Evaluation of the potential of triethanolamine to alter hepatic choline levels in female B6C3F1 mice.

Triethanolamine (TEA), a widely used nongenotoxic alcohol-amine, has recently been reported to cause an increased incidence of liver tumors in female B6C3F1 mice, but not in males nor in Fischer 344 rats. Choline deficiency induces liver cancer in rodents, and TEA could compete with choline uptake into tissues. The potential of TEA to cause choline deficiency in the liver of these mice as a mode of tumorigenesis was investigated. Groups of female B6C3F1 mice were administered 0 (vehicle) or a maximum tolerated dosage (MTD) of 1000 mg/kg/day TEA (Trial I) and 0, 10, 100, 300, or 1000 mg/kg/day TEA (Trial II) in acetone vehicle via skin painting 5 days/week for 3 weeks. Female CDF(R) rats were also administered 0 or an MTD dosage of 250 mg/kg/day TEA (Trial II) in a similar manner. No clinical signs of toxicity were noted, and upon sacrifice, levels of hepatic choline, its primary storage form, phosphocholine (PCho), and its primary oxidation product, betaine, were determined. A statistically significant decrease in PCho and betaine, was observed at the high dosage (26-42%) relative to controls and a dose-related, albeit variable, decrease was noted in PCho levels. Choline levels were also decreased 13-35% at the high dose level in mice. No changes in levels of choline or metabolites were noted in treated rats. A subsequent evaluation of the potential of TEA to inhibit the uptake of (3)H-choline by cultured Chinese Hamster Ovary Cells revealed a dose-related effect upon uptake. It was concluded that TEA may cause liver tumors in mice via a choline-depletion mode of action and that this effect is likely caused by the inhibition of choline uptake by cells.

An in vitro screening paradigm for extracts of whole foods for detection of potential toxicants.

The application of organic, conventional and biotechnology techniques can alter the intrinsic levels of natural toxicants in crop foods and methods are needed to screen for unexpected changes in toxicant levels. We evaluated crude, aqueous preparations of 37 foods purchased from a local market in a battery of four in vitro mammalian toxicity screens. The foods were evaluated in one or more of the following tests: (1) cytotoxicity (37 foods) and (2) chromosomal aberration test (nine foods), both in Chinese hamster ovary cells, (3) limb bud micromass assay (nine foods) using 11-day old CD-1 mouse embryos and (4) estrogenicity (MCF-7 cells transfected with estrogen receptor and lucerifase reporter constructs, 12 foods). IC50s for cellular proliferation ranged from < 1% (v/v, garlic) to > 10% (v/v, 18 foods), the maximal concentration tested. Five of nine preparations (soybeans, broccoli, garlic, snow peas and corn) were clastogenic and two (soybeans and snow peas) inhibited chrondrogenesis in the limb bud micromass assay. Five of nine preparations (soybeans, snow peas, cumin, asparagus and bean sprouts) produced significant estrogenic responses. Overall, the 12 foods evaluated in two or more of the tests showed different patterns of response. These preliminary data indicate that screening for potential toxicants is possible with fast, relatively inexpensive in vitro tests. These in vitro tests, while potentially useful to detect unexpected toxicants in plants that may signal the need for further evaluation, are not directly useful to predict human or animal risk from eating these plants.

Evaluation of the genotoxicity of 2,4-dichlorophenoxyacetic acid and its derivatives in mammalian cell cultures.

2,4-dichlorophenoxyacetic acid and its derivatives (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. The genetic toxicity of an ester (2,4-D 2-butoxyethylester) and two salts (2,4-D isopropylamine and 2,4-D triisopropanolamine) was investigated in cultured mammalian cells. The end points used were the induction of chromosomal aberrations in primary cultures of rat lymphocytes and forward mutations at the HGPRT locus of Chinese hamster ovary cells. There was no evidence of genotoxicity for the test materials in the experimental systems used. These results were consistent with the general lack of genotoxic potential for 2,4-D in a number of other test systems.

Clastogenicity of isoamylene oxide to rat lymphocytes in culture.

