Bone Marrow Polychromatic Erythrocytes Research Articles

In vivo effects of chlorophyllin on the antitumour agent cyclophosphamide.

Cyclophosphamide (CP) is a potent antitumour agent used against many forms of cancer and against certain other diseases. Chlorophyllin (CHL), which is obtained by hydrolysis of chlorophyll to remove phytyl alcohol, is an efficient antimutagenic agent and has been used as a dietary supplement or to diminish the intensity of the discomforting side effects of CP therapy. We undertook to determine the antimutagenic effectiveness of CHL against CP in a mouse model and to determine whether the antitumour efficacy of CP was compromised in vivo by CHL treatment. Experiments utilised CHL administered either in drinking water (1%) for 2 days before treatment, or by gavage (200 mg/kg) 2 hr before treatment with CP (220 mg/kg). Urinary mutagenicity following CP treatment, as determined by the Salmonella/microsome assay, was decreased by both regimes of CHL co-treatment. Similarly, the increase in micronuclei in bone marrow polychromatic erythrocytes in response to CP was reduced by concomitant CHL treatment. In contrast, antitumour efficacy, as determined by growth delay of Colon 38 adenocarcinomas, was not diminished by CHL treatment. We conclude that CHL may have beneficial effects when used in combination with CP therapy.

An evaluation of micronucleus induction in bone marrow and in hepatocytes isolated from collagenase perfused liver or from formalin-fixed liver using four-week-old rats treated with known clastogens.

The bone marrow (BM) micronucleus (MN) test is a sensitive assay for identifying clastogens. However, some clastogenic compounds and metabolites may never reach the BM. The liver has been suggested as an alternative tissue to BM but adult rat liver has a low mitotic index that increases the difficulty of evaluating hepatocytes (HEP) for MN induction. Chemical mitogens and partial hepatectomy have been used to increase HEP proliferation to improve the sensitivity for detection of clastogenic compounds, but these practices raise concerns for the evaluation of drug candidates. The use of 4-wk-old rats provides an alternative to mitogenic stimulation because livers from these animals have approximately 5.4% of their HEP in S-phase. HEP were isolated by collagenase perfusion, or from formalin-fixed tissue, from 4-wk-old treated rats. Six compounds were evaluated for the incidence of MN in HEP that were isolated by both methods. The results for MN induction by these compounds were similar for the two methods and confirmed that formalin-fixed tissue is an acceptable source of cells for evaluating MN induction in HEP. BM polychromatic erythrocytes (PCE) also were harvested at the end of the live phase for each study and then evaluated for the incidence of MN. Diethylnitrosamine and 2-nitrofluorene induced MN in HEP but had no effect in PCE. 2-Acetylaminofluorene, cyclophosphamide and 7,12-dimethylbenz[a]anthracene did not induce MN in HEP but were positive in PCE. The direct-acting clastogen, mitomycin C, was positive in both HEP and PCE. These results indicate that this modified liver micronucleus test, using 4-wk-old rats, offers an alternative to existing methods that use mitogens or partial hepatectomy to stimulate cell replication. Analysis of MN from formalin-fixed tissue provides additional flexibility by allowing the investigator to assess MN induction at a later time.

Comparative study of DNA adduct formation in mice following inhalation of smoke from cigarettes that burn or primarily heat tobacco

