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Ribonucleoprotein complexes are dynamic assemblies of RNA with RNA-binding proteins (RBPs), which can modulate the fate of the RNA molecules from transcription to degradation. Vice versa, RNA can regulate the interactions and functions of the associated proteins. Dysregulation of RBPs is linked to diseases such as cancer and neurological disorders. RNA and RBPs are present in mitotic structures like the centrosomes and spindle microtubules, but their influence on mitotic spindle integrity remains unknown. Thus, we applied the R-DeeP strategy for the proteome-wide identification of RNA-dependent proteins and complexes to cells synchronized in mitosis versus interphase. The resulting atlas of RNA-dependent proteins in cell division can be accessed through the R-DeeP 3.0 database (R-DeeP3.dkfz.de). It revealed key mitotic factors as RNA-dependent such as AURKA, KIFC1 and TPX2 that were linked to RNA despite their lack of canonical RNA-binding domains. KIFC1 was identified as a new interaction partner and phosphorylation substrate of AURKA at S 349 and T 359 . In addition, KIFC1 interacted with both, AURKA and TPX2, in an RNA-dependent manner. Our data suggest a riboregulation of mitotic protein-protein interactions during spindle assembly, offering new perspectives on the control of cell division processes by RNA-protein complexes., Competing Interests: Declaration of interests S.D. is co-owner of siTOOLs Biotech, Martinsried, Germany, without relation to this work. The other authors disclose no conflicts of interest. This study is part of the PhD thesis of V.R.

Coronaviruses (CoVs) are common among humans and many animals, causing respiratory or gastrointestinal diseases. Currently, only a few antiviral drugs against CoVs are available. Especially for SARS-CoV-2, new compounds for treatment of COVID-19 are urgently needed. In this study, we characterize the antiviral effects of two high-sulfated glycosaminoglycan (GAG) derivatives against SARS-CoV-2 and bovine coronaviruses (BCoV), which are both members of the Betacoronavirus genus. The investigated compounds are based on hyaluronan (HA) and chondroitin sulfate (CS) and exhibit a strong inhibitory effect against both CoVs. Yield assays were performed using BCoV-infected PT cells in the presence and absence of the compounds. While the high-sulfated HA (sHA3) led to an inhibition of viral growth early after infection, high-sulfated CS (sCS3) had a slightly smaller effect. Time of addition assays, where sHA3 and sCS3 were added to PT cells before, during or after infection, demonstrated an inhibitory effect during all phases of infection, whereas sHA3 showed a stronger effect even after virus absorbance. Furthermore, attachment analyses with prechilled PT cells revealed that virus attachment is not blocked. In addition, sHA3 and sCS3 inactivated BCoV by stable binding. Analysis by quantitative real-time RT PCR underlines the high potency of the inhibitors against BCoV, as well as B.1-lineage, Alpha and Beta SARS-CoV-2 viruses. Taken together, these results demonstrated that the two high-sulfated GAG derivatives exhibit low cytotoxicity and represent promising candidates for an anti-CoV therapy.

Purpose: This study evaluated the musculoskeletal injury (MSKI) self-reporting behaviors among active-duty Air Force Special Warfare personnel to explore potential limitations of injury surveillance approaches., Methods: Participants completed a 47-item survey between December 2018 and March 2019 regarding their MSKI history. Participants were asked if they sought medical care for symptoms consistent with MSKIs and reasons they did or did not report their injuries. Injury reporting rates were calculated with descriptive statistics and rank ordering was utilized to determine frequency., Results: A total of 398 airmen reported 1,057 injuries occurring in the previous 12-month period, including 508 (48%) injuries identified as not reported to medical personnel. Approximately 55% (N = 579) of all injuries were described as gradual onset. The most common reason for not reporting injuries (28.8%, N = 62) was "fear of potential impact on future career opportunities.", Conclusion: Approximately half of MSKIs in this sample of US Air Force Special Warfare personnel were not reported to medical personnel. The underreporting of injuries may pose unknown levels of risk and negatively impact military readiness levels., (2021.)

Human cytomegalovirus (HCMV) has been implicated in the development of human malignancies, for instance in colon cancer. Proteasome inhibitors were developed for cancer therapy and have also been shown to influence HCMV infection. The aim of this study was to investigate if proteasome inhibitors have therapeutic potential for colon carcinoma and how this is influenced by HCMV infection. We show by immunofluorescence and flow cytometry that the colon carcinoma cell line Caco-2 is susceptible to HCMV infection. Growth curve analysis as well as protein expression kinetics and quantitative genome analysis further confirm these results. HCMV has an anti-apoptotic effect on Caco-2 cells by inhibiting very early events of the apoptosis cascade. Further investigations showed that HCMV stabilizes the membrane potential of the mitochondria, which is typically lost very early during apoptosis. This stabilization is resistant to proteasome inhibitor Bortezomib treatment, allowing HCMV-infected cells to survive apoptotic signals. Our findings indicate a possible role of proteasome inhibitors in colon carcinoma therapy.

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses., Competing Interests: The authors have declared that no competing interests exist.

Merkel cell polyomavirus (MCPyV) is the only polyomavirus known to be associated with tumorigenesis in humans. Similarly to other polyomaviruses, MCPyV expresses a l arge t umor antigen (LT-Ag) that, together with a s mall t umor antigen (sT-Ag), contributes to cellular transformation and that is of critical importance for the initiation of the viral DNA replication. Understanding the cellular protein network regulated by MCPyV early proteins will significantly contribute to our understanding of the natural MCPyV life cycle as well as of the mechanisms by which the virus contributes to cellular transformation. We here describe KRAB-associated protein 1 (Kap1), a chromatin remodeling factor involved in cotranscriptional regulation, as a novel protein interaction partner of MCPyV T antigens sT and LT. Kap1 knockout results in a significant increase in the level of viral DNA replication that is highly suggestive of Kap1 being an important host restriction factor during MCPyV infection. Differently from other DNA viruses, MCPyV gene expression is unaffected in the absence of Kap1 and Kap1 does not associate with the viral genome. Instead, we show that in primary normal human dermal fibroblast (nHDF) cells, MCPyV DNA replication, but not T antigen expression alone, induces ataxia telangiectasia mutated (ATM) kinase-dependent Kap1 S824 phosphorylation, a mechanism that typically facilitates repair of double-strand breaks in heterochromatin by arresting the cells in G 2 We show that MCPyV-induced inhibition of cell proliferation is mainly conferred by residues within the origin binding domain and thereby by viral DNA replication. Our data suggest that phosphorylation of Kap1 and subsequent Kap1-dependent G 2 arrest/senescence represent host defense mechanisms against MCPyV replication in nHDF cells. IMPORTANCE We here describe Kap1 as a restriction factor in MCPyV infection. We report a novel, indirect mechanism by which Kap1 affects MCPyV replication. In contrast with from other DNA viruses, Kap1 does not associate with the viral genome in MCPyV infection and has no impact on viral gene expression. In MCPyV-infected nHDF cells, Kap1 phosphorylation (pKap1 S824) accumulates because of genomic stress mainly induced by viral DNA replication. In contrast, ectopic expression of LT or LT MCPyV mutants, previously shown to be important for induction of genotoxic stress, does not result in a similar extent of pKap1 accumulation. We show that cells actively replicating MCPyV accumulate pKap1 (in a manner dependent on the presence of ATM) and display a senescence phenotype reflected by G 2 arrest. These results are supported by transcriptome analyses showing that LT antigen, in a manner dependent on the presence of Kap1, induces expression of secreted factors, which is known as the senescence-associated secretory phenotype (SASP)., (Copyright © 2020 Siebels et al.)

