Scientific assessment of chemical's toxicity - 28 chemicals tested
with toxicogenomics
http://www.antidote-europe.org/substances_gb.htmScientific assessment of chemical's toxicity
28 chemicals tested with toxicogenomics
Introduction
The aim of this work is to introduce a method that is considered reliable
for assessing the toxicity of chemical substances. This goal of safety for
human health cannot be achieved through the use of standard toxicology
methods, which are still largely based on animal experiments. No animal
species is a biological model for another species, including man. This was
clearly demonstrated in the second half of the 20th century's discoveries in
the field of genetics. We can now define a species in terms of its
reproductive isolation which results from the fact that genes and
chromosomes are unique to each species and hence preclude reproduction
between different species. Genes determine protein synthesis and biological
functions. As any given species has a unique set of genes, it follows that
those biological functions will be species-specific.
Pennicillin kills guinea pigs whereas it has saved millions of humans ;
aspirin can cause birth defects in rats or dogs whereas this has not been
observed in humans. Many more examples illustrate the fact that no animal
species is a reliable model for another. Claude Bernard, the physiologist
who, in the 19th century, promulgated animal experiments as the main source
of knowledge for human medicine, stressed the similarities between animals
and humans, rather than the differences. He also justified his use of
animals on ethical grounds, as wanting to avoid experiments that could harm
humans.
We are now in the 21st century and possess many non-invasive methodologies
for the study of human medicine. Toxicological studies can be performed on
cultured human cells. In addition, for the past 15 years, we also possess
powerful new tools with which to monitor cells exposed to a given chemical -
DNA chips. These chips can reveal which genes are active in a cell at a
given time, even as soon as 24 hours after the cells have been exposed to
the test chemical. Knowing the function of these genes, we can observe the
response of the cell to the chemical and whether this response drives the
cell towards a pathological state. Toxicogenomics is the study of the genes
expressed following exposure to a chemical.
Antidote Europe has developed a novel approach to toxicogenomics by using
minituarized DNA chips, in combination with sequential exposure of two
different cell types, approximating what would occur in a whole body. We
have termed this approach "Scientific Toxicology Program" (STP) and what
follows are the results of 28 substances tested according to the STP.
Selection of test substances
France is the second largest consumer of pesticides in the world. One
hundred and fifty thousand people a year die in France from cancer. A
further 600,000 persons suffer from Alzheimer's disease and, in common with
other European countries, 15% of couples are infertile. Several renowned
researchers and medical scientists have pointed out that the widespread use
of chemicals is very likely the main cause of these shocking health
statistics. Greenpeace and the WWF have shown that dozens of chemicals are
present in the air we breathe in our homes. Worse still, chemicals in the
blood of pregnant mothers are 'off-loaded' on to the unborn foetus in the
womb. Yet, chemical manufacturers maintain that the link between chemical
use and disease has not been established.
In 2001, the European Commission launched the REACH (Registration,
Evaluation and Authorization of CHemicals) project, claiming that little or
no safety data existed with respect to almost 100,000 chemical substances
produced by industry. Following mounting pressure from manufacturers, this
number was subsequently reduced to 30,000 chemicals.
While acknowledging the need for such a project, Antidote Europe cautions
against the use of unreliable test methods for assessing toxic risk.
Classical toxicological methods still largely rely on animal experiments,
which, as we have explained, are not reliable for humans. Instead, we urge
the European Commission to incorporate STP as an integral part of its
testing strategy, based on human exposure data. Many observations have been
made following occupational or accidental exposure to chemicals. This should
be employed as a reference with respect to both the toxicity of these
chemicals and as a standard for the validation of relevant toxicological
methods. Since our aim was not to present the regulatory authorities with
data of unknown chemicals but simply to demonstrate the validity of STP, we
selected 28 chemicals already known or suspected of being toxic. We
therefore selected 15 pesticides (abamectin, aldicarb, aldrin, carbaryl,
chlorpyriphos, dicofol, fenazaquin, fipronil, heptachlor, lindane,
methoxychlor, paraquat, permethrin, phosmet and rotenone), 2 food additives
(benzoic acid -E210- and quinoline -E104-), 5 cosmetic ingredients
(3-aminophenol, 4-aminobiphenyl, 2-butoxyethanol, benzophenone-3 and propyl
paraben -E214-), 1 prescription drug (acetaminophen -paracetamol-) and 5
additional substances commonly used in industry (1,4-dioxane, acetonitril,
acrylamide, bisphenol A and ethylene glycol). Some of these substances are
multi-purpose. Although some of the pesticides have been banned in many
European countries, they may still be present in our environment and in our
water supply, thereby contaminating our bodies.
