Toxicological and cytogenetic assessment of a Salacia oblonga extract in a rat subchronic study.

Salacia oblonga holds potential as a natural method to mitigate the blood glucose response for people with diabetes by inhibiting the activity of intestinal alpha-glucosidases. As part of a safety evaluation of novel ingredients for use in blood glucose control, the toxicity of a S. oblonga root extract (SOE) was evaluated in a subchronic 90-day feeding study in rats. An in vivo-in vitro rat peripheral blood lymphocyte chromosomal aberrations assay was added at termination of the subchronic rat study to examine cultured lymphocytes for possible chromosomal aberration induction. This was conducted due to a previous weak; although reproducible, positive chromosomal aberrations response in cultured peripheral blood human lymphocytes after acute in vitro treatment with SOE. The present study results indicate that SOE was negative for the induction of chromosomal aberrations in cultured rat peripheral blood lymphocytes after 90 consecutive days of treatment with SOE. The no observable adverse effect level (NOAEL) was determined to be 2,500 mg/kg/day following daily subchronic oral gavage administrations to rats.

Genotoxicity testing of a Salacia oblonga extract.

Salacia oblonga has been used for thousands of years in Ayurvedic medicine for the oral treatment of diabetes. The root extract has been shown to inhibit the activity of intestinal alpha-glucosidases, therefore S. oblonga holds potential as a natural method to mitigate the blood glucose response for people with diabetes. As part of a safety evaluation of novel ingredients for use in blood glucose control, the potential genotoxicity of a S. oblonga root extract (SOE) was evaluated using the standard battery of tests (reverse mutation assay; chromosomal aberrations assay; mouse micronucleus assay) recommended by US Food and Drug Administration (FDA) for food ingredients. SOE was determined not to be genotoxic under the conditions of the reverse mutation assay and mouse micronucleus assay, and weakly positive for the chromosomal aberrations assay. A reproducible, although weak, positive chromosomal aberrations response in human lymphocytes is of concern and further toxicity research is recommended. Use of SOE is presently expected to be safe, as anticipated intake is small compared to the doses administered in the genotoxicity assays and may, after further toxicity research, may prove be a useful ingredient in foodstuffs.

Application of simplified in vitro screening tests to detect genotoxicity of aristolochic acid.

Aristolochic acid (AA), the active compound found in Aristolochia extracts, has been used as a traditional medicine. However, products containing AA were withdrawn from the market in the early 1980s because AA was found to be a potent carcinogen. Some genotoxicity studies of AA were conducted after the carcinogenicity of AA was reported. The purpose of this study was to check the ability of simplified, screening tests for genotoxicity to indicate the genotoxic activities of AA. Four commonly used in vitro genotoxicity endpoints were examined. In a bacterial mutation screening test, AA was mutagenic to tester strains TA98 and TA100 with and without rat liver S9. In the L5178Y mouse lymphoma cell gene mutation test, mutagenic activity was observed at > or = 25 microg/ml with or without S9. A concentration-dependent increase in structural chromosome aberrations was observed in CHO cells, with significant increases at 50 microg/ml without S9 and at 25 microg/ml with S9. Significant increases in micronucleated binucleated cells were observed in CHO cells treated with AA at > or = 25 microg/ml with or without S9. These results demonstrated that the genotoxicity of AA would have been easily detected if simple screening versions of in vitro genotoxicity assays had been used during early product development. It is suggested that simplified screening tests such as those used in this study would be a rapid and economical way of obtaining the preliminary genotoxicity profiles of new substances or products as an aid to decision-making for further development.

Genotoxicity testing of a fenugreek extract.

Fenugreek seeds have been used in traditional medicines as a remedy for diabetes. Rich in protein, fenugreek seeds contain the unique major free amino acid 4-hydroxyisoleucine (4-OH-Ile), which has been characterized as one of the active ingredients for blood glucose control. Current use of fenugreek in foodstuff has been limited to its role as a flavoring agent, and not as an ingredient to help mitigate the blood glucose response for people with diabetes. As part of a safety evaluation of novel ingredients for use in blood glucose control, the potential genotoxicity of a fenugreek seed extract (THL), containing a minimum of 40% 4-OH-ILE, was evaluated using the standard battery of tests (reverse mutation assay; mouse lymphoma forward mutation assay; mouse micronucleus assay) recommended by US Food and Drug Administration (FDA) for food ingredients. THL was determined not to be genotoxic under the conditions of the tested genetic toxicity battery. The negative assay results provide support that addition of THL to foodstuffs formulated for people with diabetes is expected to be safe. A wide safety margin is established, as anticipated doses are small compared to the doses administered in the assays.

