In vivo genotoxicity assessment of N-(-9 acridinyl)-b-alanine hydrochloride (S-300) using a validated Pig-a mutagenesis assay.

BACKGROUND: N-(-9 acridinyl)-b-alanine hydrochloride (S-300) is the main byproduct of red blood cell (RBC) amustaline/glutathione(GSH) pathogen reduction, currently undergoing phase III US clinical trials following successful European studies(1-3). Phosphatidylinositol glycan, class A (Pig-a) X-linked gene mutagenesis is a validated mammalian in vivo mutation assay for genotoxicity, assessed as clonal loss of glycosylphosphatidylinositol-linked CD59 cell-surface molecules on reticulocytes (RETs) and RBCs. METHODS: Male Sprague-Dawley rats received continuous infusion of S-300 up to the maximum feasible dose (240 mg/kg/day-limited by solubility and volume) for 28 days. Positive controls received a known mutagen by oral gavage on Days 1-3. Plasma levels of S-300 were assessed by HPLC before, during and after infusion. CD59-negative RBCs and RETs were enumerated in pre-dose and Day 28 samples, using a flow cytometric method. Outcome was evaluated by predetermined criteria using concurrent and historical controls. Toxicity was assessed by laboratory measures and necropsy. RESULTS: S-300 reached maximum, dose-dependent levels (3-15 µmol/L) within 2-8 h that were sustained for 672 h and undetectable 2 h after infusion. Circulating RET levels indicated a lack of hematopoietic toxicity. Necropsy revealed minimal-mild observations related to poor S-300 solubility at high concentrations. Pig-a assessment met the preset acceptability criteria and revealed no increase in mutant RBCs or RETs. CONCLUSIONS: Maximum feasible S-300 exposure of rats by continuous infusion for 28 days was not genotoxic as assessed by an Organization for Economic Cooperation and Development-compliant, mammalian, in vivo Pig-a gene mutation assay that meets the requirements of International Conference on Harmonization (ICH) S2(R1) and FDA guidances on genotoxicity testing.

Effect of life stage and target tissue on dose-response assessment of ethyl methane sulfonate-induced genotoxicity.

In order to investigate the possibility that treatment age affects the genotoxic response to ethyl methane sulfonate (EMS) exposure, we dosed gpt-delta neonatal mice on postnatal days 1-28 with 5-100 mg/kg/day of EMS and measured micronucleus (MN) induction in peripheral blood and gpt gene mutation in liver, lung, bone marrow, small intestine, spleen, and kidney. The data were compared to measurements from similarly exposed adult gpt-delta mice. Our results indicate that the peripheral blood MN frequencies in mice treated as neonates are not substantially different from those measured in mice treated as adults. There were, however, differences in tissue-specific gpt mutation responses in mice treated with EMS as neonates and adults. Greater mutant frequencies were seen in DNA isolated from kidney of mice treated as neonates, whereas the mutant frequencies in bone marrow, liver, and spleen were greater in the animals treated as adults. Benchmark dose potency ranking indicated that the differences for kidney were significant. Our data indicate that there are differences in EMS-induced genotoxicity between mice treated as adults and neonates; the differences, however, are relatively small.

Pig-a gene mutation assay study design: critical assessment of 3- versus 28-day repeat-dose treatment schedules.

The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organisation for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the details that will need to be articulated in an eventual guideline are recommended treatment and harvest schedules. With this in mind, experiments reported herein were performed with Wistar Han rats exposed to aristolochic acid I (AA), 1,3-propane sultone, chlorambucil, thiotepa or melphalan using each of two commonly used treatment schedules: 3 or 28 consecutive days. In the case of the 3-day studies, blood was collected for Pig-a analysis on days 15 or 16 and 29 or 30. For the 28-day studies blood was collected on day 29 or 30. The effect of treatment on mutant reticulocytes and mutant erythrocytes was evaluated with parametric pair-wise tests. While each of the five mutagens increased mutant phenotype cell frequencies irrespective of study design, statistical significance was consistently achieved at lower dose levels when the 28-day format was used (e.g. 2.75 vs 20 mg/kg/bw for AA). To more thoroughly investigate the dose-response relationships, benchmark dose (BMD) analyses were performed with PROAST software. These results corroborate the pair-wise testing results in that lower BMD values were obtained with the 28-day design. Finally, mutagenic potency, as measured by BMD analyses, most consistently correlated with the mutagens' tumorigenic dose 50 values when the lengthier treatment schedule was used. Collectively, these results suggest that both 3- and 28-day treatment schedules have merit in hazard identification-type studies. That being said, for the purpose of regulatory safety assessments, there are clear advantages to study designs that utilise protracted exposures.

