CELL STRUCTURE AND FUNCTION 27: 173–180 (2002)
© 2002 by Japan Society for Cell Biology
Ultrastructural Morphometrical and Immunocytochemical Analyses of
Hepatocyte Nuclei from Mice Fed on Genetically Modified Soybean
Manuela Malatesta1∗, Chiara Caporaloni1,2, Stefano Gavaudan2, Marco B.L. Rocchi3, Sonja Serafini4,
Cinzia Tiberi1, and Giancarlo Gazzanelli1
1Istituto di Istologia e Analisi di Laboratorio, via Zeppi s.n., University of Urbino, 61029 Urbino (PU), Italy,
2Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, via Salvemini 1, 06126 Perugia, Italy,
3Istituto di Biomatematica, Località Crocicchia, University of Urbino, 61029 Urbino (PU), Italy, and 4Istituto
di Chimica Biologica “G. Fornaini”, via Saffi 2, University of Urbino, 61029 Urbino (PU), Italy
has been reported so far; however, the scientific literature in this field is still quite poor. Therefore, we carried out
an ultrastructural morphometrical and immunocytochemical study on hepatocytes from mice fed on GM soybean,
in order to investigate eventual modifications of nuclear components of these cells involved in multiple metabolic
pathways related to food processing. Our observations demonstrate significant modifications of some nuclear
features in GM-fed mice. In particular, GM fed-mice show irregularly shaped nuclei, which generally represents
an index of high metabolic rate, and a higher number of nuclear pores, suggestive of intense molecular trafficking.
Moreover, the roundish nucleoli of control animals change in more irregular nucleoli with numerous small
fibrillar centres and abundant dense fibrillar component in GM-fed mice, modifications typical of increased
metabolic rate. Accordingly, nucleoplasmic (snRNPs and SC-35) and nucleolar (fibrillarin) splicing factors are
more abundant in hepatocyte nuclei of GM-fed than in control mice. In conclusion, our data suggest that GM
soybean intake can influence hepatocyte nuclear features in young and adult mice; however, the mechanisms
responsible for such alterations remain unknown.
No direct evidence that genetically modified (GM) food may represent a possible danger for health
cell nucleus/liver/genetically modified soybean
Humans have been altering the genome of animals and
plants for centuries and selective breeding has been used
to produce some desirable characteristics such as yield
increase, quality modifications or resistance to diseases.
Recently, genetic modification has become the domain of
molecular biology and genetic engineering, and genetically
modified (GM) organisms have been produced in which
new genes have been inserted into the original genome. In
particular, genetic engineering has been widely applied in
agriculture, thus creating GM crops which are nowadays
distributed all over the world.
No direct evidence that GM food may represent a possi-
ble danger for health has been reported so far; however, the
scientific literature in this field is still quite poor (Schubbert
et al., 1994, 1997, 1998; Ewen and Pustzai, 1999; Chiter et
al., 2000; Edwards et al., 2000; Halford and Shewry, 2000),
especially as to the possible effect of a diet involving a
significant amount of GM plants.
The liver is a primary site for biotransformation of the
products of digestion and is strategically located between
the intestinal tract and the general circulation. Moreover, it
degrades and detoxifies toxic compounds received from the
intestines or from the general circulation and excretes them
in the bile. Finally, it synthesizes many protein components
of blood plasma and exercises an important degree of con-
trol over the general metabolism. Therefore, hepatocytes
*To whom correspondence should be addressed: Dr. Manuela Malatesta,
Istituto di Istologia e Analisi di Laboratorio, University of Urbino, via
Zeppi s.n., 61029 Urbino, Italy.
Tel: +39–0722–320168,Fax: +39–0722–322370
Abbreviations: ALT, alanine aminotransferase; AST, aspartate amino-
transferase; CP4 EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase of
Agrobacterium sp. strain CP4; DFC, dense fibrillar component; FC, fibrillar
centre; GC, granular component; GGT, gamma-glutamyltranspeptidase;
GM, genetically modified; IG, interchromatin granule; LDH, lactic
dehydrogenase; N/C, nucleus/cytoplasm; NGS, normal goat serum; NP,
nuclear pore; PBS, phosphate buffered saline; PF, perichromatin fibril;
PG, perichromatin granule; RER, rough endoplasmic reticulum; RNP,
ribonucleoprotein; snRNP, small nuclear ribonucleoprotein.