Lack of in vivo clastogenic activity of grape seed and grape skin extracts in a mouse micronucleus assay.

Meganatural brand grape seed extract (GSE) and grape skin extract (GSKE), containing proanthocyanidin polyphenolic compounds, are intended for use in food as functional ingredients exhibiting antioxidant activity. Proanthocyanidins, as well as the minor constituent phenolic compounds in GSE and GSKE, are present naturally in many foods such as fruits, vegetables, chocolate, tea, etc., and on average people consume 460-1000 mg/day of these combined substances. While some polyphenolic compounds, tested individually, have demonstrated antitumorigenic or antipromotional activity, at least one minor component of GSE and GSKE, quercitin, has exhibited positive activity in Salmonella and other in vitro mutagenicity assays. As part of a program to investigate the safety of GSE and GSKE, these products were tested for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in Crl:CD-1(ICR) BR mouse bone marrow. The appropriate test article was dissolved in 0.5% carboxymethylcellulose and dosed by oral gavage to five males/test article/dose level/harvest time point. Animals were dosed at 500, 1000 and 2000 mg/kg. Five animals dosed with either test article at 500, 1000 and 2000 mg/kg dose levels and five animals dosed with the cyclophosphamide (80 mg/kg) positive control were euthanized approximately 24 h after dosing for extraction of bone marrow. Five animals dosed with either test article at the 2000 mg/kg dose level and five animals dosed with the vehicle control article were euthanized approximately 24 and 48 h after dosing for extraction of bone marrow. At least 2000 PCEs per animal were analyzed for frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 erythrocytes for each animal. For both GSE and GSKE, no statistically significant increase in micronucleated PCEs was observed at any dose level or harvest time point. GSE produced indication of cytotoxicity (decreased PCE:NCE ratio) at the 2000 mg/kg dose level for the 48-h harvest time point, confirming that the test article reached the target bone marrow in significant amount. Meganatural GSE and Meganatural GSKE were evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.

Differential sensitivity of Chinese hamster V79 and Chinese hamster ovary (CHO) cells in the in vitro micronucleus screening assay.

Both the V79 and CHO cell lines are routinely used in the in vitro MN screening assay for the detection of possible genotoxicants. The CHO cell line is the predominant cell line currently used in the genetic toxicology testing industry. However, some laboratories routinely utilize the V79 cell line since the in vitro MN screening assay was initially developed using V79 cells. Our laboratory has historically used the CHO cell line. Therefore, our laboratory was interested in comparing the two cell lines with regard to possible similarities or differences in MN induction sensitivity after exposure to cyclophosphamide (CPA) and mitomycin C (MMC), the two standard positive control chemicals routinely used in this assay. Three exposure conditions in the presence of CPA and MMC were examined in both cell lines. Replicate cultures of CHO cells in McCoy's 5A and V79 cells in both McCoy's 5A and E-MEM were established and treated with 5 microg CPA/ml (4h exposure with S9), 0.5 microg MMC (4h exposure without S9) and 0.5 microg MMC (24h exposure without S9). A total of 400 cytochalasin B-blocked binucleated cells and 200 consecutive cells were analyzed from each culture for MN and cell cycle kinetics, respectively. Analysis of the data demonstrated that CHO cells were up to approximately five-fold more sensitive to the induction of CPA- and MMC-induced MN than V79 cells. Both cell lines exhibited similar average generation times among identical exposure groups. Therefore, the difference in MN sensitivity cannot be attributed to possible differences in cell cycle kinetics and is possibly related to inherent cellular differences in the processing of and/or repair of CPA- and MMC-induced damage by V79 and CHO cells.