Integrated In Vivo Genotoxicity Assessment of Procarbazine Hydrochloride Demonstrates Induction of Pig-a and LacZ Mutations, and Micronuclei, in MutaMouse Hematopoietic Cells.

Procarbazine hydrochloride (PCH) is a DNA-reactive hematopoietic carcinogen with potent and well-characterized clastogenic activity. However, there is a paucity of in vivo mutagenesis data for PCH, and in vitro assays often fail to detect the genotoxic effects of PCH due to the complexity of its metabolic activation. We comprehensively evaluated the in vivo genotoxicity of PCH on hematopoietic cells of male MutaMouse transgenic rodents using a study design that facilitated assessments of micronuclei and Pig-a mutation in circulating erythrocytes, and lacZ mutant frequencies in bone marrow. Mice were orally exposed to PCH (0, 6.25, 12.5, and 25 mg/kg/day) for 28 consecutive days. Blood samples collected 2 days after cessation of treatment exhibited significant dose-related induction of micronuclei in both immature and mature erythrocytes. Bone marrow and blood collected 3 and 70 days after cessation of treatment also showed significantly elevated mutant frequencies in both the lacZ and Pig-a assays even at the lowest dose tested. PCH-induced lacZ and Pig-a (immature and mature erythrocytes) mutant frequencies were highly correlated, with R2 values ≥0.956, with the exception of lacZ vs. Pig-a mutants in mature erythrocytes at the 70-day time point (R2 = 0.902). These results show that PCH is genotoxic in vivo and demonstrate that the complex metabolism and resulting genotoxicity of PCH is best evaluated in intact animal models. Our results further support the concept that multiple biomarkers of genotoxicity, especially hematopoietic cell genotoxicity, can be readily combined into one study provided that adequate attention is given to manifestation times. Environ. Mol. Mutagen. 60:505-512, 2019. © 2018 Her Majesty the Queen in Right of Canada.

Assessment of the Pig-a, micronucleus, and comet assay endpoints in rats treated by acute or repeated dosing protocols with procarbazine hydrochloride and ethyl carbamate.

The utility and sensitivity of the newly developed flow cytometric Pig-a gene mutation assay have become a great concern recently. In this study, we have examined the feasibility of integrating the Pig-a assay as well as micronucleus and Comet endpoints into acute and subchronic general toxicology studies. Male Sprague-Dawley rats were treated for 3 or 28 consecutive days by oral gavage with procarbazine hydrochloride (PCZ) or ethyl carbamate (EC) up to the maximum tolerated dose. The induction of CD59-negative reticulocytes and erythrocytes, micronucleated reticulocytes in peripheral blood, micronucleated polychromatic erythrocytes in bone marrow, and Comet responses in peripheral blood, liver, kidney, and lung were evaluated at one, two, or more timepoints. Both PCZ and EC produced positive responses at most analyzed timepoints in all tissue types, both with the 3-day and 28-day treatment regimens. Furthermore, comparison of the magnitude of the genotoxicity responses indicated that the micronucleus and Comet endpoints generally produced greater responses with the higher dose, short-term treatments in the 3-day study, while the Pig-a assay responded better to the cumulative effects of the lower dose, but repeated subchronic dosing in the 28-day study. Collectively, these results indicate that integration of several in vivo genotoxicity endpoints into a single routine toxicology study is feasible and that the Pig-a assay may be particularly suitable for integration into subchronic dose studies based on its ability to accumulate the mutations that result from repeated treatments. This characteristic may be especially important for assaying lower doses of relatively weak genotoxicants. Environ. Mol. Mutagen. 60:56-71, 2019. © 2018 Wiley Periodicals, Inc.

