Cytotoxic and genotoxic potential of geraniol in peripheral blood mononuclear cells and human hepatoma cell line (HepG2).

Geraniol is an acyclic monoterpene alcohol present in the essential oil of many aromatic plants and is one of the most frequently used molecules by the flavor and fragrance industries. The literature also reports its therapeutic potential, highlighting itself especially as a likely molecule for the development of drugs against cancer. In view of these considerations, this study was designed to evaluate the cytotoxic and genotoxic potential of geraniol, in an in vitro protocol, using two types of human cells: one without the ability to metabolize (peripheral blood mononuclear cells - PBMC), and the other with this capability (human hepatoma cell line - HepG2) through the comet assay and the micronucleus test. Four concentrations (10, 25, 50, and 100 µg/mL) were selected for the genotoxic assessment for PBMC and three (1.25, 2.5, and 5 µg/mL) for HepG2 cells based on cytotoxicity tests (MTT assay). Results showed that geraniol did not present genotoxic or clastogenic/aneugenic effects on both cell types under the conditions studied. However, caution is advised in the use of this substance by humans, since a significant reduction in viability of HepG2 and a marked decrease in cell viability on normal PBMC were verified.

The Comet assay for detection of DNA damage in canine sperm.

Sperm DNA integrity is a fundamental prerequisite in fertilization and embryo development. Among DNA integrity tests, the Comet assay is an accurate and sensitive test for the detection of sperm oxidative damage. The aim of this work was to evaluate sperm oxidative damage using the Comet assay and to study the correlation between Comet and routine assays for the evaluation of semen quality. Dogs were divided in two groups: group A (n = 6), comprising dogs with abnormal spermiogram, that is astheno-, terato- or oligoasthenoteratozoospermic (OAT); and group B (n = 8), comprising normospermic dogs. The distribution of sperm oxidative damage was significantly different between the two groups (p = .001): group A-median: 31.55%, interquartile range (IQR): 30.18-38.01; group B-median: 0.90%, IQR: 0.65-1.96. The correlation between oxidative damage and abnormal morphology was high (r = .846; p < .001). There was a negative correlation between progressive motility and oxidative damage (r = -.792; p = .001). Basal and oxidative DNA damage of spermatozoa are increased in dogs with non-normospermic semen. In conclusion, and considering the elevated correlation with classical tests of sperm quality, the Comet assay has ample potential for clinical and research purposes in dogs.

First cytotoxic, genotoxic, and antigenotoxic assessment of Euterpe oleracea fruit oil (açaí) in cultured human cells.

Euterpe oleracea Mart., popularly known as "açaí", is a tropical fruit from the Amazon region where it has considerable economic importance. Açaí has been used as food and for several medicinal purposes. Despite the widespread use of this fruit, there is a lack of data regarding the safety of using this fruit oil exclusively. Therefore, we evaluated the in vitro cytotoxic, genotoxic, and antigenotoxic effects of E. oleracea fruit oil (EOO) in cultured human lymphocytes (non-metabolizing cells) and HepG2 cell line (human hepatoma) (metabolizing cells) by using MTT, comet, and micronucleus assays. A wide range of EOO concentrations was tested with a preliminary MTT assay, which allowed selecting five concentrations for comet and micronucleus assays: 2.5, 10, 100, 500, and 1000 µg/mL. The results showed that none of the EOO tested concentrations presented cytotoxic effects. The genotoxic assessment revealed an absence of significant DNA and chromosome damage in human lymphocytes and HepG2 cells but did not show chemoprotection against the DNA damage induced by methyl methanesulfonate and benzo[a]pyrene, used as DNA-damaging agents.

Pb low doses induced genotoxicity in Lactuca sativa plants.

