CaMV 35S Promoter in GM Feed that Sickened Rats Transferred into Rat Blood, Liver, and Brain Cells
The rats were fed an ordinary rat chow
found to contain GMOs on PCR analysis using probes for the cauliflower mosaic
virus 35S promoter, a gene control element in more than 80 % of commercial GM
crops grown with potential health hazards predicted since 1999 Dr Mae-Wan Ho
Researchers led by Hanaa Oraby at Egypt’s
National Research Center in Cairo are not the first to look for horizontal
transfer of genetically modified (GM) DNA into animal cells, but certainly
among the first to do an experiment aimed at detecting it and succeeded .
Horizontal gene transfer is the direct uptake of DNA (or RNA) into cells and
integration of the sequence into the cell’s genome. Some of us regard horizontal
gene transfer as the most serious hidden hazard of genetically modified
organisms (GMOs) released into the environment ( Horizontal Gene Transfer - The
Hidden Hazards of Genetic Engineering (ISIS special report). But a
prevailing culture of denial by vested interests and regulators has obstructed
proper investigation until very recently (see  Horizontal
Transfer of GM DNA Widespread, SiS 64).
A GMO is an
organism with synthetic foreign DNA gene sequences inserted into its genome in
a laboratory process of artificial genetic modification that bypasses normal
reproduction. Part of the foreign DNA is a control element called a promoter
that is necessary for expressing the foreign genes. The most widely used is
the cauliflower mosaic virus (CaMV) 35S promoter (which is what enables the
virus to hijack the cell for making endless copies of the virus). The CaMV 35S
promoter is now in more than 80 % of all GM plants , and is the first test
for the presence of GMOs in unknown samples.
Probing for CaMV 35S promoter in the rat
diet and in rat tissues
The Cairo researchers used three pairs of
primers - specific short anchoring sequences that bind by specific base-pairing
to the opposite ends of the DNA segment of interest – so as to amplify
different segments from the CaMV 35S promoter with PCR (polymerase chain
reaction). The amplified segments can then be isolated and detected on
electrophoresis. The primers together amplify nearly 80 % of the entire
promoter sequence. The experimental diet was an ordinary lab chow containing 60
% yellow maize and 34 % soybean, but unlabelled as to whether it is GM or not.
The presence of GM material in the diet was ascertained using PCR assay with
the three pairs of primers, which all gave the expected positive results,
indicating that the diet contained GM material (up to a maximum of 94 %, if
both the soybean and maize were completely GM).
animals consisted of 29 male Wistar albino rats immediately after weaning (age
three to four weeks), which were divided into two main groups. One was fed on the
lab chow containing GM ingredients for three months, and were further divided
into three subgroups of 5, 5, and 7 animals, euthanized after 30, 60, and 90
days. The other control group was fed on a balanced non-GM diet for the same
period and euthanized at the end of the experiment.
The lab chow diet
used all through this experiment gave positive results when screened with primers
for CaMV 35S promoter. The expected 195 bp (base pairs) amplified product was
detected in all samples of the GM diet, but was absent in the control diet made
up with non-GM material to match the nutritional content of the GM diet. As
further confirmation, the PCR product obtained from the GM diet was sequenced,
and shown to have 100 % identity with the CaMV 35S promoter at nucleotide
coordinate 7190-7380 of the CaMV on sequence alignment analysis using the
GenBank database. It also showed 100 % sequence identity with a number of
binary vectors used for transferring genes in the lab that also contain the
CaMV 35S promoter.
PCR analysis of
the different tissues showed amplified sequence segments of the expected sizes
for the three pairs of primers - 70, 88 and 195 bp - in some of the DNA samples
of blood, liver and brain of rats fed the GM diet after 30, 60 and 90 days. None
of the three primers gave amplification product in DNA samples of tissues from
the controls fed the non-GM diet.
