Chinese Hamster Bone Marrow Research Articles

In vivo method for sister chromatid exchanges in Chinese hamster foetal and bone marrow cells.

An in vivo BrdU labelling method for sister chromatid staining in Chinese hamster foetal and bone marrow cells is reported. The mitotic index in bone marrow cells was many times higher than that in foetal cells (2.7 to 11.3 and 0.1 to 2.1, respectively) whereas both tissues produced metaphases of high quality. Sister chromatid exchanges (SCE) in the foetal cells of 3 non-treated hamsters varied from 5.4 to 7.4, and in the bone marrow cells of 11 non-treated hamsters, from 2.9 to 5.4.

Review of the genotoxicity of 4-chloro-2-methylphenoxyacetic acid

4-Chloro-2-methylphenoxyacetic acid (MCPA) has been examined for genotoxicity in a range of in vitro and in vivo assays, including assays for gene mutation and clastogenicity. MCPA is non-mutagenic in bacterial and mammalian cell gene mutation assays. Increases in percentage aberrant cells were found on analysis of metaphases of human peripheral lymphocytes treated in vitro in the presence of auxiliary metabolic activation (S9), but only at doses approaching 10 mM and causing significant cytotoxicity. These increases may therefore be non-specific. No evidence for clastogenicity in vivo was found in the mouse bone marrow micronucleus assay or the Chinese hamster bone marrow metaphase assay. No evidence for either increases in sister chromatid exchange (SCE) frequency or DNA binding was found in the rat. Very small (less than 1.5 times controls) increases in SCE were observed in vivo in the hamster at toxic or maximum tolerated dose levels. MCPA is not alerting for likely genotoxic activity using established structure-activity relationship principles and it is concluded that, on the weight of evidence from the available data, MCPA is not genotoxic in vivo. This is consistent with its lack of carcinogenicity in rats and mice.

Toxicological Evaluation of u-hEGF

The toxicological evaluation of urinary human epidermal growth factor (u-hEGF) included mutagenicity, single and repeated dose general toxicity, and teratogenicity studies in various animal species. The mutagenic potential of u-hEGF was tested in vitro (Ames test, chromosome aberration in human lymphocytes, unscheduled DNA synthesis in HeLa cells) and in vivo (chromosome aberration in Chinese hamster bone marrow and micronucleus test in rat bone marrow). No mutagenic or clastogenic effects were found. The acute toxicity of u-hEGF was evaluated in mice and rats, using single subcutaneous (sc) or intravenous (i.v.) injection of 15 mg/kg. No toxic effects were observed Four-week i.v. daily administration of u-hEGF at the doses of 0.3, o.9, and 3 mg/kg in the SD rat followed by 2 wk of compound withdrawal induced pronounced and generally dose-related effects (i.e., epithelial hyperplasia) in a wide range of tissues and organs, at all doses. However, these effects were not apparently detrimental to the general health of the rats. The repeated sc administration of u-hEGF to cynomolgus monkeys for 4 wk at the same doses as used in the rat study resulted in lethality after about 7 days of treatment in the 2 higher dose groups or after 14 days at the lowest dose. The main clinical signs observed were gastrointestinal effects, respiratory distress, sedation, marked loss of body weight, and cutaneous desquamation. At histology, hyperplasia of most epithelia was seen in all groups. In addition, atrophy of the ovarian follicles and necrosis of the uterine endometrium were noted. Changes considered secondary to physical distress were atrophy of the hemopoietic and lymphatic system and hepatic steatosis. The embryofetal toxicity and teratogenicity of u-hEGF was tested, using the i.v. route in the SD rat and the i.v. and sc routes in the New Zealand White rabbit. In both species, the compound was administered at the doses of 0, 0.3, 0.9, and 3 mg/kg/day, from day 6 to 15 of pregnancy in rats and 6-18 in rabbits. In the rat, an increase in body weight was noted in the dams and fetuses at the 2 high doses. No embryotoxic or teratogenic effects were observed. In the rabbit studies, mortality and severe clinical signs involving various systems, with marked effects on the eyes, were observed at all doses tested during the first days of treatment by both routes. From the reproductive point of view, most of the surviving treated gravid females showed only resorptions.

