Evaluation of phosphorus characterization in broiler ileal digesta, manure, and litter samples: (31)P-NMR vs. HPLC.

Using 31-phosphorus nuclear magnetic resonance spectroscopy ((31)P-NMR) to characterize phosphorus (P) in animal manures and litter has become a popular technique in the area of nutrient management. To date, there has been no published work evaluating P quantification in manure/litter samples with (31)P-NMR compared to other accepted methods such as high performance liquid chromatography (HPLC). To evaluate the use of (31)P-NMR to quantify myo-inositol hexakisphosphate (phytate) in ileal digesta, manure, and litter from broilers, we compared results obtained from both (31)P-NMR and a more traditional HPLC method. The quantification of phytate in all samples was very consistent between the two methods, with linear regressions having slopes ranging from 0.94 to 1.07 and r(2) values of 0.84 to 0.98. We compared the concentration of total monoester P determined with (31)P-NMR with the total inositol P content determined with HPLC and found a strong linear relationship between the two measurements having slopes ranging from 0.91 to 1.08 and r(2) values of 0.73 to 0.95. This suggests that (31)P-NMR is a very reliable method for quantifying P compounds in manure/litter samples.

Relationships Between Oleic and Linoleic Acid Content and Seed Colonization by Cercospora kikuchii and Diaporthe phaseolorum.

Compared with standard cultivars, seed of mid-oleic soybean genotypes sometimes have shown increased colonization by Cercospora kikuchii in the field as judged by increased levels of purple-stained seed. To examine relationships between oleic and linoleic acid levels in soybean seed and postharvest seed colonization by two fungal seed pathogens, we inoculated seed with differing oleic:linoleic acid (O/L) ratios. Seed with defined O/L ratios were produced by allowing seed development of two isogenic soybean lines to occur in three different air temperature environments. Seed produced in these environments were harvested, individually analyzed for fatty acid composition, and inoculated with mycelium preparations of the fungal seed pathogens C. kikuchii or Diaporthe phaseolorum var. sojae. Fungal biomass of infected seed was quantified by measuring in vitro ergosterol content. For both soybean lines, colonization by C. kikuchii was positively correlated with the O/L ratio (r = 0.55, P < 0.03) and oleic acid content (r = 0.61, P < 0.02), and negatively correlated with linoleic (r = -0.60, P < 0.02) and linolenic (r = -0.58, P < 0.03) acid content. No association was found between the extent of seed colonization by D. phaseolorum and the seed O/L ratio. Our data suggest that the O/L ratio may be related to soybean seed colonization by C. kikuchii, but there is no evidence of a relationship with D. phaseolorum var. sojae colonization.

Nutrient excretion, phosphorus characterization, and phosphorus solubility in excreta from broiler chicks fed diets containing graded levels of wheat distillers grains with solubles.

Increased interest in ethanol production in North America has led to increased production of distillers dried grains with solubles (DDGS), the majority of which are fed to livestock. To determine the impact of including wheat DDGS in broiler diets on nutrient excretion and P characterization and solubility, 125 one-day-old male broiler chicks were fed wheat- and soybean meal-based diets containing 0, 5, 10, 15, or 20% wheat DDGS. There were 5 replicate pens per treatment, with 5 birds per pen arranged in a randomized block design. Apparent retention of both N and P were determined by using the indicator method. Nutrients excreted per kilogram of DM intake were also calculated. Characterization of excreta P was determined by (31)P-solution nuclear magnetic resonance spectroscopy, and water-soluble P (WSP) was determined by extraction of excreta with deionized water. The apparent retention of both N (P < 0.001) and P (P < 0.008) decreased linearly with increasing inclusion rates of DDGS from 0 to 20%. The nutrient output per kilogram of DM intake increased linearly with increased DDGS inclusion rate for N (P < 0.04), P (P < 0.0001), and WSP (P < 0.0003). As the inclusion rate of DDGS increased, the P concentration in excreta increased (P < 0.008), whereas excreta phytate P concentrations decreased (P < 0.01), which led to an increase in WSP and the fraction of total P that was soluble. Because the inclusion of DDGS in poultry diets increased N and P output, as well as the solubility of P excreted, care should be taken when including high levels of DDGS in poultry diets, because increases in N and P excretion are a concern from an environmental standpoint.

Interaction of calcium and phytate in broiler diets. 2. Effects on total and soluble phosphorus excretion.