[Optimization ofPig-aGene Mutation in Rats and Assessment of Time-dependent and Dose-response Relationship of N-ethyl-N-nitrosourea].

OBJECTIVES: To optimize the method of Pig-a mutation assay, and to explore the time-dependent and dose-response relationship of N-ethyl-N-nitrosourea (ENU). METHODS: Thirty rats were randomly assigned to 5 groups: treated with PBS (control group)or different doses of ENU (10, 20, 40 and 80 mg/kg) for 3 d by oral gavage. Blood samples were collected at 0 d, 15 d, 30 d, 45 d, 60 d, 75 d and 90 d. After enrichment, erythrocytes were incubated with Anti-CD59-APC and SYTO 13 nucleic acid dye solution. Mutant phenotype erythrocytes (RBCCD59-) and mutant phenotype reticulocytes (RETCD59-) were measured by flow cytometry to analyze mutant frequencies, and the RET percentage was determined as well. RESULTS: The RBCCD59- mutation frequency in 4 ENU groups were significantly increased in a dose- and time-dependent manner. The RETCD59- mutation frequency increased to a stable high level with a slight fluctuation, and decreased at 45 d , with the peak values observed at 30 d. The RETCD59- mutation frequency showed a dose-dependent trend in 4 ENU groups. The RET percentage in all 5 groups declined at 30 d, to a stable low level thereafter, but the trends showed no significant differences by time or group. CONCLUSIONS: The optimized in vivo Pig-a mutation assay could detecte the mutagen, such as ENU, induces mutation in RBC in a time- and dose-dependent manner.

Empirical analysis of BMD metrics in genetic toxicology part II: in vivo potency comparisons to promote reductions in the use of experimental animals for genetic toxicity assessment.

Genotoxicity tests have traditionally been used only for hazard identification, with qualitative dichotomous groupings being used to identify compounds that have the capacity to induce mutations and/or cytogenetic alterations. However, there is an increasing interest in employing quantitative analysis of in vivo dose-response data to derive point of departure (PoD) metrics that can be used to establish human exposure limits or margins of exposure (MOEs), thereby supporting human health risk assessments and regulatory decisions. This work is an extension of our companion article on in vitro dose-response analyses and outlines how the combined benchmark dose (BMD) approach across included covariates can be used to improve the analyses and interpretation of in vivo genetic toxicity dose-response data. Using the BMD-covariate approach, we show that empirical comparisons of micronucleus frequency dose-response data across multiple studies justifies dataset merging, with subsequent analyses improving the precision of BMD estimates and permitting attendant potency ranking of seven clastogens. Similarly, empirical comparisons of Pig-a mutant phenotype frequency data collected in males and females justified dataset merging across sex. This permitted more effective scrutiny regarding the effect of post-exposure sampling time on the mutagenicity of N-ethyl-N-nitrosourea observed in reticulocytes and erythrocytes in the Pig-a assay. The BMD-covariate approach revealed tissue-specific differences in the induction of lacZ transgene mutations in Muta™Mouse specimens exposed to benzo[a]pyrene (BaP), with the results permitting the formulation of mechanistic hypotheses regarding the observed potency ranking. Lastly, we illustrate how historical dose-response data for assessments that examined numerous doses (i.e. induced lacZ mutant frequency (MF) across 10 doses of BaP) can be used to improve the precision of BMDs derived from datasets with far fewer doses (i.e. lacZ MF for 3 doses of dibenz[a,h]anthracene). Collectively, the presented examples illustrate how innovative use of the BMD approach can permit refinement of the use of in vivo data; improving the efficacy of experimental animal use in genetic toxicology without sacrificing PoD precision.

Assessment of methyl methanesulfonate using the repeated-dose liver micronucleus assay in young adult rats.

