Rodent Bone Marrow Research Articles

273 Hepatocyte specific metabolic activity can be induced in adult liver stem cells isolated from rodent bone marrow

273 Hepatocyte specific metabolic activity can be induced in adult liver stem cells isolated from rodent bone marrow

Micronucleus induction in mice exposed to diazoaminobenzene or its metabolites, benzene and aniline: implications for diazoaminobenzene carcinogenicity

Diazoaminobenzene (DAAB), a manufacturing intermediate metabolized primarily to the known carcinogens benzene and aniline, has been identified as an impurity in a number of dyes and coloring agents that are components of cosmetics, food products, and pharmaceuticals. Several structural analogs of DAAB are carcinogenic as well. DAAB was selected for metabolism and toxicity studies by the National Toxicology Program (NTP) based on the potential for human exposure, positive Salmonella data, and lack of adequate toxicological data. In the toxicology studies in mice, DAAB exhibited properties similar to benzene and aniline. Because both these metabolites induce micronuclei (MN) in rodent bone marrow erythrocytes, DAAB was tested for induction of micronuclei in male B6C3F 1 mice. DAAB was administered twice by corn oil gavage at 24 h intervals, at doses of 25, 50, and 100 mg/kg per day. In addition, comparative micronucleus tests were conducted with benzene, aniline, and a mixture of benzene plus aniline; doses were based on the respective molar equivalents of each metabolite to DAAB. It was hypothesized that any observed increase in micronuclei seen in DAAB-treated mice would be due primarily to the effects of the benzene metabolite, as benzene is a more potent inducer of chromosomal damage than aniline. Results of this study showed that DAAB and benzene were effective inducers of micronuclei, with stronger responses noted for DAAB at higher doses. Positive results were also obtained with the mixture of benzene and aniline, although the magnitude of the response was lower than for DAAB. Aniline gave a weak positive response at doses exceeding its molar equivalent to 100 mg/kg DAAB. Overall, the data indicated that DAAB is a potent inducer of micronuclei in mice, and its activity appears to be closely related to the activity of benzene, one of its primary metabolites. The results are consistent with a prediction of carcinogenicity for DAAB.

A re-assessment of styrene-induced clastogenicity in mice in a subacute inhalation study.

To date, a number of in vivo cytogenetic assays have studied the clastogenicity (chromosome aberrations, micronuclei formation) in bone marrow of rodents exposed to styrene by various routes. The majority of all these cytogenetic experiments yielded negative findings (Scott and Preston, Mutat Res 318:175-203, 1994). Recently published data from a micronucleus test in mice exposed via inhalation for up to 21 days showed some positive response, but was not fully conclusive (Vodicka et al., Chem Biol Interact 137:213-227, 2001). Since this exposure regimen has considerable relevance for workplace exposure, the present study was performed to further elucidate these findings. NMRI mice were exposed by whole body inhalation to styrene concentrations of 750 mg/m3 and 1,500 mg/m3 for 1, 3, 7, 14 and 21 consecutive days (6 h/day). Animals were killed directly after exposure and bone marrow was sampled for analysis of micronucleus induction. Under the experimental conditions used in the present investigation, there was no evidence of clastogenicity at any concentration or exposure interval.

Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain.

Recent studies have shown that cells from bone marrow (BM), muscle, and brain may have greater plasticity than previously known. We have identified multipotent adult progenitor cells (MAPC) in postnatal human and rodent BM that copurify with mesenchymal stem cells (MSC). BM MAPC proliferate without senescence and differentiate into mesodermal, neuroectodermal, and endodermal cell types. We hypothesized that cells with characteristics similar to BM MAPC can be selected and cultured from tissues other than BM. BM, whole brain, and whole muscle tissue was obtained from mice. Cells were plated on Dulbecco modified Eagle medium supplemented with 2% fetal calf serum and 10 ng/mL epidermal growth factor (EGF), 10 ng/mL platelet-derived growth factor (PDGF-BB), and 1000 units/mL leukemia inhibitory factor (LIF) for more than 6 months. Cells were maintained between 0.5 and 1.5 x 10(3) cells/cm(2). At variable time points, we tested cell phenotype by FACS and evaluated their differentiation into endothelial cells, neuroectodermal cells, and endodermal cells in vitro. We also compared the expressed gene profile in BM, muscle, and brain MAPC by Affimetrix gene array analysis. Cells could be cultured from BM, muscle, and brain that proliferated for more than 70 population doublings (PDs) and were negative for CD44, CD45, major histocompatibility complex class I and II, and c-kit. Cells from the three tissues differentiated to cells with morphologic and phenotypic characteristics of endothelium, neurons, glia, and hepatocytes. The expressed gene profile of cells derived from the three tissues was identical (r(2) > 0.975). This study shows that cells with MAPC characteristics can be isolated not only from BM, but also from brain and muscle tissue. Whether MAPC originally derived from BM are circulating or all organs contain stem cells with MAPC characteristics currently is being studied. Presence of MAPC in multiple tissues may help explain the "plasticity" found in multiple adult tissues.

