Evaluation of the cytotoxic effects of glyphosate herbicides in human liver,lung, and nerve

Glyphosate-based herbicides are broad-spectrum pesticides widely used in the world, which is considered a highly safe pesticide due to their target specificity, but recently, there has been an ongoing controversy regarding their carcinogenicity and possible side effects of glyphosate on human health. Commercial glyphosate-based herbicides (GBHs) consist of declared active ingredient (glyphosate salts) and a number of formulants such as ethoxylated formulants (4130®, 3780^®, and A-178^®). The aim of our study is to investigate whether the toxicity of GBHs is related to formulants. The effects of GBHs on human health were studied at the cellular level based on their toxicity to liver, lungs and nerve tissue. The inhibitory toxicity to cell viability by GBHs was examined with cell-based systems using three human cell lines: HepG2, A549, and SH-SY5Y. Data obtained showed that all tested ethoxylated formulants and their mixtures with declared active ingredient glyphosate isopropylamine salt (GP) have significant inhibitory effect on cell proliferation, while the declared active ingredient has no significant toxicity. Our study demonstrates that the toxic effect of GBH is primarily due to the use of formulants. This result suggests that GP is relatively safe and a new approach for the assessment of toxicity should be made.     

Fungal hydroxylation of polychlorinated naphthalenes with chlorine migration by wood rotting fungi

Biodegradation of the polychlorinated naphthalenes (PCNs) 1,4-dichloronaphthalene (1,4-DCN), 2,7-dichloronaphthalene (2,7-DCN), and 1,2,3,4-tetrachloronaphthalene (1,2,3,4-TCN), by the white-rot fungus Phlebia lindtneri was investigated. 1,4-DCN was metabolized to form six metabolites by the fungus. It was estimated from GC–MS fragment patterns that the metabolites were four putative hydroxylated and two dihydrodihydroxylated compounds. One of the hydroxylated products was identified as 2,4-dichloro-1-naphthol by GC–MS analysis using an authentic standard. This intermediate indicated chlorine migration in a biological system of P. lindtneri. 2,7-DCN was metabolized to five hydroxylated metabolites and a dihydrodihydroxylated metabolite. Significant inhibition of the degradation of DCNs and formation of their metabolic products was observed in incubation with the cytochrome P-450 monooxygenase inhibitor piperonyl butoxide. The formation of the dihydrodiol-like metabolites, chlorine migration and the experiment with P-450 inhibitor suggested that P. lindtneri provides hydroxyl metabolites via benzene oxide intermediates of DCNs by a cytochrome P450 monooxygenase. In addition, P. lindtneri degraded 1,2,3,4-TCN; two hydroxylated compounds and a dihydrodihydroxylated compound were formed.     

1H-NMR-based metabolomic study on toxicity of methomyl and methidathion in fish

A ¹H-nuclear magnetic resonance (NMR) spectroscopy with multivariate analysis was applied to detect the toxicity of antiacetylcholinesterase insecticides, methomyl (methyl (1E)-N-(methylcarbamoyloxy)ethanimidothioate) and methidathion (3-(dimethoxyphosphinothioyl sulfanylmethyl)-5-methoxy-1,3,4-thiadiazol-2-one), using zebrafish (Danio rerio) and Chinese bleak (Aphyocypris chinensis). Generally, methomyl and methidathion have been believed not to highly accumulate in fish tissues. However, these pesticides showed their toxicity by altering patterns of whole-body metabolites in neurotransmitter balance, energy metabolism, oxidative stress, and muscle maintenance in low concentrations. We used Pearson correlation analysis to contextualize the metabolic markers in pesticide treated groups. We observed that the positive correlations of choline with acetate and betaine in untreated control were shifted to null correlations showing acetylcholinesterase specific toxicity. This research demonstrated the applicability and potential of NMR metabolomics in detecting toxic effects of insecticide with a modicum of concentrations in aquatic environment.     

