Costus spiralis extract restores kidney function in cisplatin-induced nephrotoxicity model: Ethnopharmacological use, chemical and toxicological investigation.

ETHNOPHARMACOLOGICAL RELEVANCE: Costus spiralis (Jacq.). Roscoe (Costaceae) is traditionally used in Brazil for the treatment of kidney diseases such as pyelonephritis, urethra inflammation, kidney stones, and inflammatory conditions. There are reports of its use by Brazilian Indians since the 17th century when it was known as "pacocatinga." Currently, the use of the Costus species in Brazil is widespread, which was evidenced by the inclusion of the genus in the Brazilian National List of Medicinal Plants of Interest to the Unified Health System (RENISUS). AIM OF THE STUDY: This study aimed to confirm the ethnopharmacological use of Costus spiralis in the treatment of kidney diseases, toxicity study using animal models, and the phytochemistry of the species. MATERIALS AND METHODS: The chemical profile of Costus spiralis leaves extract (CSLE) was obtained for the hydroethanolic extract by ultra-performance liquid chromatography coupled to a mass spectrometer and ultraviolet detector with diode array (UPLC-UV/DAD-ESI-MS). The acute oral toxicity of the extract was predicted using the neutral red uptake cytotoxicity assay. Wistar rats were used in a model in vivo for confirmation of acute oral toxicity (2000 mg/kg p.o. for 14 days.) and determination of the effect on a cisplatin-induced nephrotoxicity model. RESULTS: The analysis by UPLC-UV/DAD-ESI-MS showed that the chemical composition of the extract is mostly di-glycosylated flavones of apigenin. In the extract were identified the flavones vicenin II and schaftoside. The quantification of total flavonoids by spectrometry showed 0.880%. CSLE proved to be safe for acute oral administration (2000 mg/kg) with an IC50 value of 222.9 µg/mL and predicted oral toxic dose of 523.82 µg/mL in a neutral red uptake cytotoxicity assay. The absence of death allows the classification of the extract in class 5 according to OECD 423 guidelines and therefore it can be considered as a high acute safety product, which is highly relevant, considering the wide popular use of the species. In the cisplatin-induced nephrotoxicity model, C. spiralis extract (5, 15, and 30 mg/kg) significantly improved renal function, reversing almost completely the effects on plasma creatinine levels and creatinine clearance (p < 0.001). CONCLUSIONS: This study demonstrates that oral administration of Costus spiralis extract leaves is safe and effective in restoring the renal function in rats in a cisplatin-induced nephrotoxicity. It is suggested that the observed activity is related to the flavonoids present. This hypothesis should be confirmed, and the participation of other secondary metabolites should be investigated in the future.

Plant Cell Vacuoles: Staining and Fluorescent Probes.

In plant cells, vacuoles are extremely important for growth and development, and influence important cellular functions as photosynthesis, respiration, and transpiration. Plant cells contain lytic and storage vacuoles, whose size can be different depending on cell type and tissue developmental stage. One of the main roles of vacuoles is to regulate the cell turgor in response to different stimuli. Thus, studying the morphology, dynamics, and physiology of vacuole is fundamentally important to advance knowledge in plant cell biology at large. The availability of fluorescent probes allows marking vacuoles in multiple ways. These may be fast, when using commercially available chemical dyes, or relatively slow, in the case of specific genetically encoded markers based on proteins directed either to the membrane of the vacuole (tonoplast) or to the vacuole lumen. Any of these approaches provides useful information about the morphology and physiology of the vacuole.

Isolation of Vacuoles from the Leaves of the Medicinal Plant Catharanthus roseus.

The isolation of vacuoles is an essential step to unravel the important and complex functions of this organelle in plant physiology. Here, we describe a method for the isolation of vacuoles from Catharanthus roseus leaves involving a simple procedure for the isolation of protoplasts, and the application of a controlled osmotic/thermal shock to the naked cells, leading to the release of intact vacuoles, which are subsequently purified by density gradient centrifugation. The purity of the isolated intact vacuoles is assayed by microscopy, western blotting, and measurement of vacuolar (V)-H+-ATPase hydrolytic activity. Finally, membrane functionality and integrity is evaluated by measuring the generation of a transtonoplast pH gradient by the V-H+-ATPase and the V-H+-pyrophosphatase, also producing further information on vacuole purity.

