Toxicological effects of bisphenol A, propyl paraben, triclosan, atrazine and glyphosate using Caenorhabditis elegans as a biological model
García Espiñeira, María Cecilia | 2018
The production of chemical compounds used by human beings is
growing, producing great benefits in the progress of civilization
and comfort in people's lives, while on the other hand it generates
exposure to living beings and nature, exerting a great variety of
interactions with their systems and toxicity.
Bisphenol A is a chemical compound massively used as a
plasticizer in a varied range of elements for daily use.
Propylparaben and triclosan are chemical compounds that extend
the half-life and avoid contamination with bacteria and fungi of
personal care products. Atrazine and glyphosate, in turn, are
molecules used in agriculture, as broad-spectrum herbicides.
Although several negative effects have been described in animals
and humans, its use in many Latin American countries is still
common.
Biological models are used throughout the world to determine the
acute and chronic toxicity of many chemicals and have been
effective in reproducing toxicity mechanisms. C. elegans, is a
nematode that facilitates these studies because of its multiple
advantages, among which are having a fully sequenced genome,
simple maintenance in laboratory conditions, a short life cycle,
small size, simple anatomy, transparency and sensitivity for acute
and chronic detection of toxicity, as well as an easy understanding
of the biochemical mechanisms involved, allowing a more
accurate prediction of the toxicity of studied compounds. In
addition, a large number of nematodes can be studied in a single
experiment within a short period of time, with fewer ethical
problems and comparatively cheaper compared to using other
animal models.
In this study, four stages are contemplated: the first evaluates the
toxicity of the five molecules described in the biological model
C. elegans taking into account endpoints such as mortality,
growth, fertility and progeny size. The second one studies the
amount of fatty acid deposits in the bodies of worms; the third
was the expression of C. elegans genes related to oxidative stress,
by reporter genes associated with green protein fluorescence
(GFP) and the fourth one determined the adipogenic action of one
of the molecules (BPA) in human adipoblasts cells.
The lethality of C. elegans exposed to BPA, PPB and TCS was
concentration dependent, and the LC50 after 24 h of exposure was
113.5, 261.7 and 43.2 µM, respectively. At concentrations greater
than 0.5 µM, BPA, PPB and TCS caused lethality, with statistical
differences related to the control. At lower concentrations, only
TCS (0.05 µM) was bioactive. In solutions of BPA, PPB and
TCS: body length increased slightly with BPA but did not depend
on the concentration. In contrast, PPB reduced body length,
whereas TCS did not have an effect on this parameter. The body
width increased moderately, without a clear relationship with the
concentration, although the response caused by BPA was
bimodal. The relationship between body width and body length
of the nematodes was moderately increased by exposure to the
tested chemicals, but the PPB was the most active, suggesting a
probable association with obesity in the C. elegans model.
Breeding size of nematodes exposed to BPA, PPB and TCS: the
largest brood size after exposure to BPA was reached at 5 µM;
then, it decreased in response to higher concentrations. Similarly,
the PPB increased brood size to 0.5 µM, with declining effects at
higher concentrations. In contrast, TCS decreased this feature
following a trend dependent on concentration.
The most sensitive genes, in descending order, were sod-4, hsp4, hsp-16.2 and skn-1. These genes increased their expression
after exposure to BPA, PPB and TCS, indicating a toxic response
related to the generation of reactive oxygen species (ROS). There
was no evidence of concentration dependence on these results. In
addition, low concentrations caused the overexpression of some
genes; for example, BPA at concentrations of 0.05 and 0.5 µM
caused a 3-fold expression of hsp-4. However, the high
concentrations also affected the expression of several genes such
as sod-4, which showed a 5-fold positive regulation after
exposure to PPB and TCS at a concentration of 500 µM compared
to the control.
The chemical compounds BPA, PPB and TCS caused an increase
in the accumulation of lipids in the bodies of the exposed
nematodes when staining with q-ORO. These deposits showed an
increasing tendency related to the concentration. BPA caused a
greater accumulation of lipids, followed by PPB and TCS. This
result was consistent with the changes in the body wide-long
relationship that were recorded in the worms after exposure to
these molecules.
In this study, four stages were contemplated: the first evaluates
the toxicity of the five molecules described in the biological
model C. eleganstaking into account endpoints such as mortality,
growth, fertility and progeny size. The second one studies the
amount of fatty acid deposits in the bodies of nematodes; the third
was the expression of the biomodel genes related to oxidative
stress, by reporter genes associated with green protein
fluorescence (GFP) and the fourth one determined the adipogenic
action of one of the molecules (BPA) in human adipoblasts cells.
