LC-qTOF-MS/MS phytochemical profiling of Tabebuia impetiginosa (Mart. Ex DC.) Standl. leaf and assessment of its neuroprotective potential in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tabebuia impetiginosa (Bignoniaceae) was traditionally used for memory enhancement and central nervous system (CNS) stimulation. AIM OF THE STUDY: This study aims to create a metabolic profile of the ethyl acetate fraction of T. impetiginosa (TEF) and investigate for the first time its neuroprotective potential on cyclophosphamide (CP)-induced chemobrain, validating its traditional use. MATERIALS AND METHODS: Metabolite profiling of TEF was performed using Liquid Chromatography coupled with Quadrupole Time of Flight-Mass/Mass Spectrometry (LC-qTOF-MS/MS). For the in vivo study, CP (200 mg/kg, i.p.) was administered to induce cognitive impairment in rats; TEF (30 mg/kg, p.o.) was administered throughout the 14 days of the experiment to assess its role in mitigating CP-induced neuronal deficits. Behavioral tests including locomotor, Y-maze, and passive avoidance tests were conducted. Additionally, biochemical markers such as reduced glutathione (GSH), malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), and caspase-3 immunoexpression were assessed in the hippocampus area. RESULTS: Forty-four phytoconstituents were tentatively identified in TEF, mainly iridoids and organic acids. TEF showed significant memory enhancement as evidenced by the increase in step-through latency in the passive avoidance test by 1.5 folds and the increase in sequence alternation percentage (SAP) in the Y-maze test by 67.3%, as compared to CP-group. Moreover, it showed pronounced antioxidant and anti-inflammatory potentials evidenced by the significant elevation in reduced glutathione (GSH) levels by 80% and a pronounced decline in MDA and TNF-α levels by 24% and 45%, respectively relative to the CP group. TEF treatment restored normal hippocampal histological features and attenuated apoptotic caspase-3 expression by 70% compared to the CP group. CONCLUSIONS: TEF can act as a promising natural scaffold in managing the chemobrain induced by CP in cancer patients.

Hazard assessment of antineoplastic drugs and metabolites using cytotoxicity and genotoxicity assays.

Antineoplastic drugs are among the most toxic pharmaceuticals. Their release into the aquatic ecosystems has been reported, giving rise to concerns about the adverse effects, including cytotoxicity and genotoxicity, that they may have on exposed organisms. In this study, we analyzed the cytotoxicity and genotoxicity of 5-fluorouracil (5-FU) and its metabolite alpha-fluoro-beta-alanine (3-NH2-F); gemcitabine (GEM) and its metabolite 2'-deoxy-2',2'-difluorouridine (2-DOH-DiF); as well as cyclophosphamide (CP) on the HepG2 cell line. Drug concentrations were based on those previously observed in the effluent of a major cancer hospital in Brazil. The study found that GEM, 2-DOH-DiF and 5-FU resulted in reduced cell viability. No reduction in cell viability was observed for CP and 3-NH2-F. Genotoxic assessment revealed damage in the form of nucleoplasmic bridges for CP and 3-NH2-F. The tested concentrations of all compounds resulted in significantly increased MNi and NBUDs. The results showed that these compounds induced cytotoxic and genotoxic effects in HepG2 cells at concentrations found in the environment. To the best of our knowledge, this study is the first to report on the cytogenotoxic impacts of the metabolites 3-NH2-F and 2-DOH-DiF in HepG2 cells. These findings may help in the development of public policies that could minimize potential environmental contamination.

Molecular and behavioral assessment in larval zebrafish (Danio rerio) following exposure to environmentally relevant levels of the antineoplastic cyclophosphamide.