The 195 bp segment
amplified from DNA samples of liver and brain in rats fed GM diet was subjected
to DNA sequencing, and comparison with GenBank database revealed 100 % identity
with the CaMV whole genome at the same nucleotide coordinates 7190-7384 for the
35S promoter. Furthermore, it also showed 100 % identity with the PCR segment amplified
from the GM diet, and with the binary vectors segments that are 100 % identical
to the PCR product from the GM diet.
Feeding rats with
GM diet for 30, 60, and 90 days increased the mean transfer frequency of GM
target sequences significantly from 0 in the controls to 8 + 0.0000 %,
12.3 + 1.2018 % and 16.7 + 2.4529 % respectively. Thus, there is
a cumulative effect with time of exposure.
Bearing in mind that the
three primer pairs together amplify nearly 80 % of the entire CaMV 35S promoter,
and in some animals, the three segments were all amplified in the same sample,
it suggests that the whole CaMV 35S promoter may have been transferred into the
genome of those animals. Considering that even a promoter containing only 46 bp
of the 5’ sequence from the CaMV 35S promoter was previously reported to be
sufficient for accurate initiation of gene transcription for gene expression ,
it is highly likely that the transferred CaMV35S promoter sequence would alter
the activity of some genes in the host cell genome that may have harmful
consequences (see later).
There was no significant
difference in the rate of transfer into blood, liver, or brain tissues.
Moreover, the frequency of uptake for the larger segments was greater than that
for the smaller segments; thus, the transfer frequency of the 70 bp segment was
the lowest at 1.09 + 0.4161 %, the 88 bp at 2.09 + 0.7318 %, and the 159 bp at
3.8 + 0.8069 %. This finding is consistent with previous researchers who
postulated that the shorter the fragment, the lower the uptake efficiency .
GM fed rats suffered damages to liver,
kidney and testis
In a separate report written by some of the
same researchers in Cairo, the same GM diet was fed to identical rats in a
post-market safety assessment of GMOs . Biochemical, histopathological, and
cytogenetic analyses on liver, kidney, and testis revealed that the GM diet fed
for 30, 60 and 90 days suffered significant deleterious effects.
A total of 30 rats
were fed the GM diet, 10 each for 30 days, 60 days and 90 days. The controls
were on a wheat-based non-GM nutritionally matched diet for similar periods of
aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme activities
were measured in blood serum as indicators of liver cell damage. Creatinine and
uric acid levels were determined also in blood serum as indicators of kidney
function. Malondialdehyde in liver cells resulting from lipid peroxidation is a
biomarker of oxidative stress. In addition, specimens of liver, kidney and
testis were dissected immediately after the rats were euthanized and sectioned
for histopathological and histochemical investigations. Cytogenetic and DNA
damage analyses were carried out on testis and liver. Chromosome analysis was
done on germ cells from the testis. Sperms were examined for morphological
abnormalities and DNA fragmentation was determined in liver cells.
The results were
Histopathology of liver, kidney and
Liver cells showed slight damage in rats
fed GM diet for 30 days, with damage increasing after 60 and 90 days. The
effects start as a slight dilatation and congestion of the central vein
(supplying the liver) and fragmentation of the nucleus in some cells. After 60
days, mild cellular infiltration (from the blood) was observed. After 90 days,
the blood sinusoids (spaces) also showed slight dilatation and congestion
1 Liver sections: a, control; b, 30 day GM-fed; c, 60 day GM-fed; d 90 day GM
fed (see text for details)
Kidney sections show damaging
effects of the GM diet evident even after the first 3o days as interstitial
haemorrhage (blood in spaces between cells) and a widening of the tubules. This
got worse in 60 and 90 days (Figure 2).
2 Kidney sections: a, control; b, 30 day GM-fed; c, 60 day GM-fed; d 90 day
GM fed (see text for details)
The testis of rats fed on GM
food for 30 days showed mild thickening of the basement membrane of the
seminiferous tubules (where germ cells develop) with gaps appearing between the
germinal epithelium of some tubules. At 60 and 90 days, an increase in the
connective tissue component and in the number of Leidig cells (in the
connective tissue), with a disarrangement of the germinal epithelium (Figure 3).