A key step in replication of human cytomegalovirus (HCMV) in the host cell is the generation and packaging of unit-length genomes into preformed capsids. The enzymes involved in this process are the terminases. The HCMV terminase complex consists of two terminase subunits, the ATPase pUL56 and the nuclease pUL89. A potential third component pUL51 has been proposed. Even though the terminase subunit pUL89 has been shown to be essential for DNA packaging and interaction with pUL56, it is not known how pUL89 mechanistically achieves sequence-specific DNA binding and nicking. To identify essential domains and invariant amino acids vis-a-vis nuclease activity and DNA binding, alanine substitutions of predicted motifs were analyzed. The analyses indicated that aspartate 463 is an invariant amino acid for the nuclease activity, while argine 544 is an invariant aa for DNA binding. Structural analysis of recombinant protein using electron microscopy in conjunction with single particle analysis revealed a curvilinear monomer with two distinct domains connected by a thinner hinge-like region that agrees well with the predicted structure. These results allow us to model how the terminase subunit pUL89's structure may mediate its function., Competing Interests: The authors have declared that no competing interests exist.

This article discusses (1) the ways in which law functions as a determinant of health, (2) historical collaborations between the health and legal professions, (3) the benefits of creating medical-public health-legal collaborations, and (4) how viewing law through a collaborative, population health lens can lead to health equity.

The US health care system needs effective tools to address complex social and environmental issues that perpetuate health inequities, such as food insecurity, education and employment barriers, and substandard housing conditions. The medical-legal partnership is a collaborative intervention that embeds civil legal aid professionals in health care settings to address seemingly intractable social problems that contribute to poor health outcomes and health disparities. More than three hundred health care organizations are home to medical-legal partnerships. This article draws upon national survey data and field research to identify three models of the medical-legal partnership that health care organizations have adopted and the core elements of infrastructure that they share. Financing and commitment from health care organizations are key considerations for sustaining and scaling up the medical-legal partnership as a health equity intervention.

Various methods are available for DNA isolation from environmental samples. Because the chemical and biological composition of samples such as soil, sludge, or plant material is different, the effectiveness of DNA isolation can vary depending on the method applied and thus, have a substantial effect on the results of downstream analysis of the microbial community. Although the process of biogas formation is being intensely investigated, a systematic evaluation of kits for DNA isolation from material of biogas plants is still lacking. Since no DNA isolation kit specifically tailored for DNA isolation from sludge of biogas plants is available, this study compares five commercially available kits regarding their influence on downstream analyses such denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR). The results show that not all kits are equally suited for the DNA isolation from samples of different biogas plants, but highly reproducible DGGE fingerprints as well as qPCR results across the tested samples from biogas reactors using different substrate compositions could be produced using selected kits.

Degradation of biomass in the absence of exogenous electron acceptors via anaerobic digestion involves a syntrophic association of a plethora of anaerobic microorganisms. The commercial application of this process is the large-scale production of biogas from renewable feedstock as an alternative to fossil fuels. After hydrolysis of polymers, monomers are fermented to short-chain fatty acids and alcohols, which are further oxidized to acetate. Carbon dioxide, molecular hydrogen (H2), and acetate generated during the process are converted to methane by methanogenic archaea. Since many of the metabolic pathways as well as the syntrophic interactions and dependencies during anaerobic digestion involve formation, utilization, or transfer of H2, its metabolism and the methanogenic population were assessed in various samples from three commercial biogas plants. Addition of H2 significantly increased the rate of methane formation, which suggested that hydrogenotrophic methanogenesis is not a rate-limiting step during biogas formation. Methanoculleus and Methanosarcina appeared to numerically dominate the archaeal population of the three digesters, but their proportion and the Bacteria-to-Archaea ratio did not correlate with the methane productivity. Instead, hydrogenase activity in cell-free extracts from digester sludge correlated with methane productivity in a positive fashion. Since most microorganisms involved in biogas formation contain this activity, it approximates the overall anaerobic metabolic activity and may, thus, be suitable for monitoring biogas reactor performance.

We report a systematic study of carrier dynamics in Al(x)Ga(1-x)As-passivated GaAs nanowires. With passivation, the minority carrier diffusion length (L(diff)) increases from 30 to 180 nm, as measured by electron beam induced current (EBIC) mapping, and the photoluminescence (PL) lifetime increases from sub-60 ps to 1.3 ns. A 48-fold enhancement in the continuous-wave PL intensity is observed on the same individual nanowire with and without the Al(x)Ga(1-x)As passivation layer, indicating a significant reduction in surface recombination. These results indicate that, in passivated nanowires, the minority carrier lifetime is not limited by twin stacking faults. From the PL lifetime and minority carrier diffusion length, we estimate the surface recombination velocity (SRV) to range from 1.7 × 10(3) to 1.1 × 10(4) cm·s(-1), and the minority carrier mobility μ is estimated to lie in the range from 10.3 to 67.5 cm(2) V(-1) s(-1) for the passivated nanowires.

Thin Au films (~5 nm) are known to form island-like structures with small gaps between the islands, which produce intense electric field "hot spots" under visible illumination. In this work, we perform finite difference time domain (FDTD) simulations based on experimentally observed high resolution transmission electron microscope (HRTEM) images of these films in order to study the nature of the "hot spots" in more detail. Specifically, we study the dependence of the electric field intensity in the hot spots on the surrounding film environment and on the size of the nanogaps. From our simulations, we show that the surrounding film contributes significantly to the electric field intensity at the hot spot by focusing energy to it. Widening of the gap size causes a decrease in the intensity at the hot spot. However, these island-like nanoparticle hot spots are far less sensitive to gap size than nanoparticle dimer geometries, studied previously. In fact, the main factor in determining the hot spot intensity is the focusing effect of the surrounding nano-islands. We show that these random Au island films outperform more sophisticated geometries of spherical nanoparticle clusters that have been optimized using an iterative optimization algorithm.

The activity of acid sphingomyelinase (aSMase) was previously reported to be involved in glucocorticoid-induced cell death (GICD) of T lymphocytes. This mechanism in turn is believed to contribute to the therapeutic efficacy of glucocorticoids (GCs) in the treatment of inflammatory diseases. In this study, we reassessed the role of aSMase in GICD by using aSMase knockout mice. The absence of aSMase largely abolished the partial protection that effector memory CD4(+) T cells in wild-type mice possess against GICD. Reduced IL-2 secretion by aSMase-deficient CD4(+) T cells suggested that a lack of this important survival factor might be the cause of these cells' enhanced susceptibility to GICD. Indeed, addition of IL-2 restored the protection against GICD, whereas neutralization of IL-2 abrogated the otherwise protective effect seen in wild-type effector memory CD4(+) T cells. The therapeutic implications of the altered sensitivity of aSMase-deficient T cells to GICD were assessed in models of inflammatory disorders; namely, experimental autoimmune encephalomyelitis and acute graft-versus-host disease. Surprisingly, GC treatment was equally efficient in both models in terms of ameliorating the diseases, regardless of the genotype of the T cells. Thus, our data reveal a hitherto unrecognized contribution of aSMase to the sensitivity of effector memory CD4(+) T cells to GICD and call into question the traditionally attributed importance of GICD of T cells to the treatment of inflammatory diseases by GCs.