The genotoxic potential of mainstream smoke from a test cigarette (TOB-HT) that primarily heats tobacco and a representative tobacco-burning cigarette (Kentucky reference 1R4F) was compared in male B6C3/F1 mice after nose-only inhalation exposure. Mice were exposed 1 hr per day, 5 days/ week for a 4 week period to mainstream smoke at concentrations of 0, 0.16, 0.32, and 0.64 mg total particulate matter/liter of air. Micronuclei formation in bone marrow and peripheral blood polychromatic erythrocytes (PCE) of animals exposed to either the TOB-HT or 1R4F cigarette was not significantly different compared with control animals exposed nose-only to filtered and humidified air (sham controls). DNA adduct measurement by the 32P-post-labeling method indicated an exposure-dependent increase in lung adducts of animals exposed to 1R4F cigarette smoke at all three concentrations with the mid and high exposure groups exhibiting statistically significant increases (P < 0.05) in adduct formation compared to sham-exposed animals. The concentration of DNA adducts in the lungs of animals exposed to the TOB-HT cigarette was not significantly increased (P < 0.05) at any concentration compared to sham-exposed controls. A statistically significant (P < 0.05) concentration-dependent formation of DNA adducts was also observed in the heart tissues of animals exposed to smoke from the 1R4F cigarette at all three concentrations, but no significant increase in adduct formation was observed in heart DNA of the animals exposed to the TOB-HT cigarette (P < 0.05). Under the conditions of this experiment, the mainstream smoke from the TOB-HT cigarette was demonstrated to be less genotoxic in mice than mainstream smoke from the 1R4F cigarette, which is representative of cigarettes in the current U.S. market.

Acute and Subchronic Inhalation Studies on Trifluoroiodomethane Vapor in Fischer 344 Rats

Trifluoroiodomethane (CF3I) is being considered as a replacement compound for halon fire suppressants. Its structure is similar to that of Halon 1301 (CF3Br), but it has very low ozone depletion potential compared to CF3Br. As part of the process of developing environmental and health effects criteria, acute, 2-week, and 13- week nose-only inhalation toxicity studies were conducted in Fischer 344 rats. In the acute study, three groups of 30 male rats each were exposed to 0 (control), 0.5, or 1.0% (v/v) CF3I for 4 hr and euthanized immediately following exposure, 3 days postexposure, or 14 days postexposure. There were no deaths and no clinical signs of toxicity throughout the study. Histopathologic examination of select tissues showed no lesions of pathologic significance. In the 2-week study, four groups of 5 male rats each were exposed for 2 hr/day, 5 days/week to 0, 3, 6, or 12% CF3I No deaths were observed, though lethargy and slight incoordination were noted in rats of the 6 and 12% groups at the conclusion of each daily exposure. Mean body weight gains were depressed in rats of the 6 and 12% groups. Serum thyroglobulin and reverse T3 (rT3) values were increased at all exposure levels. At necropsy, no gross lesions or differences in absolute or relative organ weights were noted. Histopathologic examination of the thyroid and parathyroid glands indicated no morphological abnormalities in the CF3I-exposed rats. In the 13-week study, four groups of 15 male and 15 female rats were exposed to 0, 2, 4, or 8% CF3I 2 hr/day, 5 days/week for 13 weeks. Rats exposed to 4 or 8% CF3I had lower mean body weights than the controls. Deaths observed in the 2 and 8% groups were attributed to accidents resulting from the restraint system employed. Hematologic alterations were minimal and considered insignificant. Increases in the frequency of micronucleated bone marrow polychromatic erythrocytes were observed in rats of all three CF3I groups. Serum chemistry alterations observed in rats of all CF3I exposure groups included decreases in T3 and increases in thyroglobulin, rT3, T4, and TSH. Relative organ weight increases (8% CF3I group) occurred in the brain, liver, and thyroid glands; decreases were observed in the thymus and testes. A decrease in relative thymus weights and an increase in relative thyroid weights were observed also in rats of the 2 and 4% groups. Histopathological findings included a mild inflammation in the nasal turbinates of rats exposed to 4 or 8% CF3I, mild atrophy and degeneration of the testes (4 and 8% CF3I groups), and a mild increase in thyroid follicular colloid content in rats of all CF3I exposure groups. Though NOAELs were observed for select target organs (e.g., nasal turbinates, testes), NOAELs were not apparent in all target organs examined (e.g., thyroid glands, bone marrow).