A repeated-dose liver micronucleus assay using young adult rats was conducted with methyl methanesulfonate (MMS) as a part of a collaborative study supported by the Collaborative Study Group for the Micronucleus Test/the Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group. MMS is a classical DNA-reactive carcinogen, but it is not a liver carcinogen. In the first experiment (14-day study), MMS was administered per os to 6-week-old male Crl:CD (SD) rats every day for 14 days at a dose of 12.5, 25, or 50mg/kg/day. In the second experiment (28-day study), 6-week-old male SD rats were treated with MMS at 7.5, 15, or 30mg/kg/day for 28 days, because the highest dose used in the 14-day study (50mg/kg/day) caused mortality. Hepatocyte and bone marrow cell specimens were prepared on the day after the final dose. The frequency of micronucleated hepatocytes (MNHEPs) in the liver and that of micronucleated immature erythrocytes (MNIMEs) in the bone marrow were evaluated. Exposure to 50mg/kg/day MMS for 14 days resulted in an increased frequency of MNHEPs, but MMS had no effect on the frequency of MNHEPs in the rats exposed to the chemical for 28 days at doses up to 30mg/kg/day. MMS induced MNIMEs production at doses of 25 and 50mg/kg/day in the 14-day study and at doses of 15 and 30mg/kg/day in the 28-day study. Overall, the effect of MMS on the frequency of MNHEPs was considered to be equivocal.

Part 2. Assessment of micronucleus formation in rats after chronic exposure to new-technology diesel exhaust in the ACES bioassay.

The formation of micronuclei (MN*) is a well-established endpoint in genetic toxicology; studies designed to examine MN formation in vivo have been conducted for decades. Conditions that cause double-strand breaks or disrupt the proper segregation of chromosomes during division result in increases in MN formation frequency. This endpoint is therefore commonly used in preclinical studies designed to assess the potential risks to humans of exposure to a myriad of chemical and physical agents, including inhaled diesel exhaust (DE). As part of the Advanced Collaborative Emissions Study (ACES) Phase 3B, which examined numerous additional toxicity endpoints associated with lifetime exposure to DE in a rodent model, this ancillary 24-month investigation examined the potential of inhaled DE to induce chromosome damage in chronically exposed rodents. The ACES design included exposure of both mice and rats to DE derived from heavy-duty engines that met U.S. Environmental Protection Agency (EPA) 2007 standards for diesel-exhaust emissions (new-technology diesel exhaust). The exposure conditions consisted of air (the control) and three dilutions of DE, resulting in four levels of exposure. At specific times, blood samples were collected, fixed, and shipped by the bioassay staff at Lovelace Respiratory Research Institute (LRRI) to Litron Laboratories (Rochester, NY) for further processing and analysis. In recent years, significant improvements have been made to MN scoring by using objective, automated methods such as flow cytometry, which allows the detection of micronucleated reticulocytes (MN-RET), micronucleated normochromatic erythrocytes (MN-NCE), and reticulocytes (RET) in peripheral blood samples from mice and rats. By using a simple staining procedure coupled with rapid and efficient analysis, many more cells can be examined in less time than was possible using traditional, microscopy-based MN assays. Thus, for each sample in the current study, 20,000 RET were scored for the presence of MN. In the chronic-exposure (12 and 24 months) bioassay, blood samples were obtained from separate groups of exposed animals at specific time points throughout the course of the study. The automated method using flow cytometry has found widespread use in safety assessment and is supported by regulatory guidelines, including International Conference on Harmonisation (ICH) S2(R1) (2011). Statistical analyses included the use of analysis of variance (ANOVA) to compare the effects of sex, exposure condition, and duration, as well asthe interactions between them. Analyses of blood samples from rats combined data from our earlier 1- and 3-month exposure studies (Bemis et al. 2012) with data from our current 12- and 24-month exposure studies. Consistent with findings from the preliminary studies, no sex-based differences in MN frequency were observed in the rats. An initial examination of mean frequencies across the treatment groups and durations of exposure showed no evidence of treatment-related increases in MN at any of the time points studied. Further statistical analyses did not reveal any significant exposure-related effects. An examination of the potential genotoxic effects of DE is clearly valuable as part of a large-scale chronic exposure bioassay. The results described in this report provide a comprehensive examination of chronic exposure to DE in a rodent model. Our investigation of chromosomal damage also plays an important role in the context of ACES, which was designed to assess the safety of emissions from 2007-compliant diesel engines.