Antineoplastics treat cancers and enter aquatic ecosystems through wastewater and hospital effluent. Risks associated with antineoplastics are not well characterized in aquatic organisms. We conducted zebrafish embryo/larvae toxicity assays to evaluate responses to cyclophosphamide (0.01-50 µM). Zebrafish survival was affected by 5 µM cyclophosphamide and deformities were noted at > 1 µM. Oxidative respiration remained unchanged in embryos with exposure up to 200 µM. Reactive oxygen species were not increased by 50 µM cyclophosphamide exposure. More than 15 oxidative stress and immune-related transcripts were measured. Superoxide dismutase 2 and heat shock protein 70 and 90a were induced in larvae by cyclophosphamide. Immune-related transcripts were assessed due to immunosuppressive properties of cyclophosphamide, and mmp9 and myd88 levels were altered in expression. Hyperactivity of larvae was noted following 5 µM cyclophosphamide exposure. There was no change in anxiety-related endpoints (light-dark preference). Risks for larval fish exposed to cyclophosphamide in the environment may be low.

In vivo assessment of antioxidant, antigenotoxic, and antimutagenic effects of bark ethanolic extract from Spondias purpurea L.

Medicinal plants have always been used for therapeutic purposes; however, some plants may contain toxic and mutagenic substances. The aim of this study was to assess the cytotoxic, genotoxic, mutagenic, antioxidant, antigenotoxic, and antimutagenic effects of the bark ethanolic extract of Spondias purpurea L. using male and female Swiss albino mice. To determine the protective effects of the extract, benzo[a]pyrene (B[a]P) and cyclophosphamide (CP) were selected as cell damage inducers. The extract was examined at doses of 500, 1000, or 1500 mg/kg body weight (BW)via gavage alone or concomitant with B[a]P or CP. Oxidative stress was measured by quantification of blood catalase activity (CAT), reduced glutathione (GSH) levels in total blood, liver, and kidney, and concentrations of malondiadehyde (MDA) in liver and kidney. Genotoxicity and antigenotoxicity were evaluated by the comet assay using peripheral blood. Cytotoxicity, mutagenicity, and antimutagenicity were determined utilizing the micronucleus test in bone marrow and peripheral blood. The S. purpurea L extract increased CAT activity and GSH levels accompanied by a decrease in MDA levels after treatment with B[a]P and CP. No genotoxic, cytotoxic, or mutagenic effects were found in mice exposed only to the extract. These results indicate that the extract of S. purpurea exhibited protective effects against oxidative and DNA damage induced by B[a]P and CP.

Mutagenicity assessment of environmental contaminations in a hospital centralized reconstitution unit.

INTRODUCTION: Cytotoxic drug exposure of hospital staff preparing intravenous chemotherapy is a major issue and related mutagenic risks should be more explored. The aim of this study was to assess the mutagenicity of several cytotoxic mixtures prepared at fixed concentrations, and the mutagenicity of environmental samples collected in a hospital centralized reconstitution unit. In parallel cytotoxic exposure in environmental samples was quantified. METHODS: Environmental samples were performed by wiping method using swabs in five critical production unit areas. Mutagenicity was assessed with a liquid microplate AMES test using two salmonella typhimurium strains (TA98 and TA100), in prepared cytotoxic mixtures containing 14 cytotoxic drugs (cyclophosphamide, cytarabine, dacarbazine, docetaxel, doxorubicin, epirubicin, etoposide, 5-fluorouracil, gemcitabine, ifosfamide, irinotecan, methotrexate, paclitaxel and pemetrexed) according a dichotomous strategy and in environmental samples. Cytotoxic drugs were quantified in samples using liquid chromatography coupled to mass tandem spectrometry. RESULTS: Mutagenesis was observed for the mix of 14 cytotoxic drugs with TA98 strain ±â€¯S9 fraction but not TA100 strain. After dichotomous approach, only doxorubicin and epirubicin exposure were associated to mutagenesis. The mutagenesis observed was expressed at lower concentrations with the mix of the 14 drugs than with anthracyclins alone, assuming a synergistic effect. Despite measurable level of cytotoxic contamination in environmental samples, no mutagenesis was highlighted in Ames tests performed on these environmental samples. CONCLUSIONS: The analyses carried out show the conservation of the mutagenicity of cytotoxic drugs found in very low quantities in the environment. The traces of cytotoxic drugs found in our unit regularly exceed the limits given by some authors. This approach may be considered as a new tool to monitor environmental contamination by cytotoxic drugs.