Flow Cytometry and Fluorescence Microscopy as Tools for Structural and Functional Analysis of Vacuoles Isolated from Yeast and Plant Cells.

A series of optimized protocols to isolate vacuoles from both yeast and plant cells, and to characterize the purified organelles at a functional and structural level, are described. For this purpose, we took advantage of the combined use of cell fractionation techniques with different fluorescence-based approaches namely flow cytometry, fluorescence microscopy and spectrofluorimetry. These protocols altogether constitute valuable tools for the study of vacuole structure and function, as well as for the high-throughput screening of drug libraries to identify new molecules that target the vacuole.

Using Vital Dyes to Trace Uptake of dsRNA by Green Peach Aphid Allows Effective Assessment of Target Gene Knockdown.

RNA interference (RNAi) is an effective tool to study gene function. For in vitro studies of RNAi in insects, microinjection of double-stranded (ds)RNA may cause stress. Non-persuasive oral delivery of dsRNA to trigger RNAi is a better mode of delivery for delicate insects such as aphids because it mimics natural feeding. However, when insects feed ad libitum, some individuals may not feed. For accurate measurement of gene knockdown, analysis should only include insects that have ingested dsRNA. The suitability of eleven dyes was assessed to trace ingestion of dsRNA in an artificial feeding system for green peach aphids (GPA, Myzus persicae). Non-toxic levels of neutral red and acridine orange were suitable tracers: they were visible in the stylet and gut after feeding for 24 h, and may also attract aphids to feed. Nymphs stained with neutral red (0.02%) were analysed for target gene expression after feeding on sucrose with dsRNA (V-ATPase, vha-8). There was a greater reduction in vha-8 expression and reproduction compared to nymphs fed the diet without dye. The results confirm the importance of identifying aphids that have ingested dsRNA, and also provide evidence that the vha-8 gene is a potential target for control of GPAs.

Neutral red cytotoxicity assays for assessing in vivo carbon nanotube ecotoxicity in mussels--Comparing microscope and microplate methods.

The purpose of the present study was to compare two neutral red retention methods, the more established but very labour-intensive microscope method (NRR) against the more recently developed microplate method (NRU). The intention was to explore whether the sample volume throughput could be increased and potential operator bias avoided. Mussels Mytilus sp. were exposed in vivo to 50, 250 and 500 µg L(-1) single (SWCNTs) or multi-walled carbon nanotubes (MWCNTs). Using the NRR method, SWCNTs and MWCNTs caused concentration dependent decreases in neutral red retention time. However, a concentration dependent decrease in optical density was not observed using the NRU method. We conclude that the NRU method is not sensitive enough to assess carbon nanotube ecotoxicity in vivo in environmentally relevant media, and recommend using the NRR method.

Antioxidant potential, cytotoxic activity and total phenolic content of Alpinia pahangensis rhizomes.

BACKGROUND: Alpinia pahangensis, a wild ginger distributed in the lowlands of Pahang, Malaysia, is used by the locals to treat flatulence. In this study, the antioxidant and cytotoxic activities of the crude aqueous methanol and fractionated extracts of Alpinia pahangensis against five different cancer and one normal cell lines were investigated. The total phenolic content of each extract and its fractions were also quantified. This is the first report on the antioxidant and cytotoxic activities of Alpinia pahangensis extract. METHODS: In the current study, the crude methanol and fractionated extract of the rhizomes of Alpinia pahangensis were investigated for their antioxidant activity using four different assays namely, the DPPH scavenging activity, superoxide anion scavenging, ß-carotene bleaching and reducing power assays whilst their phenolic contents were measured by the Folin-Ciocalteu's method.In vitro neutral red cytotoxicity assay was employed to evaluate the cytotoxic activity against five different cancer cell lines, colon cancer (HCT 116 and HT-29), cervical cancer (Ca Ski), breast cancer (MCF7) and lung cancer (A549) cell lines, and one normal cell line (MRC-5). The extract that showed high cytotoxic activity was further investigated for its chemical constituents by GC-MS (gas chromatography-mass spectrometry) analysis. RESULTS: The ethyl acetate fraction showed the strongest DPPH radical scavenging (0.35 ± 0.094 mg/ml) and SOD activities (51.77 ± 4.9%) whilst the methanol extract showed the highest reducing power and also the strongest antioxidant activity in the ß-carotene bleaching assays in comparison to other fractions. The highest phenolic content was found in the ethyl acetate fraction, followed by the crude methanol extract, hexane and water fractions. The results showed a positive correlation between total phenolic content with DPPH radical scavenging capacities and SOD activities. The hexane fraction showed potent cytotoxic effect against KB, Ca Ski and HCT 116 cell lines with IC50 of 5.8 ± 0.1 and 9.1 ± 2.0 ug/ml, respectively. The major components of hexane fraction analysed by GC-MS analysis were mostly methyl esters. CONCLUSIONS: The current study suggests that the methanol extract and ethyl acetate fraction of A. pahangensis is a potential source of natural antioxidant for protective as well as prevention of life-threatening diseases. The hexane fraction of A. pahangensis may have the potential to be developed into therapeutic option for treating cancer.