B cell development in gut associated lymphoid tissues

B lymphocyte development can occur in a variety of anatomical sites. While typically considered to be a process that occurs in the bone marrow throughout life, it is becoming clear that gut associates sites of B cell development are critically important in many species of veterinary importance. Among these sites, the bursa of Fabricius in chickens and the ileal Peyer’s patches of sheep are among the best studied. In these organs, it has become clear that many of the properties associated with B cell development in rodent and primate bone marrow do not apply. Thus while bone marrow B cell development typically involves an ongoing maturation of mature B cells from immature B lineage precursors that lack the expression of a surface immunoglobulin complex, gut associated lymphoid tissues (GALTs) may be colonized by a single wave of precursor cells during embryo development. Nonetheless, molecular analysis of the requirements for B lymphocyte development in GALTs reveals some striking parallels with requirements identified for B cell development in bone marrow. This article will discuss differences between B cell development in the bone marrow and GALTs and recent evidence emerging that yields insights into how these processes are regulated.

Expression of Neural Markers in Human Umbilical Cord Blood

A population of cells derived from human and rodent bone marrow has been shown by several groups of investigators to give rise to glia and neuron-like cells. Here we show that human umbilical cord blood cells treated with retinoic acid (RA) and nerve growth factor (NGF) exhibited a change in phenotype and expressed molecular markers usually associated with neurons and glia. Musashi-1 and β-tubulin III, proteins found in early neuronal development, were expressed in the induced cord blood cells. Other molecules associated with neurons in the literature, such as glypican 4 and pleiotrophin mRNA, were detected using DNA microarray analysis and confirmed independently with reverse transcriptase polymerase chain reaction (RT-PCR). Glial fibrillary acidic protein (GFAP) and its mRNA were also detected in both the induced and untreated cord blood cells. Umbilical cord blood appears to be more versatile than previously known and may have therapeutic potential for neuronal replacement or gene delivery in neurodegenerative diseases, trauma, and genetic disorders.

Continuing ability of the rodent bone marrow micronucleus assay to act as a predictor of the possible germ cell mutagenicity of chemicals

Continuing ability of the rodent bone marrow micronucleus assay to act as a predictor of the possible germ cell mutagenicity of chemicals

Further evidence for the rodent bone marrow micronucleus assay acting as a sensitive predictor of the possible germ cell mutagenicity of chemicals

Further evidence for the rodent bone marrow micronucleus assay acting as a sensitive predictor of the possible germ cell mutagenicity of chemicals

Practical aspects of mutagenicity testing strategy: an industrial perspective

Genetic toxicology studies play a central role in the development and marketing of new chemicals for pharmaceutical, agricultural, industrial, and consumer use. During the discovery phase of product development, rapid screening tests that require minimal amounts of test materials are used to assist in the design and prioritization of new molecules. At this stage, a modified Salmonella reverse mutation assay and an in vitro micronucleus test with mammalian cell culture are frequently used for screening. Regulatory genetic toxicology studies are conducted with a short list of compounds using protocols that conform to various international guidelines. A set of four assays usually constitutes the minimum test battery that satisfies global requirements. This set includes a bacterial reverse mutation assay, an in vitro cytogenetic test with mammalian cell culture, an in vitro gene mutation assay in mammalian cell cultures, and an in vivo rodent bone marrow micronucleus test. Supplementary studies are conducted in certain instances either as a follow-up to the findings from this initial testing battery and/or to satisfy a regulatory requirement. Currently available genetic toxicology assays have helped the scientific and industrial community over the past several decades in evaluating the mutagenic potential of chemical agents. The emerging field of toxicogenomics has the potential to redefine our ability to study the response of cells to genetic damage and hence our ability to study threshold phenomenon.