What is the STP ?
The first report on STP was published in Biogenic Amines, 2003, vol. 18, pp
41-54. STP is based on human cell cultures, DNA chips and knowledge of
genetics.
Culturing human cells has been routine in labs since about 1930. Some
centers, especially in Germany, the UK and the US, keep many different cell
lines obtained from human tissues and organs. These cells lines are
commercially available. In spite of being cancerous cells, their
characteristics are well known and they are used as biological models for
research worldwide. Our study used HepG2 liver cells and SH-SY5Y neuronal
cells. Once the culture box was covered by one layer of cells (a monolayer
of cells), the chemical to be tested was introduced and left in the culture
medium for 24 or 48 hours, at two different concentrations.
We chose liver cells because the liver, with its detoxifying function, is
crucially in contact with all chemicals circulating in the blood, and will
metabolize these chemicals in order to facilitate their elimination,
although this metabolism can result in substances becoming even more
dangerous than the original, depending on the enzymatic capability of the
liver. It is important to note that the enzymatic capability can differ
significantly between different animal species, which is one of the reasons
for the failure of animal-based toxicology with respect to humans. We chose
neuronal cells because many insecticides target the nervous system of their
victims and wanted to observe this effect on human cells.
A novel idea of our scientific team was to expose liver and neuronal cells
sequentially rather than in parallel, thus approximating the whole body
physiological response, in which the nervous system and other tissues and
organs are exposed to the metabolites circulating in the blood once the
liver has modified the original substances. And indeed we observed that some
of the tested chemicals did not elicit much response from the liver while
they (or rather their metabolites) significantly affected the neuronal
cells.
Another novel idea was to construct minituarized DNA chips containing 6
families of genes known to be implicated in the 6 metabolic pathways we had
selected for our study. These pathways were already known for their
association to particular diseases. Extensive literature already exists
regarding the role of each of these genes in normal cell metabolism, cycle
and function, as well as in pathological states. In contrast with the huge
number (many thousands of genes), expensive and time-consuming processes of
DNA chips developed in the US, for example, our cost-effective and very easy
to process minituarized DNA chips provide a key solution to the problem of
dealing with large automated platforms capable of testing tens of thousands
of substances (and many of their combinations) in reasonable time and cost
frames.
Our DNA chips contained 51 genes comprising 6 families, and designed for the
study of :
1. Cell stress : 5 genes for monitoring the response to oxidative stress
(GSS, GPX1, SOD1, GSTM3 and EPHX1), 2 genes implicated in the survival of a
cell in a stress situation (TRPM2 and HSPA9B) and 2 genes implicated in the
inflammatory response (PTGS2 -Vioxx's target- and NOS2A). If these genes are
elicited following the introduction of the substance to the culture medium,
it means the cell is under duress, and making attempts to repair cell
damage. The cell may subsequently 'commit suicide' if it cannot cope with
the damaging effects of the substance. According to the kind of damage and
if systemic mechanisms are not available for repair, exposed individuals
could develop different diseases : cancer, due to free radical activity,
inflammation or auto-immune disease, etc.
2. DNA damage : 3 genes implicated in DNA repair (RAD50, RAD51 and NFKB1), 3
genes preventing cell cycle (cell life and reproduction) following damage
(CDC25C, CDK4 and CDKN1) and 3 genes triggering apoptosis (cell death) if
the damage is not repaired (APAF1, ATM and BAX). If these genes are
themselves impaired, cells with damaged DNA will eventually grow and
multiply, resulting in cancer or developmental abnormalities, for example,
if systemic repair mechanisms are not available.
