Toxicological effects of bisphenol A, propyl paraben, triclosan, atrazine and glyphosate using Caenorhabditis elegans as a biological model

• Autores: María Cecilia García Espiñeira
• Directores de la Tesis: Jesús Olivero Verbel (dir. tes.)
• Lectura: En la Universidad de Cartagena (Colombia) ( Colombia ) en 2023
• Idioma: inglés
• Enlaces
  • Tesis en acceso abierto en: hdl.handle.net
• Resumen

  • 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.