A robust procedure for distinctively visualizing zebrafish retinal cell nuclei under bright field light microscopy.

To simultaneously visualize individual cell nuclei and tissue morphologies of the zebrafish retina under bright field light microscopy, it is necessary to establish a procedure that specifically and sensitively stains the cell nuclei in thin tissue sections. This necessity arises from the high nuclear density of the retina and the highly decondensed chromatin of the cone photoreceptors, which significantly reduces their nuclear signals and makes nuclei difficult to distinguish from possible high cytoplasmic background staining. Here we optimized a procedure that integrates JB4 plastic embedding and Feulgen reaction for visualizing zebrafish retinal cell nuclei under bright field light microscopy. This method produced highly specific nuclear staining with minimal cytoplasmic background, allowing us to distinguish individual retinal nuclei despite their tight packaging. The nuclear staining is also sensitive enough to distinguish the euchromatin from heterochromatin in the zebrafish cone nuclei. In addition, this method could be combined with in situ hybridization to simultaneously visualize the cell nuclei and mRNA expression patterns. With its superb specificity and sensitivity, this method may be extended to quantify cell density and analyze global chromatin organization throughout the retina or other tissues.

Investigation of landfill leachate toxic potency: an integrated approach with the use of stress indices in tissues of mussels.

The present study investigates the harmful impacts of landfill leachate release and/or disposal into the marine environment, as well as its ability to induce lethal and pre-pathological alterations in marine organisms, such as the mussel Mytilus galloprovincialis. In specific, mortality test (96 h), performed first in order to estimate leachate lethal endpoints, showed increased levels of mussel mortality after exposure to leachate higher than 0.5%, v/v (96 h LC(50)=0.526%, v/v), while the exposure to 0.01 and 0.1% (v/v) of leachate showed negligible levels of mortality (96 h LC(10)=0.167%, v/v). Furthermore, the estimation of lysosomal membrane integrity in hemocytes of exposed mussels (Neutral Red Retention Time assay) showed increased levels of lysosomal destabilization in cells of mussels exposed to sub-lethal concentrations of leachate (0.01, 0.1 and 0.5%, v/v) for 4 days. In order to exclude parameters, such as mussel mortality and cell death, which could interfere with the obtained results, leachate at final concentrations of 0.01 and 0.1% (v/v) were finally used for the estimation of a battery of stress indices in target tissues of mussels, such as hemolymph, gills and digestive gland. According to the results, leachate-exposed mussels showed a significant inhibition of acetylcholinesterase activity, increased levels of nuclear abnormalities, as well as increased levels of metallothionein, superoxide anion and lipid peroxides (in terms of malondialdehyde equivalents) in each tissue tested. The results of the present study clearly indicate leachate-induced lethal effects as well as the ability of leachate to induce disturbances on different levels of organism function before mortality occurs.

Preconcentration of aqueous dyes through phase-transfer liquid-phase microextraction with a room-temperature ionic liquid.

In this study, we employed the room-temperature ionic liquid [bmim][PF(6)] as both ion-pair agent and an extractant in the phase-transfer liquid-phase microextraction (PTLPME) of aqueous dyes. In the PTLPME method, a dye solution was added to the extraction solution, comprising a small amount of [bmim][PF(6)] in a relatively large amount of CH(2)Cl(2), which serves as the disperser solvent to an extraction solution. Following extraction, CH(2)Cl(2) was evaporated from the extractant, resulting in the extracted dyes being concentrated in a small volume of the ionic liquid phase to increase the enrichment factor. The enrichment factors of for the dye Methylene Blue, Neutral Red, and Methyl Red were approximately 500, 550 and 400, respectively; their detection limits were 0.014, 0.43, and 0.02 µg L(-1), respectively, with relative standard deviations of 4.72%, 4.20%, and 6.10%, respectively.