Dietary Ca has been reported to influence the amount of phytate excreted from broilers and affect the solubility of P in excreta. To address the effects of dietary Ca and phytate on P excretion, 12 dietary treatments were fed to broilers from 16 to 21 d of age. Treatments consisted of 3 levels of phytate P (0.10, 0.24, and 0.28%) and 4 levels of Ca (0.47, 0.70, 0.93, and 1.16%) in a randomized complete block design. Feed phytate concentrations were varied by formulating diets with 3 different soybean meals (SBM): a low-phytate SBM, a commercial SBM, and a high phytate Prolina SBM having phytate P concentrations of 0.15 to 0.51%. Fresh excreta was collected from cages during 2 separate 24-h periods; collection I commenced after the start of dietary treatments (16 to 17 d) and collection II followed a 3-d adaptation period (19 to 20 d). Ileal samples were also collected at 21 d. Excreta samples were analyzed for total P, water soluble P (WSP), and phytate P, whereas ileal samples were analyzed for total P and phytate P. Results indicated that excreta total P could be reduced by up to 63% and WSP by up to 66% with dietary inclusion of low-phytate SBM. There was a significant effect of dietary Ca on both the excreta WSP and the ratio of WSP:total P. As dietary Ca increased, the excreta WSP and WSP:total P decreased, with the effects being more pronounced following a dietary adaptation period. There was a linear relationship between the slope of the response in WSP to dietary Ca and feed phytate content for excreta from collection II (r(2) = 0.99). There was also a negative correlation between excreta phytate concentration and excreta WSP during both excreta collections. The response in WSP to dietary manipulation was important from an environmental perspective because WSP in excreta has been related to potential for off-site P losses following land application.

Interaction of calcium and phytate in broiler diets. 1. Effects on apparent prececal digestibility and retention of phosphorus.

Phytate P utilization from soybean meal (SBM) included in broiler diets has been shown to be poor and highly dependent on dietary Ca intake. However, the effect of Ca on P utilization and on the optimal ratio of Ca to nonphytate P (Ca:NPP) when diets contained varying levels of phytate has not been clearly shown and was the objective of this research. A factorial treatment structure was used with 4 dietary Ca levels from 0.47 to 1.16% and 3 levels of phytate P (0.28, 0.24, and 0.10%). Varying dietary phytate P levels were obtained by utilizing SBM produced from 3 varieties of soybeans with different phytate P concentrations. Ross 508 broiler chicks were fed 1 of 12 diets from 16 to 21 d of age. Excreta were collected from 16 to 17 d and from 19 to 20 d of age and ileal digesta was collected at 21 d of age. Apparent prececal P digestibility decreased when dietary Ca concentration increased and was higher when diets contained low-phytate SBM. The apparent digestibility of Ca and percentage of phytate P hydrolysis at the distal ileum were not reduced when dietary phytate P concentration increased. Including low-phytate SBM in diets reduced total P output in the excreta by 49% compared with conventional SBM. The optimum ratio of Ca:NPP that resulted in the highest P retention and lowest P excretion was 2.53:1, 2.40:1, and 2.34:1 for diets with 0.28, 0.24, and 0.10% phytate P. These data suggested that increased dietary Ca reduced the extent of phytate P hydrolysis and P digestibility and that the optimum Ca:NPP ratio at which P retention was maximized was reduced when diets contained less phytate P.

What aspect of dietary modification in broilers controls litter water-soluble phosphorus: dietary phosphorus, phytase, or calcium?

Environmental concerns about phosphorus (P) losses from animal agriculture have led to interest in dietary strategies to reduce the concentration and solubility of P in manures and litters. To address the effects of dietary available phosphorus (AvP), calcium (Ca), and phytase on P excretion in broilers, 18 dietary treatments were applied in a randomized complete block design to each of four replicate pens of 28 broilers from 18 to 42 d of age. Treatments consisted of three levels of AvP (3.5, 3.0, and 2.5 g kg(-1)) combined with three levels of Ca (8.0, 6.9, and 5.7 g kg(-1)) and two levels of phytase (0 and 600 phytase units [FTU]). Phytase was added at the expense of 1.0 g kg(-1) P from dicalcium phosphate. Fresh litter was collected from pens when the broilers were 41 d of age and analyzed for total P, soluble P, and phytate P as well as P composition by (31)P nuclear magnetic resonance (NMR) spectroscopy. Results indicated that the inclusion of phytase at the expense of inorganic P or reductions in AvP decreased litter total P by 28 to 43%. Litter water-soluble P (WSP) decreased by up to 73% with an increasing dietary Ca/AvP ratio, irrespective of phytase addition. The ratio of WSP/total P in litter decreased as the dietary Ca/AvP ratio increased and was greater in the phytase-amended diets. This study indicated that while feeding reduced AvP diets with phytase decreased litter total P, the ratio of Ca/AvP in the diet was primarily responsible for effects on WSP. This is important from an environmental perspective as the amount of WSP in litter could be related to potential for off-site P losses following land application of litter.