3. Cell cycle control : DNA replicates before cell division gives birth to
two cells, each receiving one copy of the original DNA. Many proteins have
crucial roles in the accurate and quality control of DNA replication as well
as in authorizing and controlling the progression of the cell cycle from
step to step. Our chips harboured 2 genes controlling cell proliferation
(FOS and JUN) and 7 genes implicated in stopping cell division and apoptotic
signal (BCL2, GADD45A, MDM2, TP53, EGF, PPARA and TUBA1). Abnormal
expression of these genes has been observed in cancer.
4. Neurotoxicity : 8 genes implicated (but not exclusively) in nerve
transmission and nervous system development (ACHE, CTSB, DRD2, TH, BZRP,
THBS1, HOXD1 and ROBO1). Abnormal expression of these genes, in the absence
of compensating mechanisms, could lead to neurological or even psychiatric
disorders, or malformations of nervous, muscular or cardiac systems if gene
expression is affected during foetal or early childhood development.
5. Hormonal response : ranging from glycaemia to reproduction, hormones
regulate many physiological functions. We have selected 10 genes under
hormonal control (TFF1, CTSD, PGR, RAN, AR, CREB1, ESR1, CALR, CYP19A1 and
ALB). Some of these genes code for steroid hormone receptors and their
deregulation could explain some of the increase in cases of genital
malformation, reduced male fertility and even breast or prostate cancer.
6. Protein conformation : the tridimensional structure of proteins is
crucial for their ability to perform their function. Correct amino acid
sequence but wrong folding is thought to lead to accumulation of non
functional proteins contributing to conformational diseases such as
Alzheimer's, type I diabetes, etc. Our chips harboured 6 genes implicated in
quality control of newly synthesized and cell loading of proteins (HSPA5,
XBP1, ATF6, ERN1, C12orf8 and A2M).
Results
Full results are available on our website or upon request from Antidote
Europe. Here, we present a brief summary of the reactions elicited by the
tested chemicals.
1. 1,4-dioxane : 3 out of the 6 marker families were impaired in liver
cells, all 6 families were impaired in neuronal cells, especially the cell
stress markers. This substance can lead to all 6 of the pathological
pathways explored.
2. 2-butoxyethanol : 41 of the 51 genes present on our DNA chips were
significantly repressed (for some of them, gene expression was reduced a
hundred-fold). All studied cellular functions were severely compromised in
both cell lines.
3. 3-aminophenol : 36 of the 51 genes were repressed, ten-fold for some of
them. All studied cellular functions were compromised in both cell lines.
4. 4-aminobiphenyl : 4 genes were overexpressed in liver cells (markers for
cell stress, DNA damage, cell cycle control and hormonal response) ; 25
genes were repressed in neuronal cells, in all 6 pathological pathways
explored.
5. Abamectin : 44 of the 51 genes were strongly repressed, a hundred-fold in
some cases. 27 genes were repressed in both cell lines. This insecticide
(targeting GABA synapses) has an unequivocal pathological potential.
6. Acetaminophen (paracetamol) : 30 genes were repressed quite strongly, 2
genes were overexpressed, affecting all cellular functions under study in
both cell lines.
7. Acetonitril : 2 genes were repressed and 3 overexpressed in liver cells
(markers for cell stress, DNA damage, cell cycle control, neurotoxicity and
hormonal response). 15 genes were repressed in neuronal cells, affecting all
studied cellular functions.
8. Benzoic acid (E210) : 38 of the 51 genes were repressed, significantly in
some cases. All studied cellular functions were disturbed in both cell
lines.
9. Acrylamide : 3 genes were overexpressed, 30 were repressed, significantly
in some cases. In neuronal cells, the response was mainly observed after
exposure to the highest dose. In contrast, liver cells were also affected at
low doses. All studied functions were affected in both cell lines.
10. Aldicarb : expression of 20 genes was affected in both cell lines and a
further 24 genes were affected in one or the other cell line, totalling 44
markers elicited. All studied cellular functions were affected in both cell
lines.
11. Aldrin : 14 genes were affected in liver cells, mainly overexpressed. 11
genes were affected in neuronal cells, of which, 2 were dramatically
overexpressed. All studied cellular functions were affected in both cell
lines, with very different reactions for each gene.
12. Benzophenone-3 : 2 genes were repressed (cell stress and neurotoxicity)
and 1 overexpressed (DNA damage) in liver cells. 16 genes were repressed in
neuronal cells, affecting all studied functions in this cell line.