Genotoxicity assessment of melamine in the in vivo Pig-a mutation assay and in a standard battery of assays.

The genotoxicity of melamine was evaluated with the combined Pig-a mutation/micronucleus assay, the bacterial reverse mutation assay, and the in vitro cytokinesis-block micronucleus assay (CBMN). Five groups of six- to eight-week-old male Sprague-Dawley (SD) rats were given three daily doses of vehicle control (100% pure sesame oil), melamine (500, 1000, and 2000 mg/kg) or positive control (N-ethyl-N-nitrosourea, ENU, 20 mg/kg) by oral gavage. Peripheral blood was sampled pre-dose (day -1) and at time points up to day 60. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies, on days -1, 15, 29 and 60, and micronucleus frequencies were measured in RETs on day 4. No significant increases in RBC(CD59-) or RET(CD59-) frequencies were observed for the melamine-treated group at any of the time points studied, but the positive control, ENU, induced statistically significant increases compared with the vehicle control. Similar results were obtained in the micronucleus assay. Melamine did not induce statistically significant increases in %MN-RET. In the bacterial reverse mutation assay, melamine was tested from 62.5 to 1000 µg/plate in tester strains TA97a, TA98, TA100, TA102, and TA1535, with and without metabolic activation, and no evidence of toxicity or mutagenicity was observed at any dose tested. In the in vitro CBMN assay, in Chinese hamster ovary (CHO) cells, melamine was tested (75, 150, and 300 µg/mL) in the presence and absence of S9 mix, and no positive increases in the number of cells containing micronuclei were seen. These results suggest that melamine does not exhibit significant genotoxic potential. These data could be valuable for risk assessment purposes and also for further characterizing the new in vivoPig-a gene mutation assay.

Assessment of 5-fluorouracil and 4-nitroquinoline-1-oxide in vivo genotoxicity with Pig-a mutation and micronucleus endpoints.

Genotoxicity assessments were conducted on male Sprague Dawley rats treated with 5-fluorouracil (5-FU) and 4-nitroquinoline-1-oxide (4NQO) as part of an international validation trial of the Pig-a mutant phenotype assay. Rats were orally exposed to 0, 11.5, 23, or 46 mg/kg/day 5-FU for three consecutive days (Days 1-3); blood was sampled on Days -1, 4, 15, 29, and 45. Pig-a mutant phenotype reticulocyte (RET(CD59-)) and mutant phenotype erythrocyte (RBC(CD59-)) frequencies were determined on Days -1, 15, 29, and 45, and percent micronucleated reticulocytes (%MN-RET) were measured on Day 4. Rats were treated with 4NQO for 28 consecutive days by oral gavage, at doses of 1.5, 3, or 6 mg/kg/day. RBC(CD59-) and RET(CD59-) frequencies were determined on Days -1, 15, and 29, and MN-RET were quantified on Day 29. Whereas 5-FU was found to increase %MN-RET, no significant increases were observed for RBC(CD59-) or RET(CD59-) at any of the time points studied. The high dose of 4NQO (6 mg/kg/day) was observed to markedly increase RBC(CD59-) and RET(CD59-) frequencies, and this same dose level caused a weak but significantly elevated increase in MN-RET (approximately twofold). Collectively, the results provide additional support for the combination of Pig-a mutation and MN-RET into acute and 28-day repeat-dose studies.

Assessment of genotoxicity induced by 7,12-dimethylbenz(a)anthracene or diethylnitrosamine in the Pig-a, micronucleus and Comet assays integrated into 28-day repeat dose studies.