Research Articles

The metabolism of phenanthrene was studied both in cell suspension cultures of wheat (Triticum aestivum) and soybean (Glycine max), and in intact plants of the water mossFontinalis antipyretica. Metabolism in cell suspension cultures strongly differed between the monocotyle and the dicotyle plant. Only small amounts oftrans-phenanthrene-9,10-dihydrodiole and phenanthrene-9,10-dione were detectable in the wheat culture. Soybean cultures, in contrast demonstrated a strong turnover resulting in a 75% reduction of the initial phenanthrene concentration. Metabolites were phenanthrene-9,10-dione, not further characterized polar metabolites and bound residues. Intact plants ofFontinalis antipyretica metabolized only small amounts of phenanthrene. Data obtained from cell cultures did not provide information for the metabolic potential in intact plants. Therefore standardized tests with model systems like suspension cultures lead to inadequate assessment of the ecological risk of certain xenobiotics.     

Metabolism of the Nonylphenol Isomer [Ring-U-14c]-4-(3′,5′-Dimethyl-3′-Heptyl)-Phenol by Cell Suspension Cultures of Agrostemma githago and Soybean

Abstract

The biotransformation of the nonylphenol isomer [ring-U-¹⁴C]-4-(3′,5′-dimethyl-3′-heptyl)-phenol (4-353-NP, consisting of two diastereomers) was studied in soybean and Agrostemma githago cell suspension cultures. With the A. githago cells, a batch two-liquid-phase system (medium/n-hexadecane 200:1, v/v) was used, in order to produce higher concentrations and amounts of 4-353-NP metabolites for their identification; 4-353-NP was applied via the n-hexadecane phase. Initial concentrations of [¹⁴C]-4-353-NP were 1 mg L^?1 (soybean), and 5 and 10 mg L^?1 (A. githago). After 2 (soybean) and 7 days (A. githago) of incubation, the applied 4-353-NP was transformed almost completely by both plant species to four types of products: glycosides of parent 4-353-NP, glycosides of primary 4-353-NP metabolites, nonextractable residues and unknown, possibly polymeric materials detected in the media. The latter two products emerged especially in soybean cultures. Portions of primary metabolites amounted to 19–22% (soybean) and 21–42% of applied ¹⁴C (A. githago). After liberation from their glycosides, the primary 4-353-NP metabolites formed by A. githago were isolated by HPLC and examined by GC-EIMS as trimethylsilyl derivatives. In the chromatograms, eight peaks were detected which due to their mass spectra, could be traced back to 4-353-NP. Seven of the compounds were side-chain monohydroxylated 4-353-NP metabolites, while the remaining was a (side-chain) carboxylic acid derivative. Unequivocal identification of the sites of hydroxylation/oxidation of all transformation products was not possible. The main primary metabolites produced by A. githago were supposed to be four diastereomers of 6′-hydroxy-4-353-NP (about 80% of all products identified). It was concluded that plants contribute to the environmental degradation of the xenoestrogen nonylphenol; the toxicological properties of side-chain hydroxylated nonylphenols remain to be examined.     

Distribution of14C-TNT and derivatives in different biochemical compartments ofPhaseolus vulgaris

¹⁴C-TNT was used to quantify the uptake rate and metabolic turnover of TNT inPhaseolus vulgaris. Seventeen plants were analysed by a special cell fractionation method with polar and nonpolar solvents and enzymes. We obtained three cytoplasmic fractions and five cell wall derived fractions. The recovery rate was 72% as measured by liquid scintillation counting.¹⁴C partitioned almost in equal amounts with approximately 50% in the cytoplasm and in the cell wall. The majority of the TNT-metabolites are present in the cytoplasm as was shown by GC/ ECD and thin layer chromatography. The¹⁴C in the cell wall is bound probably resulting in long-term immobilisation of these metabolites. We conclude that plants may also be a model for nitroaromatic turnover and immobilisation in soil components.     