Effects of Nesting Material on the Toxicologic Assessment of Cyclophosphamide in Crl:CD1(ICR) Mice.

The provision of nesting material benefits mice by reducing cold stress, improving feed conversion, increasing litter size, and improving adaptive immunity. The effects of toxins are sensitive to environmental changes, and the introduction of novel items can alter results in some toxicologic studies. We hypothesized that nesting material would reduce stress and positively alter immunologic parameters in Crl:CD1(ICR) mice, thus changing typical results from a well-studied immunomodulating drug, cyclophosphamide. A 13-wk study assessed the following treatments in a factorial design (n = 4; 32 cages total): nesting (0 or 10 g) and drug (50 mg/kg cyclophosphamide or 10 mL/kg saline; IP weekly). Detailed examinations and body weights were recorded weekly, and nests were scored twice weekly. Fecal pellets were collected at 0, 4, 6, and 12 wk for analysis of corticosterone metabolites. At study termination, clinical pathology and immune parameters were collected, a necropsy performed, and lymphoid organs and adrenal glands were submitted for histopathology. All expected results due to cyclophosphamide were observed. Nesting reduced the proportion of mice with piloerection, and body weights were highest in saline-nested male mice. No differences in hematology, clinical chemistry, or absolute lymphocyte counts were observed. Corticosterone metabolites in all nested groups were not different from baseline levels but all nonnested groups had higher levels than baseline. Nested cyclophosphamide-treated groups had significantly lower corticosterone levels than nonnested cyclophosphamide-treated groups. This study illustrates that nesting material does not alter the results of a standard toxicology study of cyclophosphamide but alleviates study-related stress and improves mouse welfare.

Algorithm of Molecular and Biological Assessment of the Mechanisms of Sensitivity to Drug Toxicity by the Example of Cyclophosphamide.

Comparative study of the liver, blood, and spleen of DBA/2JSto and BALB/cJLacSto mice sensitive and resistant to acute toxicity of the cyclophosphamide allowed us to reveal basic toxicity biomarkers of this antitumor and immunosuppressive agent. Obtained results can be used for the development of an algorithm for evaluation of toxic effects of drugs and food components.

Acute aquatic toxicity assessment of six anti-cancer drugs and one metabolite using biotest battery - Biological effects and stability under test conditions.

Available ecotoxicological data for anti-cancer drugs and their metabolites are incomplete, and only some studies have been accompanied by chemical analysis. Therefore, the main aim of this study was to evaluate the acute toxicity of the six most commonly used cytostatics, namely cyclophosphamide (CF), ifosfamide (IF), 5-fluorouracil (5-FU), imatinib (IMT), tamoxifen (TAM) and methotrexate (MET) and its metabolite - 7-hydroxymethotrexate (7-OH-MET), towards selected aquatic organisms, namely bacteria Vibrio fischeri, algae Raphidocelis subcapitata, crustaceans Daphnia magna and duckweed Lemna minor. All ecotoxicological tests were accompanied by chemical analysis to determine the differences between nominal and actual concentrations of investigated compounds and their stability under test conditions. For unstable compounds, tests were performed in static and semi-static conditions. It was observed that L. minor was the most sensitive organism. The compounds that were most toxic to aquatic organisms were 5-FU (highly toxic to algae, EC50 = 0.075 mg L-1), MET and TAM (very toxic to highly toxic to duckweed depending on the test conditions; EC50MET 0.08-0.16 mg L-1, EC50TAM 0.18-0.23 mg L-1). It is suspected that MET and 5-FU mainly affected algae and plants most probably because the exposure time was long enough for them to cause a specific effect (they inhibit DNA replication and act predominantly on actively dividing cells). Furthermore, the obtained results also suggest that the toxicity of the metabolites/potentially produced degradation products of MET towards duckweed is lower than that of the parent form, whereas the toxicity of TAM degradation products is in the same range as that of TAM.