We study the effect of polarized laser annealing on the crystalline structure of individual crystalline-amorphous core-shell silicon nanowires (NWs) using Raman spectroscopy. The crystalline fraction of the annealed spot increases dramatically from 0 to 0.93 with increasing incident laser power. We observe Raman lineshape narrowing and frequency hardening upon laser annealing due to the growth of the crystalline core, which is confirmed by high resolution transmission electron microscopy (HRTEM). The anti-Stokes:Stokes Raman intensity ratio is used to determine the local heating temperature caused by the intense focused laser, which exhibits a strong polarization dependence in Si NWs. The most efficient annealing occurs when the laser polarization is aligned along the axis of the NWs, which results in an amorphous-crystalline interface less than 0.5 µm in length. This paper demonstrates a new approach to control the crystal structure of NWs on the sub-micron length scale.

We demonstrate a method for fabricating arrays of plasmonic nanoparticles with separations on the order of 1 nm using an angle evaporation technique. Samples fabricated on thin SiN membranes are imaged with high-resolution transmission electron microscopy (HRTEM) to resolve the small separations achieved between nanoparticles. When irradiated with laser light, these nearly touching metal nanoparticles produce extremely high electric field intensities, which result in surface-enhanced Raman spectroscopy (SERS) signals. We quantify these enhancements by depositing a p-aminothiophenol dye molecule on the nanoparticle arrays and spatially mapping their Raman intensities using confocal micro-Raman spectroscopy. Our results show significant enhancement when the incident laser is polarized parallel to the axis of the nanoparticle pairs, whereas no enhancement is observed for the perpendicular polarization. These results demonstrate proof-of-principle of this fabrication technique. Finite difference time domain simulations based on HRTEM images predict an electric field intensity enhancement of 82400 at the center of the nanoparticle pair and an electromagnetic SERS enhancement factor of 10(9)-10(10).

Using Raman spectroscopy, we measure the optical phonon energies of suspended graphene before, during, and after thermal cycling between 300 and 700 K. After cycling, we observe large upshifts ( approximately 25 cm(-1)) of the G band frequency in the graphene on the substrate region due to compression induced by the thermal contraction of the underlying substrate, while the G band in the suspended region remains unchanged. From these large upshifts, we estimate the compression in the substrate region to be approximately 0.4%. The large mismatch in compression between the substrate and suspended regions causes a rippling of the suspended graphene, which compensates for the change in lattice constant due to the compression. The amplitude (A) and wavelength (lambda) of the ripples, as measured by atomic force microscopy, correspond to an effective change in length Deltal/l that is consistent with the compression values determined from the Raman data.

We exploit the strong plasmon resonance of gold nanoparticles in the catalytic decomposition of CO to grow various forms of carbonaceous materials. Irradiating gold nanoparticles in a CO environment at their plasmon resonant frequency generates high temperatures and strong electric fields required to break the CO bond. By varying the laser power, exposure time, and gas flow rate, we deposit amorphous carbon, graphitic carbon, and carbon nanotubes. The formation of iron oxide nanocrystals catalyzes the growth of carbon nanotubes. Predefined microstructure geometries are patterned by moving the focused laser spot during the growth process, forming suspended single-walled carbon nanotube structures. Raman spectroscopy, energy dispersive X-ray spectroscopy, and transmission electron microscopy are used to characterize the resulting material. The localized nature of the plasmonic heating enables growth of these materials, while the underlying substrate remains at room temperature.

The carcinogenic potential of nelfinavir mesylate (nelfinavir) was evaluated in a 2-year oral (gavage) study on Sprague-Dawley rats at dose levels of 0 (control), 0 (vehicle control), 100, 300 and 1000 mg/kg per day. At the end of the treatment, increased incidences of thyroid follicular cell hyperplasia and neoplasms were observed at 300 (males) and 1000 mg/kg per day (both sexes). There were no other treatment-related effects and no tumors at other sites. Results from previous studies indicated a number of effects in the liver and thyroid, as well as metabolic profiles that suggested nelfinavir might cause thyroid hyperplasia/neoplasia secondary to hormone imbalance by altering thyroid hormone disposition. To investigate this hypothesis, the effects of nelfinavir on gene expression in rat hepatocytes and liver slices (in vitro), thyroxine plasma clearance, and thyroid gland function were evaluated. Compared to controls, gene expression analyses demonstrated an increased expression of glucuronyltransferase (UDPGT) and CYP450 3A1 in nelfinavir-treated rat hepatocytes and liver slices. In rats treated with nelfinavir (1000 mg/kg per day) for 4 weeks, liver weights and centrilobular hepatocellular hypertrophy were increased and minimal to mild diffuse thyroid follicular cell hypertrophy and follicular cell hyperplasia were evident in the thyroid gland. Thyroid-stimulating hormone (TSH) levels were significantly increased (three-fold), while tri-iodothyronine (T3)/tetra-iodothyronine (T4) and reverse T3(rT3) levels were unchanged, indicating that a compensated state to maintain homeostasis of T3/T4 had been achieved. Plasma 125I-thyroxine clearance was increased and the plasma thyroxine AUC0-48 was decreased (24%) compared to control. In conclusion, these data indicate that thyroid neoplasms observed in the nelfinavir-treated rats were secondary to thyroid hormone imbalance. Increased thyroxine clearance contributes to the effects of nelfinavir on thyroid gland function and is probably a result of UDPGT induction that leads to elevated TSH levels in the rat and eventual thyroid neoplasia. These results are consistent with a well-recognized rat-specific mechanism for thyroid neoplasms.

Coherent vortices in geostrophic turbulence grow in size and become fewer as they merge. It is shown that the deformation radius L(D) has no effect on the growth of the average vortex radius a as a grows from a L(D) to a>> L(D) . Its growth is algebraic, given by a proportional to t(xi/4) , where xi=0.72 . However, the deformation radius does have an effect on the decay of the number of vortices or vortex density rho, given by rho proportional to t(-xi) for a <> L(D) . Thus the decay of rho becomes slower once a has grown to a size comparable to that of L(D) . One scaling theory for the entire range from a<< L(D) to a>> L(D) is presented and verified by numerical experiments. A special method for quadruplicating the numerical domain when the vortices become too few is proposed, which keeps the computation inexpensive. This work generalizes and agrees with previous work, in which the two special cases a<> L(D) are independently investigated.

The ability to interpret metabolic responses to toxic insult as expressed in altered urine composition and measured by NMR spectroscopy is dependent upon a database of proton NMR spectra of urine collected from both control and treated animals. Pattern recognition techniques, such as principal component analysis (PCA), can be used to establish whether the spectral data cluster according to a dose response. However, PCA will be sensitive to other variables that might exist in the data, such as those arising from the NMR instrument itself. Thus, studies were conducted to determine the impact that NMR-related variables might impart on the data, with a view towards understanding and minimizing variables that could interfere with the interpretation of a biological effect. This study has focused on solvent suppression methods, as well as instrument-to-instrument variability, including field strength. The magnitude of the NMR-induced variability was assessed in the presence of an established response to the nephrotoxin bromoethanamine. Changes caused by the model toxin were larger and easily distinguished from those caused by using different solvent suppression methods and field strengths.