Sub-lethal effects induced by a mixture of three non-steroidal anti-inflammatory drugs (NSAIDs) on the freshwater bivalve Dreissena polymorpha.

Non-steroidal anti-inflammatory drugs (NSAIDs) are the sixth top-selling drugs worldwide and are commonly found in freshwater ecosystems in the high ng/l to low µg/l range. Recent studies have investigated both the acute and the chronic toxicity of single NSAIDs on different biological models, but these studies have completely neglected the fact that, in the environment, non-target organisms are exposed to mixtures of drugs that have unforeseeable toxicological behavior. This work investigated the sub-lethal effects induced by a mixture of three common NSAIDs, namely, diclofenac, ibuprofen and paracetamol, on the freshwater bivalve, the zebra mussel (Dreissena polymorpha). The mussels were exposed to three different environmental concentrations of the mixture (Low, Mid and High). A multi-biomarker approach was used to highlight cyto-genotoxic effects and the imbalance of the oxidative status of the treated specimens. The Neutral Red Retention Assay (NRRA) was used as a biomarker of cytotoxicity, whereas the activities of catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase were measured to assess the role played by the oxidative stress enzymes. In addition, the single cell gel electrophoresis assay, the DNA Diffusion assay and the micronucleus test were used to investigate possible genotoxic effects. According to our NRRA results, each treatment was able to induce a significant cellular stress in bivalves, probably due to the raise of oxidative stress, as indicated by the alteration of enzyme activities measured in treated specimens. Moreover, the mixture induced significant enhancements of DNA fragmentation, which preluded fixed genetic damage, as highlighted by the increase of both apoptotic and micronucleated cells.

Application of neutral red retention assay to caged clams (Ruditapes decussatus) and crabs (Carcinus maenas) in the assessment of dredged material.

Dredged material management is a key issue for the protection of aquatic environments. The in situ approach using caged bioindicator species has been chosen lately as a new methodology for the assessment of dredged material. In a tier testing approach, neutral red retention (NRR) assay has been applied as a screening tool to detect adverse changes in health status associated with contamination. Nevertheless, to authors' knowledge, little is known about the application and validation of this technique in sediment bioindicator species and under field conditions. Caged Ruditapes decussatus and Carcinus maenas were exposed during 28 days to potentially contaminated sediments at three sites in Algeciras Bay (SW Spain) and one site in Cádiz Bay (SW Spain). Lysosomal membrane stability was measured over time in haemolymph samples of exposed clams and crabs using the NRR assay. Sediment characterization of the study sites was performed in parallel. NRR time did not vary significantly (p > 0.05) over time in organisms from Cádiz Bay. Conversely, significant differences (p < 0.05) in NRR time were found in clams and crabs exposed to sediments from Algeciras Bay, which exhibited a 30-70% decrease in haemocyte lysosome membrane stability compared to day 0. Statistical analysis showed a strong correlation between the drop of haemocyte lysosome membrane stability, in both crabs and clams, and the presence of metals (p < 0.05) and PAHs (p < 0.01) in the studied sediments. The results obtained confirmed the use of NRR assay as a suitable and sensitive method to be used in the assessment of sediment quality using as bioindicator species the clam R. philippinarum and the crab C. maenas.

Mutagenic, cytotoxic, and genotoxic properties of tobacco smoke produced by cigarillos available on the Canadian market.