Limitations of Murine Models for Assessment of Antibody-Mediated Therapies or Vaccine Candidates against Staphylococcus epidermidis Bloodstream Infection.

Staphylococcus epidermidis is normally a commensal colonizer of human skin and mucus membranes, but, due to its ability to form biofilms on indwelling medical devices, it has emerged as a leading cause of nosocomial infections. Bacteremia or bloodstream infection is a frequent and costly complication resulting from biofilm fouling of medical devices. Our goal was to develop a murine model of S. epidermidis infection to identify potential vaccine targets for the prevention of S. epidermidis bacteremia. However, assessing the contribution of adaptive immunity to protection against S. epidermidis challenge was complicated by a highly efficacious innate immune response in mice. Naive mice rapidly cleared S. epidermidis infections from blood and solid organs, even when the animals were immunocompromised. Cyclophosphamide-mediated leukopenia reduced the size of the bacterial challenge dose required to cause lethality but did not impair clearance after a nonlethal challenge. Nonspecific innate immune stimulation, such as treatment with a Toll-like receptor 4 (TLR4) agonist, enhanced bacterial clearance. TLR2 signaling was confirmed to accelerate the clearance of S. epidermidis bacteremia, but TLR2(-/-)mice could still resolve a bloodstream infection. Furthermore, TLR2 signaling played no role in the clearance of bacteria from the spleen. In conclusion, these data suggest that S. epidermidis bloodstream infection is cleared in a highly efficient manner that is mediated by both TLR2-dependent and -independent innate immune mechanisms. The inability to establish a persistent infection in mice, even in immunocompromised animals, rendered these murine models unsuitable for meaningful assessment of antibody-mediated therapies or vaccine candidates.

Degradation of cyclophosphamide and 5-fluorouracil by UV and simulated sunlight treatments: Assessment of the enhancement of the biodegradability and toxicity.

The presence of pharmaceuticals in the environment has triggered concern among the general population and received considerable attention from the scientific community in recent years. However, only a few publications have focused on anticancer drugs, a class of pharmaceuticals that can exhibit cytotoxic, genotoxic, mutagenic, carcinogenic and teratogenic effects. The present study investigated the photodegradation, biodegradation, bacterial toxicity, mutagenicity and genotoxicity of cyclophosphamide (CP) and 5-fluorouracil (5-FU). The photodegradation experiments were performed at a neutral to slight pH range (7-7.8) using two different lamps (medium-pressure mercury lamp and a xenon lamp). The primary elimination of the parent compounds was monitored by means of liquid chromatography tandem mass spectrometry (LC-IT-MS/MS). NPOC (non-purgeable organic carbon) analyses were carried out in order to assess mineralization rates. The Closed Bottle Test (CBT) was used to assess ready biodegradability. A new method using Vibrio fischeri was adopted to evaluate toxicity. CP was not degraded by any lamp, whereas 5-FU was completely eliminated by irradiation with the mercury lamp but only partially by the Xe lamp. No mineralization was observed for the experiments performed with the Xe lamp, and a NPOC removal of only 18% was registered for 5-FU after 256 min using the UV lamp. Not one of the parent compounds was readily biodegradable in the CBT. Photo transformation products (PTPs) resulting from photolysis were neither better biodegradable nor less toxic than the parent compound 5-FU. In contrast, the results of the tests carried out with the UV lamp indicated that more biodegradable and non-toxic PTPs of 5-FU were generated. Three PTPs were formed during the photodegradation experiments and were identified. The results of the in silico QSAR predictions showed positive mutagenic and genotoxic alerts for 5-FU, whereas only one of the formed PTPs presented positive alerts for the genotoxicity endpoint.

In vitro tests aiding ecological risk assessment of ciprofloxacin, tamoxifen and cyclophosphamide in range of concentrations released in hospital wastewater and surface water.

