Toxicity assessment of the herbicide metolachlor comparative effects on bacterial and mitochondrial model systems

Abstract

a b s t r a c tMetolachlor is one of the most intensively used chloroacetamide herbicides. However, its effects on the environment and on non-target animals and humans as well as its interference at a cell/molecular level have not yet been fully elucidated. The aim of this study was: firstly, to evaluate the potential toxicity of metolachlor at a cell/subcellular level by using two in vitro biological model systems (a strain of Bacillus stearothermophilus and rat liver mitochondria); secondly, to evaluate the relative sensibility of these models to xenobiotics to reinforce their suitability for pollutant toxicity assessment. Our results show that metolachlor inhibits growth and impairs the respiratory activity of B. stearothermophilus at concentrations two to three orders of magnitude higher than those at which bacterial cells are affected by other pesticides. Also at concentrations significantly higher than those of other pesticides, metolachlor depressed the respiratory control ratio, membrane potential and respiration of rat liver mitochondria when malate/glutamate or succinate were used as respiratory substrates. Moreover, metolachlor impaired the respiratory activity of rat liver mitochondria in the same concentration range at which it inhibited bacterial respiratory system (0.4-5.0 lmol/mg of protein). In conclusion, the high concentration range at which metolachlor induces toxicity in vitro suggests that this compound is safer than other pesticides previously studied in our laboratory, using the same model systems. The good parallelism between metolachlor effects on both models and the toxicity data described in the literature, together with results obtained in our laboratory with other compounds, indicate the suitability of these systems to assess toxicity in vitro.

Herbicides: the Face and the Reverse of the Coin. An in Vitro Approach to the Toxicity of Herbicides in Non-Target Organisms

“…Studies of growth or respiration performed in a broad concentration range have allowed the determination of toxicity indices as EC50, NOAEL and/or LOEC. A very good correlation has been observed between toxicity data provided by B. stearothermophilus and by other model systems, namely rat liver mitochondria (Monteiro et al, 2008;Pereira et al, 2009). B. stearothermophilus presents several advantages to be used as a toxicological model system, in addition to the general ones of bacterial cells.…”

“…In our laboratory, a strain of the thermophile Bacillus stearothermophilus, grown in a liquid medium (L-Broth) at 65ºC (within the optimal temperature range), has been extensively used as a model system for assessing the cytotoxicity of different groups of environmental pollutants, as herbicides (Pereira et al, 2009;, insecticides (Donato et al, 1997a,b;Martins et al, 2003;Monteiro et al, 2005) and organometals (Martins et al, 2005). Toxicity indicators have been taken from parameters of growth (lag time, specific growth rate and bacterial yield in the stationary phase) when growth is followed by turbidity measurements or by counting the colony-forming units (c.f.u.)…”

“…However, an accurate determination of the levels at which xenobiotics act in the bacterial oxidative phosphorylation system is technically difficult, since there are not known inhibitors for the respiratory complexes, except for the cytochrome oxidase (CN -as inhibitor). In spite of this limitation and the restricted number of substrates that efficiently support the respiration of protoplasts, very good correlations have been obtained regarding the concentration range of xenobiotics that impair the electron transfer system in both bacterial and eukaryotic models (Donato et al, 1997b;Monteiro et al, 2005;Pereira et al, 2009). Taking together growth, respiration and membrane physical data collected with B. stearothermophilus, a remarkable parallelism may be established with results from studies carried out with other toxicological model systems, revealing differential toxic effects exerted by xenobiotics and their metabolites (Donato et al, 1997b and2000), xenobiotic isomers Monteiro et al, 2003) or different molecules of the same family of compounds (Pereira et al, 2009 and.…”

“…The low stimulatory effect of state 4 respiration suggest a low ability of the compound to induce inner membrane permeabilization. Therefore, the toxicological effects of metolachlor on rat liver mitochondrial bioenergetics, expressed by its inhibitory action on mitochondrial respiratory chain for complexes I and II, may be relevant to understand the mechanism responsible for its toxic action (Pereira et al, 2009). Just as metolachlor, the herbicide alachlor [2-chloro-N-(2,6-diethyphenyl)-N-(methoxymethyl acetamide)], in the concentration range of 400-1200 nmol/mg protein (0.4-1.2 mM), interferes with rat liver mitochondrial bioenergetics by inhibiting mitochondrial respiratory chain activity at complexes I and II levels (Pereira et al, 2010).…”

“…Metolachlor (Pereira et al, 2009) and alachlor (Pereira et al, 2010) inhibit the growth of a strain of B. stearothermophilus in the concentration range of 100 to 600 µM. The herbicides added to cultures of this Gram-positive bacterium, grown in a complex liquid growth medium (L-Broth) at 65 ºC, affect the bacterial growth in the dependence of the concentration, inducing an increase of the lag time, decrease of the specific growth rate and reduction of the maximal optical density attained in the stationary phase of growth.…”