Assessment of genotoxic effects by Lindane

The well known and previously widespread insecticide lindane has been re-assessed for DNA-damaging activity. A first group of investigations using standard in vitro and in vivo mutagenicity assays did not indicate any genotoxic effects of lindane at all. The assay systems used were for the induction of HPRT mutations and sister chromatid exchanges in CHO cells cultured in vitro, and for micronuclei induction in vivo in bone marrow cells of rats, hamsters and mice. Also, lindane was assessed for its potential to induce sister chromatid exchanges in vivo in the bone marrow of Chinese hamsters. These specific assay systems had not been used previously for elucidating the genotoxic effects of this compound, but they are basically similar to other standard mutagenicity assays in which lindane has been shown to be devoid of genotoxic activity. The second part of the investigations was directed at re-evaluating a previously reported positive effect of the compound in primary rat hepatocytes in vitro. We performed in vitro and in vivo studies with hepatocytes from the rat liver and used alkaline elution to detect DNA damage. However, we could not demonstrate that lindane induced genotoxicity, unless considerable concomitant cytotoxicity was apparent as well. Finally, since lindane can be ingested and inhaled by humans, we also measured the induction of DNA damage in local target organs of absorption using single cell microgelelectrophoresis (the comet assay). In these cases lindane was genotoxic in cells of the gastric and nasal mucosa in vitro and also in vivo following appropriate routes of application (oral and inhalational exposure).

1-Methyl-2-pyrrolidinone (NMP) does not induce structural and numerical chromosomal aberrations in vivo

1-Methyl-2-pyrrolidinone induces aneuploidy in yeast, but only under special treatment conditions. Other genotoxic effects have not been found in vitro, and in vivo no data are available in the literature. Therefore, NMP was investigated in the mouse micronucleus test and the Chinese hamster bone marrow test for structural and numerical chromosomal aberrations. These tests can detect both types of alterations as demonstrated by appropriate positive control substances (cyclophosphamide, vincristine sulfate and benomyl). NMP at single oral doses up to 3800 mg/kg body weight (∼ 80% of the LD 50) did not lead to an increase either in micronucleated erythrocytes or in structural or numerical chromosomal aberrations when bone marrow was sampled 16, 24 and 48 h after treatment in the micronucleus test or after 24 and 48 h for karyotype analysis.

Comparative investigation of cyclophosphamide action on bone marrow cells of the Armenian hamster and 4 other species of rodents

We studied the clastogenic action of cyclophosphamide (CP) on bone marrow cells of the Armenian hamster (AH), Cricetulus migratorius. CP induced a dose-dependent linear increase in aberrant cells. The maximal cytogenetic action was observed 12 h after CP treatment. Male and female AHs were similarly sensitive to the clastogenic action of CP. We compared CP clastogenicity at a dose of 25 mg/kg on bone marrow cells of AHs, mice, rats, guinea pigs and Chinese hamsters 24 h after treatment. We observed that this dose of CP induced only 2.8% aberrant cells in bone marrow of AHs, but 42.8%, 32.2%, 25% and 14.6% aberrant cells in bone marrow of guinea pigs, rats, mice and Chinese hamsters respectively. AHs are much more resistant to the metaphase-arresting action of colchicine than other species of rodents (e.g., the colchicine dose for AHs is 100-fold more than for rats). Thus AHs are the most resistant of all rodent species studied to the clastogenic action of CP.

The possible role of probiotics as dietary antimutagens

Possible antimutagenic actions of probiotics - mainly lactic acid bacteria - were examined using in vitro and in vivo test systems. In the Ames test with Salmonella typhimurium TA1538 beef extract and nitrosated beef extract were used as mutagens. L. casei showed high antimutagenic activity on mutagenicity induced by nitrosated beef extract only without S9 mix, whereas Omniflora (a lyophilized preparation of lactobacilli and E. coli and its cellfree culture broth exhibited antimutagenic action only on beef extract. The actions of probiotics were more homogeneous when living animais were used in the tests. Using busulfan as a mutagen both the chromosome aberration test (with Chinese hamster bone marrow cells) and the micronucleus test (with bone marrow cells of Chinese hamsters and mice) showed strong anticlastogenic action when L. casei, Omniflora or yoghurt (with living bifiobacteria) were given orally at the same time as the mutagen. Lactobacilli were effective also after i.p. injection. Cell-free culture broths had no or only weak antimutagenic effects. Mutagen-induced chromosome aberrations and micronuclei were reduced by up to 80% by the lactobacilli.