Ciprofloxacin (CIP), tamoxifen (TAM) and cyclophosphamide (CP) which are often used in anticancer treatment are released in hospital effluent and into the environment. Although the concentrations are low (from ng/L to µg/L), no data exist concerning their ecotoxicological impact. In this study two biomarkers of early effect were performed on hepatic cells (HepG2): cell viability and genotoxicity (DNA breaks) using cell proliferative assay and comet assay, respectively. These data were compared with two standardized ecotoxicological tests: algaltoxkit F™ and microtox®. Cells were exposed to an increasing amount of an individual drug or in a mixture for 24, 48 or 72h. The time-exposure of bacteria and algae ranged between 5 and 30min and 72h, respectively. A non-monotonic dose-response on cell viability was observed when HepG2 cells were exposed to TAM alone or in the presence of CIP. The same scheme was observed with microtox® when the bacteria were exposed to the mixtures. On the other side, an individual drug does not induce any DNA breaks on hepatic cells, whereas a mixture leads to a dose dependent increase of DNA breaks. Similarly a positive response was observed with algaltoxkit F™ only with mixtures. Synergistic effects observed when drugs are in a mixture highlight the importance of investigating the ecotoxicological effects of contaminants at low concentrations and in mixtures.

Interlaboratory assessment of mitotic index by flow cytometry confirms superior reproducibility relative to microscopic scoring.

A flow cytometric procedure for determining mitotic index (MI) as part of the metaphase chromosome aberrations assay, developed and utilized routinely at Pfizer as part of their standard assay design, has been adopted successfully by Covance laboratories. This method, using antibodies against phosphorylated histone tails (H3PS10) and nucleic acid stain, has been evaluated by the two independent test sites and compared to manual scoring. Primary human lymphocytes were treated with cyclophosphamide, mitomycin C, benzo(a)pyrene, and etoposide at concentrations inducing dose-dependent cytotoxicity. Deming regression analysis indicates that the results generated via flow cytometry (FCM) were more consistent between sites than those generated via microscopy. Further analysis using the Bland-Altman modification of the Tukey mean difference method supports this finding, as the standard deviations (SDs) of differences in MI generated by FCM were less than half of those generated manually. Decreases in scoring variability owing to the objective nature of FCM, and the greater number of cells analyzed, make FCM a superior method for MI determination. In addition, the FCM method has proven to be transferable and easily integrated into standard genetic toxicology laboratory operations.

Risks to health professionals from hazardous drugs in Japan: a pilot study of environmental and biological monitoring of occupational exposure to cyclophosphamide.

PURPOSE: In Japan, concerns exist regarding the dangers inherent when handling cytotoxic drugs, particularly drugs such as 5-FU, Thiotepa, Cytarabine, Tegaful, and Sizofiran which are contained in ampoules or vials, since nurses and medical doctors have been preparing these cytotoxic drugs in the open spaces of wards in the absence of appropriate garments and personal protective equipment. In addition, the administration tubes for these dangerous drugs have been exchanged at the patients' bedside, typically in rooms shared by several patients. To gain insight into the severity of the occupational hazards posed by these practices, we conducted a pilot study of environmental and biological monitoring of occupational exposure to cyclophosphamide (CP). SETTING: At Nagoya University Hospital, Nagoya, Japan, in February 2006, two departments, A and B, were monitored with surface-wipe, and urine samples were analyzed using the Sessink method (exposure control, The Netherlands). Department A had a preparation room with biological safety cabinet (BSC) where the pharmacists prepare cytotoxic drugs. Department B did not have a BSC. RESULTS: Many areas of the treatment rooms were contaminated with CP. CP was detected on tables and telephone stands where cytotoxic drugs were not used as well as tables used to prepare cytotoxic drugs. Significant differences in CP concentrations were detected from the urine of two of the three nurses who cared for the same patients without gloves. The nurses rotated and inherited the patient who had the highest risk of contamination. CP was detected only once from the urine of the medical doctor who prepared CP. He was not wearing any PPE other than gloves. All of the pharmacists wearing PPE were free from contamination of CP. DISCUSSION: Regardless of the use of BSC, wards were contaminated with CP. The contamination may not occur due to the sealing used in CP containers and administration tubes when discarding them. CP was detected only once in the urine of a medical doctor who prepared CP by warming it. The cause may be inhalation of CP gas from the injector. The contamination of the nurses may be from dermal absorption because absorption continued even after the shift ended and the nurses left the facility. CP was not detected in pharmacists who followed the guidelines for preparation of CP. All of the medical staff should follow the guidelines when they handle CP.