Fluoroquinolone antiinfective drugs exhibit phototoxic, photogenotoxic, and photocarcinogenic activities in experimental systems which may be interrelated. Clinafloxacin (CLX), a new fluoroquinolone, is a potent antiinfective agent being developed for use in life-threatening infections. While this drug has previously been demonstrated to be phototoxic, this report evaluated the photogenotoxic and photocarcinogenic potential of CLX. When Skh-1 mice were administered CLX in the presence of ultraviolet light (UVA) at the maximum tolerated dose expected for a photocarcinogenicity bioassay, induction of DNA strand breakage was noted in keratinocytes isolated from these animals. When compared with other well-studied fluoroquinolones in vitro, CLX and Lomefloxacin (LMX) were equally effective in producing chromosome damage and DNA strand breakage in Chinese hamster ovary (CHO) cells exposed to UVA. Treatment of CHO cells with CLX in the presence of UVA also resulted in hydroxyl radical formation. However, coincubation of CHO cells with CLX and various antioxidants markedly reduced hydroxyl radical formation, but inhibited photogenotoxicity only to a limited extent. Thus, while reactive oxygen species contribute to the photogenotoxic activity of CLX, other factors may be involved. Since CLX exhibits both phototoxic and photogenotoxic activity, we predict that CLX would be photocarcinogenic in vivo. The present study suggests that under conditions of human exposure, the potential risk for CLX-induced photocarcinogenicity is small., (Copyright 1999 Academic Press.)

The micronucleus assay is a widely accepted method for evaluation of clastogens and aneugens. In the current study, acridine orange (AO) supravital staining was adapted for flow cytometric usage to assess micronucleated cells in rat bone marrow and spleen. Cyclophosphamide was used as a positive control test compound and results were compared to manual scoring in Wright-stained slides. In bone marrow, both manual and flow cytometric methods demonstrated positive dose response-trends for micronucleated polychromatic erythrocytes (MNPCE). Significant elevations in MNPCE were observed at all doses of cyclophosphamide, and comparisons between methods in bone marrow were not statistically different. The flow cytometric method was more sensitive in spleen samples, showing dose- and time-related increases in micronuclei compared with manual scoring. AO proved to be a sensitive discriminator of RNA and DNA, allowing distinct separation of polychromatic erythrocytes (PCE), normochromic erythrocytes (NCE), total nucleated cells (TNC), and micronucleated populations within both PCE and NCE regions. These results support the use of AO-based flow cytometry to provide a rapid and sensitive indicator of micronuclei inducers., (Copyright 1998 Elsevier Science B. V.)

Cytotoxic insult to bone marrow frequently impairs the proliferating and maturational abilities of erythroid cells. Typically, a ratio of enucleated, immature polychromatic erythrocytes (PCE) to mature normochromatic erythrocytes (NCE) is used to assess cytotoxicity in the micronucleus (MN) assay. The effects of cyclophosphamide (CP) on PCE/NCE ratio in rat bone marrow and spleen were assessed by a newly developed flow cytometric procedure using glutaraldehyde-fixed, acridine orange (AO)-stained cells, and compared to manual scoring of PCE/NCE in Wright stained slides. Comparison of methods showed that manual and flow cytometric determination of PCE were not statistically different. Several other parameters of cytotoxicity could be simultaneously assessed because the method allowed use of unfractionated whole bone marrow/spleen cell samples. Absolute numbers of total nucleated cells (TNC), a ratio of TNC to total erythrocytes (TE), and determination of RNA content within the PCE population demonstrated dose- and time-dependent effects with CP treatment. Shifts in RNA content were particularly sensitive, correctly identifying all CP-treated from control specimens, even in those samples where PCE/NCE ratio was similar. The AO methodology provided a more rapid, statistically-superior, and thorough approach in the assessment of bone marrow and spleen cytotoxicity than the conventional manual method of scoring PCE/NCE ratio alone., (Copyright 1998 Elsevier Science B.V.)

Tacrine (tetrahydroaminoacridine) is a reversible cholinesterase inhibitor used for the treatment of Alzheimer's disease. This drug causes an elevation of serum aminotransferases in a limited population of patients. Several in vivo studies failed to elucidate the mechanism for the enzyme elevation but previous in vitro studies have indicated defects in mitochondrial function. In this study, electron microscopic, histochemical, and confocal microscopy techniques were used with primary hepatocyte cultures from humans and rats to examine the sequence of early cellular changes after tacrine exposure. Changes included ribosome alterations as early as 1-2 h following tacrine exposure at concentrations ranging between 0.1 and 1.0 mM. Mitochondrial membrane potential was also altered as indicated by decreased rhodamine 123 uptake with time. Cellular lysosome content increased as indicated by increased staining of fluorescein isothiocyanate (FITC)-conjugated dextran. The results of acid phosphatase histochemistry correlated with the FITC-dextran findings. Additionally, tacrine-related degranulation and vesiculation of the endoplasmic reticulum paralleled the ribosomal and mitochondrial changes. These subcellular changes were reproducible in rat and human hepatocytes, showing for the first time that human hepatocytes can be altered by tacrine. The molecular mechanism of the organelle changes is unknown at this time. Also, the relationship between these subcellular changes in isolated hepatocytes and the transaminase elevation noted in human populations treated with tacrine needs to be clarified.

In this article, an integrated in vivo genotoxicity testing philosophy and a practical approach, as applied to pharmaceuticals, are described. Recently, there has been an effort to integrate the rodent (primarily rat) micronucleus assay with routine 2-4-week toxicokinetic studies. This approach has several advantages: 1) it utilizes the general principles of toxicology that govern the overall toxicity profile of a test substance; 2) factors such as the dose and/or route of drug administration, drug metabolism, principles of toxicokinetics, and saturation of defense mechanisms are considered in evaluating genotoxicity; 3) it uses the concept of administering multiple tolerable doses aiding in achieving steady state plasma drug levels, which is more relevant for risk assessment compared to high acute doses; and 4) it helps minimize the amount of drug, number of animals used, and other resources. This integration approach can be extended to other toxicology studies and other relevant genotoxicity endpoints may be assessed. Based on the experience in our laboratory, integrating micronucleus assessment in routine toxicology testing is promising and should be utilized when practical.

Acute, subacute, and chronic toxicity studies, carcinogenicity bioassays, and reproductive and genetic toxicology studies were performed with quinapril, an ACE inhibitor used in the treatment of hypertension. Acute toxicity is minimal in rodents, and repeated dosing elicits gastric irritation, juxtaglomerular apparatus (JGA) hypertrophy and hyperplasia and tubular degenerative changes in the kidney, and reduced red cell parameters and heart weights in rodents and/or dogs. Other manifestations of toxicity, including hepatic lesions in dogs, reduced offspring weights in rats, marked sensitivity of the rabbit, and clastogenic effects at cytotoxic doses in the in vitro V79 chromosome aberration assay, have been reported with other drugs of this class.

The bacterial gyrase inhibitors, ciprofloxacin and PD 124816, were tested for clastogenic and aneugenic activity in V79 Chinese hamster lung cells in vitro. Cells were exposed for 3 h, washed free of drug, and subcultured for assessment of various endpoints. For structural chromosomal aberration (SCA) analysis, cells were incubated for 18 h, and treated with Colcemid for 2 h before harvest. For micronucleus (MN) analysis, treated cells were incubated with cytochalasin B (CYB) for 16 h. Aneugenicity was assessed by utilizing antikinetochore antibody to detect kinetochore-containing (K +) MN. Both quinolones induced significant increases in SCAs and MN, indicating clastogenic activity. With both compounds, however, the MN response was apparent at lower doses, and remained much higher throughout the dose range than the SCA response. The induced MN were predominantly K --, indicating that aneugenicity was not playing a major role in their induction. A possible explanation for the chromosome effects is that cross-reactivity of the gyrase inhibitors with mammalian topoisomerase II interferes with the separation of chromatids at anaphase leading to chromosome breaks and MN. Quinolones are known to inhibit resolution of the normally transient topoisomerase II-DNA cleavable complex, which may result in chromosome stickness. Thus, SCAs detected in metaphase cells may be attributed to quinolone-induced inhibition of topoisomerase II prior to mitosis while MN arise in binucleated cells as a result of this effect which interferes with chromatid separation during anaphase.

