Longitudinal multi-omic changes in the transcriptome and proteome of peripheral blood cells after a 4 Gy total body radiation dose to Rhesus macaques.

BACKGROUND: Non-human primates, such as Rhesus macaques, are a powerful model for studies of the cellular and physiological effects of radiation, development of radiation biodosimetry, and for understanding the impact of radiation on human health. Here, we study the effects of 4 Gy total body irradiation (TBI) at the molecular level out to 28 days and at the cytogenetic level out to 56 days after exposure. We combine the global transcriptomic and proteomic responses in peripheral whole blood to assess the impact of acute TBI exposure at extended times post irradiation. RESULTS: The overall mRNA response in the first week reflects a strong inflammatory reaction, infection response with neutrophil and platelet activation. At 1 week, cell cycle arrest and re-entry processes were enriched among mRNA changes, oncogene-induced senescence and MAPK signaling among the proteome changes. Influenza life cycle and infection pathways initiated earlier in mRNA and are reflected among the proteomic changes during the first week. Transcription factor proteins SRC, TGFß and NFATC2 were immediately induced at 1 day after irradiation with increased transcriptional activity as predicted by mRNA changes persisting up to 1 week. Cell counts revealed a mild / moderate hematopoietic acute radiation syndrome (H-ARS) reaction to irradiation with expected lymphopenia, neutropenia and thrombocytopenia that resolved within 30 days. Measurements of micronuclei per binucleated cell levels in cytokinesis-blocked T-lymphocytes remained high in the range 0.27-0.33 up to 28 days and declined to 0.1 by day 56. CONCLUSIONS: Overall, we show that the TBI 4 Gy dose in NHPs induces many cellular changes that persist up to 1 month after exposure, consistent with damage, death, and repopulation of blood cells.

Liquid Chromatography-Mass Spectrometry-Based Metabolomics of Nonhuman Primates after 4 Gy Total Body Radiation Exposure: Global Effects and Targeted Panels.

Rapid assessment of radiation signatures in noninvasive biofluids may aid in assigning proper medical treatments for acute radiation syndrome (ARS) and delegating limited resources after a nuclear disaster. Metabolomic platforms allow for rapid screening of biofluid signatures and show promise in differentiating radiation quality and time postexposure. Here, we use global metabolomics to differentiate temporal effects (1-60 d) found in nonhuman primate (NHP) urine and serum small molecule signatures after a 4 Gy total body irradiation. Random Forests analysis differentially classifies biofluid signatures according to days post 4 Gy exposure. Eight compounds involved in protein metabolism, fatty acid ß oxidation, DNA base deamination, and general energy metabolism were identified in each urine and serum sample and validated through tandem MS. The greatest perturbations were seen at 1 d in urine and 1-21 d in serum. Furthermore, we developed a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) method to quantify a six compound panel (hypoxanthine, carnitine, acetylcarnitine, proline, taurine, and citrulline) identified in a previous training cohort at 7 d after a 4 Gy exposure. The highest sensitivity and specificity for classifying exposure at 7 d after a 4 Gy exposure included carnitine and acetylcarnitine in urine and taurine, carnitine, and hypoxanthine in serum. Receiver operator characteristic (ROC) curve analysis using combined compounds show excellent sensitivity and specificity in urine (area under the curve [AUC] = 0.99) and serum (AUC = 0.95). These results highlight the utility of MS platforms to differentiate time postexposure and acquire reliable quantitative biomarker panels for classifying exposed individuals.

Delayed effects of radiation exposure in a C57L/J mouse model of partial body irradiation with ~2.5% bone marrow shielding.