The lethality of C. elegans exposed to BPA, PPB and TCS was
concentration dependent, and the LC50 after 24 h of exposure was
113.5, 261.7 and 43.2 µM, respectively. At concentrations greater
than 0.5 µM, BPA, PPB and TCS caused lethality, with statistical
differences related to the control. At lower concentrations, only
TCS (0.05 µM) was bioactive. In solutions of BPA, PPB and
TCS: body length increased slightly with BPA but did not depend
on the concentration. In contrast, PPB reduced body length,
whereas TCS did not have an effect on this parameter. The body
width increased moderately, without a clear relationship with the
concentration, although the response caused by BPA was
bimodal. The relationship between body width and body length
of the nematodes was moderately increased by exposure to the
tested chemicals, but the PPB was the most active, suggesting a
probable association with obesity in the C. elegans model.
The most sensitive genes, in descending order, were sod-4, hsp4, hsp-16.2 and skn-1. These genes increased their expression
after exposure to BPA, PPB and TCS, indicating a toxic response
related to the generation of reactive oxygen species (ROS). There
was no evidence of concentration dependence on these results. In
addition, low concentrations caused the overexpression of some
genes; for example, BPA at concentrations of 0.05 and 0.5 µM
caused a 3-fold expression of hsp-4. However, the high
concentrations also affected the expression of several genes such
as sod-4, which showed a 5-fold positive regulation after
exposure to PPB and TCS at concentrations of 500 µM compared
to the control.
The chemical compounds BPA, PPB and TCS caused an increase
in the accumulation of lipids in the bodies of the exposed
nematodes when staining with q-ORO, the deposits showed an
increasing tendency related to the concentration. BPA caused a
greater accumulation of lipids, followed by PPB and TCS. This
result was consistent with the changes in the body wide-long
relationship that were recorded in the worms after exposure to
these molecules.
The mean lethal concentration value for atrazine was> 600 µM,
and the NOAEL and LOAEL were 0.006 and 0.06 µM,
respectively. The lethality percentages for the 0.0006 and 0.006
µM solutions had no significant differences with the control, but
concentrations higher than 0.06 µM induced significant lethality,
reaching up to 18 % at 600 µM. The BBF decreased to 5 for a 600
µM solution following a concentration-dependent route. The
length of the body did not follow a strict pattern dependent on
concentration. However, concentrations higher than 60 µM
inhibited body growth. In comparison with the control, the
inhibition of the size of the brood was greater to a solution of 6
µM, reaching almost 100 %. However, it increased again to 60
and 600 µM, showing a modest U-shaped graph.
Mean lethal concentration value for glyphosate was 6.4 µM (the
NOAEL and LOAEL were 0.001 and 0.01 µM, respectively). At
concentrations greater than 100 µM, total lethality was achieved.
The BBF decreased from 38.2 bends in 20 s (control) to 5 at 10
µM, following a concentration-dependent trend. The body length
did not register any significant change up to 1 µM, but it
statistically decreased at 10 µM. The brood size followed a
concentration-dependent curve, with maximum inhibition at 10
µM solution.
Glyphosate concentration-response behavior was similar to that
elicited by atrazine, the first been more potent. The expression of
sod-1, sod-4, and gpx-4 increased at least 2-fold than the control
at just 10 µM solution.
The lethality for the exposure mixtures of atrazine-glyphosate
was concentration-dependent. Maximum lethality occurred with
glyphosate 1000 µM + atrazine 600 µM, reaching 80 %. The BBF
was inhibited at the lowest tested concentrations. It followed a
concentration-dependent tendency similar to that experienced by
glyphosate alone. The body length was also affected by the
minimum herbicide concentration, but the slope of the
concentration-response curve was minimal.
In the concentration addition model assay, the lethality of the
mixture showed that at low concentrations the effect is additive,
whereas at high concentrations the lethality of the mixture was
lower than the effect of GBF alone.
The gene expression behavior for tested genes of the mixture of
atrazine-glyphosate was similar to that observed for individual
molecules.
In summary, with the obtained results it is demonstrated that the
molecules atrazine, glyphosate, bisphenol A, propylparaben and
triclosan exert toxicity C. elegans and its endocrine disruption
effects are qualitative and quantitatively measurable on the on the
biological model.
LEER