Ergosterol as a Quantifiable Biomass Marker for Diaporthe haseolorum and Cercospora kikuchi.

The relationship between ergosterol content and biomass was determined for the soybean fungal pathogens Diaporthe phaseolorum (Cooke & Ellis) Sacc. var. sojae, causal agent of Phomopsis seed decay, and Cercospora kikuchii (Matsumoto & Tomoy.), causal agent of leaf blight and purple seed stain. Biomass was manipulated by varying incubation period, and ergosterol was quantified by high-pressure liquid chromatography. Fungal dry mass was linearly correlated with ergosterol content (r2 = 0.90, P < 0.05 for D. phaseolorum, and r2 = 0.95, P < 0.01 for C. kikuchii). In vitro ergosterol content of fungi was 3.16 µg/mg for D. phaseolorum and 2.85 µg/mg for C. kikuchii. Ergosterol content of inoculated seed was qualitatively correlated with observed seed colonization by both pathogens. Soybean variety had a significant effect on fungal colonization by D. phaseolorum and ergosterol content. Results show that ergosterol content can be used to quantify colonization of soybean seed by both pathogens.

Subunit and amino acid composition of diacylglycerol acyltransferase from germinating soybean cotyledons.

The subunit and amino acid composition of the enzyme that catalyses triacylglycerol synthesis was determined for the first time from plant tissues. Diacylglycerol acyltransferase (acyl-CoA:1,2-diacylglycerol O-acyltransferase, EC 2.3.1.20) purified from germinating soybean (Glycine max L. Merr. cv. Dare) cotyledons, dissociated into three nonidentical subunits having apparent molecular masses of 40.8, 28.7, and 24.5 kDa. The respective subunits occurred in a 1:2:2 molar ratio in the native enzyme. Five peptides in that molar ratio were assumed to constitute a monomer having a putative molecular mass of 153.1 kDa. Based upon the apparent molecular mass of purified diacylglycerol acyltransferase after delipidation (1539 kDa), there was a high probability that the complete structure of the native enzyme from soybean contained ten identical monomers. The polarity index of each subunit was less than 21%, far below the 40% boundary reported for membrane bound proteins. Hydrophobic amino acids accounted for greater than 48% of the composition in each subunit. It was predicted from these data that the native enzyme contained 12,525 amino acid residues, and that the two smaller subunits were more deeply embedded in the membrane than the 40.8 kDa subunit. Attempts to reactivate the denatured or delipidated protein were not successful.

Nuclear DNA codes for Nicotiana ferredoxin.

Ferredoxin was purified from 10 species of Nicotiana and spinach leaves. Fingerprints showed all to contain five major tryptic peptides. Some of the spinach peptides were different in RF and mobility from the Nicotiana peptides, but none of the Nicotiana ferredoxins had peptides which could distinguish one species of ferredoxin from another. Electrofocusing S-carbaminomethylcysteinyl ferredoxins showed spinach ferredoxin to have a more acidic and N. glutinosa ferredoxin a slightly more acidic isoelectric point than the other 9 Nicotiana species which were alike. Electro-focusing ferredoxin from the hybrid N. glutinosa female times N. glauca male resolved two bands or isozymes of ferredoxin, one corresponding to N. glutinosa, the other to N. glauca, the code for the latter having come from the DNA in the N. glauca pollen used to form the hybrid plant. N. glutinosa ferredoxin does not contain methionine and is different from N. tabacum and N. glauca ferredoxins which contain methionine. The N. glutinosa female times N. glauca male ferredoxin contained one-half the methionine found in N. glauca ferredoxin, thus confirming that some of the genetic information for ferredoxin in the hybrid was originally contained in the nuclear DNA of N. glauca.

Visualization and quantitation of peroxisomes using fluorescent nanocrystals: treatment of rats and monkeys with fibrates and detection in the liver.

