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 However, products containing AA were withdrawn from the market in the early 1980s because AA was found to be a potent carcinogen.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.

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.

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 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.

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.

Interspecies cytogenetic comparisons: studies with X-radiation and bleomycin sulfate.

A series of in vitro experiments were conducted to determine if there are innate differences in the sensitivity of peripheral blood lymphocytes (PBLs) from different mammalian species to clastogens. Mouse, rat, and human whole blood samples were exposed to either 0, 0.38, 0.75, 1.5, or 3.0 Gy x-radiation or 0, 5, 10, 20, 40, or 80 micrograms/ml bleomycin for 4 hr. Bromodeoxyuridine-containing cultures were initiated and the PBLs stimulated to divide with phytohemagglutinin. All cultures were harvested following a 3-hr colcemid treatment. Slides were made and differentially stained, and first-division metaphases were scored for chromosome aberrations. In the x-radiation studies human PBLs were significantly more sensitive than mouse PBLs which were in turn more sensitive than rat PBLs as measured by either the total percent aberrant cells or the number of dicentrics. Data from all three species could be fitted to a linear-quadratic model. Results with bleomycin suggest that the mouse and human PBLs are equally sensitive to the clastogenic effects of bleomycin. Both appeared to be more sensitive than the rat PBLs, but the variation between experiments was such that the results among species were not significantly different. These results indicate that there may be inherent differences in sensitivity among PBLs of mammalian species; however, more studies are needed to determine if the differences presented here hold for other agents.

Induction of micronuclei by X-radiation in human, mouse and rat peripheral blood lymphocytes.

We compared the radiosensitivity of human, rat and mouse peripheral blood lymphocytes (PBLs) by analyzing micronuclei (MN) in cytochalasin B-induced binucleated (BN) cells. For each species and dose 4-ml aliquots of whole blood were X-irradiated to obtain doses of 38, 75, 150 or 300 cGy. Controls were sham-irradiated. After exposure to X-rays, mononuclear leukocytes were isolated using density gradients and cultured in RPMI 1640 medium containing phytohemagglutinin to stimulate mitogenesis. At 21 h cytochalasin B was added to produce BN PBLs, and all cultures were harvested at 52 h post-initiation using a cytocentrifuge. Significant dose-dependent increases in the percentage of micronucleated cells and the number of MN per BN cell were observed in all three species. The linear-quadratic regression curves for the total percentage of micronucleated cells for the three species were similar; however, the curve for the mouse PBLs had a larger quadratic component than either of the curves for the rat or human PBLs. Although the correlation between the percentage of cells with MN and those with chromosome aberrations was high (r2 greater than 0.95), the mouse and rat PBLs were over twice as efficient as human PBLs in forming MN from presumed acentric fragments. These data indicate that the induction of MN in BN cells following ionizing radiation is similar in human, rat and mouse PBLs, but care must be taken in using the MN results to predict frequencies of cells with chromosomal aberrations.

Effect of acrolein on phosphoramide mustard-induced sister chromatid exchanges in cultured human lymphocytes.

Although phosphoramide mustard (PM) is generally recognized as being the most genotoxic metabolite of cyclophosphamide (CP), the contribution of acrolein to the cytogenetic toxicity of CP is unclear. Besides covalently binding to DNA, acrolein can inactivate critical proteins necessary for replicative DNA synthesis, RNA transcription, cell membrane integrity, and metabolism of xenobiotic and endogenous substrates. Because enzymatic processes are involved in sister chromatid exchange (SCE) formation and DNA excision repair, we hypothesized that acrolein might modulate SCE induction by PM due to acrolein's high binding affinity for proteins and low molecular weight sulfhydryl compounds. Human mononuclear leukocytes were isolated on a Ficoll-Hypaque density gradient, and 10(6) cells were inoculated into 1.9 ml of complete medium. T-cells were stimulated to grow with 4 micrograms concanavalin A/ml, and 5-bromo-2'-deoxyuridine (5 microM) was added 24 h later. The cultures were then treated with PM (0.069 microM) in the absence or presence of diethyl-4'-hydroperoxy-CP (DEHP-CP), an activated acrolein-generating compound, at concentrations of 0.1, 1, or 10 microM for 48 h. Demecolcine (1.35 microM) was added for the final 4 h of culture. PM alone induced about a 2-fold increase in the SCE frequency (PM, 14.1 +/- 0.5 (SD) versus control, 7.7 +/- 0.4) without cell cycle inhibition or reduced mitotic activity. DEHP-CP induced a concentration-related increase in the SCE frequency of up to 1.6-fold without any significant cell cycle inhibition or lowered mitotic activity. When PM and DEHP-CP were combined, SCE induction was additive for all three DEHP-CP concentrations. Except at the highest molar ratio of DEHP-CP:PM (145:1), there was no evidence of cytotoxicity in the other treatment groups. These results suggest that acrolein has a diminished role in mediating the cytogenetic and cytotoxic effects of CP. In addition, enzymes associated with SCE formation and, by inference, DNA excision repair may not be particularly susceptible to acrolein-induced inactivation.

Effect of treatment protocol and sample time on the frequencies of micronucleated polychromatic erythrocytes in mouse bone marrow and peripheral blood.