M. Malatesta et al.
represent a useful model for monitoring one of the targets of
In the present study, we carried out ultrastructural mor-
phometrical and immunocytochemical analyses on hepa-
tocyte nuclei from mice fed on GM soybean, in order to
investigate eventual modifications of nucleoplasmic and
nucleolar constituents. Such nuclear components represent
the structural counterpart of transcription and processing of
messenger and ribosomal RNAs and therefore constitute
fine and highly sensitive indicators of cellular activity.
Materials and Methods
Animals and tissue processing
Pregnant Swiss mice were fed ad libitum on a standard laboratory
chow (Mulino & Frantoio del Trasimeno, Castiglione del Lago,
PG, Italy) containing wheat, barley, maize, alfa alfa, skimmed
milk, minerals and 14% GM soybean obtained by the insertion of
the bacterial CP4 EPSPS (5-enolpyruvylshikimate-3-phosphate
synthase, an enzyme obtained from Agrobacterium sp. strain CP4)
gene conferring a high level of tolerance to glyphosate, the active
ingredient of the herbicide Roundup (Padgette et al., 1995). In
parallel, other pregnant mice were fed on the same diet with wild
soybean. The respective litters were allowed to grow in standard
cages (four animals each) under constant environmental conditions
(21±1°C, 50±5% moisture, 12L:12D daylight cycle) for different
periods on the parental diet. Both animal groups started their re-
spective diets at weaning. Twenty-four female mice from the litter
were used: twelve of them were control animals while twelve ate
GM food. The mice were weighed and then sacrificed by cervical
dislocation when 1, 2, 5 or 8 months old. The liver was quickly
removed, weighed and cut in small fragments. For conventional
ultrastructural morphology samples of liver from the right lobe
were fixed by immersion in a mixture of 2.5% glutaraldehyde and
2% paraformaldehyde in 0.1M Sörensen phosphate buffer, pH 7.4
for 3 h, washed, post-fixed with 1% OsO4 and 1.5% potassium
ferrocyanide at 4°C for 1 h, dehydrated with acetone and embedded
in Epon. For morphometrical and immunocytochemical studies on
cell nuclei other samples of liver from the same lobe were fixed
with 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1M
Sörensen buffer at 4°C for 2 h. After washing in the same buffer
and in phosphate buffered saline (PBS), free aldehydes were
blocked in 0.5 M NH4Cl in PBS for 45 min at 4°C. Following
washing in PBS, the specimens were dehydrated through graded
concentrations of ethanol and embedded in LRWhite resin poly-
merised with U.V. light. Semithin sections (2 µm in thickness)
were stained with 1% toluidine blue and observed with a Leitz
Orthoplan light microscope. Ultrathin sections (70–90 nm in
thickness) were placed on grids coated with a Formvar-carbon
layer; Epon-embedded samples were conventionally contrasted
with uranyl acetate and lead citrate, while LRWhite-embedded
samples were stained with the EDTA method (Bernhard, 1969),
which reduces chromatin contrast, thus revealing ribonucleoprotein
(RNP) constituents. The specimens were observed in a Philips EM
300 electron microscope operating at 80 kV.