As part of the Stage 3 of the Pig-a international trial, we evaluated 7,12-dimethylbenz(a)anthracene (DMBA) for induction of Pig-a gene mutation using a 28-day repeat dose study design in Sprague-Dawley rats. In the same study, chromosomal damage in peripheral blood and primary DNA damage in liver were also investigated by the micronucleus (MN) assay and the Comet assay, respectively. In agreement with previously published data (Dertinger et al., [2010]: Toxicol Sci 115:401-411), DMBA induced dose-dependent increases of CD59-negative erythrocytes/reticulocytes and micronucleated reticulocytes (MN-RETs). However, there was no significant increase in DNA damage in the liver cells when tested up to 10 mg/kg/day, which appears to be below the maximum tolerated dose. When tested up to 200 mg/kg/day in a follow-up 3 dose study, DMBA was positive in the liver Comet assay. Additionally, we evaluated diethylnitrosamine (DEN), a known mutagen/hepatocarcinogen, for induction of Pig-a mutation, MN and DNA damage in a 28-day study. DEN produced negative results in both the Pig-a mutation assay and the MN assay, but induced dose-dependent increases of DNA damage in the liver and blood Comet assay. In summary, our results demonstrated that the Pig-a mutation assay can be effectively integrated into repeat dose studies and the data are highly reproducible between different laboratories. Also, integration of multiple genotoxicity endpoints into the same study not only provides a comprehensive evaluation of the genotoxic potential of test chemicals, but also reduces the number of animals needed for testing, especially when more than one in vivo genotoxicity tests are required.

Genotoxicity assessment of an energetic propellant compound, 3-nitro-1,2,4-triazol-5-one (NTO).

3-Nitro-1,2,4-triazol-5-one (NTO) is an energetic explosive proposed for use in weapon systems, to reduce the sensitivity of warheads. In order to develop toxicity data for safety assessment, we investigated the genotoxicity of NTO, using a battery of genotoxicity tests, which included the Ames test, Chinese Hamster Ovary (CHO) cell chromosome aberration test, L5178Y TK(+/-) mouse lymphoma mutagenesis test and rat micronucleus test. NTO was not mutagenic in the Ames test or in Escherichia coli (WP2uvrA). NTO did not induce chromosomal aberrations in CHO cells, with or without metabolic activation. In the L5178Y TK(+/-) mouse lymphoma mutagenesis test, all of the NTO-treated cultures had mutant frequencies that were similar to the average frequencies of solvent control-treated cultures, indicating a negative result. Confirmatory tests for the three in vitro tests also produced negative results. The potential in vivo clastogenicity and aneugenicity of NTO was evaluated using the rat peripheral blood micronucleus test. NTO was administered by oral gavage to male and female Sprague-Dawley rats for 14 days at doses up to 2g/kg/day. Flow cytometric analysis of peripheral blood demonstrated no significant induction of micronucleated reticulocytes relative to the vehicle control (PEG-200). These studies reveal that NTO was not genotoxic in either in vitro or in vivo tests and suggest a low risk of genetic hazards associated with exposure.

[Blood toxicity assessment of subchronic exposure to hydroxylammonium nitrate in wistar rats ].

OBJECTIVE: To investigate the effects of hydroxylammonium nitrate (HAN) on the blood system in rats. METHODS: A total of 160 rats were randomly divided into three HAN treated groups and one negative control group, each group included 40 rats. Different doses of HAN at 6.97, 13.93 and 27.86mg/kg were administered to the three treated groups by intraperitoneal injection, and the control group was treated with 0.9% saline, the treatment had been performed every other day for 13 weeks. After the last treatment, 30 rats of each group were sacrificed and the rest were kept for another 4 weeks for recovery. Hematological parameters, reticulocyte and marrow cells were observed and measured. RESULTS: For hematological parameters, the main changes were the decrease of RBC and HGB level and the increase of WBC at the end of the administration (P < 0.05). The reticulocyte values were obviously increase along with the increasing HAN dosage compared with those of control group (P < 0.01). For bone marrow cells observation, the ratio of erythrocytic line increased, and the ratio of granulocytes/erythrocyte was lower than that of control group (P < 0.05). After 4 weeks' recovery period, no obviously changes were observed for hematological parameters, reticulocyte and marrow cells. CONCLUSION: HAN has toxic effect on the blood system in rats for long-term exposure.

An assessment of recombinant human erythropoietin effect on reticulocyte production rate and lifespan distribution in healthy subjects.

