Alfred F. McFee

Improved sister-chromatid differentiation using paraffin-coated bromodeoxyuridine tablets in mice.

Abstract A quick and easy method for partially coating 5-bromo-2′-deoxyuridine tablets with paraffin before they were subcutaneously implanted in mice increased availability of the chemical from 8 to more than 24 h and significantly reduced the baseline level of sister-chromatid exchanges. Accurate identification of first-, second- and third-division metaphases was possible for 30 h postimplantation.

The effect of agent treatment time on the induction of sister-chromatid exchanges in mouse bone marrow cells in vivo.

The effect of time of agent administration, via intraperitoneal injection, on the yield of SCEs in bone marrow cells of male B6C3F1 mice was determined for cyclophosphamide (CP), 7,12-dimethylbenz[a]anthracene (DMBA) and mitomycin C (MMC). Animals were treated with several doses of each carcinogen/mutagen at 3 different treatment times: -1, +1 and +8 h in relation to the onset of BrdUrd administration. The results of these studies indicate that the optimal treatment time for inducing a maximal SCE response is agent-specific. For CP, the slope of the SCE response was greatest at the +8 h treatment time while the maximal response for DMBA occurred at the -1 h treatment time. For MMC, the slope of the SCE response was independent of treatment time and of the method of bromodeoxyuridine administration (intravenous infusion vs. tablet implantation) but dependent on the laboratory conducting the study (Brookhaven National Laboratory vs. Oak Ridge Associated Universities). Based on the results of these studies, the +1 h acute treatment time is considered optimal for the in vivo cytogenetic evaluation of suspect chemicals for genotoxic activity when bone marrow is used as the target cell population.

Influence of treatment to sacrifice time and the presence of BrdUrd on chemically-induced aberration rates in mouse marrow cells

4 chemicals, with various modes of clastogenic action were used to evaluate induced chromosomal aberrations in mouse bone marrow at different times after intraperitoneal injection. Aberration frequencies induced by mitomycin C, cyclophosphamide and dimethylbenz[a]anthracene increased with increasing time between treatment and sampling until those time points (approximately 18 h) when significant proportions of second-division metaphases were among the cells being scored; this increase was not obvious following treatment with 4-nitroquinoline 1-oxide. When BrdUrd tablets were implanted prior to treatment and scoring was restricted to first-division metaphases, aberration rates continued to increase for as long as 24 h post-treatment. The presence of BrdUrd did not affect significantly the rate of aberration induction by the chemicals. Our data indicate that the sensitivity of the in vivo mouse marrow assay for clastogenic chemicals can be greatly increased by utilizing BrdUrd to insure the scoring of only first-division metaphases at post-treatment times of approx. 18 h.

In vivo SCE analysis using bromodeoxyuridine, iododeoxyuridine, and chlorodeoxyuridine☆

Sister-chromatid exchange (SCE) frequencies were determined for mouse and rabbit bone-marrow cells following incorporation of 3 different halogenated analogues of thymidine. For both species the SCE frequency was highest for chlorodeoxyuridine, lowest for iododeoxyuridine and intermediate for bromodeoxyuridine. The study demonstrates that halogenated pyrimidine analogues other than brominated compounds can be used for in vivo SCE analysis and that their effects on baseline SCE frequencies are qualitatively similar to results of previous in vitro studies.

Induction of chromosomal damage in mammalian cells in vitro and in vivo by sulfapyridine or 5-aminosalicylic acid

Sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA) are the two primary metabolites of the anti-inflammatory drug salicylazosulfapyridine (SASP). These two metabolites were studied for induction of chromosomal damage in mammalian cells, in vitro and in vivo, in an attempt to understand better the genetic effects produced by SASP in humans and laboratory mice. To this end, SP and 5-ASA were tested for induction of sister-chromatid exchanges (SCE) and chromosomal aberrations (Abs) in Chinese hamster ovary (CHO) cells in vitro. In addition, they were tested in vivo for induction of micronuclei (MN) in mouse bone marrow polychromatic erythrocytes (PCE). SP gave positive results in the in vitro SCE test and the in vivo MN test, and negative results in the in vitro Abs test. 5-ASA was negative in all three tests. These results indicate that it is the SP metabolite of SASP that is necessary for the induction of chromosomal damage reported to occur in humans and mice after treatment with SASP.