Morphometrical analyses were performed both at light and elec-
tron microscopic level on LRWhite-embedded samples. Semithin
sections of liver were photographed (final print magnification
×400) by the Leitz Orthoplan light microscope and 100 hepato-
cytes per each animal were considered. By using a computerised
image analysis system (Image Pro-Plus for Windows 95), nuclear
and cellular areas were measured; this allowed us to calculate the
nucleus/cytoplasm (N/C) ratio. In order to evaluate quantitatively
the fine nuclear features, further morphometrical analyses were
performed on a total of 200 randomly selected electron micro-
graphs (final magnification ×18,000) of hepatocyte nuclei (10 mi-
crographs from each animal). Areas and perimeters of nuclei were
measured to compute an index of nuclear shape irregularity, ex-
pressed as the ratio between the perimeter and the circumference
of the equivalent circle (I=P/2πr, where P is the observed perime-
ter, r is the radius of equivalent circle having the same area A; thus
r=). Moreover, areas of nucleoli as well as of each nucleolar
component – fibrillar centres (FCs), dense fibrillar component
(DFC) and granular component (GC) – were measured and the
percentage of FC, DFC and GC area per nucleolus was calculated.
Finally, the nuclear pores (NPs) were counted and their density
expressed as the ratio between their number and the nuclear mem-
brane length (NP/µm).
For each biological variable a two-way ANOVA test (with age
and food factors) was performed. The ANOVA models included an
interaction term between the factors. A correction term for multi-
level design was introduced to take into account the fact that cellu-
lar data from different animals were pooled. When necessary, data
were transformed to achieve either normalisation or variance stabi-
lisation, as appropriate. Significance level was fixed at α=0.05.
In order to investigate the fine intranuclear distribution of some
splicing factors in GM-fed and control mice, samples of LRWhite-
embedded liver were processed for immunocytochemistry. Since
our ultrastructural observations have revealed similar nuclear fea-
tures among control animals as well as among the GM-fed mice re-
gardless of age, we carried out the immunocytochemical study on
5 month-old animals, as a sample. Mouse monoclonal antibodies
directed against the (Sm)snRNP (small nuclear ribonucleoprotein)
core protein (Lerner et al., 1981), the non-snRNP splicing factor
SC-35 (Sigma-Aldrich, Buchs, Switzerland) and the nucleolar pro-
tein fibrillarin (Cytoskeleton Inc., Denver, CO) were used. Sec-
tions were floated for 3 min on normal goat serum (NGS) diluted
1:100 in PBS and then incubated for 17 h at 4°C with the primary
antibodies diluted with PBS containing 0.1% bovine serum
albumin (Fluka, Buchs, Switzerland) and 0.05% Tween 20. After
rinsing, sections were floated on NGS, and then reacted for 20 min
at room temperature with the secondary gold-conjugated antibody
(Jackson ImmunoResearch Laboratories Inc., West Grove, PA,
USA) diluted 1:10 in PBS. Finally, the sections were rinsed and
GM Soybean Effects on Hepatocyte Nuclei
air-dried. As controls, some grids were treated with the incubation
mixture without the primary antibody, and then processed as
In order to assess the presence of the splicing factors quantita-
tively, the labelling density over some nuclear compartments was
evaluated on sections treated in the same immunolabelling experi-
ment. The surface area of each compartment considered – nucleo-
plasm, nucleolus and, in the case of the anti-fibrillarin antibody,
DFC – was measured on fifteen randomly selected electron micro-
graphs (×20,000) from each animal by using a computerised image
analysis system (Image Pro-Plus for Windows 95). For back-
ground evaluation resin outside the tissue was considered. The
gold grains present over the investigated compartments were
counted and the labelling density was expressed as the number of
gold grains per square micrometer.
The data for each variable were then pooled according to the ex-
perimental groups and the meansstandard error of the mean (SE)
values calculated. Statistical comparisons were performed by the
Kruskal-Wallis one-way ANOVA test. Statistical significance was
set at P≤0.05.
At the time of sacrifice some liver samples from the right lobe
were quickly removed into ice-cold homogenisation buffer (in
mM): 280 mannitol, 10 KCl, 1 MgCl2, 0.2 Pefabloc SC, 10 Hepes,
pH 7.0 adjusted with Tris (Thevenod et al., 1999). The tissue was
minced into a fine paste and homogenised manually. The crude
homogenate was centrifuged at 10,000 rpm for 10 min in a
Microfuge 18 Centrifuge (Beckman Coulter, Inc.), total protein
content was determined according to Bradford (1976) and as-
partate aminotransferase (AST), alanine aminotransferase (ALT),
lactic dehydrogenase (LDH) and gamma-glutamyltranspeptidase
(GGT) activities were evaluated enzymatically by means of a Vitros
System Chemistry 950 (Ortho-Clinical Diagnostics, Johnson &
Statistical comparisons were performed by the non-parametric
Mann-Whitney U-test and the significance level was set at P≤0.05.