Acute and subchronic inhalation studies on trifluoroiodomethane vapor in Fischer 344 rats.

Trifluoroiodomethane (CF3I) is being considered as a replacement compound for halon fire suppressants. Its structure is similar to that of Halon 1301 (CF3Br), but it has very low ozone depletion potential compared to CF3Br. As part of the process of developing environmental and health effects criteria, acute, 2-week, and 13-week nose-only inhalation toxicity studies were conducted in Fischer 344 rats. In the acute study, three groups of 30 male rats each were exposed to 0 (control), 0.5, or 1.0% (v/v) CF3I for 4 hr and euthanized immediately following exposure, 3 days postexposure, or 14 days postexposure. There were no deaths and no clinical signs of toxicity throughout the study. Histopathologic examination of select tissues showed no lesions of pathologic significance. In the 2-week study, four groups of 5 male rats each were exposed for 2 hr/day, 5 days/week to 0, 3, 6, or 12% CF3I. No deaths were observed, though lethargy and slight incoordination were noted in rats of the 6 and 12% groups at the conclusion of each daily exposure. Mean body weight gains were depressed in rats of the 6 and 12% groups. Serum thyroglobulin and reverse T3 (rT3) values were increased at all exposure levels. At necropsy, no gross lesions or differences in absolute or relative organ weights were noted. Histopathologic examination of the thyroid and parathyroid glands indicated no morphological abnormalities in the CF3I-exposed rats. In the 13-week study, four groups of 15 male and 15 female rats were exposed to 0, 2, 4, or 8% CF3I 2 hr/day, 5 days/week for 13 weeks. Rats exposed to 4 or 8% CF3I had lower mean body weights than the controls. Deaths observed in the 2 and 8% groups were attributed to accidents resulting from the restraint system employed. Hematologic alterations were minimal and considered insignificant. Increases in the frequency of micronucleated bone marrow polychromatic erythrocytes were observed in rats of all three CF3I groups. Serum chemistry alterations observed in rats of all CF3I exposure groups included decreases in T3 and increases in thyroglobulin, rT3, T4, and TSH. Relative organ weight increases (8% CF3I group) occurred in the brain, liver, and thyroid glands; decreases were observed in the thymus and testes. A decrease in relative thymus weights and an increase in relative thyroid weights were observed also in rats of the 2 and 4% groups. Histopathological findings included a mild inflammation in the nasal turbinates of rats exposed to 4 or 8% CF3I, mild atrophy and degeneration of the testes (4 and 8% CF3I groups), and a mild increase in thyroid follicular colloid content in rats of all CF3I exposure groups. Though NOAELs were observed for select target organs (e.g., nasal turbinates, testes), NOAELs were not apparent in all target organs examined (e.g., thyroid glands, bone marrow).

Validation of an automated image analysis micronucleus scoring system

The Loats Automated Micronucleus Scoring System was developed to assist with the evaluation of compounds for the ability to induce micronuclei in mouse bone marrow polychromatic erythrocytes (PCE). This image analysis system can identify PCE as well as normochromatic erythocytes (NCE) and calculate the PCE/NCE ratio as an index for bone marrow toxicity. Two studies were conducted to provide slides for a comparison of micronucleated PCE values collected manually to those collected by the automated system. Mitomycin C was used as a micronucleus-inducing agent to elicit a positive response and Lilly compound 303497 was used as an example of a compound negative for the induction of micronuclei. No statistically significant differences were observed between micronucleus counts obtained manually and those obtained by the automated system. The PCE/NCE ratios calculated by the automated system were also similar to those determined from the manually collected PCE and NCE counts for the vehicle and positive controls, however, differences in the ratios were observed in compound treatment groups. These differences were attributed to a larger population of transitional cells in the treated groups. These results confirm that the Loats Automated Micronucleus Scoring System is an acceptable alternative to manual evaluation of mouse bone marrow slides for the incidence of micronucleated PCE.