Mutagenicity testing of doxylamine succinate, an antinauseant drug

Doxylamine succinate (DA), a compound which was formerly used as an antinauseant during pregnancy, showed no substantial mutagenicity in mouse embryos following transplacental exposure. A small dose-dependent induction of chromosomal aberrations was found in mouse embryos on day 11 of gestation. No induction of sister chromatid exchanges (SCE) was found in embryos on day 11 of gestation. A micronucleus test with fetal blood on day 17 of gestation was negative. Additionally, DA was negative in Chinese hamster bone marrow in vivo (micronuclei) and in human lymphocyte cultures in vitro (SCE).

Sister-chromatid exchanges induced by triethylenemelamine: in vivo and in vivo/in vitro studies in mouse and Chinese hamster bone marrow and spleen cells

This study was designed to obtain sister-chromatid exchange (SCE) frequencies in bone marrow and spleen cells of mice and Chinese hamsters under in vivo and in vivo/in vitro systems following treatment of animals with varying doses (15–405 μg/kg) of triethylenemelamine (TEM). A dose-related SCE response was found in both species, tissues, and systems analyzed following TEM treatment. In vivo, similar responses were noted for both tissues in both species. However, in vivo/in vitro, the response was lower than in vivo and it varied with the tissue. The spleen cells were more sensitive and gave higher numbers of SCEs than bone marrow of both species at the two highest doses tested (135 and 405 μg/kg). These differences may be attributed to cell-culturing effects, type of cells analyzed, species and tissue specificities, and pharmacokinetic properties of the chemical. This study lends support to recently established in vivo/in vitro cell culture methodologies employing mice and Chinese hamsters for comparative cytogenetic analysis.

Preparation of Chinese hamster bone marrow and spleen primary cell cultures for sister chromatid exchange and chromosomal aberration studies

Procedures for preparing and culturing Chinese hamster bone marrow and spleen cells for cytogenetic studies are described. Animals are killed by cervical dislocation, then the bone marrow is flushed from femora and tibia with Ham's F12 medium into centrifuge tubes. Bone marrow cells are collected by low-speed centrifugation (285 ×g). Approximately one million cells are cultured in a 30-ml Falcon flask with 5 ml complete medium containing 3.95 ml Ham's F12 medium, 1.0 ml fetal bovine serum (FBS), and 0.05 ml penicillin-streptomycin (5000 U/ml and 5000 µg/ml stock). Spleens are obtained aseptically, transferred to centrifuge tubes containing 2 ml of RPMI 1640 and smashed with a sterile spatula. The debris is removed, cells are collected by low-speed centrifugation (285 ×g), and washed three times with phosphate buffered saline supplemented with 2% heat inactivated FBS. Approximately one million cells are suspended in a 30-ml Falcon flask with 5 ml complete medium containing 3.70 ml RPMI 1640. 1.0 ml heat inactivated FBS, 0.05 ml penicillin-streptomycin, 0.05 ml of 200 mMl-glutamine solution, 1 × 10−5M 2-mercaptoethanol, and 0.20 ml lipopolysaccharide. For sister chromatid exchange analysis, 20 µM of 5-bromo-2′ -deoxyuridine is also added to the culture medium for 24 to 28 h for bone marrow cells and 36 to 40 h for spleen cells. These culture systems can also be used for chromosomal aberration studies.

Species differences in mutagenicity testing. II. Sister-chromatid exchange and micronucleus induction in rats, mice and Chinese hamsters treated with cyclophosphamide

Comparative investigations of sister-chromatid exchange (SCE) and micronucleus induction in the bone marrow of rats, mice and Chinese hamsters with the cytostatic alkylating mutagen cyclophosphamide (CP) revealed remarkable species differences in their mutagenic responses. With both test systems the sensitivities of the three species can be ranked into the order rat greater than mouse greater than Chinese hamster. More explicit results were obtained with the SCE test than with the micronucleus test within the same dose range. This may be due to the influence of species-related differences in the cytotoxic response to CP in the micronucleus test. These results show that clearly different mutagenic responses in different test species may be obtained in standard assays even with a compound which is metabolized in a very similar manner in all animal species.