The mutagenic activity of the aliphatic epoxide isoamylene oxide (2-methyl-2,3-epoxybutane) is not readily detectable in the standard Ames test. In this study, the clastogenic potential of isoamylene oxide was evaluated using an in vitro mammalian cell culture system. Approximately 48 h after establishing primary cultures of rat lymphocyte cultures, the cells were treated for 4 h with various concentrations of isoamylene oxide (50, 166.7, 500,, 1666.7, and 5000 micrograms/ml in the initial assay and 500, 1000, 2000, 3000, 4000, and 5000 micrograms/ml in the confirmatory assay). The cultures were harvested 24 h after termination of the treatment. Based upon the mitotic indices, cultures treated with the three highest concentrations in both the initial and confirmatory assays were evaluated to estimate the chromosomal aberration frequencies. Isoamylene oxide demonstrated a strong clastogenic activity in this assay: up to 29% aberrant cells (without gaps) were observed at the highest concentration analyzed. The presence of an external metabolic activation system (S9) did not seem to influence the magnitude of the response at the dose levels analyzed.

Evaluation of the genetic toxicity of the organophosphate insecticide chlorpyrifos.

The genetic toxicity of chlorpyrifos [O,O,-diethyl-O-(3,5,6-trichloro-2- pyridinyl)phosphorothioate, C.A.S. Number: 2921-88-2)], an organophosphate insecticide, was examined by employing several end points such as gene mutations in bacteria (Ames test) and mammalian cell cultures (CHO/HGPRT assay), cytogenetic abnormalities in mammalian cells both in vitro (rat lymphocyte chromosomal aberration test, RLCAT) and in vivo (mouse bone marrow micronucleus test) and induction of DNA damage and repair in rat hepatocytes in vitro. There was no indication of genotoxic activity for chlorpyrifos in any of these assays. These results are consistent with the reported lack of carcinogenic potential for chlorpyrifos in both mice and rats.

Chromosomal aberrations in bone marrow and peripheral blood cells from the same rats.

Spontaneous cytogenetic aberrations were analyzed in bone-marrow cells and cultured peripheral lymphocytes from the same animals. No significant differences in the total number of cells with aberrations or total aberrations were detected between the bone-marrow cells and cultured lymphocytes. It was concluded that short-term culture does not contribute significantly to in vivo aberration yield within the experimental conditions used.

Utilization of rat lymphocytes for the in vitro chromosomal aberration assay.

In vitro cytogenetic assays are widely conducted to assess the mutagenic potential of chemicals. Chinese hamster ovary (CHO) cells or human lymphocytes are often used for these assays; however, these cell types have certain limitations. In order to evaluate an alternate cell system, cultured rat lymphocytes were treated for 4 h at 48 h of incubation with a variety of direct- and indirect-acting clastogens in the presence or absence of an exogenous mammalian activation system. Cytogenetic effects of in vitro physiological alterations such as medium hypertonicity or pH changes were also evaluated. The background aberration rate of rat lymphocytes is approximately 2%, and they respond positively to both direct- and indirect-acting clastogens. In contrast to CHO cells, however, neither the hyperosmolality nor pH changes in the treatment media have significant effect on background aberrations. Unlike samples of human blood, rat blood can be collected under well-controlled environmental conditions. Because of the easy access to rat blood samples, the simplicity of culture, the reproducible nature of its in vitro growth, the positive response to known clastogens and negative response to media pH changes or hyperosmolalities, the rat lymphocyte in vitro chromosomal assay presented is an optimal system to assess the mutagenic potential of chemicals.

Assessment of cell cycle duration on the incidence of sister chromatid exchanges in somatic and spermatogonial cells of the rat.

In order to examine the influence of the length of cell cycle on the incidence of sister chromatid exchanges (SCEs), average generation times and SCEs were studied in the presence of bromodeoxyuridine from spontaneously dividing rat lymph node, bone marrow, spleen, and spermatogonial cells. Average generation time differences among the three somatic cell types (lymph node, 7.6 h; bone marrow, 12.0 h; spleen, 14.9 h) were statistically significant as were the differences between the germinal cell (37.4 h) and each of the somatic cells. The SCE per cell frequencies were significantly higher in the somatic cells (lymph node, 6.8; bone marrow, 5.8; spleen, 6.1) as compared to the spermatogonial cells (1.6). However, no difference in SCE incidence was detected among the cells from the different somatic tissues. It was concluded that there was no simple relationship between cell cycle duration time and SCE formation.

Effect of food and water deprivation on the parameters of the mouse bone marrow micronucleus test.

This study reports the influence of acute starvation on spontaneous and cyclophosphamide (CP) induced micronucleus (MN) frequencies in the bone marrow polychromatic erythrocytes (PCE) of CD-1 mice. Groups of mice (5/sex) were deprived of either food alone or food and water for 0, 24, 48 and 72 h prior to sacrifice. Although there was no evidence of a significant increase in MN-PCE frequencies among the starved groups, a significant depressant effect of starvation on erythropoietic activity was observed. Peak levels of CP-induced (40 mg/kg b.w.) MN-PCE's appeared later in male mice deprived of food and water after treatment compared to mice given food and water ad libitum. The results indicate that starvation is detrimental to bone marrow erythropoiesis and that starvation may alter the response of mice to clastogens.

