In Plant-associated Toxins; Agricultural, Phytochemical and Ecological Aspects
(Eds. S.M. Colegate & P.R. Dorling.
CAB International, Wallingford, United Kingdom.p.201-206
The Effect of Gossypol on the Growth and
Total Protein of Animal Cells in Culture
Ismartoyo and T. Acamovic
School of Agriculture, Scottish Agricultural College, 581 King Street,
Aberdeen AB9 IUD, UK
Gossypol (2,2’-bis(8-formyl)-1,6,7-trihydroxy-5-isopropyl-3-methylnaphthalene) inhibits the
growth of rumen microorganisms (Ismartoyo, et al., 1993) and also appears to depress the growth
rate of cells and sperm count in mammals, especially monogastics (Randel, 1991; Randel et al.,
1992), and causes increased fragility of red blood cells. Mitochondria and germinal epithelium
cells are also damaged by gossypol. The inclusion of pure gossypol in the diet resulted in
depression of body weight of chicks (Boatner et. al., 1948); Lillie and Bird, 1950) and rats (Eagle
and Bialek, 1950; Ambrose and Robbins, 1951); Eagle and Davies, 1957). Recent studies
(Huston et. al., 1990) indicated that daily, oral administration acetic acid (GAA) to young lambs,
at a level of 10 mg kg-1 day-1 resulted in death. Anorexia preceded death and at postmortem
examination there were macroscopic lesions in the gastrointestinal tract. Kramer et al., (1988)
found that gossypol inhibited testicular development in pubertal rams. Young bulls fed whole
cottonseed and cottonseed meat containing gossypol (Arshami and Ruttle, 1988) had
seminiferous tubules with enlarged lumina, decreased wall thickness, reduced number of cell
layers and damaged spermatogenic cells. Osmotic fragility of erythrocytes was also increased in
animals given a diet containing gossypol (Calhoun et al.,1990; Huston et al., 1990; Maksudi et
al., 1991;Wyse et al.,1991; Calhoun et.al., 1992). However, other studies (Gray and Williams,
1991) have shown that long term (150 days) inclusion of pure gossypol (10g day-1) in the diet of
mature beef cows, had no detrimental effects on ovarian steroidogenic potential and reproductive
endocrine characteristics. Gambil and Humprey (1992) found that prepubertal heifers, given
gossypol in their diet at six to 14g day-1, showed no change in their reproductive performance or
erythrocyte fragility.
Whilst it is apparent that gossypol has an adverse effect on animals, its effect on the cell is
unclear. In the present study an animal cell culture was established to examine the effect of
gossypol on cell growth and total protein.
Establishment of Cell Culture
Standard media was made from 400ml autoclaved, double distilled water, 50ml Dulbeccos MEM
10x, 50ml New Born Calf Serum, 25ml sodium bicarbonate (75g I-1), 5ml standar antibiotic
penstrep and 5ml glutamic acid 100x.
High concentration gossypol media was made by diluting 25µl of stock solution (4mg
gossypol in 2ml ethanol) with 5ml fresh media, giving a final gossypol concentration of 10µg
ml-1 media. A low gossypol concentration (2.5µg ml-1 media) was prepared by diluting 0.5 ml
stock solution with 1.5 ml ethanol and then 25µl from this solution was diluted with 5ml fresh
media.
A cell culture was established according to the method of Freshney (1987). The cell
culture was initiated by seeding secondary 3T3 mouse fibroblasts in plastic culture flasks with
5ml fresh media. The cells were incubated at 36.5C with 5 % CO2 gas phase. After 48 hours
In Plant-associated Toxins; Agricultural, Phytochemical and Ecological Aspects
(Eds. S.M. Colegate & P.R. Dorling.
CAB International, Wallingford, United Kingdom.p.201-206
incubation the cells were subcultured. The cells washed carefully with 5ml of phosphate buffered
saline (PBS). Trypsin solution (3ml) was added to the flasks and incubated at 36C for the
minutes until the cells were “rounded up”. The cells were transferred immediately into two
plastic tubes containing 5ml of fresh media. The tubes were then centrifuged at 850rpm at 4C
for 11 minutes. The supernatant was removed and the cells mixed thoroughly with 5ml media.
The cells were transferred immediately into the culture flasks and incubated as described above.