Soil and water contamination by lead (Pb) remains a topic of great concern, particularly regarding crop production. The admissible Pb values in irrigation water in several countries range from ≈0.1 to ≈5 mg L-1. In order to evaluate putative effects of Pb within legal doses on crops growth, we exposed Lactuca sativa seeds and seedlings to increasing doses of Pb(NO3)2 up to 20 mg L-1. The OECD parameter seed germination and seedling/plant growth were not affected by any of the Pb-concentrations used. However, for doses higher than 5 mg L-1 significant DNA damage was detected: Comet assay detected DNA fragmentation at ≥ 5 mg L-1 and presence of micronuclei (MN) were detected for 20 mg L-1. Also, cell cycle impairment was observed for doses as low as 0.05 mg L-1 and 0.5 mg L-1 (mostly G2 arrest). Our data show that for the low doses of Pb used, the OECD endpoints were not able to detect toxicity, while more sensitive endpoints (related with DNA damage and mitotic/interphase disorders) identified genotoxic and cytostatic effects. Furthermore, the nature of the genotoxic effect was dependent on the concentration. Finally, we recommend that MN test and the comet assay should be included as sensitive endpoints in (eco)toxicological assays.

Are DNA-damaging effects induced by herbicide formulations (Roundup® and Garlon®) in fish transient and reversible upon cessation of exposure?

Owing to the seasonality of crop cultivation and subsequent periodic/seasonal application of herbicides, their input to the aquatic systems is typically intermittent. Consequently, exposure of fish to this type of contaminants can be short and followed by a period of permanence in non-contaminated areas. Thus, the assessment of genotoxic endpoints in fish after removal of the contamination source appears as a crucial step to improve the knowledge on the dynamics of herbicide genotoxicity, as well as to determine the actual magnitude of risk posed by these agrochemicals. Therefore, the present study intended to shed light on the ability of fish to recover from the DNA damage induced by short-term exposures to the herbicide formulations Roundup(®) (glyphosate-based) and Garlon(®) (triclopyr-based) upon the exposure cessation. European eel (Anguilla anguilla) was exposed to the above commercial formulations for 3 days, and allowed to recover for 1, 7 and 14 days (post-exposure period). The comet assay was used to identify the DNA damage in blood cells during both exposure and post-exposure periods. As an attempt to clarify the DNA damaging mechanisms involved, an extra-step including the incubation of the nucleotides with DNA lesion-specific repair enzyme was added to the standard comet. The genotoxic potential of both herbicides was confirmed, concerning the exposure period. In addition, the involvement of oxidative DNA damage on the action of Roundup(®) (pointed out as pyrimidine bases oxidation) was demonstrated, while for Garlon(®) this damaging mechanism was less evident. Fish exposed to Garlon(®), though presenting some evidence towards a tendency of recovery, did not achieve a complete restoration of DNA integrity. In what concerns to Roundup(®), a recovery was evident when considering non-specific DNA damage on day 14 post-exposure. In addition, this herbicide was able to induce a late oxidative DNA damage (day 14). Blood cells of A. anguilla exposed to Roundup(®) appeared to be more successful in repairing damage with a non-specific cause than that associated to base oxidation. Overall, the present findings highlighted the genetic hazard to fish associated to the addressed agrochemicals, reinforcing the hypothesis of long-lasting damage.

DNA and chromosomal damage induced in fish (Anguilla anguilla L.) by aminomethylphosphonic acid (AMPA)--the major environmental breakdown product of glyphosate.

The assessment of the direct impact of breakdown products of pesticide components on aquatic wildlife is ecotoxicologically relevant, but frequently disregarded. In this context, the evaluation of the genotoxic hazard posed by aminomethylphosphonic acid (AMPA--the major natural degradation product of glyphosate) to fish emerges as a critical but unexplored issue. Hence, the main goal of the present research was to assess the AMPA genotoxic potential to fish following short-term exposures (1 and 3 days) to environmentally realistic concentrations (11.8 and 23.6 µg L(-1)), using the comet and erythrocytic nuclear abnormalities (ENA) assays, as reflecting different levels of damage, i.e. DNA and chromosomal damage, respectively. Overall, the present findings pointed out the genotoxic hazard of AMPA to fish and, subsequently, the importance of including it in future studies concerning the risk assessment of glyphosate-based herbicides in the water systems.