Micronuclei and gene mutations in transgenic big Blue((R)) mouse and rat fibroblasts after exposure to the epoxide metabolites of 1, 3-butadiene.

1,3-Butadiene (BD) is a commodity compound and by-product in the manufacture of synthetic rubber that elicits a differential carcinogenic response in rodents after chronic exposure. Mice are up to approximately 1000-fold more sensitive to the tumorigenicity of inhaled BD than rats, thereby confounding human risk assessment analyses. Rodent transgenic in vivo and in vitro models have been recently utilized for generating genetic toxicology data in support of risk assessment studies. However, studies have not been extended to investigate multiple endpoints of genetic damage using in vitro transgenic models. The goal of this study was to evaluate possible differences in the production of genetic damage in transgenic Big Blue((R)) mouse (BBM1) and rat (BBR1) fibroblasts exposed to three predominant epoxide metabolites of BD. Analyses of cytotoxicity, micronucleus (MN) formation, cII mutant frequency (MF) and apoptosis were assessed after in vitro exposure of BBM1 and BBR1 cells exposed to various concentrations of butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). Both BMO and DEB reduced cell survival in BBM1 and BBR1 cells. However, BDE decreased cell survival only in BBM1 cells at the concentrations evaluated. Concentration-dependent increases in the formation of MN was observed in both BBM1 and BBR1 cells, with DEB being the most potent followed by BDE and then BMO. The dose-response for mutations induced at the cII locus was essentially equal after DEB exposure of BBM1 and BBR1 fibroblasts. In contrast, the cII MF was significantly increased only in BBM1 cells after exposure to either BMO or BDE. These data demonstrate a differential genetic response for gene mutations but not for MN formation in transgenic BBM1 and BBR1 fibroblasts and suggest a rodent species-specific difference in the persistence of DNA damage that results in gene mutations. In addition, apoptosis was observed in BBR1 cells but not in BBM1 cells when treated with any of the three BD epoxide metabolites. This response may partially explain the differential response to mutations induced by BMO and BDE. These data offer insight into specific differences in mouse and rat cells with respect to their response to BD epoxide metabolites. Such data may help to explain the different tumorigenicity results observed in rodent BD carcinogenicity studies.

Reduction of diepoxybutane-induced sister chromatid exchanges by glutathione peroxidase and erythrocytes in transgenic Big Blue mouse and rat fibroblasts.

We have investigated the effect of glutathione peroxidase (GSH-Px) and mammalian erythrocytes (RBCs) on spontaneous and diepoxybutane (DEB)-induced sister chromatid exchange (SCE) in primary Big Blue(R) mouse (BBM1) and Big Blue(R) rat (BBR1) fibroblasts. DEB is the putative carcinogenic metabolite of 1,3-butadiene (BD) for which inhalation exposure yields a high rate of malignancies in mice but not in rats. BD is metabolized differently in mice and rats, producing much higher levels of DEB in mice than in rats, which may partly explain the different carcinogenic responses. However, other factors may contribute to the observed differences in the rodent carcinogenic response to BD. DEB is a highly reactive compound. Upon epoxide hydrolysis, DEB can covalently bind to DNA bases. Likewise, DEB generates reactive oxygen species that, in turn, can either damage DNA or produce H(2)O(2). Reduced glutathione (GSH) is known to play a role in the metabolism and detoxification of DEB; and GSH is reduced by GSH-Px in the presence of H(2)O(2). GSH-Px is a constitutive enzyme that is found at high concentrations in mammalian RBCs. Therefore, we were interested in examining the role of RBCs and GSH-Px on DEB-induced SCE in rat and mouse cells for detection of possible differences in the species response. Transgenic BBM1 and BBR1 fibroblasts were treated with either 0, 2 or 4 microM DEB plus 0, 2 or 20 units of GSH-Px with and without 2x10(8) species-specific RBCs. DEB effectively induced SCEs in both rat and mouse cells. The relative induction of SCEs in both cell types was comparable. Both GSH-Px and RBCs alone and in combination were effective in significantly reducing DEB-induced SCEs in both mouse and rat fibroblasts, although there was more variability in the SCE response in rat cells. The present study suggests that GSH-Px may be important in the detoxification of DEB-induced DNA damage that results in the formation of SCEs.