Investigation on the distribution of SCEs in metaphase chromosomes of Chinese hamster bone marrow cells

Investigation on the distribution of SCEs in metaphase chromosomes of Chinese hamster bone marrow cells

An in vitro and in vivo study of a BrdU-sensitive fragile site in the Chinese hamster

The frequencies of chromosome aberrations and development of the bromodeoxyuridine (BrdU)-sensitive fragile site were studied in vitro in Chinese hamster kidney and bone marrow cells and in vivo in Chinese hamster bone marrow cells. Chromosome aberrations in these cell systems were measured in response to different concentrations of BrdU, fluorodeoxyuridine, or both. The fragile site was found in both homologues of chromosome 1 at 1q22. Treatment with BrdU in vitro but not in vivo produces significant chromosome aberrations. About 50% of chromosome aberrations found after treatment in vitro were at the BrdU-sensitive fragile site compared with 12.5% after treatment in vivo. These results show that BrdU is much more potent in vitro than in vivo in inducing both chromosome aberrations and the expression of the BrdU-sensitive site.

N-Butyl acrylate: Cytogenetic investigations in the bone marrow of Chinese hamsters and rats after 4-day inhalation

Chinese hamsters and rats were exposed to an average concentration of 817 ppm and 820 ppm n-butyl acrylate (acrylic acid n-butyl ester), respectively for 4 days. In both animal species, the exposure led to distinct signs of toxicity; four of ten male Chinese hamsters died during the exposure period. The chromosome analysis carried out in the bone marrow of the animals after the 4-day inhalation did not indicate any chromosome-damaging effect of n-butyl acrylate. Under the experimental conditions chosen the percentage of metaphases damaged was always in the same range as that of the controls for both Chinese hamsters and rats.

Preclinical toxicology studies with acyclovir: Genetic toxicity tests

Acyclovir (ACV), an antiviral drug active in the treatment of oral and genital Herpes infections, has been evaluated for mutagenic and carcinogenic potential in a battery of in vitro and in vivo short-term assays. Negative results were obtained in the following in vitro tests: Ames Salmonella, plate incorporation and preincubation modification assays; E. coli polA +/polA − DNA repair; yeast ( S. cerevisiae D4) gene conversion; Chinese hamster ovary cells (HGPRT, APRT loci and ouabain-resistance marker); L5178Y mouse lymphoma cells (HGPRT locus and ouabain-resistance marker); and C3H/10T1/2 mouse fibroblast neoplastic transformation assay. All except the last assay were performed in the presence and absence of an exogenous metabolic activation system. ACV was positive at high concentrations × exposure times in the absence of exogenous metabolic activation in the following in vitro systems and at the indicated concentrations: BALB/c-3T3 neoplastic transformation (50 μg/mL, 72 h exposure); human lymphocyte cytogenetics (250–500 μg/mL, 48 h exposure); and L5178Y mouse lymphoma cells (TK locus, 400–2400 μg/mL, 4 h exposure; predominantly small colony mutants of chromosomal origin produced). No effects were seen in vivo (mouse dominant lethal assay; rat and Chinese hamster bone marrow cytogenetics) at up to maximum tolerated doses (MTD). An unusual clastogenic effect was seen in Chinese hamsters at 5 times the MTD. Overall, positive effects were seen only at either high concentrations (≥250 μg/mL in vitro or plasma levels) or prolonged exposure (72 hr in the BALB/c-3T3 neoplastic transformation assay). These studies support the view that ACV is a chromosomal mutagen, i.e., one which causes multi-locus damage but not single gene effects. The significance of these results for the genetic risk of ACV to man is discussed.

