A modified mouse peripheral blood lymphocyte culture system for cytogenetic analysis

Arsenic is a prevalent human carcinogen whose mutagenicity has not been characterized fully. Exposure to either form of inorganic arsenic, As(III) or As(V), can result in the formation of at least four organic metabolites: monomethylarsonic acid, monomethylarsonous acid (MMA(III)), dimethylarsinic acid, and dimethylarsinous acid (DMA(III)). The methylated trivalent species, as well as some of the other species, have not been evaluated previously for the induction of chromosome aberrations, sister chromatid exchanges (SCE), or toxicity in cultured human peripheral blood lymphocytes; for mutagenicity in L5178Y/Tk(+/-) mouse lymphoma cells or in the Salmonella reversion assay; or for prophage-induction in Escherichia coli. Here we evaluated the arsenicals in these assays and found that MMA(III) and DMA(III) were the most potent clastogens of the six arsenicals in human lymphocytes and the most potent mutagens of the six arsenicals at the Tk(+/-) locus in mouse lymphoma cells. The dimethylated arsenicals were also spindle poisons, suggesting that they may be ultimate forms of arsenic that induce aneuploidy. Although the arsenicals were potent clastogens, none were potent SCE inducers, similar to clastogens that act via reactive oxygen species. None of the six arsenicals were gene mutagens in Salmonella TA98, TA100, or TA104; and neither MMA(III) nor DMA(III) induced prophage. Our results show that both methylated As(V) compounds were less cytotoxic and genotoxic than As(V), whereas both methylated As(III) compounds were more cytotoxic and genotoxic than As(III). Our data support the view that MMA(III) and DMA(III) are candidate ultimate genotoxic forms of arsenic and that they are clastogens and not gene mutagens. We suggest that the clastogenicity of the other arsenicals is due to their metabolism by cells to MMA(III) or DMA(III).

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“…tonic solution, fixed, and slides were prepared and differentially stained using standard cytogenetic procedures [Erexson and Kligerman, 1987].…”

Section: Genotoxicity Assays Using Human Lymphocytesmentioning

confidence: 99%

Methylated trivalent arsenicals as candidate ultimate genotoxic forms of arsenic: Induction of chromosomal mutations but not gene mutations

Kligerman

Doerr

Tennant

et al. 2003

Environ and Mol Mutagen

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188

137

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“…It consisted of RPMI 1640 medium with 25 mM HEPES buffer (Invitrogen Corp, Carlsbad, CA, USA) containing 20% fetal calf serum, 0.03% L-glutamine, 5 · 10 )5 M mercaptoethanol (Invitrogen Corp, Carlsbad, CA, USA) and kanamycin (50 lg/ml). The effects of mitogens on mitotic indices of peripheral lymphocyte were examined using 5 lg/ml PHA (HA-16, Murex Biotech Ltd., Dartford, UK), 6 lg/ml CON-A (Type IV, Sigma, St Louis, MO, USA) and 50 lg/ ml LPS (Sigma, St Louis, MO, USA), each of which were reported as being an optimal dose [4]. Regarding splenic lymphocytes, 3 lg/ml CON-A and 10 lg/ml LPS were used as mitogens according to Matsuda et al [8].…”

Section: Cell Culture and Harvestmentioning

confidence: 99%

“…Using this technique, we examined the cell reactions to mitotic stimulators such as phytohemaggulutinin (PHA), concanavalin A (CON-A) and lipopolysaccharide (LPS), and compared them with the corresponding data of the previous study using mouse PBL cultures [4] or those using human PBL cultures [5]. Then we subjected the improved mouse PBL culture system to cytogenetic analysis using conventional and modern techniques.…”

Section: Introductionmentioning

confidence: 99%

“…However, many of these culture techniques are laborious and complex and some appear to have enjoyed only limited success. Among these studies, a few studies had somewhat better success obtaining metaphase chromosomes from mouse PBLs using whole blood culture systems [1,2], which were later modified by isolating lymphocytes using Ficoll-Hypaque density gradient [3,4].…”

Section: Introductionmentioning

confidence: 99%

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An Improved Culture System of Mouse Peripheral Blood Lymphocytes for Analysis of Radiation-Induced Chromosome Aberrations

Kanda¹,

Shang²,

Tsuji³

et al. 2004

Bioscience Reports

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There is an incentive to develop a culture system of mouse peripheral blood lymphocytes (PBLs) to serve as models for studying genotoxic effects in humans exposed to mutagens, including ionizing radiation. However, many past approaches have been laborious, complex and only partly reproducible. In the present study, we established an improved culture system of mouse PBLs by removing blood and/or plasma, which was found to inhibit in vitro mitotic stimulation or proceeding cell cycles of lymphocytes. We compared the reactions of isolated PBLs to mitogens between the classical method and the present improved one. Then, we applied this method to the cytogenetic analysis using chemically induced premature chromosome condensation (PCC) as well as the conventional analysis, and demonstrated that the frequency of excess fragments observed in PCC cells might be useful to quantify the radiation-induced damages on chromosomes.

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“…Lymphocytes, because they are generally in a nondividing state, can accumulate damage throughout a chronic exposure period. However, rodent lymphocytes are, in general, difficult to culture and only a few laboratories conduct lymphocyte studies routinely (for example, Kligerman et al, 1981;Sinha et al, 1985;Erexson and Kligerman, 1988). Bone marrow, by virtue of its proliferative status, offers insight primarily into acutely induced damage.…”

Section: Introductionmentioning

confidence: 99%

The cytogenetic evaluation of in vivo genotoxic and cytotoxic activity using rodent somatic cells

Tice

1988

Cell Biol Toxicol

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With the growing realization that in vitro short-term tests for genotoxicity can never fully mimic in vivo conditions, the evaluation of genotoxic damage in somatic cells of rodents has played an increasingly important role in assessing the carcinogenic potential of suspect compounds. Among the various genotoxic endpoints assessed in in vivo somatic cell assays, cytogenetic endpoints (e.g., chromosomal aberrations, micronuclei, sister chromatid exchanges) continue to be used most frequently. The purpose of this paper is to demonstrate the utility of evaluating different cytogenetic endpoints in the same animal, using as examples studies to evaluate the in vivo genotoxic potential of benzene, of methylisocyanate, and of butadiene, chloroprene and isoprene.

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A modified mouse peripheral blood lymphocyte culture system for cytogenetic analysis

Cited by 49 publications

References 16 publications

Methylated trivalent arsenicals as candidate ultimate genotoxic forms of arsenic: Induction of chromosomal mutations but not gene mutations

Methylated trivalent arsenicals as candidate ultimate genotoxic forms of arsenic: Induction of chromosomal mutations but not gene mutations

An Improved Culture System of Mouse Peripheral Blood Lymphocytes for Analysis of Radiation-Induced Chromosome Aberrations

The cytogenetic evaluation of in vivo genotoxic and cytotoxic activity using rodent somatic cells

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