Studies were conducted to evaluate the effect of experimental protocol on the ability of benzidine (BZD), dimethylbenzanthracene (DMBA) and mitomycin C (MMC), administered by intraperitoneal injection, to induce micronuclei in polychromatic erythrocytes (PCE) of B6C3F1 mice. Three different treatment/sampling protocols were used, involving from one to three consecutive daily treatments and from three to one, respectively, consecutive daily samplings beginning 24 h after the last injection. DMBA and MMC elicited a significant micronucleus response in all three experimental protocols, while BZD induced a significant response only in the multiple injection protocols. Of the three protocols, the 3-day injection/single sample time protocol offers the greatest efficiency in minimizing the number of animals required in a study, in decreasing the time needed for scoring and in simplifying the statistical analysis. In addition, a comparison of the frequency of micronucleated PCE in peripheral blood and bone marrow following the treatment of mice with either BZD or DMBA suggests that, following a three injection protocol, either tissue can be used with equal efficacy.

The induction of micronucleated polychromatic erythrocytes in mice using single and multiple treatments.

Studies are reviewed in which the effect of treatment/sample protocol on the induction of micronucleated polychromatic erythrocytes (MN-PCE) in male B6C3F1 mice by 3 carcinogens (benzidine, dimethylbenzanthracene and mitomycin C) were evaluated. 3 different treatment/sampling protocols were used, involving from 1 to 3 consecutive daily treatments and from 3 to 1, respectively, consecutive daily samplings beginning 24 h after the last injection. The results indicate that the 3-day injection/single sample time protocol eliminates the need for multiple sample times, minimizes the number of animals required in a study, decreases the time needed for data collection and simplifies data analysis. A comparison of the frequency of induced MN-PCE in peripheral blood and bone marrow suggests that, following a 3-injection protocol, either tissue can be used with equal efficiency.

Sensitivity of rat and mouse peripheral blood lymphocytes to BaP adduction and SCE formation.

Both mice and rats were injected i.p. with doses of benzo[a]pyrene (BaP) ranging from 10 to 100 mg/kg to compare species sensitivity to and the relationship between sister chromatid exchange (SCE) induction and DNA adduct formation. Twenty-four hours after injection, blood was removed by cardiac puncture and the peripheral blood lymphocytes (PBLs) were analyzed for both DNA adduct formation by 32P-postlabeling and SCE induction following lymphocyte culture. BaP induced similar, but not identical, SCE dose-response curves for each species. After BaP administration, the major DNA adduct, N2-[10 beta-(7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene)yl]deoxyguanosine (BPDEI-dGuo), was approximately 10-fold more prevalent in the PBLs of the mouse than those of the rat. Thus, for equivalent amounts of BPDEI-dGuo, a greater number of SCEs are induced in the rat than the mouse.

Micronuclei in binucleated lymphocytes of mice following exposure to gamma radiation.

Experiments were designed to investigate the induction of micronuclei (MN) in mouse peripheral blood lymphocytes (PBLs) after in vitro or in vivo exposure to 60Co gamma radiation. For the in vitro experiments, 4 ml of blood from male C57BL/6J mice were either irradiated in 6 ml Falcon culture tubes as whole blood or isolated to obtain mononuclear leukocytes (MNLs) that were pelleted by centrifugation and then irradiated in RPMI 1640. For the in vivo analysis, mice received whole body irradiation, blood was obtained by cardiac puncture, and the MNLs were isolated for each mouse. Exposures were at a rate of 0.82 to 0.90 Gy/min to yield doses of 0.5, 1, 2, 3, or 4 Gy. MNLs were cultured using cytochalasin B for MN analysis in binucleated PBLs. There was a significant dose-dependent increase in MN observed at all doses. Dose-response curves for the in vivo and in vitro whole blood experiment were not significantly different. However, for isolated pelleted MNLs irradiated in vitro, the MN frequency at 4 Gy was less than one-half that seen in the in vivo experiment. The large difference in MN response is thought to be due to the radioprotective effect of hypoxia.

A cytogenetic comparison of the responses of mouse and human peripheral blood lymphocytes to 60Co gamma radiation.

Experiments were conducted to compare the chromosome damaging effects of 60Co gamma radiation on mouse and human peripheral blood lymphocytes (PBLs). Either whole blood or isolated and pelleted mononuclear leucocytes (MNLs) were irradiated with a 60Co unit to yield exposures of 1, 2, 3, or 4 Gy. In addition, mice were whole-body irradiated in vivo with the same doses so that an in vitro-in vivo comparison could be made. The results indicate that mouse PBLs irradiated in whole blood, whether in vivo or in vitro, respond similarly to 60Co gamma rays as measured by dicentric chromosome formation. In addition, mouse and human PBLs showed a similar radiosensitivity, but because the mouse PBL data were best fitted to an exponential function and the human PBL data to a quadratic function, direct comparisons were difficult to make. Pelleted MNLs from mice were much less sensitive to the clastogenic effects of gamma radiation than whole blood. This is believed to be due to hypoxic conditions that developed during irradiation and transport. Human PBLs did not show a marked difference whether irradiated in whole blood or as pelleted MNLs in tissue culture medium.