Genetic and non-genetic biomarkers related to carcinogenesis in evaluating toxicological risk from Fenarimol

A multibiomarker approach based on the study of toxicity mechanisms at both genetic and metabolic levels has been applied to Fenarimol. With regard to genotoxicity, particular attention was given to assays for chromosomal aberration and micronuclei; clastogenic potential was assessed in human peripheral blood lymphocytes in vitro, while the induction of micronuclei was studied in male CD1 mouse bone marrow polychromatic erythrocytes (PCE). Fenarimol did not induce any significant increase in chromosomal aberrations in vitro, either in the presence or absence of rat liver S9 fraction. In contrast, a significant dose-related increase in micronucleated PCEs, up to 4-fold above the control level at a single dose of 75 mg/kg b.w., was observed 24 h after treatment. Using selective biochemical markers of effect Fenarimol was found to induce CYP 2B1 isoforms in liver, kidney and lung microsomes of Swiss Albino CD1 male and female mice, as shown by the significant increase in specific 2B1-probe pentoxyresorufin O-dealkylase activity. On the contrary, CYP 3A, probed by N-demethylation of aminopyrine, were only induced in the liver. Results were corroborated by by means of Western immunoblotting using rabbit polyclonal antibodies anti-CYP 2B1 and 3A. Northern blotting analysis with CYP 2B1 and 3A cDNA biotinylated probes showed that the expression of such isoforms is regulated at mRNA level. Taken as a whole, these data indicate the possible (mutagenic) cotoxic/cocarcinogenic and promoting potential of this fungicide.

Subchronic toxicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5 H)-furanone (MX) in Wistar rats

The subchronic (14–18 wk) toxicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a mutagenic by-product in chlorinated drinking water, was evaluated in Wistar rats. In a range-finding study, MX was administered daily for 14 days by gavage in deionized water to male rats (five animals per group) at doses of 12.5, 25, 50, 100 or 200 mg/kg body weight. The doses above 50 mg/kg were lethal and three out of five animals also died during treatment at 50 mg/kg. The range-finding study was repeated with doses of 5, 10 and 20 mg MX/kg, given on 5 days a week, to both males and females (10 animals per group). These doses were not overtly toxic but caused several changes in plasma clinical chemistry at 10 and 20 mg MX/kg in comparison with the controls. These included increased urea, creatinine and bilirubin and decreased inorganic phosphate and potassium in females and increased cholesterol in males. In the subchronic toxicity study, rats (15 per group) were given MX by gavage, on 5 days a week, at doses of 0 (controls) or 30 mg/kg (low dose) for 18 wk, or, in the high-dose group, at doses increasing from 45 to 75 mg/kg over 14 wk. The high dose was finally lethal (two males and one female died) and caused hypersalivation, wheezing respiration, emaciation and tangled fur in animals. The body weights of the high-dose males decreased by 15%, and food consumption was decreased by 15 to 20%, but the water consumption increased by 15% to 60%. Plasma cholesterol and triglycerides were elevated and urine excretion was increased. Urine specific gravity was decreased and the relative weights of the liver and kidneys were increased in both sexes at both doses in comparison with the controls. At both doses, duodenal hyperplasia occurred in males and females, and slight focal epithelial hyperplasia in the forestomach was observed in males. Splenic atrophy and haemosiderosis were seen in two high-dose females, and epithelial cell atypia in the urinary bladder of one high-dose male and female. The frequency of bone marrow polychromatic erythrocytes with micronuclei was slightly increased in low-dose males. The results indicate that repeated administration of MX disturbs the fluid-electrolyte balance and induces diuresis, causes mucosal hyperplasia in the gastro-intestinal tract as a local effect, and affects lipid metabolism.