Historical vehicle and positive control micronucleus data in mice and rats

The rodent bone marrow micronucleus (MN) assay has been widely used as part of an in vivo genotoxicity test battery in product safety evaluation. In this assay, the historical vehicle and positive control data form an important component in the assay performance and data interpretation. Also, in light of minimizing animal use in research and still obtain required data from a study, the routine use of positive control in every MN assay has been questioned by the scientific community, especially in laboratories which have demonstrated assay reproducibility and conduct studies under Good Laboratory Practice regulations. In this paper, mouse and rat vehicle and positive control MN data, collected manually, are described as a reference for a period of 12 years (1987–1998) in our laboratory. The vehicles generally included a variety of aqueous solutions and suspensions and cyclophosphamide dosed intraperitoneally at 20 mg/kg (rats) or 40 mg/kg (mice) served as positive control, in all studies. Based on combined sex data (430 animals), for CD 1 mice, the vehicle control MN polychromatic erythrocyte (PCE) range was 0.9–3.1 with a mean of 1.75 per 1000 PCE and the positive control range (220 animals) was 8.8–42.1 with a mean of 23.1 MNPCE per 1000 PCE. Similarly, for Wistar rats, the vehicle control range (360 animals) was 1.3–5.3 with a mean of 2.6 MNPCE per 1000 PCE and the positive control range (240 animals) was 10.4–33.8 MNPCE per 1000 PCE. Vehicle control ranges reported here are comparable to the literature database and the positive control response was ≥4-fold over vehicle control, in all studies. These data demonstrate the reproducibility of positive control response in MN assay in our laboratory and support the MN Assay Expert Panel’s view that the use of positive control may not be necessary in every study.

Avoidance of bone marrow suppression using A-5021 as a nucleoside analog for retrovirus-mediated herpes simplex virus type I thymidine kinase gene therapy.

Gene therapy using the herpes simplex virus thymidine kinase (HSV-TK) gene combined with an anti-herpes drug, ganciclovir (GCV), has been applied for human diseases, especially for cancer treatment. However, bone marrow toxicity has been the most consistent adverse effect of GCV treatment in clinical settings. We evaluated the cytotoxic activity of a novel guanosine analog, (1'S,2'R)-9[[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanin e (A-5021), against retrovirus-mediated HSV-TK gene-transduced human lung cancer cells. The bone marrow toxicity of A-5021 and GCV was studied by colony formation assay in both rodent and human bone marrow specimens. We demonstrated that A-5021 had potent cytotoxic activity equal to that of GCV against the retroviral vector-mediated HSV-TK gene-transduced lung cancer cell lines. Further, phosphorylated A-5021 could be transferred to neighboring cells, and this analog killed HSV-TK- neighboring cells, as was the case for GCV. In contrast, A-5021 did not exhibit an inhibitory effect on bone marrow progenitor cells and colony formation (the 50% inhibitory concentration of the colony-forming units in culture = >100 microg/mL in human bone marrow specimens and >66 microg/mL in rodent bone marrow specimens). These results indicate that A-5021 has potent cytotoxic activity as a nucleoside analog for gene therapy using HSV-TK gene, and can be used much more safely than GCV.

The high production volume chemical challenge program: the relevance of the in vivo micronucleus assay.

The in vivo rodent bone marrow micronucleus assay (Mnt) has assumed a pivotal role in screening strategies for the identification of substances potentially carcinogenic to humans. The analysis of the results of the current international 5-year effort to provide toxicological data for high production volume chemicals will play a crucial role in developing future strategies for identifying health hazards. As part of that program, consideration is being given to accepting either in vitro genotoxicity data or results of the Mnt. The present analyses indicate that for hazard identification purposes that, in fact, in vitro genotoxicity test results, such as those derived from the Salmonella mutagenicity assay, may be an acceptable alternative.

Beating the odds: a cardiomyocyte cell line at last.

Beating the odds: a cardiomyocyte cell line at last.

Identification of rodent carcinogens and noncarcinogens using genetic toxicity tests: premises, promises, and performance.