Differential genotoxicity of Roundup(®) formulation and its constituents in blood cells of fish (Anguilla anguilla): considerations on chemical interactions and DNA damaging mechanisms.

It has been widely recognized that pesticides represent a potential threat in aquatic ecosystems. However, the knowledge on the genotoxicity of pesticides to fish is still limited. Moreover, genotoxic studies have been almost exclusively focused on the active ingredients, whereas the effect of adjuvants is frequently ignored. Hence, the present study addressed the herbicide Roundup®, evaluating the relative contribution of the active ingredient (glyphosate) and the surfactant (polyethoxylated amine; POEA) to the genotoxicity of the commercial formulation on Anguilla anguilla. Fish were exposed to equivalent concentrations of Roundup® (58, 116 µg L⁻¹), glyphosate (17.9, 35.7 µg L⁻¹) and POEA (9.3, 18.6 µg L⁻¹), during 1 and 3 days. The comet assay was applied to blood cells, either as the standard procedure, or with an extra step involving DNA lesion-specific repair enzymes in an attempt to clarify DNA damaging mechanisms. The results confirmed the genotoxicity of Roundup®, also demonstrating the genotoxic potential of glyphosate and POEA individually. Though both components contributed to the overall genotoxicity of the pesticide formulation, the sum of their individual effects was never observed, pointing out an antagonistic interaction. Although POEA is far from being considered biologically inert, it did not increase the risk associated to glyphosate when the two were combined. The analysis of oxidatively induced breaks suggested that oxidation of DNA bases was not a dominant mechanism of damage. The present findings highlighted the risk posed to fish populations by the assessed chemicals, jointly or individually, emphasizing the need to define regulatory thresholds for all the formulation components and recommending, in particular, the revision of the hazard classification of POEA.

DNA damage in fish (Anguilla anguilla) exposed to a glyphosate-based herbicide -- elucidation of organ-specificity and the role of oxidative stress.

Organophosphate herbicides are among the most dangerous agrochemicals for the aquatic environment. In this context, Roundup(®), a glyphosate-based herbicide, has been widely detected in natural water bodies, representing a potential threat to non-target organisms, namely fish. Thus, the main goal of the present study was to evaluate the genotoxic potential of Roundup(®) in the teleost fish Anguilla anguilla, addressing the possible causative involvement of oxidative stress. Fish were exposed to environmentally realistic concentrations of this herbicide (58 and 116 µgL(-1)) during one or three days. The standard procedure of the comet assay was applied to gill and liver cells in order to determine organ-specific genetic damage. Since liver is a central organ in xenobiotic metabolism, nucleoids of hepatic cells were also incubated with a lesion-specific repair enzyme (formamidopyrimidine DNA glycosylase - FPG), in order to recognise oxidised purines. Antioxidants were determined in both organs as indicators of pro-oxidant state. In general, both organs displayed an increase in DNA damage for the two Roundup(®) concentrations and exposure times, although liver showed to be less susceptible to the lower concentration. The enzyme-modified comet assay showed the occurrence of FPG-sensitive sites in liver only after a 3-day exposure to the higher Roundup(®) concentration. The antioxidant defences were in general unresponsive, despite a single increment of catalase activity in gills (116 µgL(-1), 3-day) and a decrease of superoxide dismutase activity in liver (58 µgL(-1), 3-day). Overall, the mechanisms involved in Roundup(®)-induced DNA strand-breaks showed to be similar in both organs. Nevertheless, it was demonstrated that the type of DNA damage varies with the concentration and exposure duration. Hence, after 1-day exposure, an increase on pro-oxidant state is not a necessary condition for the induction of DNA-damaging effects of Roundup(®). By increasing the duration of exposure to three days, ROS-dependent processes gained preponderance as a mechanism of DNA-damage induction in the higher concentration.