Carcinogenicity of N1-[Tris(hydroxymethyl)]methyl-4-nitro- o-phenylenediamine fed to mated and non-mated Sprague—Dawley rats

N 1-[tris(hydroxymethyl)]methyl-4-nitro- o-phenylenediamine was fed in the diet to groups of 30 male and 55 female Sprague-Dawley rats at levels of 0.2, 0.6 and 2.0% for up to 6 months. One mid-dose and two high-dose females developed palpable mammary masses that were subsequently diagnosed as mammary adenocarcinomas at a 13-wk interim kill involving 10 rats/sex/group. After 14 wk, 25 females per group with no apparent masses were mated in a reproduction/teratology study. Mammary tumours developed in a dose-related fashion both in the pregnant rats and in the remaining 20 females/group that continued on treatment for 6 months. On gestation day 20 (wk 17–18) the final incidences of mammary adenocarcinomas in the low-, mid- and high-dose mated dose groups were 20, 60 and 84%, respectively, while the corresponding incidences in the non-mated females at 6 months were 5, 40 and 85%. Most mammary tumours were encapsulated but, at 6 months, lung metastases were noted in four rats, and four females also had Zymbal's gland tumours. Non-neoplastic changes in male and female rats considered to be related to treatment included increases in thyroid follicular cell size accompanied by an accumulation of golden-brown pigment, multifocal hepatic necrosis with non-suppurative inflammation, and renal tubular pigmentation. Increases in foetal variations in the mid- and high-dose groups were considered to be related non-specifically to retarded growth. Malformations observed in the high-dose group were found primarily in single foetuses and were not considered to be treatment related. Although the mean numbers of micronucleated polychromatic erythrocytes in bone marrow obtained from high-dose treated females after 13 wk slightly exceeded historical negative control values, the data were not considered indicative of a genotoxic effect because of the absence of either a dose relationship or a substantive increase in the frequency of micronucleated cells.

The genotoxicity profile of atorvastatin, a new drug in the treatment of hypercholesterolemia

While HMG-CoA reductase inhibitors such as fluvastatin, lovastatin, pravastatin and simvastatin demonstrate lack of in vitro and in vivo mutagenicity and clastogenicity in bacterial and mammalian cells, long term rodent carcinogenicity studies resulted in an increased incidence in neoplasms at high doses. These effects may be attributable to an exaggeration of the desired biochemical effect of the drug and/or a tumor promoting effect. The genotoxicity of atorvastatin, a newly developed HMG-CoA reductase inhibitor, was evaluated in a variety of test systems. In bacterial mutagenicity tests, the E. coli tester strain WP2(uvrA) and S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 were exposed to concentrations of atorvasatin as high as 5000 μg/plate both in the absence (S9-) and presence (S9+) of metabolic activation. Atorvastatin was not mutagenic in either E. coli or S. typhimurium. Chinese hamster lung V79 cell cultures were exposed to atorvastatin at concentrations of 50–300 μg/ml (S9-) and 100–300 μg/ml (S9+) and structural chromosome aberrations were assessed. Mutation at the hgprt locus was assessed at concentrations of 100–300 μg/ml (S9-) and 150–275 μg/ml (S9+). Atorvastatin was neither mutagenic nor clastogenic in the absence or presence of S9. The lack of in vitro genotoxicity was corroborated in vivo in a mouse micronucleus study in which single oral doses of atorvastatin were administered to male and female CD-1 mice at 1, 2500, or 5000 mg/kg. No biologically significant increases in the frequency of micronucleated polychromatic erythrocytes in bone marrow at 24, 48, or 72 h postdosing were observed. Thus, atorvastatin, as with the other tested HMG-CoA reductase inhibitors, is not genotoxic.