Dose-response assessment of four genotoxic chemicals in a combined mouse and rat micronucleus (MN) and Comet assay protocol.

The in vivo micronucleus (MN) assay has proven to be an effective measure of genotoxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic damage using the MN assay provides a limited genotoxicity profile. The in vivo alkaline (pH >13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues following administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophosphamide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24-hr intervals (VS was administered to rats for 3 days); animals were euthanized 4 hr after the last administration. All compounds induced significant increases in micronucleated reticulocytes (MN-RET) in the peripheral blood of mice, and all but ACM induced MN-RET in rats. EMS and ACM induced significant increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced DNA damage was detected in leukocytes and duodenum cells. VS, a spindle fiber disrupting agent, was negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program (NTP) is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern.

In vivo assessment of DNA damage and protective effects of extracts from Miconia species using the comet assay and micronucleus test.

The genus Miconia comprises approximately 1000 species belonging to the Melastomataceae family. Several crude plant extracts from Miconia and their isolated compounds have shown biological activities, such as analgesic and anti-neoplastic action; however, no studies concerning their effects on DNA are available. The present study aimed to evaluate, in vivo, the genotoxic and mutagenic effects of four species of plants from Miconia genus using the comet assay and micronucleus test. Their possible protective effects were also evaluated in experiments associating the plant extracts with cyclophosphamide (CPA). The methanolic extracts of Miconia albicans, Miconia cabucu, Miconia rubiginosa, Miconia stenostachya and the chloroformic extract of M. albicans were investigated. For genotoxic and mutagenic evaluations, three concentrations were tested, 200, 400 and 540 mg/kg body weight (bw), based on the solubility limit of the extract in distilled water. For the protective effects, only the highest dose was evaluated against 40 mg/kg bw of CPA. Blood was removed from mice tails pre- (T0) and post-treatment (T1-30 h) for the micronucleus test and 24 h post-treatment for the comet assay. The Student's t-test was used to compare data obtained at T0 and T1, the analysis of variance-Tukey test was used to compare between groups in the micronucleus test and the Kruskal-Wallis and Dunn's test were used to compare different groups in the comet assay. All the extracts induced alterations in DNA migration (comet assay); however, no mutagenic effect was observed in the micronucleus assay. All extracts showed a protective effect against CPA in both assays. Our study showed that the use of crude extracts could be more advantageous than the use of isolated compounds. The interaction between phytochemicals in the extracts showed efficacy in reducing mutagenicity and improving the protective effects.

Efficacy of delayed administration of post-chemotherapy granulocyte colony-stimulating factor: evidence from murine studies of bone marrow cell kinetics.

The optimal schedule of post-chemotherapy granulocyte colony-stimulating factor (G-CSF) administration has not been determined. G-CSF is customarily started 24 hours after chemotherapy; however, clinical data demonstrated that delaying G-CSF until 5 days after completion of chemotherapy has not resulted in a longer duration of neutropenia. Here, we examined the optimal timing of post-chemotherapy G-CSF administration in a mouse model, to show that delayed administration does not postpone the appearance of mature granulocytes in the peripheral blood. We also investigated the mechanism of decreased efficacy of the early G-CSF application after chemotherapy by characterizing the changes in bone marrow cellular composition. To our knowledge, we demonstrate for the first time, that early after chemotherapy, the bone marrow is predominantly composed of mature residual granulocytes and very few progenitors and precursors, on which G-CSF would act to generate granulocytes. The point when immature progenitors reappear does not occur in murine bone marrow until 48 hours after a single dose of cyclophosphamide. Our results indicate that the bone marrow cellular composition early after discontinuation of chemotherapy is not optimal for G-CSF action on acceleration of myeloid recovery. Given the high cost of G-CSF prophylaxis, its delayed administration may potentially result in substantial economic benefits.