Cigarillos (aka little cigars) have been increasing in popularity unlike cigarettes; but relatively little is known about the toxicology of the mainstream smoke (MSS) from such products. Therefore, the objective of this work was to compare the toxicological properties of the MSS (Health Canada Intensive smoking conditions) from a range of cigarillo products with the toxicological properties of MSS of cigarettes. Three in vitro assays were used to evaluate the toxicities of the MSS total particulate matter (TPM): (1) mutagenicity using Ames assay with Salmonella strains TA98 and TA100 with S9 metabolic activation (+S9); (2) cytotoxicity using the Neutral Red Uptake (NRU) assay with CHO (Chinese Hamster Ovary) cells; and (3) genotoxicity using the micronucleus assay with CHO cells and short-term exposures (3-h ± S9). The Ames assay (TA100+S9) and the NRU assay were also applied to the gas/vapour phase of the MSS that passed through the Cambridge pad. On a per-milligram-nicotine basis, the preferred way of comparing toxicities of different types of tobacco products, the MSS from cigarillos was not less toxic, and in some cases more toxic (TPM fraction TA98+S9, NRU), than the MSS from cigarettes. Thus, our findings support our prior work on smoke mutagenicity that showed MSS from cigarillos was not less toxic than MSS from cigarettes.

Spectrofluorometry assays for oxidative stress and apoptosis, with cell viability on the same microplates: a multiparametric analysis and quality control.

Our objective was to compare neutral red (NR) tests performed separately and on the same microplates as hydroethidine, H2DCF-DA and YO-PRO®-1 assays, and to compare toxicity ratios calculated with the two methods. Human trabecular meshwork cells (HTM-3) were exposed to different concentrations of benzalkonium chloride (BAC), to PBS and to diluted solutions of latanoprost and bimatoprost. Microplate fluorescence spectrophotometry assays were performed: NR uptake for cell viability evaluation, hydroethidine and H2DCF-DA for reactive oxygen species (ROS), and YO-PRO®-1 for apoptosis. The four NR assays presented identical toxicological profiles and correlation coefficients between them were close to one. There was no difference between toxicity ratios for ROS assays calculated by the two methods, with high correlation coefficients. However, in the apoptosis assay, ratios calculated with the double staining technique were more accurate. Thus, it is possible and recommended to perform NR assays on the same microplates as the other assays. This double staining procedure could constitute a quality control procedure and would allow multiparametric analysis.

Cell activity analysis by capillary zone electrophoresis combined with specific cell staining.

Capillary zone electrophoresis (CZE) was used to determine Hela cells activity with Hela treated by 0-46 µM methylmercury (MeHg) as the apoptosis model. The treated and untreated cells were stained by four different dyes (Janus Green B, Rhodamine 123, Neutral Red and Trypan Blue) and analyzed by CZE with UV/Vis detection. The absorbance of cells at 214 nm could indicate the degree of cell shrinkage and component leakage induced by MeHg. The intensity of cell absorbance at maximum visible absorption wavelength of dyes represented mitochondrial activity, lysosome phagocytosis ability and cell membrane integrity. For different concentrations of MeHg treatment, the change of cell activity was in good agreement with Janus Green B uptake colorimetric assay (R² =0.914) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-di-phenyltetrazolium bromide (MTT) assay (R² =0.892). 80% of RSD (n=3) values were in the range of 0.5-15.0%. The established CZE method could be used to analyze intact cells with only UV-Vis detector. The CZE method has some features equivalent to the existing universal method, and it has the potential to be a universal tool for cell activity determination.

In situ identification of follicles in ovarian cortex as a tool for quantifying follicle density, viability and developmental potential in strategies to preserve female fertility.

BACKGROUND: Ovarian tissue cryopreservation, in combination with autotransplantation or long-term culture, has been proposed as a means of fertility preservation. However follicle density within ovarian cortex has a profound impact on the success of in vivo and in vitro systems designed to support follicle growth and restore fertility. The objective of this study was to investigate the dye neutral red (NR) as a tool to quantify follicle density in situ, without compromising follicle viability and developmental potential. METHODS: In the first experimental series thin slices of cryopreserved and fresh ovine cortical tissue were incubated in 50 µg/ml NR and assessed for the presence of red colouration. Slices were then used for follicular structure isolation and viability evaluation using 5-(and 6)-carboxyfluoresceindiacetate succinimidylester (CFDA-SE), or prepared histologically for follicle counting or evaluation of apoptosis via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL). An additional subset of slices were cultured for 8 days, followed by re-evaluation of follicle viability. NR staining was further assessed in a pilot study using thin slices of cryopreserved human ovarian tissue donated by 17 patients undergoing laparoscopic sterilisation or elective Caesarean section. RESULTS: In both ovine and human ovarian cortex NR concentrated in follicular structures within weakly stained stroma. NR colouration was observed in 41.7 ± 4.6% of cryopreserved and 49.3 ± 6.5% of the fresh ovine tissue slices, and NR staining was consistently predictive of the presence of follicles. Non-stained ovine slices contained highly apoptotic follicles, while lower levels of apoptosis were observed in NR positive slices, indicating preferential detection of viable follicles by NR. Following culture the majority of ovine slices re-stained with NR, no significant increases in the levels of apoptosis were observed and 94.6 ± 3.1% of follicles were viable by CFDA-SE. In the human study, NR identified follicles in 19.3 ± 3.7% of tissue slices, and follicle density tended to decrease with advancing patient age. CONCLUSIONS: NR predicts viable follicle density in situ in slices of ovine and human ovarian cortex. Furthermore incubation of tissue in NR prior to culture does not compromise subsequent follicle survival in vitro, indicating the potential suitability of this approach in fertility preservation regimes.

