Elucidating the effects of pure glyphosate and a commercial formulation on early life stages of zebrafish using a complete biomarker approach: All-or-nothing!

The search for new methods in the toxicology field has increased the use of early life stages of zebrafish (Danio rerio) as a versatile organism model. Here, we use early stages of zebrafish to evaluate glyphosate as pure active ingredient and within a commercial formulation in terms of oxidative stress. Biomarkers involved in the oxidative status were evaluated along with other markers of neurotoxicity, genotoxicity, cytotoxicity, energy balance and motor performance, and the selected tools were evaluated by its sensitivity in determining early-warning events. Zebrafish embryos exposed to glyphosate active ingredient and glyphosate-based formulation were under oxidative stress, but only the commercial formulation delayed the embryogenesis, affected the cholinergic neurotransmission and induced DNA damage. Both altered the motor performance of larvae at very low concentrations, becoming larvae hypoactive. The energy balance was also impaired, as embryos under oxidative stress had lower lipids reserves. Although data suggest that glyphosate-based formulation has higher toxicity than the active ingredient itself, the most sensitive biomarkers detected early-warning effects at very low concentrations of the active ingredient. Biochemical biomarkers of defense system and oxidative damage were the most sensitive tools, detecting pro-oxidant responses at very low concentrations, along with markers of motor performance that showed high sensitivity and high throughput, suitable for detecting early effects linked to neurotoxicity. Alterations on morphology during embryogenesis showed the lowest sensitivity, thus morphological alterations appeared after several alterations at biochemical levels. Tools evaluating DNA damage and cell proliferation showed mid-sensitivity, but low throughput, thus they could be used as complementary markers.

Niacin prevents mitochondrial oxidative stress caused by sub-chronic exposure to methylmercury.

Humans and animals can be exposed to different chemical forms of mercury (Hg) in the environment. For example, methylmercury (MeHg)-contaminated fish is part of the basic diet of the riparian population in the Brazilian Amazon Basin, which leads to high total blood and plasma Hg levels in people living therein. Hg induces toxic effects mainly through oxidative stress. Different compounds have been used to prevent the damage caused by MeHg-induced reactive oxygen species (ROS). This study aims to investigate the in vivo effects of sub-chronic exposure to low MeHg levels on the mitochondrial oxidative status and to evaluate the niacin protective effect against MeHg-induced oxidative stress. For this purpose, Male Wistar rats were divided into four groups: control group, treated with drinking water on a daily basis; group exposed to MeHg at a dose of 100 µg/kg/day; group that received niacin at a dose of 50 mg/kg/day in drinking water, with drinking water being administered by gavage; group that received niacin at a dose of 50 mg/kg/day in drinking water as well as MeHg at a dose of 100 µg/kg/day. After 12 weeks, the rats, which weighed 500-550 g, were sacrificed, and their liver mitochondria were isolated by standard differential centrifugation. Sub-chronic exposure to MeHg (100 µg/kg/day for 12 weeks) led to mitochondrial swelling (p < 0.05) and induced ROS overproduction as determined by increased DFCH oxidation (p < 0.05), increased gluthatione oxidation (p < 0.05), and reduced protein thiol content (p < 0.05). In contrast, niacin supplementation inhibited oxidative stress, which counteracted and minimized the toxic MeHg effects on mitochondria.

Toxicity of brominated flame retardants, BDE-47 and BDE-99 stems from impaired mitochondrial bioenergetics.