Selenium monosulfide (SeS) was reported to be carcinogenic to livers of male and female rats and livers and lungs of female mice. However, its genotoxicity profile in short-term assays is somewhat equivocal. A multiple endpoint/multiple tissue approach to short-term genetic toxicity testing has been developed in our laboratory. In the present paper, the effect of SeS in in vivo and in vivo/in vitro micronucleus and chromosome aberration assays in rat bone marrow and spleen are reported. In the in vivo assay, small but statistically significant increases in bone marrow micronucleated polychromatic erythrocytes (MNPCEs) were observed 24 h after treatment of rats with 50 mg/kg SeS and 48 h after treatment with 12.5 mg/kg. A significant decrease in the PCE/total erythrocyte (TE) ratio, indicative of cytotoxicity, was observed at the 50 mg/kg dose at the 24-h timepoint. In spleen, no increases in MNPCEs or decreases in the PCE/TE ratios were observed. No evidence of a significant increase in aberrations was observed in bone marrow or spleen. In the in vivo/in vitro assay, no increase in micronucleated binucleated cells or cells with aberrations was observed in SeS-treated rats. The small but statistically significant increases in MN observed in the in vivo study are considered likely not to be biologically significant since no dose-response was observed and all the values obtained were within historical control range in our laboratory. Given the overall genetic toxicity profile of SeS, it appears that SeS may be a weak mutagen and that differences between testing protocols may be very important in determining whether or not it is found to be negative or positive. Histological evidence was obtained in this study that suggests that the liver is the acute target organ of SeS in rats. Given the fact that SeS is selectively hepatocarcinogenic, we are currently testing the hypothesis that the genotoxicity of SeS in rats may be more readily detectable in liver than in bone marrow or spleen.

Tacrine is the first drug approved for the treatment of Alzheimer's disease. Approximately 50% of patients treated with tacrine develop elevated serum aminotransferase levels, as an indication of potential hepatotoxicity. However, acute and chronic studies with a limited number of animal models have not demonstrated hepatotoxicity. The present study compared the cytotoxicity in hepatocyte cultures of tacrine with structurally (proflavine and 9-aminoacridine) or pharmacologically similar compounds (physostigmine), as well as structurally modified tacrine to determine if there was a structure activity relationship with regards to toxicity. Cytotoxicity was assessed by determination of extra- and intracellular amounts of lactate dehydrogenase. Cytotoxicity was assessed after a four-hour exposure over a test compound concentration range of 0 to 3 mM. Concentration-dependent cytotoxicity occurred with tacrine and all structurally related compounds. Physostigmine which is pharmacologically similar, but structurally different, did not induce cytotoxicity. Cytotoxic potency did not appear to be related to acetylcholinesterase inhibitory activity, while compounds with acridine structures induced cytotoxicity. Thus, in this in vitro model, cytotoxicity appears to be related to structure and not pharmacological action. Results of this study indicate that compounds structurally related to tacrine are cytotoxic because of the heterocyclic ring structure. Neither unsaturation of an aromatic ring of the heterocyclic compound, amino substitution of the heterocyclic rings, N-hydroxylation of the amino group, nor ring hydroxylation dramatically alter cytotoxicity.

Tacrine is the first drug approved for the treatment of Alzheimer's disease. Approximately 50% of patients treated with tacrine develop elevated serum aminotransferase levels, an indication of potential hepatotoxicity. The mechanism of human hepatoxicity has been difficult to study, because of the absence of an animal model. Therefore, this study compared the cytotoxicity induced by tacrine in primary rat, mouse, monkey, dog, rabbit and human hepatocytes to determine differences in response to tacrine between species in vitro. Cytotoxicity was assessed by determination of extra- and intracellular lactate dehydrogenase. The ratio of intracellular enzyme to total enzyme (i.e. intracellular and extracellular) was used to represent the viabilities of the cultures. Concentration-dependent cytotoxicity occurred after four and 24-hour exposure over a tacrine concentration range of 0 to 380 micrograms/ml. Cytotoxic potency of tacrine in hepatocytes from human, dog, mouse and rat was not significantly different; monkey hepatocytes appeared slightly more sensitive, while rabbit hepatocytes appeared slightly less sensitive than human hepatocytes. Increased time of exposure to tacrine decreased the concentration necessary to induce a cytotoxic response. This in vitro model suggests only minimal differences in sensitivity to tacrine-induce cytotoxicity; therefore, cytotoxicity in primary cultures of hepatocytes from various species would appear to be related to common metabolite(s) and/or mechanism of cellular injury.

Analyses of dominant lethal (DL) mutations and micronuclei (MN) are 2 important and widely used genotoxicity assays to measure drug-induced chromosome damage in germ cells and somatic cells, respectively. Cyclophosphamide (CP) has been widely used as a positive control in the single-dose mouse MN assay; however, its utility as a positive control for the DL assay has not been fully studied. In the present study, CP was tested in both assays under similar experimental conditions and MN seen in somatic tissue (bone marrow) were correlated with DL mutations seen in germinal tissue. In a dose-range finding study, groups of 5 male mice were dosed i.p. daily for 5 days at 0, 30 or 40 mg/kg CP and bone marrow was harvested 24 h later for MN assay. CP induced a dose-related increase (7- and 11-fold over control at 30 and 40 mg/kg) in micronucleated polychromatic erythrocytes (MNPCEs) and decreased %PCEs (to 60% and 54% of controls at 30 and 40 mg/kg, respectively). Based on this, a definitive DL and MN study was conducted using separate groups of 30 male mice at 0 and 40 mg/kg CP with a daily times 5 dosing regimen. For the MN assay, bone marrow was collected 24 h after the last dose from 5 animals and evaluated for MNPCEs and %PCEs. For the DL assay, each male was caged with 2 untreated females per week for 8 weeks to cover the postmeiotic germ cell stages. On day 17 after the initiation of breeding, the females were evaluated for the number of implantation sites and live, dead and resorbed implants. The results indicated that CP induced about a 17-fold increase in MNPCEs and a 46% decrease in PCEs in relation to controls. In the DL assay, CP produced a slight (13%) but statistically significant reduction in fertility index at week 7 of mating. Also, the total number of implants was significantly lower during weeks 1, 2, 3, 6 and 7 and the numbers of dead implants and postimplantation loss (PIL) were increased for weeks 1, 2 and 3 (55%, 71% and 34% PIL, respectively) over controls. These data clearly show that CP produced clastogenicity and some toxicity in both somatic tissue and germinal tissue. It was concluded that a dose of 40 mg/kg CP can be used as a positive control compound in the DL assay and in the multiple-dose marrow MN assay.