European eel (Anguilla anguilla) genotoxic and pro-oxidant responses following short-term exposure to Roundup--a glyphosate-based herbicide.

The glyphosate-based herbicide, Roundup, is among the most used pesticides worldwide. Due to its extensive use, it has been widely detected in aquatic ecosystems representing a potential threat to non-target organisms, including fish. Despite the negative impact of this commercial formulation in fish, as described in literature, the scarcity of studies assessing its genotoxicity and underlying mechanisms is evident. Therefore, as a novel approach, this study evaluated the genotoxic potential of Roundup to blood cells of the European eel (Anguilla anguilla) following short-term (1 and 3 days) exposure to environmentally realistic concentrations (58 and 116 microg/l), addressing also the possible association with oxidative stress. Thus, comet and erythrocytic nuclear abnormalities (ENAs) assays were adopted, as genotoxic end points, reflecting different types of genetic damage. The pro-oxidant state was assessed through enzymatic (catalase, glutathione-S-transferase, glutathione peroxidase and glutathione reductase) and non-enzymatic (total glutathione content) antioxidants, as well as by lipid peroxidation (LPO) measurements. The Roundup potential to induce DNA strand breaks for both concentrations was demonstrated by the comet assay. The induction of chromosome breakage and/or segregational abnormalities was also demonstrated through the ENA assay, though only after 3-day exposure to both tested concentrations. In addition, the two genotoxic indicators were positively correlated. Antioxidant defences were unresponsive to Roundup. LPO levels increased only for the high concentration after the first day of exposure, indicating that oxidative stress caused by this agrochemical in blood was not severe. Overall results suggested that both DNA damaging effects induced by Roundup are not directly related with an increased pro-oxidant state. Moreover, it was demonstrated that environmentally relevant concentrations of Roundup can pose a health risk for fish populations.

The w/w+ SMART assay of Drosophila melanogaster detects the genotoxic effects of reactive oxygen species inducing compounds.

The somatic mutation and recombination w/w+ eye assay has been used for genotoxic evaluation of a broad number of chemicals with different action mechanisms yielding high values of sensitivity, specificity and accuracy. The aim of this work was to determine the utility of this assay in the evaluation of reactive oxygen species inducers. For this, we have tested eight compounds: diquat, paraquat, menadione, juglone, plumbagin, streptonigrin, tert-butyl hydroperoxide and 4-nitroquinoline 1-oxide, using the Drosophila Oregon K strain which had previously shown advantageous conditions to test this type of compounds. Diquat was the only chemical for which the results were clearly negative, probably because its high toxicity, whereas indications of a marginal genotoxicity raised for menadione. The remaining compounds were evaluated as positives. The conclusion of these experiments is that the w/w+ assay is capable to detect genotoxic effects induced by compounds that generate reactive oxygen species through different action mechanisms.

The w/w+ somatic mutation and recombination test (SMART) of Drosophila melanogaster for detecting reactive oxygen species: characterization of 6 strains.

The w/w+ somatic mutation and recombination test (SMART) of Drosophila melanogaster is a fast and low cost in vivo assay that has shown excellent results in the assessment of genotoxicity of a large number of compounds. However, recent studies have revealed that, when procarcinogens are to be evaluated, the performance of the assay is largely dependent on the genetic background of the strains used. To determine which one of the strains available for this test would be advisable to evaluate agents producing reactive oxygen species we have used two approaches. Firstly, the w/w+ assay was carried out using 6 different strains and two compounds: menadione and paraquat. Secondly, 3 biochemical traits were determined for the 6 strains: superoxide dismutase and catalase activities, and their capacity to induce reactive oxygen species. The results suggest that the strains Oregon K and Haag 79 would be usable when potential inducers of reactive oxygen species are to be investigated.