Natural variability and correlations in the metabolic profile of healthy Eisenia fetida earthworms observed using ¹H NMR metabolomics

¹H NMR metabolomics can be used to assess the sub-lethal toxicity of contaminants to earthworms by identifying alterations in the metabolic profiles of contaminant- exposed earthworms in contrast to those of healthy (control) individuals. In support of this method this study sought to better characterize the baseline metabolic profile of healthy, mature earthworms of the species, Eisenia fetida, which is recommended for both acute and sub-lethal toxicity testing for soil contaminants. Profiles of D₂O-buffer extracted metabolites were determined using ¹H NMR spectroscopy and both inter-individual metabolic variability and pair-wise metabolic correlations were assessed. The control earthworm extracts exhibited low overall inter-individual metabolic variability, with a spectrum-wide median relative standard deviation (%RSD = standard deviation/mean × 100) of 14%, which suggests that the metabolic profile of E. fetida earthworms is well controlled in laboratory conditions and supports further use of this organism in environmental metabolomics research. In addition, strong positive correlations were detected between the levels of maltose, betaine, glycine, and glutamate as well as between the levels of lactate, valine, leucine, alanine, lysine, tyrosine, and phenylalanine which had not previously been reported. Since comparison of pair-wise metabolic correlations between control and treated organisms can reveal changes in the underlying pattern of biochemical relationships between the metabolites, identification of these significant metabolic correlations in control earthworms provides an additional characteristic that may be applied to delineate between control and treated earthworms in future NMR-based metabolomic studies.     

Surfactant-based oil dispersant toxicity to developing nauplii of Artemia: effects on ATPase enzymatic system

The paper deals with the toxicity of a surfactant-based oil dispersant to the ATPase activities of two naupliar stages of Artemia (instar I & II). Both instars were exposed to sub-lethal and lethal concentrations derived from acute toxicity data. The chosen concentrations were near to LOECs and NOECs. An eightfold difference indicated between the instars was instar-exposure time dependent. The most prominent effects were the inhibition and the stimulation of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activities, respectively. The cause of these effects was related to the dispersant components, the surfactants. The pattern stimulation/inhibition of Mg²⁺-ATPase and Na⁺/K⁺-ATPase activities could be used to indicate toxic stress by surfactant-based oil dispersants since previous studies with other contaminants have shown different ATPase activity patterns.     

Cytotoxic activity of the bioactive principles from Ficus burtt-davyi

Ficus burtt-davyi (Moraceae) is a medicinal plant species indigenous to Southern Africa. In this study, a phytochemical and cytotoxic investigation on F. burtt-davyi was conducted to evaluate its ethno-medicinal use. The phytochemical study of the fruits yielded triterpenoids (lupeol and α-amyrin). The cytotoxic evaluation was done on the methanolic extracts and selected compounds, lupeol, α-amyrin, lupeol acetate and (+)-catechin isolated from F. burtt-davyi stem bark and fruits. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay was carried out against two human cancer cell lines, breast adenocarcinoma (MCF-7) and colorectal adenocarcinoma (Caco-2), and normal human embryonic kidney cells (HEK293). The methanol extract from the stem bark was significantly cytotoxic to MCF-7 and Caco-2 cell lines (p < 0.05) in a concentration-dependent manner with IC₅₀ values of 6.6 and 8.1 µg mL^?1, respectively relative to the control. Lupeol and (+)-catechin showed cytotoxic activity against MCF-7 cell lines with IC₅₀ values of 22.6 and 29.8 µg mL^?1, respectively and greater cytotoxic activity against Caco-2 cell lines with IC₅₀ values of 10.7 and 9.0 µg mL^?1, respectively. Data from this study suggests that F. burtt-davyi exhibits cytotoxicity with no significant inhibitory effects against HEK293. The results also indicate that (+)-catechin and lupeol, the most abundant bioactive principles in the stem bark, are responsible for the synergistic cytotoxic effects against tested human cancer cell lines. This study provides evidence on the pharmaceutical potential of the medicinal plant, F. burtt-davyi, as a chemotherapeutic agent against cancer.     

Acute and chronic effects of nano- and non-nano-scale TiO2 and ZnO particles on mobility and reproduction of the freshwater invertebrate Daphnia magna

