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3-Food: Effects of GM-lectin potatoes on rat guts - the Lancet article

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TITLE:  Effect of diets containing genetically modified potatoes
        expressing Galanthus nivalis lectin on rat small
        intestine - Research Letter
SOURCE: The Lancet, 354 (9187)
        by Stanley W. B. Ewen & Arpad Pusztai
DATE:   October 16, 1999

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Effect of diets containing genetically modified potatoes
expressing Galanthus nivalis lectin on rat small intestine -
Research Letter

Diets containing genetically modified (GM) potatoes expressing
the lectin Galanthus nivalis agglutinin (GNA) had variable
effects on different parts of the rat gastrointestinal tract.
Some effects, such as the proliferation of the gastric mucosa,
were mainly due to the expression of the GNA transgene. However,
other parts of the construct or the genetic transformation (or
both) could also have contributed to the overall biological
effects of the GNA-GM potatoes, particularly on the small
intestine and caecum.

Genetically modified (GM) plant products are becoming
increasingly common in the human food-chain, yet in contrast to
the general acceptance of the need for the biological testing of
novel foods and feedstuffs, few studies have been carried out on
the possible effects of GM products on the mammalian ut mucosa.
GM potatoes expressing a snowdrop lectin (Galanthus nivalis
agglutinin [GNA]) under the CaMV35s promoter have been developed
to increase insect and nematode resistance. (1) GNA was selected
for insertion into potatoes because the initial effect of this
mannose-specific lectin on the rat small bowel has been shown to
be minimal, (2) and because its binding to mannose present on the
epithelial surface of rat jejunal villi is demonstrable only
after feeding for 10 days. We compared the histological indices
of the gut of rats fed potato diets containing GM potatoes, non
GM potatoes, or non-GM potatoes supplemented with GNA, to find
out whether GNA gene insertion had affected the nutritional and
physiological impact of potatoes on the mammalian gut.

ELISA analysis confirmed that the expression level of GNA in raw
GM potatoes was 25.4 µg/g dry matter; the concentration was
decreased to 4.9 µg/g after boiling for 1 h. Six rats were
randomly allocated to each group, and were fed diets containing
either raw or boiled GNA-GM potatoes, parent potatoes (Desiree),
or parent-line potatoes supplemented with 25.4 µg/g GNA for 10
days. All potato diets were isocaloric and contained an average
of 6% protein. Histological samples of stomach, jejunum, ileum,
caecum, and colon were taken 10 days after the start of feeding.
The samples, each 2 cm in length, were opened along the
antimesenteric border. The serosal surface was allowed to adhere
to card for 3 min and was then fixed in 10% neutral buffered
formalin for 18 h at 20°C. Paraffin sections (4 µm) were stained
with haematoxylin and eosin, and mucosal thickness (stomach) or
crypt length (jejunum, ileum, caecum, and colon) was measured by
video-image analysis. Intraepithelial lymphocytes are equally
distributed in all parts of the small intestine, and are known to
increase when non-specific intestinal damage occurs. Thus, to
assess potential damage, intraepithelial lymphocytes were counted
in eight jejunal villi from each of the six rats fed diets
containing GNA-GM potatoes or parent potatoes, both raw and
boiled. No such measurements were made for the group fed parent
potatoes spiked with GNA because dietary GNA or other lectins do
not induce lymphocyte infiltration. GNA binding to the jejunum
and ileum was measured by elution with 0.1 mol/L mannose,
followed by ELISA.