Apoptosis or programmed cell death is a biological event that is biochemically and morphologically distinct from cellular necrosis. Nonetheless, its relationship has not been studied in terms of a cytogenetic endpoint such as micronucleus formation. In the present study, based on cytological observations, the incidence of dexamethasone-induced apoptotic cells was related to the frequency of micronucleated cells in vitro. Rat primary spleen cells were grown in 6-well plates with RPMI 1640 media using concanavalin A and lipopolysaccharide as mitogens. At culture initiation, the test agent dexamethasone (10, 20 or 40 microM) and a cytokinesis inhibitor cytochalasin B (3 micrograms/ml) were added. Cultures were harvested 18 h and 40 h later. Slides were prepared and stained with Diff-Quik stain. Frequencies of apoptotic cells and micronucleated binucleate cells were enumerated cytologically based on 500 cells per treatment from the same slides. The results showed a dose-dependent increase in the number of apoptotic cells in rat spleen cultures treated with dexamethasone. At 18 h, the percentages of apoptotic cells were 0.8, 1.6, 3.4 and 4.4 with 0, 10, 20 and 40 microM dexamethasone, respectively. The corresponding percentages of apoptotic cells at 40 h were: 2.8, 2.6, 5.6 and 10.4. However, at the same concentrations of dexamethasone, the micronucleus frequency in binucleate cells remained relatively unchanged. The phenomenon of apoptosis induced by dexamethasone was confirmed biochemically based on a characteristic DNA 'ladder' pattern by gel electrophoresis. These data suggest that dexamethasone at the concentrations which induced apoptosis did not produce cytogenetic damage. Also, these findings indicate that micronucleus formation and nuclear changes leading to apoptosis are separate events and these endpoints may not be closely correlated for dexamethasone.

The mouse micronucleus assay has long been used as an indicator of in vivo genotoxicity. Recently, it was shown that no single protocol is adequate to detect all clastogens. As a first step in developing a potentially more sensitive assay, micronucleus induction by cyclophosphamide (CP) was assessed in an in vivo/in vitro system using rat bone marrow and spleen cells. In each of two independent experiments, two rats/dose were treated i.p. with 0, 20, or 40 mg CP/kg and killed 6 h later. Cultures were then established in the presence of growth stimulants (interleukin-3 and granulocyte-macrophage colony stimulating factor for bone marrow; lipopolysaccharide and concanavalin A for spleen) and cytochalasin B, a cytokinesis inhibitor. Bone marrow cells were harvested and slides prepared 24 h after initiation, while spleen cells were harvested at 48 h. One thousand cells/tissue/group were scored for cell cycle kinetics and 1000 binucleate (BN) cells were scored for micronuclei. In addition, spleen cells were concurrently assayed for chromosome aberrations. A dose-related cell cycle delay was observed in both tissues in both experiments. Bone marrow showed a 6% average background frequency of micronucleated BN cells, while the low dose induced an average of 20%, and the high dose 31%. For spleen, the average control frequency of micronucleated BN cells was 3%, the low dose induced a 40% average frequency, and the high dose 65%. Also in splenocytes, a dose-dependent increase in chromosome aberrations was observed, with an almost 40-fold increase observed over the control value at the high dose. Thus, the in vivo/in vitro approach described here shows great potential in detecting drug induced genotoxicity. Also, spleen appears more sensitive than bone marrow to CP.

An in vivo/in vitro system using rat bone marrow cells and spleen cells to assess micronucleus (MN) and structural chromosome aberrations (SCA) simultaneously (Moore et al., 1995) was further developed. In two separate experiments, two rats/dose/experiment were treated i.p. with 0, 5, 10 and 15 mg chlorambucil (CA)/kg or with mitomycin C (MMC) at 0, 1, 2, 4 mg/kg (experiment 1) or 0, 4, 6, and 8 mg/kg (experiment 2) and killed 6 h later. Cultures were then established in the presence of growth stimulants (interleukin-3 and granulocyte-macrophage colony stimulating factor for bone marrow; lipopolysaccharide and concanavalin A for spleen) and cytochalasin B, a cytokinesis inhibitor. Bone marrow cells were harvested 24 h after establishment of cultures, while spleen cells were harvested at 48 h. In addition, spleen cells were concurrently assayed for chromosome aberrations. With the MN endpoint, spleen cells appeared more sensitive than bone marrow cells to the effects of CA due both to a lower background and an increased response. For MMC, bone marrow cells exhibited both a higher background of MN and a greater numerical response than did spleen cells. However, on the basis of a fold-increase over control values, spleen cells were more sensitive than bone marrow cells. In general, the MN endpoint appeared more sensitive than the SCA in spleen cells after treatment with CA or MMC. Thus, the approach described here shows greater potential in detecting genotoxicity.

While HMG-CoA reductase inhibitors such as fluvastatin, lovastatin, pravastatin and simvastatin demonstrate lack of in vitro and in vivo mutagenicity and clastogenicity in bacterial and mammalian cells, long term rodent carcinogenicity studies resulted in an increased incidence in neoplasms at high doses. These effects may be attributable to an exaggeration of the desired biochemical effect of the drug and/or a tumor promoting effect. The genotoxicity of atorvastatin, a newly developed HMG-CoA reductase inhibitor, was evaluated in a variety of test systems. In bacterial mutagenicity tests, the E. coli tester strain WP2(uvrA) and S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 were exposed to concentrations of atorvastatin as high as 5000 micrograms/plate both in the absence (S9-) and presence (S9+) of metabolic activation. Atorvastatin was not mutagenic in either E. coli or S. typhimurium. Chinese hamster lung V79 cell cultures were exposed to atorvastatin at concentrations of 50-300 micrograms/ml (S9-) and 100-300 micrograms/ml (S9+) and structural chromosome aberrations were assessed. Mutation at the hgprt locus was assessed at concentrations of 100-300 micrograms/ml (S9-) and 150-275 micrograms/ml (S9+). Atorvastatin was neither mutagenic nor clastogenic in the absence or presence of S9. The lack of in vitro genotoxicity was corroborated in vivo in a mouse micronucleus study in which single oral doses of atorvastatin were administered to male and female CD-1 mice at 1, 2500, or 5000 mg/kg. No biologically significant increases in the frequency of micronucleated polychromatic erythrocytes in bone marrow at 24, 48, or 72 h postdosing were observed. Thus, atorvastatin, as with the other tested HMG-CoA reductase inhibitors, is not genotoxic.

Simultaneous assessment of in vivo micronucleus and chromosome aberration endpoints has been described in the rat and the mouse. Bone marrow and spleen cells were utilized for genotoxicity assessment. A cellulose column methodology was used in the micronucleus assay (where applicable) to eliminate nucleated cells and facilitate cytogenetic scoring. The test agents--cyclophosphamide, chlorambucil, and acrylamide--produced qualitatively comparable results between micronucleus and chromosome aberration endpoints and varied slightly on a quantitative basis depending on the type of test agent and tissue studied. The results from test agents such as cyclophosphamide, chlorambucil, acrylamide, dimethylnitrosamine, vincristine, and x-rays indicated that bone marrow cytogenetic results are similar to spleen and that the spleen tissue is at least as sensitive as the bone marrow. The concurrent analysis of cytogenetic damage in vivo using a multiple endpoint-multiple tissue approach described here has the following advantages: a) reducing the overall animal usage, b) clarifying marginal micronucleus and/or chromosome aberration data, c) correlating cytogenetic results from multiple endpoints and multiple tissues, and d) helping the investigation of the mechanism of action of test agents and their potential target organs. Also, the multiple endpoint-multiple tissue approach can be extended to other endpoints, tissues, and species (where appropriate and practical) to obtain detailed genotoxicity information.