Introduction: Mouse models of radiation injury are critical to the development of medical countermeasures (MCMs) against radiation. Now that MCMs against hematopoietic acute radiation syndrome (H-ARS) have achieved regulatory approval, attention is shifting to develop MCMs against the adverse effects of gastrointestinal acute radiation syndrome (GI-ARS) and delayed effects of acute radiation exposure (DEARE). The C57L/J mouse model of partial body irradiation (PBI) with 2.5% bone marrow shielding (BM2.5) is being leveraged to examine both GI-ARS and DEARE effects. Within days of PBI, mice may develop H- and GI-ARS followed several months later by DEARE as a multi-organ injury, which typically involves the lung and kidney (L- and K-DEARE, respectively). The objective of this manuscript is to describe the dose response relationship and progression of radiation injury in the C57L/J mouse and to evaluate its suitability for use in DEARE MCM testing. Materials and methods: In two separate studies conducted over 2 years, male and female C57L/J mice were exposed to PBI BM2.5 with one hindlimb shielded from radiation, representing ~2.5% bone marrow shielding/sparing. Mice were X-ray irradiated at doses ranging from 9 to 13 Gy at 10 to 12 weeks of age for the purposes of assessing ARS survival at 30 days and DEARE survival at 182 days post-irradiation. Clinical indicators of ARS and DEARE were determined by clinical observations, body weights, hematology, clinical chemistry, magnetic resonance imaging (MRI) of lung, and histopathology of selected tissues. Results: C57L/J mice developed canonical ARS responses of hematopoietic atrophy and gastrointestinal injury resulting in dose dependent mortality at doses ≥11 Gy between 1- and 15-days post-irradiation. In animals that survived ARS, DEARE associated mortality occurred in dose dependent fashion at ≥9 Gy for both sexes between 60- and 159-days post-irradiation with histopathology examinations indicating lung injury as the primary cause of death in moribund animals. Conclusion: The PBI BM2.5 C57L/J mouse model reliably produced known H- and GI-ARS effects at doses greater than those resulting in DEARE effects. Because of this, the C57L/J mouse can be used to test MCMs against L-DEARE injury, while avoiding ARS associated mortality.

The progression of radiation injury in a Wistar rat model of partial body irradiation with ∼5% bone marrow shielding.

PURPOSE: To describe the dose response relationship and natural history of radiation injury in the Wistar rat and its suitability for use in medical countermeasures (MCM) testing. MATERIALS & METHODS: In two separate studies, male and female rats were exposed to partial body irradiation (PBI) with 5% bone marrow sparing. Animals were X-ray irradiated from 7 to 12 Gy at 7-10 weeks of age. Acute radiation syndrome (ARS) survival at 30 days and delayed effects of acute radiation exposure (DEARE) survival at 182 days were assessed. Radiation effects were determined by clinical observations, body weights, hematology, clinical chemistry, magnetic resonance imaging of lung, whole-body plethysmography, and histopathology. RESULTS: Rats developed canonical ARS responses of hematopoietic atrophy and gastrointestinal injury resulting in mortality at doses ≥8Gy in males and ≥8.5 Gy in females. DEARE mortality occurred at doses ≥8Gy for both sexes. Findings indicate lung, kidney, and/or liver injury, and persistent hematological dysregulation, revealing multi-organ injury as a DEARE. CONCLUSION: The Wistar rat PBI model is suitable for testing MCMs against hematopoietic and gastrointestinal ARS. DEARE multi-organ injury occurred in both sexes irradiated with 8-9Gy, also suggesting suitability for polypharmacy studies addressing the combination of ARS and DEARE injury.

Survival and Hematologic Benefits of Romiplostim After Acute Radiation Exposure Supported FDA Approval Under the Animal Rule.

PURPOSE: Patients exposed to acute high doses of ionizing radiation are susceptible to dose-dependent bone marrow depression with resultant pancytopenia. Romiplostim (RP; Nplate) is a recombinant thrombopoietin receptor agonist protein that promotes progenitor megakaryocyte proliferation and platelet production and is an approved treatment for patients with chronic immune thrombocytopenia. The goal of our study was to evaluate the postirradiation survival and hematologic benefits of a single dose of RP with or without pegfilgrastim (PF; Neulasta, granulocyte colony stimulating factor) by conducting a well-controlled, treatment-concealed, good laboratory practice-compliant study in rhesus macaques that was compliant with the United States Food and Drug Administration Animal Rule regulatory approval pathway. METHODS AND MATERIALS: Irradiated male and female rhesus macaques (20/sex in each of 3 groups: control, RP, and RP + PF) were subcutaneously administered vehicle or RP (5 mg/kg, 10 mL/kg) on day 1 in the presence or absence of 2 doses of PF (0.3 mg/kg, 0.03 mL/kg, days 1 and 8). Total body radiation (680 cGy, 50 cGy/min from cobalt-60 gamma ray source) occurred 24 ± 2 hours previously at a dose targeting 70% lethality for the control cohort over 60 days. The study examined 60-day survival postirradiation as the primary endpoint. Secondary endpoints included incidence, severity, and duration of thrombocytopenia and neutropenia, other hematology parameters, coagulation parameters, and body weight change to provide insights into potential mechanisms of action. RESULTS: Compared with sham-treated controls, treated animals demonstrated a 40% to 55% survival benefit compared with controls, less severe clinical signs, reduced incidence of thrombocytopenia and/or neutropenia, earlier hematologic recovery, and reduced morbidity from bacterial infection. CONCLUSIONS: These results were pivotal in obtaining Food and Drug Administration approval in January 2021 for RP's new indication as a single administration therapy to increase survival in adults and pediatric patients acutely exposed to myelosuppressive doses of radiation.