Body weight of mice at the time of sacrifice ranged from 26
to 38 g without significant differences between control and
GM soybean-fed animals; moreover, no macroscopic modi-
fications of liver was observed, its weight ranging from 0.8
g to 1.9 g in all animals.
Electron microscopic examination of Epon-embedded
liver samples demonstrated similar structural features of
hepatocyte cytoplasmic organelles in control and GM
soybean-fed mice (Fig. 1) at all ages considered: the RER
was arranged in irregularly oriented cisternae; the Golgi
apparata were well developed; mitochondria exhibited
ovoid shapes and well-developed transversal cristae;
with transversal cristae, Golgi apparata (arrows) are well developed and RER cisternae (thick arrows) are irregularly arranged. G: glycogen; L: lipid
droplets. Bars: 0.5 µm.
Hepatocyte cytoplasm from control and GM soybean fed mice. In both control (a) and GM-fed (b) mice the mitochondria (M) show ovoid shapes
M. Malatesta et al.
glycogen particles were numerous and mostly gathered in
lakes, sometimes in association with lipid droplets.
On the other hand, the ultrastructural observations carried
out on LRWhite-embedded EDTA-stained samples revealed
modifications of some nuclear features of hepatocytes from
mice fed on GM soybean in comparison to control animals.
Hepatocyte nuclei from both 1, 2, 5 and 8 month-old con-
trol mice (Fig. 2a) generally showed roundish shapes and
contained clumps of condensed chromatin distributed
both at the nuclear periphery and inside the nucleus. In the
nucleoplasm, abundant perichromatin fibrils (PFs) and
perichromatin granules (PGs) were distributed along the
borders of the condensed chromatin, while interchromatin
granule (IG) clusters occurred in the interchromatin space.
the nuclear irregular shape of the nucleus from the GM-fed mouse (b) in
comparison to the nuclear roundish shape of the control animal (a). C:
condensed chromatin; Nu: nucleolus; IG: interchromatin granules; thick
arrows: perichromatin granules; arrows: perichromatin fibrils. Bars: 1µm.
Hepatocyte nuclei from control and GM soybean fed mice. Note
nucleolus of the control animal (a) shows large fibrillar centres (asterisks)
surrounded by dense fibrillar component (thick arrows), while the nucleo-
lus of the GM-fed mouse (b) displays small fibrillar centres (asterisks) and
abundant dense fibrillar component (thick arrows). Bars: 0.5 µm.
Hepatocyte nucleoli from control and GM soybean fed mice. The
GM Soybean Effects on Hepatocyte Nuclei
The nucleoli generally displayed roundish shapes, with
some FCs surrounded by DFC and abundant GC (Fig.3a).
Hepatocyte nuclei from 1 month-old mice fed on GM
soybean showed roundish shapes similar to those of control
animals (not shown), whereas hepatocyte nuclei from 2, 5
and 8 month-old mice fed on GM soybean (Fig. 2b) fre-
quently showed irregular shapes. This nuclear shape irregu-
larity appeared as a fine waving and was not due to cyto-
plasmic inclusions distorting the nuclear surface. Moreover,
GM-fed mice showed irregular and less compact nucleoli,
with many small FCs and abundant DFC (Fig. 3b). On the
other hand, the nucleoplasmic components did not show any
Morphometric results are described in Table I. In detail,
the nuclear area was generally larger in control than in GM-
fed mice; the N/C ratio was lower in 8 month-old animals in
comparison to younger mice; the shape index was generally
higher in GM-fed than in control mice. The nucleolar area
did not change; FC area and GC percentage were generally
lower in GM-fed mice than in controls, while the DFC per-
centage was always higher in GM-fed mice. The FC per-
centage drastically decreased in older animals with respect
to younger ones. Finally, the nuclear pore density was al-
ways higher in GM-fed mice than in controls.