As part of the International Workshop on Standardization of Genotoxicity Test Procedures, in Melbourne, 27-28 February 1993, various international guidelines were examined with respect to protocol issues in the area of mammalian cell gene mutation assays. The working group on mammalian cell gene mutation assays discussed a wide range of protocol issues related to study design; in most cases the recommendations are reasonably consistent with existing guidelines. Agreement was reached on several issues as follows. The upper limit of concentration for testing non-toxic substances should be 10 mM or 5 mg/ml, whichever is lower. For testing toxic substances the criteria of an acceptable upper limit of concentration should yield 10-20% survival. Any of several established mammalian cell mutation assays (L5178Y TK+/-, CHO/HPRT, AS52/XPRT, V79/HPRT) can be used to evaluate mutagenesis in mammalian cells; the ouabain (Na/K-ATPase) system is not an acceptable mutation assay for routine evaluation of mutagenesis in mammalian cells. Ability to recover small colonies must be convincingly demonstrated when using the L5178Y TK+/- mouse lymphoma assay. In the mouse lymphoma assay (L5178Y TK+/-), colonies in positive controls and at least two (if available) representative positive doses of the test compound should be sized if a positive response is seen; in the event of a negative response due to the test compound, colony sizing of the positive control is necessary to validate the conduct of the assay. Testing both in the presence and absence of S9 metabolic activation is necessary. It was not possible to come to a firm conclusion about the length of treatment. There was a general agreement that extended treatment times (> 2 cell cycles) often bear more disadvantages than advantages and should only be used with adequate justification. It is not necessary to repeat clear positive or clear negative tests when the assay has been adequately performed; this recommendation differs significantly from the UK guidelines. If treatment groups are not replicated, the numbers of doses tested should be increased; this recommendation differs significantly from the UK guidelines. Each laboratory should establish a historical database for the performance of a given assay in that laboratory.

Bone marrow and spleen toxicity, clastogenicity and aneugenicity were analyzed in the CD1 mouse using an antikinetochore antibody (AKA) procedure (Krishna et al., Mutation Res., 282, 159-169, 1992). Further, to verify the fluorescence micronucleus (MN) analysis, additional slides were stained with Wright's Giemsa and results were compared. 5 mice per sex were treated with cyclophosphamide (CP) (40 mg/kg) or vincristine (VC) (0.1 or 0.2 mg/kg). Slides were prepared 24 h postdose using a column fractionation procedure. Per animal, 400 total erythrocytes (TEs) for toxicity and 2000 polychromatic erythrocytes (PCEs) for MN per tissue were analyzed. In the fluorescent method, the clastogen, CP, produced MNPCEs predominantly devoid of kinetochores (K) and the aneugen, VC, produced mostly MNPCEs containing K. The MNPCE frequency did not differ significantly between tissues; however, it differed statistically between sexes. On an overall basis, spleen had significantly lower PCE to TE ratios compared to bone marrow. In general, CP and VC caused a small, but statistically significant decrease in PCE frequencies compared to controls, suggesting possible toxicity to these tissues at the given doses. The data on Wright's stain indicated that the proportion of PCEs and MNPCEs in general, were comparable to those using fluorescent stain. This study further confirms the usefulness of an AKA-staining technique in a multiple genetic endpoint evaluation under a single set of microscopic conditions.

Chemical methods for the degradation of 11 antineoplastic drugs [etoposide, teniposide, bleomycin, mitomycin C, cisplatin, cis-dichloro-trans-dihydroxy-bis(isopropylamine) platinum IV (CHIP), cyclophosphamide, ifosfamide, carmustine, lomustine, and methotrexate] were investigated. The success of the degradation procedures was assessed by HPLC and degree of biological inactivation by mutagenicity assays. The most widely applicable procedure was oxidation with potassium permanganate or 5.25% sodium hypochlorite solution (bleach). Oxidation completely degraded and inactivated etoposide, teniposide, bleomycin, mitomycin C, and methotrexate. In addition, oxidation followed by nucleophilic substitution resulted in the complete degradation and inactivation of cyclophosphamide and ifosfamide. Although carmustine and lomustine were chemically degraded by treatment with acidic potassium permanganate, the resulting reaction mixtures remained mutagenic. Therefore, this procedure cannot be recommended. The platinum-containing compounds, cisplatin and CHIP, were rendered nonmutagenic by reaction with sodium diethyldithiocarbamate. These easily performed, relatively safe procedures can be used to prevent exposure to mutagenic wastes and spills in the hospital setting.

In the present study, cellulose-column fractionation methodology which has been used to eliminate nucleated cells in bone marrow was verified for its usefulness in micronucleus analysis and compared to standard smear methodology using cyclophosphamide as the test compound. Also, the possibility of using column-fractionated cells in the evaluation of micronucleus frequency by flow cytometry has been explored and comparative results are reported. The results indicated that column fractionation was effective in removing nucleated cells from mouse bone marrow and provided clean preparations of polychromatic and normochromatic erythrocytes (PCEs and NCEs). An initial comparison of manual scoring of cyclophosphamide-induced (10, 20 or 40 mg/kg) micronucleus frequency between standard whole bone-marrow smear and column-fractionated cytospun smears from the same animals showed comparable results. In a definitive study, manual scoring of micronuclei in whole bone marrow was compared with the column-fractionated cell preparations quantified manually and using flow cytometry. Statistically significant positive dose-related trends were detected with all 3 methods, with each treatment group having significantly elevated micronucleated PCEs (MNPCEs) compared to the control group. The 3 methods provided comparable MNPCE values for the lower dose groups but diverged somewhat for the high dose group. The flow method yielded similar individual animal variability in the data when compared to the other two methods. These results support the use of column fractionation in the enumeration of MNPCEs and indicate that coupling this technique with flow cytometry may provide a rapid and sensitive method for the conduct of mouse bone-marrow micronucleus studies.

A high capacity in vitro micronucleus assay was developed to evaluate the ability of selected 6-fluorinated quinolone and naphthyridone antibacterial compounds to induce micronuclei (MN) in vitro in V79 Chinese hamster lung cells. Log-phase cells in six-well cluster dishes were exposed for 3 h in the absence of S9 to 34 compounds. After treatment, cells were refed with media containing cytochalasin B, incubated for 16 h, and harvested for cell-cycle kinetics (CCK) and MN analyses. The quinolones tested were grouped according to the substituent at the 8-position. All 4 compounds having a halogen substitution at position 8, five of the six 8-trifluoromethyl quinolones, and all eight 8-methoxy-substituted compounds induced a significant increase in MN. Only 5 of the 10 naphthyridone compounds tested, having a variety of substituents at the 7-position, were inducers of MN and the overall magnitude of the response was less than with the quinolones. The minimum clastogenic concentration for the quinolones ranged from 4 to 400 micrograms/ml and for the naphthyridones this range was from 22.5 to 100 micrograms/ml. In the groups examined, napthyridone compounds were less likely than quinolones to induce in vitro MN, particularly when the substituent at the 7-position in the naphthyridone contains some bulk (methyl groups) around the amine side-chain. Most of the quinolones tested induced MN, irrespective of the substituents at positions 7 or 8.

An improved antikinetochore antibody technique was established in the mouse micronucleus assay to simultaneously evaluate toxicity, clastogenicity and aneugenicity induced by various test agents. The procedure involved the use of cellulose column fractionated cytospun slides for analysis. The staining method consisted of sequential treatment of slides with crest serum, fluorosceinated goat-antihuman and swine-antigoat antibodies, and propidium iodide. In this method, polychromatic erythrocytes (PCEs, dark red), normochromatic erythrocytes (NCEs, green), chromosome(s)/fragments/micronuclei (orange), and kinetochores (yellow), are identified using the same filter setting under blue excitation (440-490 nm) with a barrier filter at 520 nm. Using this method, three agents, cyclophosphamide, X-rays and vincristine were tested for micronucleus/aneuploidy induction and bone marrow toxicity. The aneugen, vincristine, and clastogens, X-rays and cyclophosphamide, induced predominantly kinetochore positive (K+) and negative (K-) micronucleated PCEs, respectively. At the doses tested, cyclophosphamide caused a slight but statistically significant decrease in PCEs in females, and other agents did not produce any severe bone-marrow toxicity in either male or female mice. These results are comparable with the results reported in the literature on these compounds with various methods and thus demonstrate the usefulness of this assay in distinguishing clastogenicity from aneugenicity and in evaluating toxicity.