Health Effects of Exposure to Diesel Engine Emissions: A Summary of Animal Studies Conducted by the U.S. Environmental Protection Agency's Health Effects Research Laboratories at Cincinnati, Ohio

In order to evaluate the potentially harmful effects of diesel engine emissions, inhalation exposure studies were carried out using a variety of animal species and strains, and measuring a wide range of toxicological parameters. Exhaust was provided by a 6 cylinder Nissan diesel engine operated 20 hours/day, 7 days/week, during a 2 month preliminary trial and 8 hours/day, 7 days/week during a 2 year, 4 month long-term study. The exhaust was diluted to produce a concentration of 6 mg/m3 particulate matter during the preliminary study and during the first year of the long term study and 12 mg/m3 thereafter. Exposure to diesel emissions did not result in detectable genotoxic effects as measured by increases in sister chromatid exchange (SCE), micronucleus testing and metaphase analysis in Chinese hamster and mouse bone marrow cells or in the morphology of cat sperm. SCE in lung cells from Syrian hamsters, however, was increased at the higher exposure level. The incidence of heritable mutations was not increased in mice. An increase in lung tumor incidence was detected in female Senear mice exposed from conception, but not in males, or in either male or female strain A mice exposed from 6 weeks of age. Voluntary activity was decreased in rats during exposure and learning ability was impaired in adult animals exposed during the early postnatal period. Susceptibility to an infectious challenge was increased in diesel exposed mice. Clearance of particles from the lung was found to be minimal even after 90 days recovery in clean air. Pulmonary function alterations in diesel exposed cats suggested the presence of a lesion restricting the air flow. The integrity of the lung as measured by leakage of 131I labeled protein into the alveoli did not seem to be impaired, but increased collagen synthesis suggested that fibrosis may develop with long-term exposure. The preliminary inhalation study resulted in the induction of xenobiotic metabolizing enzymes in the liver. In later studies, however, enzyme induction was found only in male mice injected with large doses of diesel particulate extract.

Chemically-induced sister-chromatid exchange in vivo in bone marrow of Chinese hamsters An evaluation of 24 compounds

Chemically-induced sister-chromatid exchange (SCE) was measured in vivo in bone marrow of Chinese hamsters. Chemicals were administered either intraperitoneally or orally and increased SCE frequencies were noted with 6 of 6 direct-acting genotoxins and with 9 of 14 activation-dependent genotoxins. Metronidazole, O-toluidine, 4-nitro- O-phenylenediamine and 2-nitro- p-phenylenediamine, compounds which have shown either mutagenic or carcinogenic activity, did not induce SCE in vivo. 4 non-genotoxins and 4 different control treatments did not induce SCE. The results show that the in vivo SCE method may be useful for the identification of genotoxins and that the outcome of the test is, for certain chemicals, dependent upon the route of exposure.

N-Butyl Acrylate: Cytogenetic Investigations in the Bone Marrow of Chinese Hamsters and Rats After 4-Day Inhalation

n-Butyl Acrylate: Cytogenetic Investigations in the Bone Marrow of Chinese Hamsters and Rats After 4-Day Inhalation

Genotoxic activity of the aminophenols as evidenced by the induction of sister chromatid exchanges.

1 Most hair dyes contain the structural isomers ortho-, meta- and para- aminophenol which were investigated concerning their ability to induce sister chromatid exchanges (SCE) in cultured human lymphocytes in vitro and in Chinese hamster bone marrow cells in vivo. 2 In vitro only ortho-aminophenol significantly induced SCE in a dose-dependent manner. 3 In vivo none of the structural isomers increased significantly the SCE frequency. 4 It is concluded that ortho-aminophenol is a weak directly acting compound which is detoxified during its metabolism.

An investigation of the genetic toxicology of irradiated foodstuffs using short-term test systems. iii- in vivo tests in small rodents and in drosophila melanogaster

Six in vivo genetic toxicity tests were carried out on irradiated or unirradiated cooked chicken, dried dates and cooked fish. The tests were as follows: sex-linked recessive lethal mutations in Drosophila melanogaster (dried dales only), chromosome aberrations in bone marrow of Chinese hamsters, micronucleus test in rats, mice and Chinese hamsters, sister-chromatid exchange in bone marrow of mice and Chinese hamsters and in spermalogonia of mice, and DNA metabolism in spleen cells of Chinese hamsters. None of the tests provided any evidence of genetic toxicity induced by irradiation. However, dried dates, whether irradiated or not, showed evidence of some genetic toxicity in their effect on DNA metabolism in spleen cells and SCE induction in bone marrow. Feeding irradiated fish affected DNA metabolism in the spleen cells of Chinese hamsters. This effect could be interpreted as an induction of an immunoactive compound, although it could also be explained by the persistence of an immunoaclive compound due to the removal by irradiation of spoilage organisms that would normally degrade it.