Immunocytochemcal analysis demonstrated that no
difference in snRNP, SC-35 and fibrillarin distribution
occurred between GM-fed and control mice. As expected,
snRNPs were mainly associated to PFs and, to lesser extent,
to IGs, (Fig. 4a), moreover, a few snRNPs were found in
nucleoli; SC-35 was specifically associated to PFs and IGs
(Fig. 4b); fibrillarin accumulated in nucleolar DFC (Fig.
4c). However, quantitative evaluation of immunolabelling
revealed a stronger labelling in GM-fed mice in comparison
to controls for all splicing factors investigated (Table II).
The background level was 1.02±0.11 gold grains/µm2.
Biochemical analyses of AST, ALT, LDH and GGT activ-
ities revealed no difference between control and GM-fed
mice at all ages considered (Table III).
Our observations carried out on hepatocyte nuclei from
control and GM soybean-fed mice demonstrate significant
modifications of some nuclear features in all GM-fed mice,
whereas cytoplasmic organelles do not show any evident
Modifications of hepatocyte nuclear size are related to
both age and food. It is known that cell nuclei become pro-
gressively larger as age increases (Schmucker, 1990), but, in
our animals, they are also generally larger in control than in
GM soybean fed mice. However, the differences in nuclear
size between control and GM-fed mice do not imply differ-
ences in N/C ratio; conversely, N/C ratio changes are related
to the age. In fact, 8 month-old mice show a lower N/C ratio
value, probably due to the increased deposition of lipids and
glycogen in hepatocyte cytoplasm observed at this age (not
The nuclear shape also is influenced by both age and
food. In fact, except for 1 month-old animals, all GM fed-
mice showed irregularly shaped nuclei, while control ani-
mals generally showed roundish nuclei. An irregular nucle-
ar shape generally represents an index of high metabolic
rate (e.g. Aziz and Barathur, 1994; Motohashi et al., 1992;
Malatesta et al., 1998); in fact, an increase in the nucleus-
cytoplasmic interface may improve the molecular traffick-
ing between the two cellular compartments. Accordingly, an
increased nuclear pore frequency has been found in the
irregularly shaped nuclei of GM fed-mice in comparison to
the roundish nuclei of control animals.
Hepatocyte nucleoli also undergo structural modifica-
MORPHOMETRIC EVALUATION OF NUCLEAR VARIABLES IN CONTROL AND GM-FED MICE
Age Nuclear area
(* † ‡§)
(* † ‡ §)
FC area (×100)
(* † §)
(* ‡ §)
(* ‡ §)
(* † ‡)
1 month (C)40.52±1.100.20±0.004 1.16±0.021.06±0.101.77±0.202.49±0.3426.16±2.12 71.45±2.270.97±0.04
1 month39.73±0.58 0.19±0.0031.18±0.021.04±0.091.17±0.182.96±0.5833.07±1.42 64.12±1.591.49±0.05
2 months (C) 44.59±0.880.22±0.0031.13±0.020.83±0.082.00±0.31 2.63±0.4926.39±1.85 70.05±2.231.01±0.04
5 months (C)50.61±1.040.25±0.0041.17±0.010.87±0.06 1.87±0.142.92±0.2731.71±1.8765.37±1.860.97±0.05
5 months49.45±1.000.24±0.003 1.31±0.021.05±0.060.74±0.031.59±0.1932.40±1.2166.01±1.33 1.59±0.06
8 months (C)54.53±1.270.20±0.0041.18±0.011.02±0.081.32±0.131.16±0.2826.81±1.46 72.03±1.541.17±0.06
Means±SE values of variables considered in hepatocyte nuclei. Different symbols indicate statistical significance for complete model (∗), age (†), food (‡)