The presence of GNA in the diets, irrespective of whether
originating from GNA-GM potatoes or from parent-potato diets
supplemented with GNA, was associated with significantly greater
mucosal thickness of the stomach when compared with parent-potato
diets (table 1). This effect was observed with both raw and
boiled potatoes. Crypt length in the jejunum of rats fed on raw
GNA-GM potato diets was significantly greater than in those given
parent-line or parent-line plus GNA potato diets. However, the
increase in jejunal crypt length was not seen in rats fed boiled
GNA-GM potatoes (table 1). GNA had no significant effects on the
ileum, but rats fed boiled potatoes had shorter ileal crypts than
rats given respective raw potato diets. Rats fed boiled GNA-GM
potatoes had significantly thinner caecal mucosae than rats given
boiled parent potatoes, with or without GNA supplementation
(table 1). Intraepithelial lymphocyte counts per 48 villi were
7.6 (SD 2.7) in rats fed on boiled parent potatoes, compared with
10.3 (3.3) in rats fed boiled transgenic potatoes (p<0.01). With
raw potato diets, the intraepithelial lymphocyte counts were
again significantly different: 5.3 (2.0) and 9.3 (2.6) in parent
and GM potatoes, respectively (p<0.01). Peyer's patches appeared
normal in all rats. GNA binding in the jejunum and ileum was
about the same, irrespective of whether spiked GNA potatoes or GM
potatoes were fed (table 2). Measurement of GNA binding by
immunocytochemistry also showed a similar pattern. (2)

Table 1: Effect of raw and cooked parent, parent+GNA, and GNA+GM
         potatoes on histological indices of rat gut

[part 1, split due to resticted line length in email text, HM]

Mean and Standard Deviation of crypt length (µm)
and difference between treatments*
       Parent   Parent     Parent  Parent+GNA  GNA-GM  Parent vs
                vs parent  +GNA    vs GNA-GM           GNA-GM
                  (p)                (p)                (p)

Boiled   294      0.29      347      0.37      339      0.02
         (46)               (42)               (36)
Raw      261      0.03      312      0.98      323      0.07
         (32)               (32)               (54)
p        0.18               0.94               0.35	 

Boiled    75      0.72       78      0.97       78      0.71
         (19)               (17)               (12)
Raw       57      0.14       64      0.01       90     <0.01
          (8)               (11)               (20)
p        0.06               0.09               0.24

Boiled    59      0.20       55      0.12       63      0.43
          (8)                (7)               (13)
Raw       71      0.24       79      0.43       87      0.15
          (9)               (13)               (25)
p        0.02              <0.01               0.06	 

Boiled    95      0.90       98      0.04       70      0.05
         (19)               (21)               (15)
Raw      132      0.02      104      0.25      119      0.35
         (19)               (17)               (25)
p       <0.01               0.55              <0.01	 

Boiled   146      0.02      177      0.02      139      0.65
         (15)               (24)               (24)
Raw      192      0.04      148     <0.01      215      0.28
         (34)               (25)               (34)
p        0·02               0·07              <0·01	 


[part 2, split due to resticted line length in email text, HM]

           Statistical analysis (p)#          Interaction (p)#
       Effect of   Effect of   Effect of   GNA-cook   Trans-cook
          GNA       cooking     trans-	 	 

Boiled   0.001       0.052       0.868       0.917       0.543
Boiled   0.029       0.171       0.041       0.035       0.037
Boiled   0.221       0.001       0.106       0.209       0.942
Boiled   0.033       0.001       0.566       0.497       0.021
Boiled   0.878       0.002       0.181       0.231       0.001


  Data are the means of six animals calculated from five
  observations for each
  GNA-cook   = interaction between GNA and cooking
  Trans-cook = interaction between transformation and cooking

*: by Student's t-test
#: by multivariate analysis with Tukey's test

We suggest that the promotion of jejunal growth was the result of
the transformation of the potato with the GNA gene, since the
jejunum of rats was shown to be stimulated only by GM potatoes
but not by dietary GNA (table 1), in agreement with a previous
study in which the dietary GNA concentration was 1000-fold higher
than the one used in this study. (2) Thus, we propose that the
unexpected proliferative effect was caused by either the
expression of other genes of the construct, or by some form of
positioning effect in the potato genome caused by GNA gene
insertion. Because caecal thickness was similar in rats given
boiled parent potatoes in the presence or absence of spiked GNA,
we suggest that the decrease in caecal mucosal thickness seen in
rats fed boiled GM-potato diets was the consequence of the
transfer of the GNA gene into the potato. Caecal mucosal
thickness in rats given raw potato diets was significantly higher
than in those given the corresponding boiled potatoes. Thus, the
main effect of boiling was to decrease mucosal thickness; this
binding was fully in line with expectations. The raw parent-line
potato diets supplemented with GNA were associated with a
significantly thinner caecal mucosa than that of rats given
parent-line potato diets. A similar trend was also observed in
rats fed raw GNA-GM potatoes, but the difference did not reach
significance (table 1).