Validation of Drosophila melanogaster as an in vivo model for genotoxicity assessment using modified alkaline Comet assay.

The single cell gel electrophoresis or Comet assay is one of the most popular techniques for genotoxicity assessment. The present study was undertaken to validate our previously modified version of the Comet assay for genotoxicity assessment in Drosophila melanogaster (Oregon R(+)) with four well-known mutagenic and carcinogenic alkylating agents, i.e. ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU) and cyclophosphamide (CP). Third instar larvae (74 +/- 2 h) of D.melanogaster were fed different concentrations of EMS, MMS, ENU and CP (0.05, 0.5 and 1.0 mM) mixed standard Drosophila food for 24 h. At 98 +/- 2 h, the anterior midgut from control and treated larvae were dissected out, single-cell suspensions were prepared and Comet assay was performed. Our results show a dose-dependent increase in DNA damage with all the four alkylating agents, in comparison to control. The lower concentration (0.05 mM) of the test chemicals, except MMS, did not induce any DNA damage in the gut cells of the exposed larvae. When comparison of Comet parameters was made among the chemicals, MMS was found to be the most potent genotoxicant and ENU the least. The present study validated our previous observation and shows that D.melanogaster is a sensitive and suitable model for the in vivo assessment of genotoxicity using our modified alkaline Comet assay.

Assessment of quantitative dual-parameter flow cytometric analysis for the evaluation of testicular toxicity using cyclophosphamide- and ethinylestradiol-treated rats.

In the drug discovery process, effects to the human spermatogenesis must be fully evaluated before the first human trial. To estimate testicular toxicity, histopathological evaluation has been recommended in addition to the traditional mating procedure. However, it is laborious and time-consuming. Flow cytometric analysis (FCM) has also been applied to estimate testicular toxicity because of its speed, simplicity, and the objectivity of the data. Using cyclophosphamide (CP)- and ethinylestradiol (EE)-treated rat testis, we attempted to validate our dual-parameter, DNA ploidy and cell-size FCM, in a high-throughput toxicity study. Our results showed that CP damaged some spermatogonia and some early meiotic spermatocytes and EE caused severe decrease of spermatogenic cells except for spermatogonia as well as marked decrease of somatic cells, most probably Leydig cells. This is the first report discriminating between the changes of spermatogonia and that of somatic cells with FCM analysis. These results demonstrate that this method is a very useful and powerful tool to assess testicular toxicity, especially in high-throughput toxicological studies.

Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes.

Two organophosphorus (OP) pesticides (chloropyriphos and acephate) and cyclophosphamide (CP) (positive control) were tested for their ability to induce in vivo genotoxic effect in leucocytes of Swiss albino mice using the single cell gel electrophoresis assay or comet assay. The mice were administered orally with doses ranging from 0.28 to 8.96 mg/kg body weight (b. wt.) of chloropyriphos and 12.25 to 392.00 mg/kg b.wt. of acephate. The assay was performed on whole blood at 24, 48, 72 and 96 h. A significant increase in mean comet tail length indicating DNA damage was observed at 24h post-treatment (P<0.05) with both pesticides in comparison to control. The damage was dose related. The mean comet tail length revealed a clear dose dependent increase. From 48 h post-treatment, a gradual decrease in mean tail length was noted. By 96 h of post-treatment the mean comet tail length reached control levels indicating repair of the damaged DNA. From the study it can be concluded that the comet assay is a sensitive assay for the detection of genotoxicity caused by pesticides.