Among the emerging literature addressing the biological effects of nanoparticles, very little information exists, particularly on aquatic organisms, that evaluates nanoparticles in comparison to non-nanocounterparts. Therefore, the potential effects of nano-scale and non-nano-scale TiO₂ and ZnO on the water flea, Daphnia magna, were examined in 48-h acute toxicity tests using three different test media, several pigment formulations – including coated nanoparticles – and a variety of preparation steps. In addition, a 21-d chronic Daphnia reproduction study was performed using coated TiO₂ nanoparticles. Analytical ultracentrifugation analyses provided evidence that the nanoparticles were present in a wide range of differently sized aggregates in the tested dispersions. While no pronounced effects on D. magna were observed for nano-scale and non-nano-scale TiO₂ pigments in 19 of 25 acute (48-h) toxicity tests (EC50 > 100 mg L⁻¹), six acute tests with both nano- and non-nano-scale TiO₂ pigments showed slight effects (EC10, 0.5–91.2 mg L⁻¹). For the nano-scale and non-nano-scale ZnO pigments, the acute 48-h EC50 values were close to the 1 mg L⁻¹ level, which is within the reported range of zinc toxicity to Daphnia. In general, the toxicity in the acute tests was independent of particle size (non-nano-scale or nano-scale), coating of particles, aggregation of particles, the type of medium or the applied pre-treatment of the test dispersions. The chronic Daphnia test with coated TiO₂ nanoparticles demonstrated that reproduction was a more sensitive endpoint than adult mortality. After 21 d, the NOEC for adult mortality was 30 mg L⁻¹ and the NOEC for offspring production was 3 mg L⁻¹. The 21-d EC10 and EC50 values for reproductive effects were 5 and 26.6 mg L⁻¹, respectively. This study demonstrates the utility of evaluating nanoparticle effects relative to non-nano-scale counterparts and presents the first report of chronic exposure to TiO₂ nanoparticles in D. magna.     

ISOPROTURON DEGRADATION AS AFFECTED BY THE GROWTH OF TWO ALGAL SPECIES AT DIFFERENT CONCENTRATIONS AND pH VALUES

Metabolism of [¹⁴C-u-phenyl]isoproturon [3-(4-isopropylphenyl)-1,1-dimethylurea] by two soil and freshwater microorganisms, green alga Chlorella kesslerei and cyanobacterium Anabaena inaequalis, was studied as a function of pH, pesticide concentration, and incubation time. Metabolized isoproturon, in the media, ranged from 0% (Chlorella at pH 5.5 after 1 d) to 22% (Anabaena at pH 5.5 after 10 d). Twenty-five percent faster degradation of isoproturon by Anabaena occurred at pH 5.5 versus pH 7.5, when measured over 10 d. Increased ¹⁴C incorporation into tissue, with time and at lower pH, was due mainly to bioaccumulation of [¹⁴C]isoproturon and/or its metabolites in the cells. Metabolic degradation resulted in four identifiable (by TLC) metabolites. Based on this, a degradation pathway is proposed, involving mono- and di-N-demethylation, hydroxylation of the isopropyl moiety, and hydrolysis to 4-isopropylaniline. Similarity in the metabolites produced suggests that the enzyme systems responsible for metabolizing isoproturon are almost identical in both photosynthetic micro-algae.     

Presence of DDT metabolites in water,sediment and fish muscle tissue from Lake Prespa,Republic of Macedonia

Organochlorine pesticides were determined in water and sediment samples collected from the littoral zone of Lake Prespa, as well as from its three main tributaries (the rivers Golema, Brajcinska and Kranska), during the period 2004 to 2006. In addition, muscle tissue samples of barbus fish (Barbus prespensis Karaman, 1928) collected from the littoral zone of Lake Prespa were also analysed. The obtained results give an overview of the contamination levels of these problematic compounds at their potential sources in the river mouths, in the potentially affected, species-rich littoral section of the lake and in the muscle tissue of one selected fish species, collected near the rivers’ deltas. Special attention was paid to the presence of some DDT metabolites (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p′–DDE); (1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (p,p′–DDD) and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p′–DDT). The extraction of pesticides from water samples was done by liquid-liquid partition in dichloromethane. For the sediment and fish tissue we used solid-liquid extraction. The extracted residues were analyzed on a gas chromatograph equipped with an electron capture detector (GC-ECD). The results of the respective studies indicated the presence of DDT metabolic forms in the samples of the three analysed matrixes. The highest levels of presence for these pollutants were found in the muscle tissue of the fish samples. The total DDTs content in the analysed muscle tissue samples range from 11.67 to 13.58 μg kg^?1of fresh tissue. The average total DDTs content for the sediment samples were within the range of 2.32 to 4.17 μg kg^?1 of dry sediment. Higher DDT metabolites content were found in the sediments collected from the rivers than in the samples from the littoral zone. The lowest average total concentrations of DDTs, on the other hand, were recorded in the water samples and ranged between 0.036 and 0.057 μg L^?1. The obtained results indicated that the dominant metabolic form in the samples of the three investigated matrixes (water, sediment and fish tissue) from Lake Prespa was p,p′-DDE. There was a very good linear correlation in this study between the content of DDT's (total DDT metabolites) detected and the percentage of total organic material in the sediment. The detected concentrations are clearly below the toxicity thresholds; consequently, severe effects on the endemic species of Lake Prespa are not very likely.     