PURPOSE: An empirical pharmacodynamic model was developed to assess the effect of recombinant human erythropoietin (rHu-EPO) treatment on the reticulocyte production rate and lifespan distribution. MATERIALS AND METHODS: Single doses of rHu-EPO at levels 20, 40, 60, 90, 120, and 160 kIU were administered to healthy volunteers (n = 8 per dose level). Erythropoietin plasma concentrations as well as hematologic responses were measured up to 42 days. The hematological data were used to determine explicit relationships between reticulocyte and red blood cell counts (RBC) and the reticulocytes' production rate and lifespan distribution. RESULTS: The parameter estimates obtained by simultaneous fitting of the model to the reticulocyte and RBC data revealed that rHu-EPO transiently increased the reticulocyte lifespan from the baseline value of 1.7 days to 3.4 days and the effect lasted for 8.3 days. The dose dependent increase in the reticulocyte production had the maximal value of 77.5 10(9) cells/l/day and was followed by a rebound that was less than 9% of the baseline value. Both reticulocyte and RBC responses were preceded by a dose-independent lag time of 1.7 days. CONCLUSIONS: The effect of rHu-EPO on the reticulocyte production rate and lifespan distribution was characterized. The results of the present study can be further utilized in building more mechanistic pharmacodynamic models of rHu-EPO stimulatory effects.

Assessment of DNA damage by extracts and fractions of Strychnos pseudoquina, a Brazilian medicinal plant with antiulcerogenic activity.

Strychnos pseudoquina St. Hil. is a native plant of the Brazilian Savannah, used in popular medicine to treat a number of conditions. Since it contains large quantities of alkaloids with proven antiulcer activity, we tested the genotoxic potential of crude extracts and fractions containing alkaloids and flavonoids from the leaves of this plant, on Salmonella typhimurium and performed the micronucleus test on peripheral blood cells of mice treated in vivo. The results showed that the methanol extract of the leaves of S. pseudoquina is mutagenic to the TA98 (-S9) and TA100 (+S9, -S9) strains of Salmonella. The dichloromethane extract was not mutagenic to any of the tested strains. Fractions enriched with alkaloids or flavonoids were not mutagenic. In vivo tests were done on the crude methanol extract in albino Swiss mice, which were treated, by gavage, with three different doses of the extract. The highest dose tested (1800 mg/kgb.w.) induced micronuclei after acute treatment, confirming the mutagenic potential of the methanol extract of the leaves of S. pseudoquina. In high doses, constituents of S. pseudoquina compounds act on DNA, causing breaks and giving rise to micronuclei in the blood cells of treated animals.

Assessment of the in vivo genotoxicity of vanadate: analysis of micronuclei and DNA damage induced in mice by oral exposure.

Vanadium compounds are able to interact with living cells exerting a variety of biological effects. The pentavalent form is the most stable and toxic form of the element. In systems in vitro pentavalent vanadium is an effective genotoxic agent, inducing DNA damage and chromosome malsegregation at low doses. On the other hand, no adequate in vivo data are available for the characterization of the genotoxic hazard following oral intake, the most relevant route of human exposure. In this study, the genotoxic effects produced by the oral intake of sodium ortho-vanadate (Na(3)VO(4)) were investigated. Male CD-1 mice were treated for 5 weeks with a range of concentrations of Na(3)VO(4) in drinking water (0.75-1500 mg/l). Both micronuclei and primary DNA lesions as detected by comet assay were assessed in several tissues. Statistically significant increases of micronuclei in bone marrow were observed in mice receiving the two highest concentrations of Na(3)VO(4) (750 and 1500 mg/l). A significant increase of comet tail length was observed in splenocytes of mice receiving Na(3)VO(4) at 1500 mg/l, whereas no effect was observed in bone marrow and testis cells. No treatment-related effect on sperm chromatin structure or on testis cell population was observed. The determination of vanadium content in mouse tissues at the end of treatment highlighted a very low internal exposure, especially in soft tissues. Overall, the results obtained indicate that the genotoxic activity of pentavalent vanadium is expressed in vivo only following high dose exposure, possibly as a consequence of the poor bioavailability of the element.

Assessment of high-affinity hybridization, RNase H cleavage, and covalent linkage in translation arrest by antisense oligonucleotides.