Scaleable manufacture of HIV-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component.

To prevent sexually transmitted HIV, the most desirable active ingredients of microbicides are antiretrovirals (ARVs) that directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals, which are costly to manufacture and deliver to resource-poor areas where effective microbicides are urgently needed. Here, we report a manufacturing breakthrough for griffithsin (GRFT), one of the most potent HIV entry inhibitors. This red algal protein was produced in multigram quantities after extraction from Nicotiana benthamiana plants transduced with a tobacco mosaic virus vector expressing GRFT. Plant-produced GRFT (GRFT-P) was shown as active against HIV at picomolar concentrations, directly virucidal via binding to HIV envelope glycoproteins, and capable of blocking cell-to-cell HIV transmission. GRFT-P has broad-spectrum activity against HIV clades A, B, and C, with utility as a microbicide component for HIV prevention in established epidemics in sub-Saharan Africa, South Asia, China, and the industrialized West. Cognizant of the imperative that microbicides not induce epithelial damage or inflammatory responses, we also show that GRFT-P is nonirritating and noninflammatory in human cervical explants and in vivo in the rabbit vaginal irritation model. Moreover, GRFT-P is potently active in preventing infection of cervical explants by HIV-1 and has no mitogenic activity on cultured human lymphocytes.

Heavy-Ion-Induced Lung Tumors: Dose- & LET-Dependence.

There is a limited published literature reporting dose-dependent data for in vivo tumorigenesis prevalence in different organs of various rodent models after exposure to low, single doses of charged particle beams. The goal of this study is to reduce uncertainties in estimating particle-radiation-induced risk of lung tumorigenesis for manned travel into deep space by improving our understanding of the high-LET-dependent dose-response from exposure to individual ion beams after low particle doses (0.03-0.80 Gy). Female CB6F1 mice were irradiated with low single doses of either oxygen, silicon, titanium, or iron ions at various energies to cover a range of dose-averaged LET values from 0.2-193 keV/µm, using 137Cs γ-rays as the reference radiation. Sham-treated controls were included in each individual experiment totally 398 animals across the 5 studies reported. Based on power calculations, between 40-156 mice were included in each of the treatment groups. Tumor prevalence at 16 months after radiation exposure was determined and compared to the age-matched, sham-treated animals. Results indicate that lung tumor prevalence is non-linear as a function of dose with suggestions of threshold doses depending on the LET of the beams. Histopathological evaluations of the tumors showed that the majority of tumors were benign bronchioloalveolar adenomas with occasional carcinomas or lymphosarcomas which may have resulted from metastases from other sites.

Total body irradiation models in NHPs - consideration of animal sex and provision of supportive care to advance model development.