and interaction term age-food (§). C: control animals
M. Malatesta et al.
tions in GM soybean fed-mice, although their size remains
unchanged. In fact, the roundish nucleoli of control animals
change in more irregular nucleoli with numerous small FCs
and abundant DFC in GM-fed mice. The nucleolus is the
site of ribosomal gene transcription and of rRNA processing
and assembly with ribosomal proteins (reviews in Smetana
and Busch, 1974; Hadjiolov, 1985). The nucleolus is a very
dynamic structure that can rapidly adapt its activity, and
consequently its architecture, to the cellular metabolic state
(for reviews see e.g. Haidjiolov, 1985; Schwarzacher and
Wachtler, 1993; Shaw and Jordan, 1995). In particular, it is
known that when the metabolic rate increases the number of
small FCs as well as the amount of DFC increase (Jordan
and McGovern, 1981; Lafarga et al., 1991; Schwarzacher
and Wachtler, 1993; Dzidziguri et al., 1994). Interestingly,
in our animals the modifications of FC size as well as of
DFC and GC amounts are related to food only.
Taken together, our morphometrical results suggest that
hepatocyte nuclei of mice fed on GM soybean modify their
metabolic activities. According to such hypothesis, the nu-
cleoplasmic and nucleolar splicing factors investigated in
our study – snRNPs, involved in early pre-mRNA splicing
(review in Luhrmann et al., 1990); SC-35, required for spli-
ceosome assembly (Fu and Maniatis, 1990) and fibrillarin, a
component of the U3 snRNP complex, involved in several
steps of rRNA processing (Kass et al., 1990) – are more
abundant in GM-fed mice than in controls. On the other
hand, biochemical analyses of major hepatic proteins did
(arrows) and, in a lesser extent, interchromatin granules (IG), while perichromatin granules (thick arrows) are devoid of gold grains. b. The anti-SC-35 anti-
body specifically labels perichromatin fibrils (arrows) and interchromatin granules (IG), whereas perichromatin granules (thick arrows) appear unlabelled.
c. The anti-fibrillarin signal is located in the dense fibrillar component (asterisks) of nucleoli. Bars: 0.2 µm.
Hepatocyte nuclei immunolabelled with antibodies against splicing factors. a. The anti-snRNP antibody specifically labels perichromatin fibrils
GM Soybean Effects on Hepatocyte Nuclei
not reveal significant differences between control and GM-
fed mice and no modification of cytoplasmic organelles has
been observed. Therefore, it seems likely that GM food
interferes with some hepatocyte nuclear activities only.
Unfortunately, at present, it is impossible to know how that
occurs. Hepatocytes are involved in numerous metabolic
pathways: they metabolise and transform most of the prod-
ucts of digestion, degrade and detoxify toxic substances and
excrete them in the bile, synthesise many protein compo-
nents of blood plasma and are able to store glycogen and to
release glucose, thus playing a primary role in the mainte-
nance of carbohydrate homeostasis. It has been demonstrat-
ed that dietary amino acids and nucleotides are able to mod-
ulate RNA and protein synthesis in the liver and that an
altered intake may induce nuclear modifications (Bailey et
al., 1976; Lopez-Navarro et al., 1996a,b). As for the scien-
tific literature about the possible effects of GM food con-
sumption, some authors have investigated the potential pas-
sage of a part of the modified genome through the gut
(Schubbert et al., 1994, 1997, 1998; Chiter et al., 2000),
while others have studied the effects on the gastrointestinal
mucosa (Ewen and Pustzai, 1999); however, the results
obtained so far are quite controversial.
In conclusion, the present work demonstrate that GM
soybean intake can influence hepatocyte nuclear features
in young and adult mice and, although the mechanisms
responsible for such alterations are still unknown, our data
encourage further investigations on this subject of relevant
Acknowledgments. We express our gratitude to Dr. T.E. Martin for pro-
viding us with the anti-(Sm)snRNP antibody and Dr. O. Stocchi for kind
help with biochemical analyses.
Aziz, D.C. and Barathur, R.B. 1994. Quantitation and morphometric anal-
ysis of tumors by image analysis. J. Cell. Biochem., 19: 120–125.