The toxicity of copper, cadmium and zinc to four different Hydra (Cnidaria: Hydrozoa)

An acute toxicity study of three metals to Hydra species carried out using two different assessment methods, (i) determination of the LC50 and (ii) measurement of progressive morphological changes, demonstrated that relative toxicity decreased from copper to cadmium with zinc the least toxic for all species. The latter method revealed more details of the effect on Hydra in terms of physical damage to the polyp but both methods indicated that H. viridissima was more sensitive to copper and cadmium than H. vulgaris¹ (Zurich strain, male clone), H. vulgaris² (a dioecious strain reproducing sexually and asexually) and H. oligactis (dioecious, reproducing sexually and asexually). The responses to zinc were similar for all Hydra. The possible role of metabolic interactions between H. viridissima and its symbiotic green algae in contributing to the greater sensitivity of this polyp is discussed.     

Biodegradation of mixed pesticides by mixed pesticide enriched cultures

This paper discusses the degradation kinetics of mixed (lindane, methyl parathion and carbofuran) pesticides by mixed pesticide enriched cultures (MEC) under various environmental conditions. The bacterial strains isolated from the mixed microbial consortium were identified as Pseudomonas aeruginosa (MTCC 9236), Bacillus sp. (MTCC 9235) and Chryseobacterium joostei (MTCC 9237). Batch studies were conducted to estimate the biokinetic parameters like the maximum specific growth rate (μ_max), Yield Coefficient (Y_T), half saturation concentration (K_s) and inhibition concentration (Ki) for individual and mixed pesticide enriched cultures. The cultures enriched in a particular pollutant always showed high growth rate and low inhibition in that particular pollutant compared to MEC. After seven weeks of incubation, mixed pesticide enriched cultures were able to degrade 72% lindane, 95% carbofuran and 100% of methyl parathion in facultative co-metabolic conditions. In aerobic systems, degradation efficiencies of lindane methyl parathion and carbofuran were increased by the addition of 2g L^{? 1} of dextrose. Though many metabolic compounds of mixed pesticides were observed at different time intervals, none of the metabolites were persistent. Based on the observed metabolites, a degradation pathway was postulated for different pesticides under various environmental conditions.     

Evaluation of the capacity of the cyanobacterium Microcystis novacekii to remove atrazine from a culture medium

The bioaccumulation of atrazine and its toxicity were evaluated for the cyanobacterium Microcystis novacekii. Cyanobacterial cultures were grown in WC culture medium with atrazine at 50, 250 and 500 μg L^?1. After 96 hours of exposure, 27.2% of the atrazine had been removed from the culture supernatant. Spontaneous degradation was found to be insignificant (< 9% at 500 μg L^?1), indicating a high efficiency for the bioaccumulation of atrazine by M. novacekii. There were no atrazine metabolites detected in the culture medium at any of the doses studied. The acute toxicity (EC₅₀) of atrazine to the cyanobacterium was 4.2 mg L^?1 at 96 hours demonstrating the potential for M. novacekii to tolerate high concentrations of this herbicide in fresh water environments. The ability of M. novacekii to remove atrazine combined with its tolerance of the pesticide toxicity showed in this study makes it a potential biological resource for the restoration of contaminated surface waters. These findings support continued studies of the role of M. novacekii in the bioremediation of fresh water environments polluted by atrazine.     