Application and evaluation of the neutral red retention (NRR) assay for lysosomal stability in mussel populations along the Iberian Mediterranean coast.

The neutral red retention (NRR) assay to determine lysosomal membrane stability (LMS) was applied to wild mussels (Mytilus galloprovincialis) sampled from fourteen sites with different degrees of chemical pollution along the Iberian Mediterranean coast in 2002 and 2003. Total body burdens for a range of contaminants were measured in pooled samples (n = 50) of whole soft tissues in order to explore possible causality. Mean LMS values (n = 15) were significantly greater in mussels from one of the selected two reference sites (San Diego) than in chemically well-characterized hotspot sites. Mean LMS values were inversely correlated with contaminant concentrations in mussel tissues (except for Zn). The multidimensional scaling ordination technique classified the sampling sites into three groups according to their chemical-physical-biological similarities and the results were statistically confirmed using ANOSIM analysis. The results show that lysosomal stability is an effective indicator of health status in mussels along the Iberian Mediterranean coast and that it is related to body burden of contaminants, particularly polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs).

Effect of temperature and season on reproduction, neutral red retention and metallothionein responses of earthworms exposed to metals in field soils.

This study investigated the short-term survival, reproduction and physiological (lysosomal membrane stability, metallothionein transcript copy number, body tissue metal concentrations) responses of Lumbricus rubellus exposed to metal contaminated field soils under different laboratory temperatures (10, 15 and 20 degrees C) and physiological responses of earthworms collected from the field in three different seasons (spring, autumn, winter). In the laboratory, metal contaminated soils had significant effects on reproduction (p<0.001), metallothionein-2 (MT-2) expression (p=0.033) and earthworm As (p=0.003), Cd (p=0.001), Pb (p<0.001) and Zn (p<0.001) concentration, but not lysosomal membrane stability and tissue Hg and Cu. No effect of temperature was found for any parameter. Principal component analysis of extractable and tissue metal concentrations indicated PC1 as a measure of metal stress. Both cocoon production (r=-0.75) and MT-2 induction (r=0.41) were correlated with PC1. A correlation was also found between cocoon production and MT-2 expression (r=-0.41). Neutral red retention and MT-2 measurements in worms collected from the field sites in three seasons confirmed the absence of a temperature effect on these responses.

Biomarkers responses of the earthworm Eisenia fetida to acetochlor exposure in OECD soil.

To examine the potential of a suite of biomarkers as early warning indicators of environmental pollution, sperm count, neutral red retention time (NRRT) and DNA damage were measured in earthworm Eisenia fetida exposed to increasing concentrations of acetochlor in OECD soil. The neutral red retention time of earthworms coelomocytes was sensitive to acetochlor pollution, and decreased significantly when the concentration was more than 10mgkg(-1) after 30 and 60 days of exposure (P<0.05). The reduced neutral red retention time correlated with the soil acetochlor residual. Sperm count decreased significantly at the concentrations of 40 and 80mgkg(-1) after 15 days of exposure (P<0.05). The DNA damage of earthworms coelomocytes increased significantly after 30 days of exposure at the highest concentration (80mgkg(-1); P<0.05). Earthworms were under physiological stress at field dose of acetochlor (10mgkg(-1)). Higher concentrations of acetochlor caused sperm count decrease and DNA damage of earthworms. Such a suite of biomarkers could serve as indicators of the health of the soil environment and to evaluate the toxicity of acetochlor on earthworms or as a means of monitoring soil acetochlor pollution.