PURPOSE: Harmonized animal models are an indispensable tool for the development of safe and effective medical countermeasures (MCMs) against radiation injury, and rhesus macaques (referred herein as NHPs) play a critical role in FDA approval of radiation medical countermeasures for acute and delayed radiation syndromes. Reliance on such models requires that they be well characterized, which consists, in part, of a reproducible dose to mortality response relationship (DRR). However, data describing the DRR for both male and female NHPs from the same study are scarce. Furthermore, the level of supportive care and the use of blood transfusions may shift the DRR, yet such information can be difficult to compare across publications. To address these knowledge gaps, the DRRs of two different NHP total body irradiation (TBI) models are compared in this paper, one which is reliant on the use of male animals provided blood transfusions, and the other which incorporates both sexes wherein animals are not provided transfusions. MATERIALS AND METHODS: Studies were conducted using NHPs (Macacca mulatta) receiving TBI, with survival reported over a 60 days. Two primary studies, incorporating both male and female animals not receiving blood transfusions as a provision of supportive care, were compared to two previously published studies, which incorporated only male animals provided blood transfusions as a part of the supportive care regimen. Criterion for euthanasia, and all other provisions of supportive care were comparable. Linear probit plots estimating the lethal dose (LD) and upper and lower limits of the 95% confidence interval (CI) for 10, 30, 50, 70 and 90% mortality, were compared between individual studies and the two models presented. RESULTS: Comparison of probit estimates reveals two important findings. (1) Females have higher mortality than males at identical radiation doses, and (2) blood transfusions increased survival of male animals at lower doses but not at high doses of radiation exposure. CONCLUSIONS: The use of single sex animal models may lead to an incomplete understanding of potential sex differences in the dose to mortality response of the TBI model. Consistent use of both sexes and type of supportive care will improve the transferability and reliability of NHP-TBI models currently in use, assist in the selection of radiation doses for single dose lethality studies, and allow investigators to determine the effectiveness of a particular MCM.

Particle radiation-induced dysregulation of protein homeostasis in primary human and mouse neuronal cells.

Space particle radiations may cause significant damage to proteins and oxidative stress in the cells within the central nervous system and pose a potential health hazard to humans in long-term manned space explorations. Dysregulation of the ubiquitin-proteasome system as evidenced by abnormal accumulation of polyubiquitin (pUb) chain linkages has been implicated in several age-related neurodegenerative disorders by mechanisms that may involve the inter-neuronal spread of toxic misfolded proteins, the induction of chronic neuroinflammation, or the inappropriate inhibition or activation of key enzymes, which could lead to dysfunction in, for example, proteolysis, or the accumulation of post-translationally-modified substrates.In this study, we employed a quantitative proteomics method to evaluate the impact of particle-radiation induced alterations in three major pUb-linked chains at lysine residues Lys-48 (K-48), Lys-63 (K-63), and Lys-11 (K-11), and probed for global proteomic changes in mouse and human neural cells that were irradiated with low doses of 250 MeV proton, 260 MeV/u silicon or 1 GeV/u iron ions. We found significant accumulation in K-48 linkage after 1 Gy protons and K-63 linkage after 0.5 Gy iron ions in human neural cells. Cells derived from different regions of the mouse brain (cortex, striatum and mesencephalon) showed differential sensitivity to particle radiation exposure. Although none of the linkages were altered after proton exposure, both K-48 and K-63 linkages in mouse striatal neuronal cells were elevated after 0.5 Gy of silicon or iron ions. Changes were also seen in proteins commonly used as markers of neural progenitor and stem cells, in DNA binding/damage repair and cellular redox pathways. In contrast, no significant changes were observed at the same time point after proton irradiation. These results suggest that the quality of the particle radiation plays a key role in the level, linkage and cell type specificity of protein homeostasis in key populations of neuronal cells.

Romiplostim (Nplate®) as an effective radiation countermeasure to improve survival and platelet recovery in mice.