Combined toxic impacts of thiamethoxam and four pesticides on the rare minnow (Gobiocypris rarus)

To examine pesticide mixture toxicity to aqueous organisms, we assessed the single and combined toxicities of thiamethoxam and other four pesticides (chlorpyrifos, beta-cypermethrin, tetraconazole, and azoxystrobin) to the rare minnow (Gobiocypris rarus). Data from 96-h semi-static toxicity assays of various developmental phases (embryonic, larval, juvenile, and adult phases) showed that beta-cypermethrin, chlorpyrifos, and azoxystrobin had the highest toxicities to G. rarus, and their LC₅₀ values ranged from 0.0031 to 0.86 mg a.i. L^?1, from 0.016 to 6.38 mg a.i. L^?1, and from 0.39 to 1.08 mg a.i. L^?1, respectively. Tetraconazole displayed a comparatively high toxicity, and its LC₅₀ values ranged from 3.48 to 16.73 mg a.i. L^?1. By contrast, thiamethoxam exhibited the lowest toxic effect with LC₅₀ values ranging from 37.85 to 351.9 mg a.i. L^?1. Rare minnow larvae were more sensitive than embryos to all the pesticides tested. Our data showed that a pesticide mixture of thiamethoxam–tetraconazole elicited synergetic toxicity to G. rarus. Moreover, pesticide mixtures containing beta-cypermethrin in combination with chlorpyrifos or tetraconazole also had synergetic toxicities to fish. The majority of pesticides are presumed to have additive toxicity, while our data emphasized that the concurrent existence of some chemicals in the aqueous circumstance could cause synergetic toxic effect, leading to severe loss to the aqueous environments in comparison with their single toxicities. Thence, the synergetic impacts of chemical mixtures should be considered when assessing the ecological risk of chemicals.

     

Study on the cytotoxicity of microcystin-LR on cultured cells

The toxicity of purified blue-green algal toxin, microcystin-LR, on permanent cell lines KB, NIH/3T3, H-4-II-E, HeLa, Vero, Hep G2, Caco-2 and HL-60 was studied. Assessment of cell viability using colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays indicated that purified microcystin-LR induced toxic effect on KB and H-4-II-E cell lines after 96 h incubation at toxin concentrations greater than 18.75 microg/ml. KB cell line was selected for further study when reproducibility, consistency and sensitivity were considered. Significant amounts of lactate dehydrogenase (LDH) were released from KB cells when incubation durations were 72 and 96 h with toxin concentrations of 18.75 microg/ml and higher. Although previous studies suggested that microcystin-LR had no cytotoxic effect on permanent cell lines, LDH release assay performed on KB cells indicated that exposure to microcystin-LR could result in damage to the cell membrane.     

Influence of pH on the sonolysis of ciprofloxacin: Biodegradability, ecotoxicity and antibiotic activity of its degradation products

The presence of antibiotics in the aquatic environment has raised concerns due to the potential risk for the emergence or persistence of antibiotic resistance. Antibiotics are often poorly degraded in conventional wastewater treatment plants. In this study, sonolysis at 520 kHz and 92 W L⁻¹ was used for the degradation of the fluoroquinolone antibiotic ciprofloxacin. In a first experiment at pH 7, 57% of the ciprofloxacin (15 mg L⁻¹) was degraded after 120 min of ultrasonic irradiation at 25 °C. pH proved to be an important parameter determining the degradation rate, since the pseudo first order degradation constant increased almost fourfold when comparing treatment at pH 7 (0.0058 min⁻¹) and pH 10 (0.0069 min⁻¹) with that at pH 3 (0.021 min⁻¹). This effect can be attributed to the degree of protonation of the ciprofloxacin molecule. The BOD/COD ratio of the solutions, which is a measure for their biodegradability, increased from 0.06 to 0.60, 0.17, and 0.18 after 120 min of irradiation depending on the pH (3, 7, and 10, respectively). The solution treated at pH 3 can even be considered readily biodegradable (BOD/COD > 0.4). The antibiotic activity against Escherichia coli (G−) and Bacillus coagulans (G+) of the treated solutions also reduced after sonolysis. The highest decrease was again found when irradiated at pH 3. In contrast, ecotoxicity of the solutions to the alga Pseudokirchneriella subcapitata increased 3- to 10-fold after 20 min of treatment, suggesting the formation of toxic degradation products. The toxicity slowly diminished during further treatment.