Table 2: GNA binding to the jejunum and ileum of rats given diets
         containing GNA-GM potatoes or parent potato diets spiked
         with GNA

                       Raw potato             Boiled potato	 
                Parent+GNA     GNA-GM     Parent+GNA     GNA-GM
GNA intake (µg)	    30           29           15           5·6
Mean and Standard Deviation bound GNA (µg)				

Jejunum        0.47 (0.28)  0.37 (0.27)  0.25 (0.21)  0.05 (0.04)
Ileum          0.28 (0.15)  0.44 (0.25)  0.17 (0.08)  0.07 (0.02)
Remainder      5.04 (2.67)  2.23 (0.63)  0.78 (0.35)  0.20 (0.17)
Total          5.79 (2.71)  3.04 (0.60)  1.20 (0.49)  0.32 (0.17)

  On the morning of day 10, rats were given 1.5 g allocated diet
  and were killed 2 h later. After dissection, oesophagus,
  pylorus, and ileocaecal junction were clipped, and small
  intestine was washed thoroughly with saline. Small intestine
  was cut into three segments: jejunum (first 20 cm), ileum (last
  20 cm), and remainder. Tissues were homogenised with phosphate
  buffered saline containing 0.1 mol/L mannose, and solutions
  were used for determination of GNA content by competitive

As expected, colonic crypt lengths were generally higher in rats
given raw potato diets than in those given boiled potatoes,
except for animals fed GNA-supplemented raw or boiled potato
diets, between which there was no significant difference. Feeding
rats on diets containing GM potatoes, irrespective of whether raw
or boiled, had no significant effect on colonic crypt length
compared with that in animals fed the corresponding parent-line
potatoes (table 1). Rats fed on GNA-supplemented parent potatoes
had significantly shorter colonic crypt lengths than those fed on
parent potatoes of GNA-GM potatoes; the reason for this finding
is not clear.

In conclusion, the stimulatory effect of GNA-GM potatoes on the
stomach was mainly due to the expression of the GNA transgene in
the potato. By contrast, the potent proliferative effect of raw
GNA-GM potatoes on the jejunum, and the antiproliferative effect
of boiled transgenic potatoes on the caecum can be attributed
only partly to GNA gene expression. Other parts of the GM
construct, or the transformation, could have contributed to the
overall effects. Once bound, GNA is internalised by endocytosis;
(2) some other component of the construct in the GNA-GM potato or
its expressed gene product might also be able to penetrate and
affect the rat mucosal cells in a similar manner. The growth
promoting effect of raw GNA-GM potatoes in the jejunum, evident
as crypt hyperplasia, is probably due to a direct stimulatory
effect on crypt cells; the increase in T lymphocyte infiltration
may be important in the elimination of damaged enterocytes. (3)
The possibility that a plant vector in common use in some GM
plants can affect the mucosa of the gastrointestinal tract and
exert powerful biological effects may also apply to GM plants
containing similar constructs, particularly those containing
lectins, such as soya beans or any plants expressing lectin genes
or transgenes.

This study was supported by Scottish Office: Agriculture,
Environment, and Fishery Department (grant number FF 818).

1 Gatehouse AMR, Down RE, Powell KS, et al. Transgenic potato
  plants with enhanced resistance to the peach-potato aphid Myzus
  persicae. Ent Exp Appl 1996; 79: 295-307.

2 Pusztai A, Ewen SWB, Grant G, et al. Relationship between
  survival and binding of plant lectins during small intestinal
  passage and their effectiveness as growth factors. Digestion
  1990; 46 (suppl 2): 306-16.

3 Marsh NM, Ensari A. The gut associated lymphoid tissue and
  immune system. In: Whitehead R, ed. Gastrointestinal and
  oesophageal pathology. 2nd edn. Edinburgh: Churchill
  Livingstone, 1995: 201-25.

Department of Pathology, University of Aberdeen, Aberdeen AB25
2ZD, UK (S W B Ewen FRCPath, A Pusztai PhD)

Correspondence to:
Dr Stanley W B Ewen (e-mail: 

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