3 Testis sections: a, control; b, 30 day GM-fed; c, 60 day GM-fed; d 90 day
GM fed (see text for details)
Protein content in liver
tissues decreased significantly after 30 or 90 days, indicating dysfunction of
some hepatocytes. Abnormal cellular activity in the kidney was also confirmed
by a statistically significant increase in the protein content.
Consistent with the liver and
kidney damages seen, AST and ALT activity increased in the serum of
experimental rats by 33 to 107 % and 33 to 92 % respectively. Blood creatine
and uric acid concentrations significantly increased by 15 to 315 % and 37 to
96 % respectively. MDA concentrations in liver, as an indicator of oxidative
stress, increased significantly in all animals fed GM diet by 286 to as high as
Mitotic index (as a measure
of cell division) was significantly reduced in the 60 and 90 days fed rats from
8.8 + 0.326 % in controls to 8.4 + 0.221 % (30 days), 6.8 +
0.466 % (60 days) and 6.6 + 0.266 % (90 days). Concomitantly, there was
a significant increase in chromosomal aberrations, from 0.4 + 0.163 % in
controls to 6.6 + 0.221 %, 13.8 + 0.326 % and 8.0 + 0.632 % respectively
for 30, 60 and 90 day GM fed rats. The frequency of morphologically abnormal
sperm increased by up to two-fold, from 3.33 +0.35 % to 5.83 +
0.60 %, 7.8 + 0.65 % and 6.6 + 0.24 %. At the same time DNA
fragmentation went up from 11.83 + 0.7 % in controls to 19.0 +
1.2 %, 28.3 + 1.6 % and 24.3+ 0.7% respectively.
The researchers concluded the
results  “indicate that there are health hazards linked to the ingestion of
diets containing genetically modified components.”
However, the investigations
have not gone further into the mechanisms whereby the genetically modified
components were hazardous to health; nor do the results directly implicate the
CaMV 35S promoter found transferred to rat blood, liver and brain in the other report
. It will be useful to review the potential hazards of the CaMV 35S
promoter, which were first pointed out 15 years ago.
Predicted hazards of cauliflower mosaic
virus 35S promoter
When first deployed, geneticists assumed
that the CaMV 35S promoter would only work in plants, as the complete virus (wrapped
in its protein coat) specifically infects only plant cells. But it soon
transpired that the isolated piece of promoter DNA without its coat is
extremely promiscuous, and works in cells across kingdoms of plants and
animals, as well as bacteria. We issued a serious warning against its use in
1999  Cauliflower Mosaic
Viral Promoter - A Recipe for Disaster (ISIS scientific publication) when
it was found to have a recombination hotspot where it tends to fragment and
join, which makes it prone to unintended (horizontal) gene transfer into cells
of all organisms exposed to the GMO, including bacteria, fungi, pollinators,
wild animals and humans (see  CaMV
35S promoter fragmentation hotspot confirmed, and it is active in animals (ISIS
scientific publication). What that implies is the CaMV 35S promoter can break
loose from the plant genome DNA and jump into the genome of all those other
cells, with the potential to mutate, activate or inactivate genes (including
those leading to cancer), reactivate dormant viruses, or create new viruses by
recombination (gene shuffling) [8, 10] (Hazards of Transgenic Plants
Containing the Cauliflower Mosaic Viral Promoter, ISIS scientific publication) But our
warnings were met with abuse and denial and ultimately ignored.
then, evidence has emerged that the CaMV 35S promoter
may enhance the multiplication of disease-associated viruses including HIV and
cytomegalovirus through the induction of proteins required for transcription of
the viruses  (New Evidence Links
CaMV 35S Promoter to HIV Transcription, ISIS scientific publication). Further, the CaMV 35S promoter overlaps with a virus gene, the
product of which is toxic to plants and likely also to animals . For a more
detailed description on the risks of the CaMV 35S promoter and indeed on GMOs
in general, see  and final chapter in  Ban GMOs Now, ISIS Report.