Antisense oligonucleotides (ONs) are designed to hybridize target mRNA in a sequence-specific manner and inhibit gene expression by preventing translation, either by activation of RNase H or steric blockage of the ribosome complex. Second-generation ONs, which possess greater binding affinity for target RNA relative to the isosequential phosphodiester (PO) ONs, have been developed and include, among others, peptide nucleic acids (PNA) and N3' P5' phosphoramidate oligonucleotides (npONs). In the present study, PNA and npON derivatives were targeted to the coding portion of the complementary mRNA of the N protein of the vesicular stomatitis virus (VSV) in order to evaluate their ability to arrest translation in an in vitro rabbit reticulocyte lysate system. High-affinity hybridization of ONs lacking RNase H activity was not sufficient to block translation in this test system. Only antisense ONs acting via an RNase H mechanism or by steric hindrance through covalent attachment (via transplatin modification) to the target mRNA were found to definitively arrest translation in this study.

Assessment of the ability of propoxur, methomyl, and aldicarb, three carbamate insecticides, to induce micronuclei in vitro in cultured Chinese hamster ovary cells and in vivo in BALB/c mice.

Three carbamate insecticides (propoxur, methomyl, and aldicarb) were evaluated for their ability to induce micronuclei (MN) in vitro using cultured Chinese hamster ovary (CHO) cells, and in vivo in mouse bone marrow erythrocytes. In vitro, all three insecticides induced a significant increase in micronucleated binucleate cells, which was generally both dose and sample time dependent. The in vivo studies involved treating male BALB/c mice by different routes, either once or on 3 consecutive days, followed by multiple or single sampling. Treatment by intraperitoneal injection or oral gavage induced a significant increase in micronucleated reticulocytes (MNRETs) in peripheral blood. For all three chemicals, the MN response depended on sample time and the number of treatments, while for aldicarb, the response depended also on the route of exposure. These positive results demonstrate that propoxur, methomyl, and aldicarb are capable of inducing structural and/or numerical chromosomal aberrations in mammalian cells either in vitro or in vivo. Furthermore, based on the results obtained, on optimal in vivo MN protocol for carbamate insecticides is a single treatment followed by blood sampling at 24 and 48 hr after treatment.

Genotoxicity of trophosphamide in mouse germ cells: assessment of micronuclei in spermatids and chromosome aberrations in one-cell zygotes.

The genotoxicity of trophosphamide (TP) in mouse germ cells was assessed by the cytogenetic analysis of micronuclei in spermatids and chromosome aberrations in one-cell zygotes and compared with the genotoxicity in somatic cells evaluated by the micronucleus reticulocyte assay. Single acute doses of 50, 75, 100 and 150 mg/kg were studied after i.p. injection. TP was only weakly mutagenic for preleptotene spermatocytes-differentiating spermatogonia, but clear-cut cytotoxic effects were demonstrated after treatment of these cells by a dose-dependent reduction of the ratio between Golgi and cap phase spermatids. Effects induced in post-meiotic stages were estimated, after mating the treated males with untreated superovulated females, by the frequencies of zygotes with chromosome aberrations: a peak of genetic damage was detected in late spermatids, with as many as 55% zygotes with aberrations, but spermatozoa and early spermatids were also clearly affected. When compared with matched solvent-injected controls, the lowest effective dose in spermatozoa and late spermatids was 100 mg/kg, although the 3- to 4-fold increases detected at 50 mg/kg were also statistically significant when compared with a pool of laboratory controls. In peripheral blood reticulocytes, the micronucleus frequencies were increased by 3-20 times the respective baseline values in the individual animals. A marked cytotoxic effect on bone marrow cells was revealed by the reduction of the proportion of early reticulocyte stages, which dropped to 20% of the control value at 150 mg/kg. Both genotoxic and cytotoxic effects were higher in bone marrow than in germ cells of the same animals, pointing to a generalized higher susceptibility of somatic cells to TP, possibly related to chemical distribution and target organ accessibility. The accurate description of stage- and dose-effect relationships in germ cells of experimental models is crucial for genetic risk assessment after chemical exposure. The approaches applied in this study may contribute to this goal.