Effect of Gossypol on Cells
Cell Counting and Measurement of Total Protein
Four treatments were examined in this experiment. These treatments were: T0 = standard media;
Te = ethanol, 1µl ml-1 of media; TgL = gossypol, 2.5µg ml-1 of media; and TgH = gossypol, 10µg
ml-1 of media. The number of cells and total protein were measured after 0, 24, 48 and 72 hours
of incubation. Cells were counted using a haemocytometer. Total protein was assyed
spectrophotometrically with coomassie blue according to the method of Bradford 91976). The
influence of gossypol on protein estimation using coomassie blue was investigated and found to
have no effect. One way ANOVA was employed (Minitab) to examine the differences between
the means of treatments. Table 37.1 shows the effect on cell numbers of the presence of gossypol
in the culture medium.
The presence of gossypol at 10µg ml-1 significantly depressed the growth of the cells and
resulted in the death of some cells which floated in the media, reducing the density of surviving
cells following this treatment. The numbers of these cells were slightly in the first 24 hours of
incubation, but significantly decreased at 48 and 72 hours after incubation. The shape of the cells,
in the dishes treated with gossypol, was distorted and appeared to be hypertrophic. The cells
appreared to adapt to gossypol at the low concentration (2.5µg ml-1).
Table 37.1 The effects of gossypol on the growth of mouse 3T3 cells in culture.
Number of cells (x 106)
T0
Te
TgL
TgH
SEM
Prob.
0 hours
0.58
0.60
0.35
0.33
0.05
P<0.01
24 hours
1.61
1.02
0.70
0.35
0.08
P<0.01
48 hours
1.96
2.53
10.7
0.31
0.19
P<0.01
72 hours
3.28
1.98
1.11
0.30
0.28
P<0.01
Mean
1.86a 1.53a 0.81a 0.33b 0.36
P<0.05
Means with different superscripts are significantly different (p<0.05, n = 16)
Times of incubation
The Effect of Gossypol on the Total Protein
The presence of gossypol at 2.5µg ml-1 had no significant influence on the total protein of the
cells (Table 37.2). However, the presence of gossypol at a concentration of 10µg ml-1
significantly depressed (p<0.05) the total protein of the cells demonstrating that gossypol has an
adverse effect on protein synthesis of the cells in culture.
The protein content of the cells (Table 37.3) increased as gossypol concentration
increased (cell numbers decrease and cell size increases). This confirm the observation that
In Plant-associated Toxins; Agricultural, Phytochemical and Ecological Aspects
(Eds. S.M. Colegate & P.R. Dorling.
CAB International, Wallingford, United Kingdom.p.201-206
gossypol inhibits cell division causing hypertrophy, by a mechanism which has yet to be
elucidated.
Conclusions
Cell growth was adversely affected by the presence of gossypol at concentration of 2.5µg ml-1,
and at 10µg ml-1 was completely inhibited. The density of surviving cells was reduced and the
shape and size of the cells was distorted and appeared to be hypertrophic. The protein content of
cells treated with gossypol was higher than control cells. The significance of these observations
on animal cell in vivo remains to be ascertained.
Aknowledgments
We wish to acknowledge the financial support from the government of Indonesia through The
British Council. The helpful advice of G. Pugh-Humphries and J.R. Scaife (Zoology Department
and School of Agriculture, respectively, Aberdeen University) with respect to tissue culture was
greatly appreciated
Table 37.2 The effect of gossypol on the total protein of mouse 3T3 cells in culture.
Total protein (µg ml-1)
To
Te
TgL
TgH
0 hours
91.5
93.5
82.0
102.0
24 hours
128.0
117.0
93.0
100.2
48 hours
141.5
126.0
111.0
72.0
72 hours
252.0
126.0
156.5
89.5
Mean
153.3a
115.6a
110.6a
91.0b
Means with different superscripts are significantly different (P<0.05, SEM = 8.21, n=8).
Times of incubation
Table 37.3 Ratio of total protein of cell number (µg[106 cells]-1)
Times of incubation
Ratio of protein to cell number
To
Te
TgL
0 hours
155.87
155.83
234.28
24 hours
47.40
114.14
132.85
48 hours
72.12
49.66
103.25
72 hours
76.66
63.41
140.99
a
a
Mean
96.01
95.76
152.84a
SD
40.01
48.70
56.66
Means with different subscripts are significantly different (P<0.01, SEM = 22.7).
TgH
302.67
287.14
230.76
298.33
279.72b
33.29
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In Plant-associated Toxins; Agricultural, Phytochemical and Ecological Aspects
(Eds. S.M. Colegate & P.R. Dorling.
CAB International, Wallingford, United Kingdom.p.201-206