13. Bisphenol A : 1 gene was repressed in liver cells after exposure to high
dose. 7 genes were overexpressed in liver cells after exposure to low dose.
20 genes were repressed in neuronal cells, affecting all studied functions.
14. Carbaryl : 4 genes were overexpressed and 7 were repressed in liver
cells, affecting all studied functions in this cell line. The effect was
dramatic on neuronal cells, in which 48 genes were significantly repressed
(a hundred-fold or more).
15. Chlorpyriphos : expression of 42 genes was affected in both cell lines
and a further 7 genes were affected in one or the other cell line, totalling
49 markers elicited out of the 51 harboured by our DNA chips. Genes were
repressed except for one neurotoxicity marker, which was overexpressed in
neuronal cells.
16. Dicofol : only one gene (a neurotoxicity marker) was slightly repressed
in liver cells. But 41 genes were repressed in neuronal cells, affecting all
studied functions in this cell line.
17. Ethylene glycol : 29 genes were repressed in liver cells, 48 genes were
repressed in neuronal cells, affecting all studied functions in both cell
lines.
18. Fenazaquin : 22 genes were strongly repressed in liver cells, 11 genes
were significantly repressed in neuronal cells, affecting all studied
functions in both cell lines.
19. Fipronil : 2 cell stress markers and 2 DNA damage markers were affected
in both cell lines ; 1 neurotoxicity and 1 hormonal response marker were
repressed in liver cells ; 1 cell cycle control, 2 hormonal response and 1
protein conformation marker were repressed in neuronal cells.
20. Heptachlor : 1 DNA damage marker was overexpressed in liver cells. 17
genes were repressed in neuronal cells, affecting all studied functions
mostly after low dose but long exposure time.
21. Lindane : 11 genes were repressed in both cell lines and another 32
genes were repressed in one or the other cell line, totalling 43 markers
repressed, thus affecting all studied functions in both cell lines.
22. Methoxychlor : 20 genes were affected in both cell lines and a further
23 genes were repressed in one or the other cell line, totalling 43 markers
elicited, thus affecting all studied functions in both cell lines. Except
for 1 overexpressed neurotoxicity marker in liver cells, fairly significant
repression was observed for all the other markers.
23. Paraquat : 46 genes were strongly repressed in both cell lines,
affecting all studied functions. Most dramatic effects were seen for DNA
damage and cell cycle control markers. For the long time exposure, most of
the elicited markers in neuronal cells were repressed a hundred-fold.
24. Permethrin : 7 genes were affected in liver cells, with respect to cell
stress, DNA damage and hormonal response functions. Neuronal cells were more
significantly affected, with 33 genes repressed, especially after long
exposure. All studied functions were affected in neuronal cells.
25. Phosmet : 2 genes wre overexpressed and 11 repressed in liver cells. 41
genes were significantly repressed in neuronal cells. All studied functions
were affected in both cell lines.
26. Propyl paraben (E214) : 6 out of 6 studied functions were affected in
both cell lines. Neurotoxicity markers were only slightly elicited in liver
cells.
27. Quinolin (E104) : 5 out 6 studied functions were affected in liver
cells, with most of elicited markers repressed two-fold. All studied
functions were affected in neuronal cells.
28. Rotenone : 29 genes repressed in liver cells, 27 in neuronal cells. The
most dramatic effects (hundred-fold repression) were observed on neuronal
cells after long exposure.
These being the first studies done with the STP, we do not have all the keys
for translating laboratory observations into specific prediction of disease
risk. However, we clearly see that each substance elicits a specific
response. In order to accurately hypothesize possible effects on the
individual, many parameters would have to be taken into account. For
example, water-soluble substances will easily circulate in body fluids and
be quickly eliminated whereas lipid-soluble ones will be stored in
adipocytes (fat cells) and can also remain in the lipidic sheaths of the
neuronal cells, thus posing a risk to potential target cells and tissues
over long periods of time. Whereas a substance capable of damaging the
central nervous system must cross the blood-brain barrier, a xenoestrogen
will easily reach its target simply by being transported in the blood, as is
the case with steroid hormones.