GMOs are once again found to be deleterious
for health in a feeding trial that last no longer than 90 days. And within that
time, the most widespread piece of transgenic DNA found in the GM diet, the
CaMV 35S promoter, was found transferred horizontally into the animals’ tissues
at high frequencies. The CaMV 35S promoter is not the only hazardous piece of
transgenic DNA, there are similar aggressive promoters designed to make genes
express out of context, as well as genes coding for antibiotics and other
dangerous functions, together with numerous recombination hotspots that enhance
horizontal gene transfer; all of which contribute to making all GMOs
unsafe. That is indeed the conclusion from research carried out by scientists
independent of the industry up to now, which fully corroborates what farmers
have been witnessing in their livestock and doctors in their patients for years
. People need to take immediate action to ban GMOs from their own home and
local communities. Governments should recall all GMOs from the market. And
companies and regulators should face prosecution for causing damages to health
and criminal negligence.
Susan Wright Comment left 8th January 2015 10:10:46 this is interesting just more and more evidence that gmo's are hazardous to our health
Tracy Comment left 8th January 2015 21:09:21 Thank you for all your hard work. Why is the date on this 7/1/15? I shared it and the question has been brought back to me.
mae-wan ho Comment left 8th January 2015 22:10:40 Tracy, thanks for your comment. In Britain we put the day before month. So the correct date, without ambiguity is 7 January 2015.
Melinda Comment left 9th January 2015 22:10:46 Is there a way for people to find out if they have been infected with either the Cauliflower virus or Agrobacterium? Any tests from commercial medical labs or researchers? I live in the USA and due to the prevalence of GMO foods, it is very hard to avoid, especially eating out. Although buying organic is a option that might limit ones exposure to GMO's and pesticides, the reality is that in the USA engineered nanoparticles were allowed as food coloring in 2002 by the FDA, and at this time not even the USDA Organic Standards Board has banned engineered nano particles. So buying organic does not currently prevent exposure to man-made nano particles in the USA. People are bushing their teeth here with nano Titanium dioxide, and applying sunscreens with NP's daily. One wonders what happens when someone has semiconductors in their skin and on their teeth and clothing from Np's, is eating GMO foods, and is forced to live in an environment with 24/7 wireless (and other) radiation. GMO's, wireless and nanotech in consumer products were all rolled out to consumers about the same time here; mid to late 1990's, but very few people are looking at the synergies of these technologies. People researching human health impacts of wireless are not considering the increase in conductivity in people caused by these nanomaterials in and on peoples bodies. Nano TiO2 may be used as a coloring (bright white) in foods, but is also used in semiconductor applications in the building industry. Nano TiO2 is also a photocatalyst and one really has to wonder about putting a photocatalyst in sun screens.
Rory Short Comment left 10th January 2015 11:11:43 There is a fundamental aspect of how society operates that is driving the appearance of practices and technologies that threaten human and environmental health. It is that sectional interests are able to benefit from these arrivals even though their impact is detrimental overall.
MaeWan Comment left 11th January 2015 11:11:02 Robyn,
Have you really read the study carefully? All the details of the controls are given in the Tables. Also, the histopathological slides shown all contain controls. Obviously, it would be impossible to show the slides from all the rats.
The authors can answer for themselves. But their findings on pathological developments of liver, kidney and testis have all been reported previously by other researchers.
Robyn Daly Comment left 11th January 2015 07:07:35 I just reread the study. The non-GM rats were not divided into three groups and dissected the way the GM rats were. In other words,there was no direct comparison made. How do we know what they would have looked like? The "standard chow" diet seems grossly inadequate to me, given that rats are omnivores.