Characterization of new transgenic Big Blue(R) mouse and rat primary fibroblast cell strains for use in molecular toxicology studies.

We have established and characterized primary mouse and rat cell strains for studies designed to complement in vivo gene mutation assays using the Big Blue(R) mouse or rat. Primary fibroblast cell strains, designated BBM1 and BBR1, were derived from a transgenic male Big Blue(R) B6C3F1 mouse and from a male Big Blue(R) Fischer-344 rat, respectively. Both BBM1 and BBR1 are genetically stable and mostly diploid. Both cell strains have low spontaneous frequencies of mutation at the lacI and cII loci as well as low frequencies of sister chromatid exchange and micronuclei formation. In addition, N-ethyl-N-nitrosourea (ENU) induces mutations at the cII locus in both BBM1 and BBR1 cells. These new primary Big Blue(R) mouse (BBM1) and rat (BBR1) fibroblast cell strains represent useful new models for molecular toxicology studies. Environ. Mol. Mutagen. 34:90-96, 1999 Published 1999 Wiley-Liss, Inc.

An evaluation of the feasibility of using cytogenetic damage as a biomarker for alachlor exposure.

Alachlor is a widely used herbicide for which there is significant human exposure, principally through groundwater contamination and inhalation. Because alachlor is purported to be carcinogenic and mutagenic, we initiated studies to determine if induced cytogenetic damage could be used as a biomarker for exposure to this herbicide. Both isolated and whole blood human lymphocytes were exposed to alachlor using several protocols. The lymphocytes were cultured for analysis of sister chromatid exchange (SCE), chromosome aberrations (CAs), micronuclei (MN) in cytochalasin B-induced binucleated cells, and proliferation kinetics using the replicative index (RI). In addition, CD rats were injected with either 10 or 50 mg kg-1 of alachlor, 2-chloro-N-(2,6-diethylphenyl) acetamide (CDEPA) or 2, 6-diethylanaline (DEA). After 24 h, the peripheral blood lymphocytes were removed and cultured for SCE and RI analysis. Alachlor did induce a concentration-related increase in SCE in vitro, but neither it nor its metabolites (CDEPA or DEA) induced a significant increase in SCEs or an alteration of RI in vivo. At the highest in vitro concentration tested, alachlor induced a statistically-significant increase in MN, but no concomitant increase in CAs was seen. From analyses of our data and the literature on alachlor clastogenicity and exposure levels, we concluded that cytogenetic damage may not be an adequately sensitive marker for evaluating human exposure to alachlor.

Cytogenetic characterization of the transgenic Big Blue Rat2 and Big Blue mouse embryonic fibroblast cell lines.

The transgenic Big Blue Rat2 and Big Blue mouse embryonic fibroblast cell lines have been used to complement the transgenic Big Blue rat and mouse in vivo mutagenesis assays. However, limited information is available regarding the karyology of these cell lines. Therefore, we have characterized the ploidy, mitotic index, spontaneous frequencies of chromosome and chromatid aberrations and rate of micronucleus (MN) formation in both cell lines. We have also characterized the frequency of sister chromatid exchange (SCE) in transgenic Big Blue mouse cells. Big Blue Rat2 cells are hyperploid and have extremely high baseline frequencies of cytogenetic damage. In addition, Big Blue Rat2 cells are BrdU-resistant, therefore, SCE frequencies cannot be assessed in these cells. We conclude that Big Blue Rat2 cells are not useful for routine cytogenetic toxicology studies. The transgenic Big Blue mouse cell line is polyploid and consistently yields a low mitotic index (approximately 1%) in untreated cells. These mouse cells also exhibited moderately high baseline frequencies of chromosome and chromatid aberrations, however, baseline frequencies of SCE and of MN were not elevated. Transgenic Big Blue mouse embryonic fibroblasts were further studied for MN induction following treatment with N-ethyl-N-nitrosourea (ENU) for 0.5 h at concentrations of 0.425, 0.85 and 1.7 mM. Concentration-dependent increases in MN were observed in these cells. Thus, while an ENU-induced cytogenetic response using transgenic Big Blue mouse cells demonstrates that this cellular model could be used to cytogenetically complement the mutagenesis assays, the low mitotic index and the high spontaneous frequency of chromosome damage confounds its use for routine genetic toxicology studies.