Effects already observed in humans
Clues about the validity of STP can be found by considering what is known
about the tested substances following human accidental or occupational
exposure. According to the
International Agency for
Research in Cancer :
- workers exposed to 4-aminobiphenyl have developed bladder cancer
- urinary tract cancer has been observed in acetaminophen users in Australia
; others have reported hepatotoxicity of this drug
- nervous system problems have been observed in workers exposed to
acrylamide
- aldicarb has already been shown to induce DNA damage and mutations in
human cultured cells
- chromosomal aberrations have been induced by aldrin in cultured human
lymphocytes (white blood cells) ; aldrin inhibited intercellular
communication in human cell systems
- slight excess of lung cancer cases was observed in workers exposed to
heptachlor
- 4 cases of leukaemia were reported in men exposed to lindane ; cases of
aplastic anaemia have also been associated with exposure to lindane.
- P. Darbre et al have found parabens in tissue samples from breast tumors
and state that these chemicals are known xenoestrogens (J Appl Toxicol,
2004, 24, 1-4).
- Professor Charles Sultan states, after a study on exposure to pesticides,
that babies born to farmers have twice the normal risk of presenting genital
malformations.
All of this data is a small part of a much bigger picture since cancer,
neurological disorders and most of other diseases under consideration may
have very long latent periods of onset, and therefore the link between the
triggering substance and the disease will not be established as exposure
will have ceased long before the appearance of the first symptoms. However
initial these observations, they are nevertheless consistent with what could
be hypothesized as the chemical's effects on the basis of our STP results.
We can improve STP
The results presented here are the very first obtained with STP based on
limited resources. Many improvements are possible.
1. Cell types. For substances likely to be absorbed through inhalation or
feeding, lung and gut cells should be tested as these organs will be exposed
first. Blood and kidney cells should also be tested routinely. For very
important chemicals (prescription drugs, for example), STP could be a first
step before microdosing tests in healthy volunteers and could give more
valuable information than animal tests, after which only 1 drug out of 12
makes it to the market. STP is potentially a more powerful tool than QSAR
and other simulations since STP is not a simulation, but a direct
observation of what is taking place in human cells.
2. Markers. The human genome contains about 25,000 genes. About 1100 are
known to respond to chemical exposure. Although the 51 set selected for our
DNA chips have key functions in the processes we intended to study, there is
still room for a greater number of markers on chips designed to monitor
unexpected effects of substances.
3. Time and dose. Our results represent a snapshot of the state of gene
expressions 24 and 48 hours after the introduction of the test substance
into the culture medium. It would be interesting to observe more snapshots
at shorter, intermediate and longer periods of exposure. The same is true
for concentrations of test compounds, especially for suspected xenoestrogens
as the endocrine system is sensitive to infinitesimal doses.
Conclusion
As imperfect as it is, STP has already proven much more reliable that
animal-based toxicology and should replace it at once. Carcinogenicity
tests, which take several months and yield unreliable results in rodents,
can be performed in a few days with STP, in conjunction with other tests
(neurotoxicity, immunotoxicity, acute toxicity, etc.), thereby sparing the
lives of countless animals. STP has the advantage of dealing with human
genes, thus allowing the identification of sensitive individuals or groups
of individuals to a particular substance, based on our knowledge of human
polymorphism, unique to humans and not predictable through animal-based
toxicology.
With STP, automatization and optimisation of tests is possible at the very
early stages of a new chemical's development, thus allowing early screening
and disqualification of dangerous or ineffective substances, with consequent
gain of time and money. The short test times employed by STP would be able
to cope with the original 100,000 chemicals initially included in the REACH
project, and even many combinations of molecules (not feasible using on
animals), in a reasonable period of time (much less than the 12 years
considered by the European Commission for only 30,000 substances with the
current means).
With toxicogenomics programs receiving substantial funding in the US and
Japan, Europe should seriously consider allocating resources to improve STP
whose novel approach could put the EU at the cutting edge of this promising
new technology. These techniques are likely to become the standard in the
coming years, and manufacturers should help to implement them rather than
spending yet money on useless animal-based tests - and before public opinion
loses confidence in an industry that avoids using the best available
technology to assess the safety of its products.
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