Purpose: Rapid depletion of white blood cells, platelets, and reticulocytes are hallmarks of hematopoietic injury of acute radiation syndrome (H-ARS) and, if left untreated, can lead to severe health consequences including death. While the granulocyte colony stimulating factors (G-CSF) filgrastim (Neupogen®), pegfilgrastim (Neulasta®), and sargramostim (Leukine®) are approved to increase survival in patients exposed to a myelosuppressive dose of radiation, no medical countermeasure is currently available for treatment of the thrombocytopenia that also results following radiation exposure. Romiplostim (Nplate®), a thrombopoietin receptor agonist, is the first FDA-approved thrombopoiesis-stimulating protein for the treatment of low platelet (PLT) counts in adults with chronic immune thrombocytopenia. Herein, we present the results of an analysis in mice of romiplostim as a medical countermeasure to improve survival and PLT recovery following acute radiation.Materials and methods: Male and female C57BL/6J mice (11 - 12 weeks of age, n = 21/sex/group) were total body irradiated (TBI) with 6.8 Gy X-rays that reduces 30-day survival to 30% (LD70/30). Vehicle, romiplostim, and/or pegfilgrastim were administered subcutaneously beginning 24 h after TBI for 1-5 days. Evaluation parameters included 30-day survival, pharmacokinetics, and hematology.Results: Full or maximal efficacy with an ∼40% increase in survival was achieved after a single 30 µg/kg dose of romiplostim. No further survival benefit was seen with higher (100 µg/kg) or more frequent dosing (3 or 5 once daily doses at 30 µg/kg) of romiplostim or combined treatment with pegfilgrastim. Pharmacodynamic analysis revealed that the platelet nadir was not as low and recovery was faster in the irradiated mice treated with romiplostim when compared with irradiated control animals (Day 8 versus 10 nadir; Day 22 versus 29 recovery to near baseline). Platelet volume also increased more rapidly after romiplostim injection. Kinetic profiles of other hematology parameters were similar between TBI romiplostim-treated and control mice. Peak serum levels of romiplostim in TBI mice occurred 4 - 24 h (Tmax) after injection with a t1/2 of ∼24 h. Cmax values were at ∼6 ng/ml after 30 µg/kg ± TBI and ∼200 ng/ml after 300 µg/kg. A 10-fold higher romiplostim dose increased the AUClast values by ∼35-fold.Conclusion: A single injection of romiplostim administered 24 h after TBI is a promising radiation medical countermeasure that dramatically increased survival, with or without pegfilgrastim, and hastened PLT recovery in mice.

Pharmacodynamics of romiplostim alone and in combination with pegfilgrastim on acute radiation-induced thrombocytopenia and neutropenia in non-human primates.

Purpose: Evaluation of the pharmacodynamics (PD) and pharmacokinetics (PK) of romiplostim alone and in combination with pegfilgrastim in a non-human primate (NHP) model of acute radiation syndrome (ARS).Materials and methods: Male and female rhesus macaques were subjected to Cobalt-60 γ irradiation, at a dose of 550 cGy 24 h prior to subcutaneous administration of either romiplostim alone as a single (2.5 or 5.0 mg/kg on Day 1) or repeat dose (5.0 mg/kg on Days 1 and 8), pegfilgrastim alone as a repeat dose (0.3 µg/kg on Day 1 and 8), or a combination of both agents (romiplostim 5.0 mg/kg on Day 1; pegfilgrastim 0.3 µg/kg on Days 1 and 8). Clinical outcome, hematological parameters and PK were assessed throughout the 45 d study period post-irradiation.Results: Administration of romiplostim, pegfilgrastim or the combination of both resulted in significant improvements in hematological parameters, notably prevention of severe thrombocytopenia, compared with irradiated, vehicle control-treated NHPs. The largest hematologic benefit was observed when romiplostim and pegfilgrastim were administered as a combination therapy with much greater effects on both platelet and neutrophil recovery following irradiation compared to single agents alone.Conclusions: These results indicate that romiplostim alone or in combination with pegfilgrastim is effective at improving hematological parameters in an NHP model of ARS. This study supports further study of romiplostim as a medical countermeasure to improve primary hemostasis and survival in ARS.

Salivary Metabolomics of Total Body Irradiated Nonhuman Primates Reveals Long-Term Normal Tissue Responses to Radiation.

PURPOSE: To identify metabolomic biomarkers of acute radiation exposure in saliva that show time-dependent changes. METHODS AND MATERIALS: Nonhuman primates were exposed to 4 Gy of total body irradiation with γ-rays. Saliva was collected from 7 animals twice before and at days 1, 3, 5, 7, 15, 21, 28, and 60 after irradiation. Profiling was conducted with liquid chromatography time-of-flight mass spectrometry. Multivariate data analysis and potential biomarker identification was conducted through random Forests and the software MetaboAnalyst. Candidate biomarkers were validated through tandem mass spectrometry, and receiver operating characteristic curves were constructed to show the diagnostic ability of the signature over time. RESULTS: Untargeted metabolomic analysis revealed significant and persistent effects up to the 60 days evaluated in this study. Biomarkers spanning primarily amino acids and nucleotides were identified, with a significant number showing long-term responses. Fifteen biomarkers showed high statistical significance in the first week after irradiation and 16 at >7 days after irradiation (false discovery rate-adjusted P < .05). The combination of the biomarkers in a single biosignature was able to accurately show the diagnostic ability of the signature in a binary classifier system with receiver operating characteristic curves. CONCLUSIONS: Radiation can alter the metabolome in saliva, and metabolomics could effectively be used to monitor radiation responses, as a biodosimetry method, in the event of a radiological incident. Saliva metabolomics also has potential relevance in a clinical setting.