Cytogenetic analyses of the in vitro and in vivo responses of murine cells to peroxyacetyl nitrate (PAN).

Peroxyacetyl nitrate (PAN) is one of a class of common air pollutant formed by the action of sunlight on volatile organic compounds and nitrogen oxides. PAN has been shown to be a bacterial mutagen. To determine if PAN can cause DNA damage in mammalian cells, we exposed murine peripheral blood lymphocytes (PBLs) to various volumes of PAN in vitro and analyzed the cells for chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and DNA damage using the single cell gel (SCG) assay. At in vitro concentrations of PAN that were cytotoxic (inhibited cell division), an increase in DNA damage was noted in the SCG assay. At lower exposure levels that permitted cell division, no increases in SCEs, CAs, or DNA damage were evident. For in vivo studies, male mice were exposed nose-only by inhalation for 1 h to 0, 15, 39 or 78 ppm PAN, and their lung cells removed and cultured for the scoring of SCEs and CAs. In addition, PBLs and lung cells were analyzed by the SCG assay. No dose-related effects were found in any of the assays. These data indicate that PAN does not appear to be a potent clastogen or DNA damaging agent in mammalian cells in vivo or in vitro.

Bleomycin sulfate-induced micronuclei in human, rat, and mouse peripheral blood lymphocytes.

The sensitivity to micronucleus (MN) induction of human, mouse, and rat peripheral blood lymphocytes (PBLs) exposed to bleomycin sulfate (BLM) in vitro was compared in cytochalasin B-induced binucleated (BN) cells. For the PBLs of each species, either 0, 5, 10, 20, 40, 60, 80, or 160 micrograms/ml BLM was added to 5 ml aliquots of whole blood for 4 hr at 37 degrees C in a 5% CO2 atmosphere. Leukocytes were isolated on a density gradient and cultured in the presence of phytohemagglutinin to stimulate blastogenesis, and cytochalasin B was added to each culture at 21 hr postinitiation to prevent cytokinesis. A total of 4,000 BNs/concentration/species was analyzed for MN in two independent experiments. In addition, multiple-MN-BNs were quantitated, and the nucleation index was determined. Significant increases both in total MN-BNs and multiple-MN-BNs were observed at all concentrations in all species. All three species' concentration-response curves gave good fits (r2 values from 0.87 to 0.95) to either a linear or a square root model (y = mx + b or y = m[x]0.5 + b, respectively; where y = the percentage of MN-BN, m is the slope, and b is the y-intercept). The MN induction in the human and rat PBLs was not statistically different, but both were significantly less sensitive than the response shown by the BLM-exposed mouse PBLs. This difference in MN susceptibility was observed only at BLM test concentrations > or = 20 micrograms/ml. The nucleation index was significantly decreased in all species at either 80 or 160 micrograms/ml.

Inhalation studies of the genotoxicity of trichloroethylene to rodents.

Trichloroethylene (TCE) (CAS No. 79-01-6) is an industrial solvent used in degreasing, dry cleaning, and numerous other medical and industrial processes. Controlled inhalation studies were performed using male C57BL/6 mice and CD rats to determine if TCE can induce cytogenetic damage in vivo. Animals were exposed in groups of five to target concentrations of either 0, 5, 500, or 5000 ppm TCE for 6 h. Tissue samples were taken between 18 and 19 h post exposure. Peripheral blood lymphocytes (PBLs) in rats and splenocytes in mice were cultured and analyzed for the induction of sister-chromatid exchanges, chromosome aberrations, and micronuclei (MN) in cytochalasin B-blocked binucleated cells. Bone marrow polychromatic erythrocytes (PCEs) were analyzed for MN. The only positive response observed was for MN in rat bone marrow PCEs. TCE caused a statistically significant increase in MN at all concentrations, inducing an approximate fourfold increase over control levels at 5000 ppm. TCE was also cytotoxic in rats, causing a significant concentration-related decrease in the ratio of PCEs/normochromatic erythrocytes. This study indicates that there may be species-specific cytogenetic effects attributed to TCE inhalation exposure. In follow-up studies, CD rats were exposed for 6 h/day over 4 consecutive days to either 0, 5, 50 or 500 ppm TCE. No statistically significant concentration-related increases in cytogenetic damage were observed. While the MN frequencies in the 4-day study were comparable to those at the equivalent concentrations in the 1-day study, they were not significantly elevated due to an unusually high MN frequency in the controls. A subsequent replication of the 1-day 5000 ppm TCE exposure with rats again showed a highly significant increase in MN frequencies compared to concurrent controls.