The basic premises that guide genetic toxicity testing for identifying carcinogens and to support administrative and regulatory decisions are: the Salmonella mutagenicity test is a necessary component of testing schemes; a chromosome aberration test is needed in addition to a gene mutation test; a mammalian cell mutagenicity test is needed in addition to the Salmonella test; in vivo tests are needed to confirm the results of in vitro tests; and test batteries are more predictive than the individual tests of the battery. Results from the Salmonella mutagenicity, in vitro chromosome aberration, mutations in mouse lymphoma cells, rodent bone marrow micronucleus, and rodent carcinogenicity tests, performed by the U.S. National Toxicology Program, were used to evaluate these premises. A positive Salmonella test was most predictive of carcinogenicity. However, the data do not support using the other tests in addition to Salmonella for predicting carcinogenicity. The genetic toxicity tests did not complement each other, and batteries or combinations of the tests were no more predictive of carcinogenicity than Salmonella alone. If a chemical is mutagenic in Salmonella it should be considered a potential rodent carcinogen, unless ancillary information suggests otherwise. Positive responses in the other in vitro or in vivo tests do not increase the probability that the chemical is a carcinogen, and negative responses in the other tests do not diminish the implications of the positive Salmonella response.

Aneuploidy: a report of an ECETOC task force.

Aneuploidy plays a significant role in adverse human health conditions including birth defects, pregnancy wastage and cancer. Although there is clear evidence of chemically induced aneuploidy in experimental systems, to date there are insufficient data to determine with certainty if chemically induced aneuploidy contributes to human disease. However, since there is no reason to assume that chemically induced aneuploidy will not occur in human beings, it is prudent to address the aneugenic potential of chemicals in the safety assessment process. A wide range of methods has been described for the detection of chemically induced aneuploidy including subcellular systems, tests with fungi, plants and Drosophila as well as in vitro mammalian systems and in vivo mammalian somatic and germ cell assays. However, none of these methods is sufficiently validated or widely used in routine screening. Underlying the efforts to develop aneuploidy-specific assays is the presumption that current genetic toxicology tests do not detected chemicals that have aneuploidy-inducing potential. To address this, we have critically evaluated data from standard genetic toxicology assays for 16 known or suspected aneugens. The conclusions from the review are listed below. 1. At present there are only nine chemicals that can be classified as definitive aneugens, as determined by positive results in in vivo rodent assays. 2. As expected, the majority of definitive and suspected aneugens are negative in the bacterial mutation assay. 3. The majority of definitive aneugens evaluated induce polyploidy in vitro. With few exception, they also induced structural chromosome aberrations in vitro. 4. All of the definitive aneugens that have been sufficiently tested induce micronuclei in rodent bone marrow cells in vivo. A number of these chemicals also induced structural chromosome aberrations in vivo. 5. There is no evidence for a unique germ cell aneugen, that is a chemical that induces aneuploidy in germ cells and not in somatic cells. Furthermore, an analysis of several databases indicates the proportion of chemicals which induce polyploidy and not chromosome aberrations in vitro is low. Based on these conclusions, the following recommendations are made: for screening purposes, a standard genotoxicity test battery (including an in vitro cytogenetic assay with an assessment of polyploidy and clastogenicity at the same harvest time) should be performed; in the absence of polyploidy induction in vitro no further evaluation of aneuploidy-inducing potential is needed; if polyploidy is observed, in vitro follow-up testing to investigate further the aneuploidy-inducing potential should be conducted; such follow-up testing will generally start with the conduct of a standard in vivo somatic cell micronucleus assay; if the in vivo somatic cell micronucleus assay is negative, with adequate evidence of exposure of the bone marrow to the test compound, no further testing of aneuploidy-inducing potential is needed; if the in vivo somatic cell micronucleus assay is positive, further information on mechanisms of micronucleus induction can be obtained by using kinetochore/centromeric staining in vitro and/or in vivo; an assessment of potential germ cell aneuploidy activity may then be considered; aneuploidy induction which does not involve the direct interaction of a chemical or its metabolite(s) with DNA is expected to have a threshold. This must be considered in the risk assessment of such chemicals; this is not addressed by current risk assessment guidelines.

