significant 10% lower mean sperm concentrations and higher oligozoospermia

with maternal smoking during pregnancy. Few other studies have also come

up with the suggestion that maternal smoking can reduce reproductive health

of male offspring (Ramlau-Hansen eta/. 2007). Earlier a study mentioned that

early exposure to maternal smoking is not associated with any measurable

impairment of semen quality in their sons but current active smoking by men

significantly decreased in the percentage of sperm with normal morphology

(Ratcliffe eta/. 1992).

There are growing evidences that oxidative stress significantly impairs

sperm functions. Due to the high content of polyunsaturated fatty acids,

spermatozoa are susceptible to damage induced by reactive oxygen species

(ROS). The observed altered spermiogram in panmasala treated groups may

if'etoto.xjcity of panmasafa 183

also be via oxidative stress. Nair et a/. ( 1987) had noted H20 2 and superoxide

radical productions during the auto oxidation of areca nut polyphenols when

the pH level was greater than 9.5. Moreover, activity of areca nut ingredients

to induce ROS and genetic damage in oral cells has been well documented

(Bagchi et a/. 2002}. Lipid peroxidation, a free radical mediated process,

induces many pathological events including bioactivation of xenobiotics,

oxidative degradation of cellular biomolecules, and modulation of chemical

carcinogenesis. The elevated level of LPO as indicated by TBARS production

in the experimental groups may further potentiate the involvement of ROS in

altered spermatogenesis. Non enzymatic and enzymatic antioxidant systems

of the cells provide a defence mechanism against the attack of ROS.

Glutathione plays an important role in protection and detoxification processes,

as well as in the regulation of cell death. The cellular thiol redox state is a

crucial mediator of multiple metabolic, signaling, and transcriptional processes

in cells, and a fine balance between oxidizing and reducing conditions is

essential for the normal function and survival of cells (Berndt et a/. 2007).

Enhancement of thiol level in testis was observed in all the panmasala treated

groups than control. Although reductions in total thiol have been associated

with oxidative stress, increase level in thiol observed might be due to

compensatory reaction to protect the cells against intensive damage.

Supplementation of GSH from Sertoli cells is required for

spermatogenic cells both as protection from ROS and source of amino acid

for spermatogenesis (Darmani and AI-Hiyasat, 2005) as GSH content of

spermatozoa is very low. The depletion of cellular glutathione makes the cell

susceptible to potential further attack by ROS. Hence, reductions in enzymatic

i.e., SOD and non enzymatic i.e., glutathione antioxidants might be

responsible for the increased TBARS production and thereby occurrence of

oxidative stress. However, panmasala induced oxidative stress and altered

spermatogenesis through modification in Sertoli cells functioning cannot be

ruled out.

q:'etotoyjcity of panmasafa 184

This study provides the first time evidence that prenatal exposure of

panmasala may cause oxidative stress in testicular tissue of offspring. Data

together with male spermiogram are consistent with increased oxidative

stress/ ROS as a component of the mechanism of altered spermatogenesis in

mice by panmasala, with potential for contributing to prenatal toxicity.

Although most of the data are statistically non significant but the results of

treated groups were distinct with respect to control. According to Lemonica

(1996), a comparison of the teratogenic effect of chemical substances

between human and animals revealed that laboratory animals are generally

more resistant than humans. The few adverse effects observed in the study

should be considered positively and pregnant women must avoid

consumption of both types of panmasala during the entire period of

pregnancy.

CHAPTER 7 Effect of panmasala on pregnant mice and their...

Documents

Transcript of CHAPTER 7 Effect of panmasala on pregnant mice and their...