Cytogenetic and germ cell effects of phosphine inhalation by rodents: II. Subacute exposures to rats and mice.

Phosphine (PH3) is a highly toxic grain fumigant to which there is significant human workplace exposure. To determine the in vivo cytogenetic effects of inhalation of PH3, male F344/N rats and B6C3F1 mice were exposed to target concentrations of 0, 1.25, 2.5, or 5 ppm PH3 for 6 hr/day for 9 days over an 11-day period. Approximately 20 hr after the termination of exposures, blood was removed from the mice and rats by cardiac puncture and the lymphocytes cultured for analyses of sister chromatid exchanges and chromosome aberrations in rats and mice, and micronuclei (MN) in cytochalasin B-induced binucleated lymphocytes from mice. In addition, bone marrow (rats) and peripheral blood (mice) smears were made for the analysis of MN in polychromatic and normochromatic erythrocytes. No significant increase in any of the cytogenetic endpoints was found at any of the concentrations examined. These results indicate that concentrations of PH3 up to 5 ppm are not genotoxic to rodents when administered by inhalation for 9 days during an 11-day period as measured by several cytogenetic assays. To evaluate the effects of PH3 on male germ cells, a dominant lethal test was conducted in male mice exposed to 5 ppm PH3 for 10 days over a 12-day period and mated to groups of untreated females (2 females/male) on each of 6 consecutive 4-day mating intervals. None of the 6 groups of females exhibited a significant increase in percent resorptions. These results indicate that exposure to 5 ppm PH3 by inhalation does not induce dominant lethality in male mouse germ cells at steps in spermatogenesis ranging from late differentiating spermatogonia/early primary spermatocytes through mature sperm.

Cytogenetic effects of phosphine inhalation by rodents. I: Acute 6-hour exposure of mice.

Phosphine (PH3) is a highly toxic grain fumigant that can be produced from the reaction of metal phosphides with water. To determine the in vivo cytogenetic effects of inhalation of PH3, male CD-1 mice were exposed to either 0, 5, 10, or 15 ppm target concentrations of PH3 for 6 hr. Twenty hours after the termination of exposure, the spleens of the mice were removed, macerated, and the splenocytes cultured for analyses of sister chromatid exchanges, chromosome aberrations, and micronuclei in cytochalasin B-induced binucleated cells. In addition, bone marrow smears were made for the analysis of micronuclei in polychromatic erythrocytes. No increase in any of the cytogenetic endpoints was found at any of the concentrations examined. The only statistically significant response was a concentration-related slowing of the cell cycle in the splenocytes.

Analyses of cytogenetic damage in rodents following exposure to simulated groundwater contaminated with pesticides and a fertilizer.

Male Fischer 344 rats and female B6C3F1 mice were each exposed through their drinking water to a mixture of pesticides and ammonium nitrate that simulated contaminated groundwater in California (California Chemical Mixture [CCM]). Exposures were for 71 or 91 days, respectively. In addition, B6C3F1 female mice were exposed for 91 days to another pesticide and ammonium nitrate mixture (Iowa Chemical Mixture [ICM]) through their drinking water. The spleens were removed from the animals, and the splenocytes were cultured for analyses of sister-chromatid exchange (SCE), chromosome aberrations (CA), and micronuclei (MN) in cytochalasin B-induced binucleate cells. A concentration-related increase in SCEs was found in the splenocytes of the rat at the 1x, 10x and 100x levels of the CCM and at the 100x concentration of the CCM in the mouse. There were no other consistent cytogenetic effects observed with the CCM, and no statistically significant cytogenetic damage was observed in mice exposed to the ICM. Evidence from the literature is discussed in order to infer which chemical or chemicals in the CCM might be responsible for the observed SCE response.