Lack of activity of estradiol in rodent bone marrow micronucleus assays

Estradiol has been evaluated in five independent rodent bone marrow micronucleus assays and has been found to be inactive. The dose-range evaluated extended from three daily doses of 20 μg/kg to the rat, a regimen that elicited a potent uterotrophic response from the animals, to single doses of between 10–150 mg/kg to the mouse. The mouse assays simulated and extended the conditions of test employed by earlier investigators who had found estradiol, and three structurally-related synthetic estrogens, to be active in mouse micronucleus assays over the dose range 1–10 mg/kg. It is concluded that estradiol is not genotoxic to the bone marrow of rodents. The top dose-level used in the present micronucleus assays (150 mg/kg) represented ∼150 000 times the minimum estrogenic dose of this chemical to rodents, and that was considered to be above the dose at which useful genetic toxicity data would be generated for this potent estrogen. The maximum tolerated dose (MTD) of estradiol to rodents remains to be established.

The genotoxicity of diaveridine and trimethoprim

We examined the genotoxicity of diaveridine and trimethoprim in the bacterial umu test, the bacterial reverse mutation test, the in vitro chromosome aberration test, the in vivo rodent bone marrow micronucleus test in two species, and the in vivo comet assay in five mouse organs. Both compounds were negative in the umu test ( Salmonella typhimurium TA1535/pSK1002) and in the reverse mutation tests ( S. typhimurium TA100, TA98, TA97, TA102, and Escherichia coli WP2 uvrA/pKM101) in the presence and absence of S9 mix. Diaveridine induced structural chromosome aberrations in cultured Chinese hamster CHL cells in the absence of a metabolic activation system, but not in the presence of a liver S9 fraction. No clastogenic activity in CHL cells was detected for trimethoprim. Bone marrow micronucleus tests in mice and rats conducted on diaveridine by single- and triple-oral dosing protocols were negative. The comet assay revealed that a single oral administration of diaveridine significantly induced DNA damage in liver, kidney, lung, and spleen cells, but not in bone marrow cells. The significant increase in migration values of DNA was reproducible with dose-response relationship. We suggest that the liver detoxifies the compound before it reaches the bone marrow, and that is why it is negative in the in vivo bone marrow micronucleus test. We concluded that diaveridine is genotoxic to mammalian cells in vitro and in vivo.

Does colchicine really induce bone formation in the rodent bone marrow?

Intravenous injection of a single dose of colchicine into inbred strains of BALB/c and CFW/L1 mice and into WAG rats did not effect rapid intramedullar bone formation and resorption, as has been claimed by the research group from Tokyo Medical and Dental University [14-17]. The applied doses of colchicine arrested metaphase during the first 4 hours postadministration and were noxious for hemopoietic tissue (necrosis of bone marrow was evident in 2 and 4 day specimens), but on longitudinal, serial sections of long bones there was no evidence of stimulation of osteogenesis at any point in time (2-26-day specimens). It is postulated that the system of ectopic osteogenesis by colchicine injection is not reproducible in mice and WAG rats, and the apparently osteogenic effect of colchicine, observed by the Ogura group [14-17], was mistakenly described as congenital osteopetrosis.

Dichotomous relationship between DNA reactivity and the induction of sister chromatid exchanges in vivo and in vitro

Structural analyses of the determinants associated with the induction of bone marrow sister chromatid exchanges in mice indicate that the phenomenon is based on an electrophilic attack on DNA. In that respect this phenomenon is different from the basis of the induction of SCE in cultured cells or of micronuclei in the bone marrow of rodents. The latter two phenomena involve other targets as well. Based on the recognition that the vast majority of recognized human carcinogens are genotoxic, the present finding indicates that the in vivo induction of SCE is a good biomarker, possibly even a biodosimeter, for exposure to potential carcinogens.

Species-specificity of pyrimethamine in the rodent bone marrow micronucleus test

An antiprotozoal agent pyrimethamine is a potent clastogen that induces structural chromosome aberrations and micronuclei in cultured Chinese hamster (CHL) cells in vitro. Our previous study on the compound, however, demonstrated no significant induction of micronuclei in the mouse bone marrow micronucleus test, even with dosings on 4 consecutive days. In the present study, we investigated the clastogenicity of pyrimethamine in the rat bone marrow micronucleus test. An obvious dose-dependent increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs) was observed when the compound was given 3 times by oral gavage at 20–120 mg/kg/day. The MNPCE frequency at the highest dose was 80 times that of the control group. Single dose of pyrimethamine at 80 and 160 mg/kg also significantly induced MNPCEs. Thus, pyrimethamine showed species-specific effects in rodent bone marrow micronucleus assays. Our finding implies that micronucleus tests using not only mouse, but also rat, may be necessary for the evaluation of clastogens detected in in vitro cytogenetic studies.