Cytogenetic analyses were conducted on bone marrow and pulmonary lavage cells from rats that received repeated inhalation exposures to formaldehyde. Male Sprague-Dawley rats were exposed to 0, 0.5, 3, or 15 ppm formaldehyde for 6 h per day, 5 days per week, for 1 and 8 weeks. There was no significant increase in chromosomal abnormalities in the bone marrow cells of formaldehyde-exposed rats relative to controls. There was a statistically significant increase in chromosomal aberrations in the pulmonary lavage cells from rats that inhaled 15 ppm. There were 7.6 and 9.2% of the scored pulmonary lavage cells that had aberrations following 1 and 8 weeks, respectively, of 15 ppm formaldehyde exposure (with control levels of 3.5 and 4.8%, respectively). The predominant damage seen was chromatid breaks. These findings indicate that marginal but statistically significant genotoxic effects could be detected locally in lung alveolar macrophages, but not distally in bone marrow, following repeated formaldehyde exposures only at a high concentration that is carcinogenic to rats. The biological significance of this effect is uncertain since formaldehyde is not considered to be a lung carcinogen in rats.

A modified antikinetochore antibody technique was established in the V79 Chinese hamster lung cells to simultaneously analyze chromosome damage and aneuploidy induced by various agents. The method involved sequential treatment of slides with crest serum, fluoresceinated goat-antihuman and swine-antigoat antibodies, and propidium iodide. In this method, cytoplasm (green), nuclei or micronuclei (red), and kinetochores (yellow), are identified using the same filter setting under blue excitation (440-490 nm) with a barrier filter at 520 nm. Using this method, three agents, vinblastine (VB), X-rays, and methyl methanesulfonate (MMS) were tested for micronucleus/aneuploidy induction. An aneugen, VB and a clastogen, X-rays, induced predominantly kinetochore positive (K+) and negative (K-) micronucleated binucleate (MNBN) cells, respectively, in a dose-dependent fashion. An alkylating agent, MMS, produced both K+ and K- MNBN cells. These results are comparable with the results reported in the literature on these compounds using various methods and thus demonstrate the usefulness of this assay in distinguishing clastogenicity from aneugenicity.

During a routine Ames assay of a potential antipsychotic drug candidate, the compound appeared to be a frameshift mutagen in Salmonella typhimurium strains TA98 and TA1538. Additional testing indicated the mutagenic activity was due to one or more contaminants incurred during synthesis. While the compound was initially shown to be greater than 98% pure by high-performance liquid chromatography, the presence of small amounts (0.01-0.1%) of a highly mutagenic impurity produced positive mutagenicity results. The need to assess for chemical purity before discontinuing development of drug candidates found positive in the Ames assay is discussed.

The genotoxicity of pirmenol was tested in the E. coli and S. typhimurium mutagenesis assay, an in vitro mammalian cell chromosome-aberration assay and an in vivo mouse micronucleus assay. The E. coli tester strain WP2s was exposed to concentrations of pirmenol as high as 10,000 micrograms/plate both in the absence (S9-) and presence (S9+) of metabolic activation. Five strains of S. typhimurium (TA98, TA100, TA1535, TA1537, TA1538) were exposed to concentrations of pirmenol as high as 5000 micrograms/plate in the absence and presence of S9. Pirmenol was not mutagenic toward either E. coli or S. typhimurium. Chinese hamster lung V79 cell cultures were exposed to pirmenol at concentrations of 500-2500 micrograms/ml (S9-) and 500-3000 micrograms/ml (S9+). Pirmenol increased the frequency of structural chromosome aberrations (SCAs). The minimum clastogenic concentration was 1500 micrograms/ml (both S9- and S9+) with a peak clastogenic response of 6% (S9-) and 34% (S9+) cells with aberrations. Although there were statistically significant results in the S9- experiment, the percent cells with aberration values for treated groups were within the historical control range (0-6%) of this laboratory. The observed effects in both the absence and presence of S9 appear at high concentrations compared to human circulating plasma levels of 1-3 micrograms/ml and the clastogenicity was confined to chromosome gaps and breaks. Consequently, this in vitro effect would not be expected to be reflected by either in vivo clastogenic or carcinogenic activity. This was supported by findings in the mouse micronucleus study of pirmenol in which single oral doses administered to male CD-1 mice at 5, 55, or 115 mg/kg (80% LD50) produced no statistically significant increases in the frequency of micronucleated polychromatic erythrocytes in bone marrow at 24, 48 or 72 h postdosing. Additionally, no evidence of carcinogenicity was seen in a mouse or rat bioassay.

Using a cellulose column fractionation procedure to eliminate nucleated cells for micronucleus assessment, micronucleus and chromosome aberration endpoints in the same animal were compared in male and female rats following i.p. injection with cyclophosphamide (CP). Groups of 5 Wistar rats per sex were given single doses of CP at 0, 20, or 40 mg/kg. Two hours prior to sacrifice, animals were given colchicine (4 mg/kg) to arrest cells in metaphase. One femur from each animal was used for micronucleus assessment and the other for chromosome aberration assessment. In the micronucleus assessment, 2000 polychromatic erythrocytes (PCEs) per animal and in the chromosome aberration assessment, 50 metaphase cells per animal were scored. This experiment was repeated once. In both experiments, significant increases in micronucleated PCEs and chromosome aberrations were noted at both doses of CP in both sexes. In general, the clastogenic effects of CP were more pronounced in males than females. Both doses of CP caused a decrease in the proportion of PCEs and in mitotic index in both experiments, indicating toxicity of CP to the bone marrow. These results show the usefulness of this rat model for simultaneous evaluation of two cytogenetic endpoints in the same animal and indicate that assessment of MNPCE frequency in the bone marrow of male rats may be an appropriate model for screening test substances for in vivo clastogenic activity in this species.

The present study was designed to obtain information on the kinetics of micronucleus (MN) formation following dimethylnitrosamine (DMN) treatment in mice. Male mice were injected once intraperitoneally with 50 or 100 mg/kg DMN. Bone marrow and spleen were obtained at various sacrifice time-points and processed for micronucleus analysis. The vehicle control group had 0.6 and 0.9 MN polychromatic erythrocytes (PCEs)/1000 PCEs in bone marrow and spleen, respectively. DMN, at 50 mg/kg, caused 3.8, 7.8, 8.5 and 10.2 MN PCEs/1000 PCEs in bone marrow and 8.0, 9.2, 19.3 and 32.8 MN PCEs/1000 PCEs in spleen at 12, 24, 36 and 48 h sacrifice times, respectively. A similar time-related elevation of micronucleus frequency was noted for 100 mg/kg DMN. At each sacrifice time-point, spleen PCEs had a higher micronucleus frequency than bone-marrow PCEs. In general, DMN decreased the proportion of PCEs to total erythrocytes, suggesting toxicity. Thus, this study demonstrates the clastogenic activity of DMN in both bone-marrow and spleen PCEs of mice and shows a time-related pattern in elevating DMN-induced MN PCE frequency.