Cytogenetic variability of lymphocytes from phenotypically normal men: influence of age, smoking, season, and sample storage.

A cytogenetic study was conducted on cultured lymphocytes from a group of 60 male volunteers to determine the baseline of chromosomal aberrations in nonchemical workers. Only males were included in the study to avoid any sex effects on the results. Blood samples were collected from each man every 13 w (quarterly) over a period of 12 m. A single batch of culture medium was used for the entire study. The influence of storing the blood samples prior to culture, donor's age, cigarette smoking, and seasonal variation on lymphocyte mitotic index and chromosomal aberration yield was analyzed. A significant decrease in mitotic activity was observed in cultures from samples stored for 3 d at room temperature (22 +/- 1 degree C). Storing of samples at refrigerator temperature (4 +/- 1 degree C) for up to 3 d prior to culture did not affect lymphocyte growth. Although the mitotic index was found to be inversely proportional to the age of the donors, a significant influence of age on total cytogenetic aberrations was not detectable. A group of 15 smokers appeared to have higher number of chromosomal aberrations; however, the difference in mean mitotic activity between lymphocytes of the two groups was not statistically significant. No detectable seasonal influence was found on any chromosomal aberration category except in the number of chromatid gaps. The mitotic indices of the first quarter cultures, on the other hand, showed significant differences from the other three quarters. The chromosomal aberration baseline of the group was not strikingly different from the ones reported by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of sister chromatid exchanges in lymphocytes cultured from 71 healthy men.

Sister chromatid exchanges (SCE) were analyzed in peripheral blood lymphocytes from a select group of 71 healthy men, 56 nonsmokers and 15 cigarette smokers. In addition to estimating baseline SCE, data were examined to seek relationships of SCE frequencies to age and smoking. The baseline value of 7.53 SCE per cell from the 56 nonsmokers was within the range (5.60 to 9.10 SCE/cell) reported for other human populations. No relationship was found between the mean SCE frequency per cell and age. However, a significant increase in the SCE mean value was observed in smokers as compared to nonsmokers. The results of this study are compared with those of other reports on SCE effects of age and smoking.

Analyses for in vitro growth conditions, cytokinetics, and sister chromatid exchanges in lymphocytes cultured from the Sprague-Dawley rat.

Peripheral blood samples from Sprague-Dawley rats gave successful lymphocyte growth in GIBCO: 1A, RPMI 1640, and Eagle's minimum essential medium (MEM) culture media. Various growth conditions, cytokinetics, and sister chromatic exchange (SCE) induction were studied using reconstituted GIBCO 1A only. Neither methoxyflurane anesthesia of the rats before sampling nor washing of the cells with phosphate buffered saline affected the mitotic index. Cultures treated with [3H]thymidine showed the lymphocytes entering into DNA synthesis after approximately 24 h. The time at which BUdR (5-bromo-2' deoxyuridine) was added, i.e. 0 vs. 24 h incubation, had minimal effect on the mitotic index of cultures harvested at 48 h. However, when harvest was extended to 72 h, mitotic activity was greater in the cultures treated with BUdR at 24 h. No significant differences in mitotic index and the number of average lymphocyte division were detected in cultures exposed to 0.3 to 0.5 microgram/ml BUdR at 24 h and harvested at 72 h. Although SCE frequencies increased in the presence of BUdR, the baseline level of SCEs was estimated to be 5 to 6/cell. Average generation time of the lymphocytes dividing between 48 and 72 h was 16.5 h. Because of its simplicity of culture and the reproducible nature of its in vitro growth kinetics, the Sprague-Dawley rat lymphocyte is a suitable model for cytogenetic investigations.

Supernumerary chromosomal elements in lymphocytes cultured from phenotypically normal human adult males.