Temporal Effects on Radiation Responses in Nonhuman Primates: Identification of Biofluid Small Molecule Signatures by Gas Chromatography⁻Mass Spectrometry Metabolomics.

Whole body exposure to ionizing radiation damages tissues leading to physical symptoms which contribute to acute radiation syndrome. Radiation biodosimetry aims to determine characteristic early biomarkers indicative of radiation exposure and is necessary for effective triage after an unanticipated radiological incident. Radiation metabolomics can address this aim by assessing metabolic perturbations following exposure. Gas chromatography-mass spectrometry (GC-MS) is a standardized platform ideal for compound identification. We performed GC time-of-flight MS for the global profiling of nonhuman primate urine and serum samples up to 60 d after a single 4 Gy γ-ray total body exposure. Multivariate statistical analysis showed higher group separation in urine vs. serum. We identified biofluid markers involved in amino acid, lipid, purine, and serotonin metabolism, some of which may indicate host microbiome dysbiosis. Sex differences were observed for amino acid fold changes in serum samples. Additionally, we explored mitochondrial dysfunction by tricarboxylic acid intermediate analysis in the first week with a GC tandem quadrupole MS platform. By adding this temporal component to our previous work exploring dose effects at 7 d, we observed the highest fold changes occurring at 3 d, returning closer to basal levels by 7 d. These results emphasize the utility of both MS-based metabolomics for biodosimetry and complementary analytical platforms for increased metabolome coverage.

Harderian Gland Tumorigenesis: Low-Dose and LET Response.

Increased cancer risk remains a primary concern for travel into deep space and may preclude manned missions to Mars due to large uncertainties that currently exist in estimating cancer risk from the spectrum of radiations found in space with the very limited available human epidemiological radiation-induced cancer data. Existing data on human risk of cancer from X-ray and gamma-ray exposure must be scaled to the many types and fluences of radiations found in space using radiation quality factors and dose-rate modification factors, and assuming linearity of response since the shapes of the dose responses at low doses below 100 mSv are unknown. The goal of this work was to reduce uncertainties in the relative biological effect (RBE) and linear energy transfer (LET) relationship for space-relevant doses of charged-particle radiation-induced carcinogenesis. The historical data from the studies of Fry et al. and Alpen et al. for Harderian gland (HG) tumors in the female CB6F1 strain of mouse represent the most complete set of experimental observations, including dose dependence, available on a specific radiation-induced tumor in an experimental animal using heavy ion beams that are found in the cosmic radiation spectrum. However, these data lack complete information on low-dose responses below 0.1 Gy, and for chronic low-dose-rate exposures, and there are gaps in the LET region between 25 and 190 keV/µm. In this study, we used the historical HG tumorigenesis data as reference, and obtained HG tumor data for 260 MeV/u silicon (LET ∼70 keV/µm) and 1,000 MeV/u titanium (LET ∼100 keV/µm) to fill existing gaps of data in this LET range to improve our understanding of the dose-response curve at low doses, to test for deviations from linearity and to provide RBE estimates. Animals were also exposed to five daily fractions of 0.026 or 0.052 Gy of 1,000 MeV/u titanium ions to simulate chronic exposure, and HG tumorigenesis from this fractionated study were compared to the results from single 0.13 or 0.26 Gy acute titanium exposures. Theoretical modeling of the data show that a nontargeted effect model provides a better fit than the targeted effect model, providing important information at space-relevant doses of heavy ions.

Proton-induced genetic damage in lacZ transgenic mice.