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants, and they have been detected in human blood, adipose tissue and breast milk, a consequence of their physicochemical and bioaccumulative properties, as well as their high environmental persistence. Many studies report liver toxicity related to exposure to PBDEs. In the present study, we investigated the toxicity of BDE-47 and BDE-99 at concentrations ranging from 0.1 to 50 µM in isolated rat liver mitochondria. We evaluated how incubation of a mitochondrial suspension with the PBDEs affected the mitochondrial inner membrane, membrane potential, oxygen consumption, calcium release, mitochondrial swelling, and ATP levels to find out whether the tested compound interfered with the bioenergetics of this organelle. Both PBDEs were toxic to mitochondria: BDE-47 and BDE-99 concentrations equal to or higher than 25 and 50 µM, respectively, modified all the parameters used to assess mitochondrial bioenergetics, which culminated in ATP depletion. These effects stemmed from the ability of both PBDEs to cause Membrane Permeability Transition (MPT) in mitochondria, which impaired mitochondrial bioenergetics. In particular, BDE-47, which has fewer bromine atoms in the molecule, can easily overcome biological membranes what would be responsible for the major negative effects exerted by this congener when compared with BDE-99.

BDE-154 induces mitochondrial permeability transition and impairs mitochondrial bioenergetics.

Brominated flame retardants are used in various consumer goods to make these materials difficult to burn. Polybrominated diphenyl ethers (PBDE), which are representative of this class of retardants, consist of two benzene rings linked by an oxygen atom, and contain between 1 and 10 bromine atoms in their chemical structure, with the possibility of up to 209 different congeners. Among these congeners, BDE-154 (hexa-BDE) is persistent in the environment and easy to detect in the biota, but no apparent information regarding the mechanism underlying action and toxicity is available. Mitochondria, as the main energy-producing organelles, play an important role in the maintenance of various cellular functions. Therefore, mitochondria were used in the present study as an experimental model to determine the effects of BDE-154 congener at concentrations ranging from 0.1 µM to 50 µM. Our results demonstrated that BDE-154 interacts with the mitochondrial membrane, preferably by inserting into the hydrophobic core of the mitochondrial membrane, which partially inhibits respiration, dissipates Δψ, and permeabilizes the inner mitochondrial membrane to deplete ATP. These effects are more pronounced at concentrations equal to or higher than 10 µM. Results also showed that BDE-154 did not induce reactive oxygen species (ROS) accumulation within the mitochondria, indicating the absence of oxidative stress. Therefore, BDE-154 impairs mitochondrial bioenergetics and permeabilizes the mitochondrial membrane, potentially leading to cell death but not via mechanisms involving oxidative stress.

Vimang (Mangifera indica L. extract) induces permeability transition in isolated mitochondria, closely reproducing the effect of mangiferin, Vimang's main component.

Mitochondrial permeability transition (MPT) is a Ca(2+)-dependent, cyclosporin A (CsA)-sensitive, non-selective inner membrane permeabilization process. It is often associated with apoptotic cell death, and is induced by a wide range of agents or conditions, usually involving reactive oxygen species (ROS). In this study, we demonstrated that Mangifera indica L. extract (Vimang), in the presence of 20 microM Ca(2+), induces MPT in isolated rat liver mitochondria, assessed as CsA-sensitive mitochondrial swelling, closely reproducing the same effect of mangiferin, the main component of the extract, as well as MPT-linked processes like oxidation of membrane protein thiols, mitochondrial membrane potential dissipation and Ca(2+) release from organelles. The flavonoid catechin, the second main component of Vimang, also induces MPT, although to a lesser extent; the minor, but still representative Vimang extract components, gallic and benzoic acids, show respectively, low and high MPT inducing abilities. Nevertheless, following exposure to H(2)O(2)/horseradish peroxidase, the visible spectra of these compounds does not present the same changes previously reported for mangiferin. It is concluded that Vimang-induced MPT closely reproduces mangiferin effects, and proposed that this xanthone is the main agent responsible for the extract's MPT inducing ability, by the action on mitochondrial membrane protein thiols of products arising as a consequence of the mangiferin's antioxidant activity. While this effect would oppose the beneficial effect of Vimang's antioxidant activity, it could nevertheless benefit cells exposed to over-production of ROS as occurring in cancer cells, in which triggering of MPT-mediated apoptosis may represent an important defense mechanism to their host.