Bailey, R.P., Vrooman, M.J., Sawai, Y., Tsukada, K., Short, J., and
Lieberman, I. 1976. Amino acids and control of nucleolar size, the
activity of RNA polymerase I, and DNA synthesis in liver. Proc. Natl.
Acad.Sci. USA, 73: 3201–3205.
Bernhard, W. 1969. A new staining procedure for electron microscopical
cytology. J. Ultrastruct. Res., 27: 250–265.
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal.l Biochem., 72: 248–254.
Chiter, A., Forbes, J.M. and Blair, G.E. 2000. DNA stability in plant tis-
sues: implications for the possible transfer of genes from genetically
modified food. FEBS Lett., 481: 164–168.
Dzidziguri, D.V., Chelidze, P.V., Zarandia, M.A., Cherkezia, E.C. and
Tumanishvili, G.D. 1994. Transcriptional activity and ultrastructure of
morphologically different types of nucleoli isolated from hepatocytes of
normal and hepatectomized rats. Epithelial Cell Biol., 3: 54–60.
Edwards, H.M., Douglas, M.W., Parsons, C.M., and Baker, D.H. 2000.
Protein and energy evaluation of soybean meals processed from geneti-
cally modified high-protein soybeans. Poult. Sci., 79: 525–527.
Ewen, S.W. and Pustzai, A. 1999. Effects of diets containing genetically
modified potatoes expressing Galanthus nivalis lecitin on rat small
intestine. Lancet, 354: 1353–1354.
Fu, X.D. and Maniatis, T. 1990. Factor required for mammalian
spliceosome assembly is localized to discrete regions in the nucleus.
Nature, 343: 437–441.
Hadjiolov, A.A. 1985. The nucleolus and ribosome biogenesis. Cell Biol.
Monographs, 12: 1–268.
Halford, N.G. and Shewry, P.R. 2000. Genetically modified crops: meth-
odology, benefits, regulation and public concerns. Br. Med. Bull., 56:
Jordan, E.G. and McGovern, J.H. 1981. The quantitative relationship of
the fibrillar centres and other nucleolar components to changes in
growth conditions, serum deprivation and low doses of actinomycin D in
cultured diploid human fibroblasts (strain MRC-5). J. Cell Sci., 52: 373–
Kass, S., Tyc, K., Steitz, J.A., and Sollner-Webb, B. 1990. The U3 small
nucleolar ribonucleoprotein functions in the first step of preribosomal
RNA processing. Cell, 60: 897–908.
Lafarga, M., Andres, M.A., Berciano, M.T., and Maquiera, E. 1991.
Organization of nucleoli and nuclear bodies in osmotically stimulated
supraoptic neurons of the rat. J. Comp. Neurol., 308: 329–339.
QUANTITATIVE EVALUATION OF SPLICING FACTOR
IMMUNOLABELING IN CONTROL AND GM-FED MICE
(Sm)snRNP2.24±0.25 (C)1.02±0.23 (C)-
Fibrillarin 0.86±0.09* (C)9.94±0.75 (C)44.51±2.96 (C)
Means±SE values of labelling densities obtained with anti-(Sm)snRNP,
anti-SC-35 and anti-fibrillarin antibodies on hepatocyte nuclei from GM-
fed and control 5 month-old mice. Values identified with common symbols
(∗, †) are not significantly different. C: control animals.
BIOCHEMICAL EVALUATION OF SOME LIVER ENZYMES IN
CONTROL AND GM-FED MICE
1 month (C) 1952.7±101.3 961.2±174.4 7964.0±683.437.3±5.7
1 month2019.3±237.3 1027.0±296.2 8650.2±736.741.6±4.5
2 months (C) 2050.2±322.1 811.0±127.3 6975.3±875.239.6±6.7
5 months (C) 2054.6±143.9815.3±70.86749.7±206.735.4±3.7
5 months1985.7±187.2 727.7±76.67185.4±165.540.2±3.8
8 months (C) 2240.3±264.1832.7±84.17338.7±462.142.0±5.8
Enzyme levels in liver tissue of the eight groups of animals (mean val-
ues±SE). Values in each column are not significantly different from each
other. C: control animals.