In a group of phenotypically normal men there were approximately 0.24% of metaphase lymphocytes with extra chromosomal elements along with the regular 46 chromosomes. They ranged in size from small acrocentric-acentric elements to elements longer than any chromosome arm. These elements have been referred to as supernumerary chromosomal elements. No significant effects due to donor's age, smoking history, season, storage of blood samples prior to culture, or culture medium, were found either in the frequency of supernumerary elements per cell or in the frequency of cells with supernumerary elements. Furthermore, the same subject did not consistently exhibit supernumerary elements. Furthermore, the same subject did not consistently exhibit supernumerary elements when sampled during four successive quarters of the year. Some of these elements in pairs were identified by G-banding technique as translocation chromosomes bearing long arms of chromosome number 2 and presumptive short arms of chromosome 8, acentric long arms of chromosome 4, and iso-acentric chromosomes for the long arms of chromosome 5. Presumably, more than one type of cytogenetic event occurred in their formation. Circumstantial evidence has been presented to show that the means of elimination of these supernumerary elements is a process of chromosomal disintegration.

The incidence of spontaneous cytogenetic aberrations in lymphocytes cultured from normal humans for 48 and 72 h.

Spontaneously occurring cytogenetic aberrations were compared in 48- and 72-h human lymphocyte cultures from a group of 20 normal male volunteers. Mitotic indices for both cultures times were also evaluated. BUdR (5-bromo, 2-deoxyuridine) labelled metaphases from three men were utilized to determine the cell cycle kinetics in the medium used. There was no statistically significant difference between 48- and 72-h cultures among the majority of aberration categories considered. Possibly, the lymphocyte populations at the two different culture times maintained a consistent yield level. Mitotic indices in 72-h cultures were invariably higher than those in 48-h cultures. An overwhelming number of first division metaphases were observed at 48 h of incubation; however, approximately 22% of the lymphocytes did not enter into mitosis until 72 h in culture. A number of difficulties with regard to utilizing BUdR-labelled first division metaphases for cytogenetic analyses are discussed.

Evaluation of the effect of food deprivation on micronucleus test results.

High doses of a test chemical sometimes affect the food consumption of the treated animals even to the point of starvation. The effect of such a non-specific toxicological stress on the bone marrow micronucleus test was investigated in male and female Sprague-Dawley rats. A 42-h starvation had a noticeable effect on the ratio of polychromatic to normochromatic erythrocytes (PCE and NCE); the values in the starved animals were approx. 77% of the negative controls. An effect of starvation on the frequency of micronucleated erythrocytes (MN-E) was not discernable from the results of the present experiment.

Evaluation of culture media for effects on cell cycle kinetics and incidence of chromosomal aberrations in human blood cells.

Peripheral blood samples from 17 apparently healthy male volunteers were set up in duplicate cultures using three commercially available media: Eagle's MEM, RPMI 1640, and TC 199. BUdR (5-bromo,2-deoxyuridine) (10 micrograms/mL) was added to one of the cultures from each person in each medium after 24 h of culture initiation. All cultures were harvested at 72 h of incubation in the presence of colcemid. RPMI 1640 stimulated the highest mitotic activity in both BUdR-treated and untreated cultures. Higher numbers of first division metaphases corresponded with the higher frequency of chromosome-type aberrations in cultures with Eagle's MEM as compared with RPMI 1640 media. On the other hand, higher numbers of chromatid-type aberrations were present in cultures with TC 199 as compared with those with Eagle's MEM. When the chromosome- and chromatid-type aberration data were pooled to score total cytogenetic abnormalities, an influence of the medium was demonstrable. While cultures with Eagle's MEM and TC 199 had the greater number of first division cells, third of subsequent division cells were most prevalent in RPMI 1640 cultures. It is inferred that the length of the cell cycle, the mitotic index, and to some degree the incidence of spontaneous cytogenetic abnormalities are variable attributes of culture media.

In vivo cytogenetic study in rats maintained for eight days on diets containing probucol.

Probucol (4,4'-(isopropylidenedithio)bis(2,6-di-t-butylphenol], a cholesterol-lowering drug, was evaluated for cytogenetic toxicity in the bone-marrow cells of Sprague-Dawley rats. Male and female rats were fed diets containing 0, 200, 400, or 800 mg/kg body weight/day for 8 consecutive days. Animals treated with trimethylphosphate served as positive controls. Femoral bone-marrow specimens were aspirated from all animals for cytogenetic evaluation. Analysis of the data generated by this study indicated that the incidence of cytogenetic aberrations in the bone marrow of rats was not affected by administration of probucol within the present treatment regimen.

A note on the utility of Fischer-344 rat in the micronucleus test.

Bone marrow smears from Fischer-344 rats possessed such a high incidence of basophilic granules that accurate detection of micronucleated erythrocytes was not possible. In contrast, preparations from Sprague-Dawley rats had far fewer such granules, so that the micronucleated marrow cells were clearly visualized and counted. The difference in the incidence of basophilic granules was attributed to strain differences in mast cell population size.