Cytogenetic studies of rodents exposed to styrene by inhalation.

Female B6C3F1 mice and Fischer 344 rats were exposed to styrene at nominal concentrations of 125, 250 and 500 ppm by inhalation for 6 h per day for 14 consecutive days. One day after the final exposure, murine peripheral blood lymphocytes, spleen and lungs were removed, and the cells were cultured for analysis of chromosomal aberrations, micronucleus induction (using the cytochalasin B-block method) and sister chromatid exchange. Peripheral blood smears were scored for micronucleus induction in normochromatic erythrocytes. For the rats, peripheral blood lymphocytes were cultured for analyses of sister chromatid exchange, chromosomal aberrations and micronuclei in cytochalasin B-induced binucleated cells and were also examined in the single-cell gel assay for analysis of DNA strand breakage under alkaline conditions. Bone-marrow smears were made from femurs of rats for analysis of micronucleus induction in normochromatic erythrocytes. Small but statistically significant concentration-related increases in the frequency of sister chromatid exchange were seen in both mice and rats in all cell types examined. No statistically significant concentration-related increase in chromosomal aberration or micronucleus induction frequencies were observed in either species, and there was no significant increase in DNA strand breakage in peripheral blood lymphocytes from exposed rats. These results indicate that styrene is a weak inducer of sister chromatid exchange in vivo when administration to rodents by inhalation.

Evaluation of a three-exposure mouse bone marrow micronucleus protocol: results with 49 chemicals.

Forty-nine chemicals were tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection. Bone marrow samples were obtained 24 hr following the final exposure. Twenty-five rodent carcinogens and 24 noncarcinogens were selected randomly from the 44 carcinogens and 29 noncarcinogens used by Tennant et al. (Science 236:933-941, 1987) to evaluate the performance of four in vitro genetic toxicity tests. As in that study of in vitro tests, the micronucleus tests were conducted with coded chemicals and test results (positive or negative) were determined prior to decoding. This study was conducted as part of an effort to assess the ability of the micronucleus test to discriminate between rodent carcinogens and noncarcinogens and to determine its potential role, in combination with other short-term tests, in identifying genotoxic chemicals that present a carcinogenic hazard. Nine chemicals were judged to be positive in the micronucleus test. This relatively low number of positive results, along with published and unpublished results from rodent micronucleus and chromosome aberration assays on several of these 49 chemicals, contributed to the conclusion that a single micronucleus test protocol is not adequate to detect all chemicals capable of inducing chromosomal damage in the bone marrow. However, a combination of two relatively simple assays such as the Salmonella and micronucleus tests can provide important information on the genetic toxicity of test chemicals and may provide guidance on the need for and the nature and extent of future toxicity studies.

DNA adducts and induction of sister chromatid exchanges in the rat following benzo[b]fluoranthene administration.

Benzo[b]fluoranthene (B[b]F) was administered (100 mg/kg by i.p. injection) to male Sprague--Dawley rats. Lungs, livers and peripheral blood lymphocytes (PBLs) were harvested 1, 3, 5, 7, 14, 28 and 56 days after treatment. Several DNA adducts were observed in each tissue, with maximal levels occurring at approximately 7 days after treatment. Lung DNA exhibited consistently higher adduct levels than liver or PBL DNA. At 56 days after B[b]F administration, the adducts in liver and PBL DNA were present at < 10 amol/microgram DNA, while in lung there were 100 amoles/microgram DNA. No significant differences were observed between tissues in the types of adducts produced. Co-chromatography with synthetic standards showed that only a minor adduct produced in vivo is derived from trans-9,10-dihydro-9,10-dihydroxybenzo[b]fluoranthene-11,12-oxide. Sister chromatid exchanges (SCEs) from whole blood cultures were significantly increased relative to concurrent controls between 1 and 14 days after B[b]F administration, with maximum levels at 14 days. By 28 days after treatment, SCEs had essentially returned to control levels. SCE induction did not correlate with the amount of B[b]F--DNA adducts remaining in the PBLs at harvest time.