M. Malatesta et al.
Lerner, E.A., Lerner, M.R., Janeway, C.A., and Steitz, J. 1981. Mono-
clonal antibodies to nucleic acid-containing cellular constituents: probes
for molecular biology and autoimmune diseases. Proc. Natl. Acad. Sci.
USA, 78: 2737–2741.
Lopez-Navarro, A.T., Bueno, J.D., Gil, A., and Sanchez-Pozo, A. 1996a.
Morphological changes in hepatocytes of rats deprived of dietary nucle-
otides. Br. J. Nutr., 76: 579–589.
Lopez-Navarro, A.T., Ortega, M.A., Peragon, J., Bueno, J.D., Gil, A., and
Sanchez-Pozo, A. 1996b. Deprivation of dietary nucleotides decreases
protein synthesis in the liver and small intestine in rats. Gastroenterolo-
gy, 110: 1760–1769.
Luhrmann, R., Kastner, B., and Bach, M. 1990. Structure of spliceosomal
snRNPs and their role in pre-mRNA splicing. Biochim. Biophys. Acta
Gene Struct. Expression, 1087: 265–292.
Malatesta, M., Mannello, F., Sebastiani, M., Cardinali, A., Marcheggiani,
F., Renò, F., and Gazzanelli, G. 1998. Ultrastructural characterization
and biochemical profile of human gross cystic breast disease. Breast
Cancer Res. Treat., 48: 211–219.
Motohashi, I., Okudaira, M., Takai, T., Kaneko, S., and Ikeda, N. 1992.
Morphological differences between hepatocellular carcinoma and
hepatocellular carcinomalike lesions. Hepatology, 16: 118–126.
Padgette, S.R., Kolacz, K.H., Delannay, X., Re, D.B., LaVallee, B.J.,
Tinius, C.N., Rhodes, W.K., Otero, Y.I., Barry, G.F., Eichholtz, D.A.,
Peschke, V.M., Nida, D.L., Taylor, N.B. and Kishore, G.M. 1995.
Development, identification and characterization of a glyphosate-
tolerant soybean line. Crop Sci., 35: 1451–1461.
Schmucker, D.L. 1990. Hepatocyte fine structure during maturation and
senescence. J. Electron Microsc. Tech., 14: 106–125.
Schubbert, R., Hohlweg, U., Renz, D., and Doerfler, W. 1998. On the fate
of orally ingested foreign DNA in mice: chromosomal association and
placental transmission to the fetus. Mol. Gen. Genet., 259: 569–576.
Schubbert, R., Lettmann, C., and Doerfler, W. 1994. Ingested foreign
(phage M13) DNA survives transiently in the gastrointestinal tract and
enters the bloodstream of mice. Mol. Gen. Genet., 242: 495–504.
Schubbert, R., Renz, D., Schmitz, B., and Doerfler, W. 1997. Foreign
(M13) DNA ingested by mice reaches perpheral leukocytes, spleen and
liver via the intestinal wall mucosa and can be covalently linked to
mouse DNA. Proc. Natl. Acad. Sci. USA, 94: 961–966.
Schwarzacher, H.G. and Wachtler, F. 1993. The nucleolus. Anat.
Embryol., 188: 515–536.
Shaw, P.J. and Jordan, E.G. 1995. The nucleolus. Ann. Rev. Cell Develop.
Biol., 11: 93–121.
Smetana, K. and Busch, H. 1974. The nucleolus and nucleolar DNA. In
The cell nucleus, vol. 1 (H. Busch, ed.). Academic Press, New York, pp
Thevenod, F., Roussa, E., Schmitt, B.M., and Romero, M.F. 1999. Clon-
ing and immunolocalization of a rat pancreatic Na+ bicarbonate
coransporter. Biochem. Biophys. Res. Commun., 264: 291–298.
(Received for publication, July 1, 2002
and in revised form, September 18, 2002)