Mouse dominant lethal and bone marrow micronucleus studies on methyl vinyl sulfone and divinyl sulfone

Methyl vinyl sulfone and divinyl sulfone were tested for the induction of dominant lethal mutations and micronucleated bone-marrow erythrocytes in male mice. These chemicals were chosen for study because of their similarities in structure and chemical reactivity to acrylamide which is known to induce both effects. Following administration of the test compounds by intraperitoneal injection at the maximum tolerated doses, no evidence of induced dominant lethal mutations or micronucleated bone-marrow cells was observed for either chemical. It is concluded that structures and Michael reactivities similar to acrylamide are not sufficient to impart similar in vivo genetic toxicity to MVS and DVS.

Caprolactam and benzoin: tests for induction of chromosome aberrations and SCEs in mouse bone marrow

CAP and ZOIN were tested in vivo for their ability to induce sister-chromatid exchanges (SCE) and chromosome aberrations in mouse bone marrow cells. Single intraperitoneal injections of ZOIN to a maximum of 3000 mg/kg body weight failed to increase the number of SCEs in metaphases recovered 24 h post-treatment, and doses of 1500 mg/kg did not induce measurable increases in chromosome-aberration levels among first-division metaphases at 18 h. Similarly, neither endpoint showed a significant increase following near-lethal doses of 700 mg/kg of CAP. Two lower doses of each chemical were also ineffective. Under the in vivo conditions of our test system, both chemicals were cytogenetically inactive.

The effect of agent dose and treatment time on the intercellular distribution of sister-chromatid exchanges induced by genotoxic agents in mouse bone marrow cells in vivo

Using two methods of bromodeoxyuridine (BrdUrd) administration and three genotoxic chemicals, the effects of dose and treatment time on the intercellular distribution of sister-chromatid exchanges (SCE) in the bone marrow of male B6C3F1 mice were evaluated. The dispersion of SCE among solvent control mice infused intravenously with BrdUrd or implanted subcutaneously with a BrdUrd tablet partially coated with paraffin was largely consistent with a Poisson model. Intraperitoneal treatment with cyclophosphamide (CP; solvent = phosphate-buffered saline), 7,12-dimethylbenzanthracene (DMBA; solvent = corn oil) and, in mice infused with BrdUrd, mitomycin C (MMC; solvent = phosphate-buffered saline) induced a significant increase in SCE, the distribution of which was not distributed as a Poisson. For CP and MMC, the increase in dispersion was dose-dependent and independent of treatment time (-1, +1 or +8 h in relation to the start of the BrdUrd treatment). The lack of a treatment time effect suggests that there were no significant differences among treatment times in the distribution of the reactive forms of these two chemicals, no variation in cell-stage sensitivity, and no cellular toxicity to modulate the response. For DMBA, the increased dispersion of induced SCE depended on treatment time and was not simply related to dose. The increase in dispersion was agent-specific; at equal levels of SCE induction, the distribution of SCE in mice treated with DMBA exhibited greater dispersion than SCE in mice treated with either CP or MMC. These differences between DMBA and CP/MMC are probably due to DMBAs slower absorption/distribution kinetics, its requirement for metabolic activation to genotoxic metabolites and its extended half-life. These data suggest that analyzing the distribution of SCE, in addition to mean frequency, is a useful method for evaluating agent specific patterns in SCE induction.

Sister-chromatid exchanges induced in swine lymphocytes by chronic oral doses of dimethylbenzanthracene

Sister-chromatid exchanges were scored at 3-week intervals in lymphocytes of female swine ingesting daily doses of 1.25 or 2.50 mg/kg of 7,12-dimethylbenzanthracene (DMBA) for 160 days. Exchanges increased with time for about 120 days then reached a plateau at approximately 2.5 times their pretreatment level. No increase in chromosome aberrations could be identified as resulting from ingestion of the chemical. Week-old progeny of animals that had ingested the chemical throughout pregnancy showed no increase of exchanges in their peripheral lymphocytes.

Chromosomal effects of theophylline measured in mouse marrow cells in vivo

Doses of 62.5, 125 and 250 mg/kg of theophylline were administered to male B6C3F1 mice by intraperitoneal injection. Chromosome aberrations were scored in first-division metaphases of marrow cells 18 and 36 h post-treatment and sister-chromatid exchanges were quantified in second-division metaphases at 24 h. A modest but statistically significant increase in the number of SCEs occurred, but chromosome aberrations were not significantly different from controls following treatment with any level of the drug at either time period.