Peroxisome proliferation in the liver is a well-documented response that occurs in some species upon treatment with hypolipidemic drugs, such as fibrates. Typically, liver peroxisome proliferation has been estimated by direct counting via electron microscopy, as well as by gene expression, enzyme activity, and immunolabeling. We have developed a novel method for the immunofluorescent labeling of peroxisomes, using an antibody to the 70-kDa peroxisomal membrane protein (PMP70) coupled with fluorescent nanocrystals, Quantum Dots. This method is applicable to standard formalin-fixed, paraffin-embedded tissues. Using this technique, a dose-dependent increase in PMP70 labeling was evident in formalin-fixed liver sections from fenofibrate-treated rats. In formalin-fixed liver sections from cynomolgus monkeys given ciprofibrate, quantitative image analysis showed a statistically significant increase in PMP70 labeling compared to control; the increase in hepatic PMP70 protein levels was corroborated by immunoblotting using total liver protein. An increase in hepatic peroxisome number in ciprofibrate-treated monkeys was confirmed by electron microscopy. An advantage of the Quantum Dot/PMP70 method is that a single common protocol can be used to label peroxisomes from several different species, and many of the common problems that arise with immunolabeling, such as fading and low signal strength, are eliminated.

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.

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.

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.

Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

Comparison of sister-chromatid exchange frequencies in mouse T- and B-lymphocytes after exposure to 4-hydroxycyclophosphamide or phosphoramide mustard.

The present study was designed to investigate the genotoxicity of 4-hydroxycyclophosphamide (4-OHCP) and phosphoramide mustard (PAM), both reactive metabolites of cyclophosphamide (CP), for possible differences in SCE-inducing activity in mouse T- and B-lymphocytes. Mouse peripheral blood lymphocytes were isolated and stimulated to divide with either phytohemagglutinin (T-cell mitogen) or lipopolysaccharide (a polyclonal B-cell activator). Significant concentration-dependent increases in SCE frequencies were observed for both 4-OHCP and PAM with both mitogens, with 4-OHCP being almost twice as potent as PAM. There was no difference in SCE response between T- and B-lymphocytes after exposure to either PAM or 4-OHCP. These data do not support the idea that the difference in SCE response in T- and B-lymphocytes by CP in vivo is due to differential responses to either of the proposed putative metabolites of CP.

A novel bacterial reversion and forward mutation assay based on green fluorescent protein.

We report the first use of green fluorescent protein (GFP) for mutation detection. We have constructed a plasmid-based bacterial system whereby mutated cells fluoresce and non-mutated cells do not fluoresce. Fluorescence is monitored using a simple hand-help UV lamp; no additional cofactors or manipulations are necessary. To develop a reversion system, we introduced a +1 DNA frameshift mutation in the coding region of GFP and the resulting protein is not fluorescent in Escherichia coli. Treatment of bacteria containing the +1 frameshift vector with ICR-191 yields fluorescent colonies, indicating that reversion to the wild-type sequence has occurred. Site-directed mutagenesis was used to insert an additional cytosine into a native CCC sequence in the coding region of GFP in plasmid pBAD-GFPuv, expanding the sequence to CCCC. A dose-related increase in fluorescent colonies was observed when the bacteria were treated with ICR-191, an agent that induces primarily frameshift mutations. The highest dose of ICR-191 tested, 16 microg/ml, produced a mutant fraction of 16 x 10(-5) and 8.8 x 10(-5) in duplicate experiments. The reversion system did not respond to MNNG, an agent that produces mainly single-base substitutions. To develop a forward system, we used GFP under the control of the arabinose PBAD promoter; in the absence of arabinose, GFP expression is repressed and no fluorescent colonies are observed. When cells were treated with MNNG or ENNG, a dose-dependent increase in fluorescent colonies was observed, indicating that mutations had occurred in the arabinose control region that de-repressed the promoter. Treating bacteria with 100 microg/ml MNNG induced mutant fractions as high as 82 x 10(-5) and 40 x 10-5 in duplicate experiments. Treating bacteria with 150 microg/ml ENNG induced a mutant fraction of 2.1 x 10(-5) in a single experiment.

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.

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 studies of mice exposed to styrene by inhalation.

The data for the in vivo genotoxicity of styrene (STY) are equivocal. To evaluate the clastogenicity and sister-chromatid exchange (SCE)-inducing potential of STY in vivo under carefully controlled conditions, B6C3F1 female mice were exposed by inhalation for 6 h/day for 14 consecutive days to either 0, 125, 250 or 500 ppm STY. One day after the final exposure, peripheral blood, spleen, and lungs were removed and cells were cultured for the analysis of micronucleus (MN) induction using the cytochalasin B-block method, chromosome breakage, and SCE induction. Peripheral blood smears were also made for scoring MN in erythrocytes. There was a significant concentration-related elevation of SCE frequency in lymphocytes from the spleen and the peripheral blood as well as in cells from the lung. However, no statistically significant concentration-related increases were found in the frequency of chromosome aberrations in the cultured splenocytes or lung cells, and no significant increases in MN frequencies were observed in binucleated splenocytes or normochromatic erythrocytes in peripheral blood smears.