The plasmid-based lacZ transgenic mouse model system was used to evaluate the mutagenic and genotoxic potential of 250 MeV/nucleon proton radiation by evaluating the frequency of micronucleated polychromatic reticulocytes in peripheral blood and bone marrow and the mutant frequencies of the lacZ reporter transgene in spleen and brain, respectively. Doses of 0.1-2 Gy produced dose- and time-dependent changes in the frequency of micronucleated polychromatic reticulocytes within 48 h, with peak induction up to sixfold above control levels. The frequency of micronucleated polychromatic reticulocytes returned to control levels within 1 week after exposure. With doses of 4 Gy, the elevation in the frequency of micronucleated polychromatic reticulocytes was delayed up to 1 week after exposure, but complete recovery to control levels was observed at 16 weeks postirradiation. Significant increase in mutant frequencies in brain tissue was observed at 8 week after proton exposure at doses as low of 0.1 Gy. Mutant frequencies in spleen increased up to twofold above spontaneous mutant frequencies at 8 weeks after exposure to 0.5-1 Gy. These effects appeared saturated at doses >1 Gy for both tissues, possibly due to elimination of damaged cells from the tissue systems. These in vivo results highlight the importance of considering tissue specificity, dose and temporal dependence when assessing radiation effects.

Genotoxicity and toxicity of the potential cancer-preventive agent polyphenon E.

The potential health benefits of green tea continue to attract public and scientific interests and are attributed in part to polyphenolic catechin constituents. Polyphenon E (Poly E) is a decaffeinated green tea catechin mixture containing about 50% epigallocatechin gallate and 30% other catechins. We evaluated the toxicity and genotoxicity of Poly E by using two in vitro assays: bacterial mutagenesis in a Salmonella typhimurium-E. coli assay and the L5178Y mouse lymphoma cell thymidine kinase (Tk) gene mutation assay. In addition, we used two in vivo genotoxicity assays: the mouse micronucleus assay and the Big Blue cII transgenic mouse mutation assay. Repeat-dose toxicity evaluations were performed in mice in parallel with the Big Blue transgenic mutation assays. No significant increases in the revertant colonies were found in the bacterial mutagenesis assay, but a significant increase in the mutant frequency (MF) at the Tk locus was observed in the mouse lymphoma test system. We observed toxicity in mice when Poly E was administered at doses of 2,000 mg/kg/day. Lower doses produced no significant increases in micronucleated erythrocytes in the bone marrow of Swiss-Webster mice and no significant increases in cII transgene MF in the liver, lung, or spleen compared with controls. These results indicate that Poly E, although toxic at high doses (2,000 mg/kg/day), poses minimal genotoxic concern. In addition, these studies highlight the importance of using both in vitro and in vivo systems in genetic toxicity screening of pharmaceuticals before they are administered to humans.

Improved oral bioavailability in rats of SR13668, a novel anti-cancer agent.

PURPOSE: SR13668, a bis-indole with potent activity in vitro and in vivo against various cancers and promising cancer chemopreventive activity, was found to have very low oral bioavailability, <1%, in rats during pilot pharmacokinetic studies. The objective of these studies was to better understand the source of low oral exposure and to develop a formulation that could be used in preclinical development studies. METHODS: An automated screening system for determining solubility in lipid-based vehicles, singly and in combination, was used to identify formulations that might enhance absorption by improving solubility of SR13668, and these results were confirmed in vivo using Sprague-Dawley rats. Pharmacokinetics of SR13668 was then determined in male and female Sprague-Dawley rats administered 1 mg/kg iv, 1, 10, and 30 mg/kg po formulated in PEG400:Labrasol (1:1 v/v). Blood was collected at time points through 24 h and the concentration of SR13668 determined using HPLC with UV and fluorescence detection. RESULTS: SR13668 was found to be resistant to plasma esterases in vitro and relatively stable to rat and human liver microsomal metabolism. SR13668 concentrates in tissues as indicated by significantly higher levels in lung compared to blood, blood concentrations ~2.5-fold higher than plasma levels, and apparent volume of distribution (V) of ~5 l/kg. A marked sex difference was observed in exposure to SR13668 with area under the curve (AUC) significantly higher and clearance (CL) lower for female compared to male rats, after both iv and oral administration. The oral bioavailability (F) of SR13668 was 25.4 ± 3.8 and 27.7 ± 3.9% (30 mg/kg), for males and females, respectively. A putative metabolite (M1), molecular weight of 445 in the negative ion mode (i.e., SR13668 + 16), was identified in blood samples from both the iv and po routes, as well as in vitro microsomal samples. CONCLUSIONS: In summary, while SR13668 does undergo metabolism, probably by the liver, the oral bioavailability of SR13668 in rats was dramatically improved by the use of formulation that contained permeation enhancers and promoted better solubilization of the drug.