Genotoxicity of quercetin in the micronucleus assay in mouse bone marrow erythrocytes, human lymphocytes, V79 cell line and identification of kinetochore-containing (CREST staining) micronuclei in human lymphocytes

Quercetin, a mutagenic flavonoid widely distributed in edible plants, was studied for the induction of micronuclei (MN). We have carried out the MN assay in bone marrow polychromatic erythrocytes in mice, in cytokinesis-blocked human lymphocytes and in cytokinesis-blocked V79 cells. MN assay in vitro was performed in the presence and in the absence of S9. To further extend the study, an antikinetochore antibody (CREST staining) was used to distinguish MN containing whole chromosomes (kinetochore positive) from those containing acentric fragments (kinetochore negative). When tested in vivo quercetin failed to induce micronuclei, a result which is in agreement with other published reports. When tested in vitro in V79 cells quercetin clearly induces micronuclei in the absence of S9 and also in the presence of S9 for the highest dose used. When tested in vitro in human lymphocytes quercetin shows a significant induction of micronuclei in the absence and in the presence of S9. The presence of s9 compared to its absence is not significant for any of the systems used. Both in the presence and absence of S9, quercetin appears to behave as a clastogenic agent in human lymphocytes inducing a significant majority of kinetochore-negative MN.

The in vivo rat micronucleus test: integration with a 14-day study

A 14-day subchronic toxicity study is routinely conducted in Fischer 344 rats Lilly Research Laboratories. This study is done to gather preliminary toxicological information about chemical entities showing efficacy in various pharmacological screens. This manuscript describes the validation of a method for evaluating micronuclei in the bone marrow polychromatic erythrocytes of animals from this test in order to obtain additional information about the genotoxic potential of these compounds without incurring the cost of additional animals or the use of additional test article, which is often in limited supply. Compounds selected for evaluation were acetylsalicylic acid, mitomycin C, cyclophosphamide, colchicine, 6-mercaptopurine, and etoposide. With the exception of colchicine, the results obtained were as expected with acetylsalicylic acid yielding negative results and the other compounds yielding positive results. These findings are consistent with those published for mice (MacGregor et al., Fund. Appl. Toxicol., 14, 513–522, 1990) and show that a bone marrow micronucleus test can be successfully integrated into a routine subchronic rat toxicology study.

Micronucleus formation induced by three polycyclic aromatic hydrocarbons in rat bone marrow and spleen erythrocytes following intratracheal instillation

Benz[ a]anthracene (BA), dibenz[ a,h]anthracene (DBA) and dibenzo[ a,i]pyrene (DBP) are polycyclic aromatic hydrocarbons (PAHs) found in incomplete combustion products of fossil fuels, coal tar, and other organic materials. Workers in related industries may be exposed to these chemicals by inhalation. The information related to the potential health hazards of these chemicals to the exposed workers, however, is very limited. In the present study, micronucleus (MN) formation in rat bone marrow and spleen polychromatic erythrocytes (PCEs) was determined following three intratracheal instillations within a 24-h period with either BA, DBA or DBP. Three doses with five rats per dose were used for each chemical. Bone marrow and spleen cells were harvested 24 h after the first dosing. Results showed that the order of toxicity for the three PAHs was DBP>DBA>BA. BA induced MN in a dose-related manner in both bone marrow and spleen PCEs at doses above 25 mg/kg. DBA caused significant increases in the frequencies of MN in both spleen and bone marrow PCEs at the dose of 8.5 mg/kg or higher. At 10 mg/kg, DBP significantly increased MN frequency in spleen PCEs, but the increase in bone marrow PCEs was not significantly different from the control. These results indicate that: (1) all three PAHs studied are absorbed through the respiratory tract and their genotoxic metabolites reach the bone marrow and/or spleen; (2) except for DBP which does not induce MN in the bone marrow, all three PAHs induced MN in both bone marrow and spleen PCEs; and (3) the sensitivity of the spleen to the three PAHs is comparable to or higher than that of the bone marrow.

A pharmacokinetic study of ethanol inhibition of micronuclei induction by urethane in mouse bone marrow erythrocytes

Urethane (ethyl carbamate) is a genotoxic carcinogen in fermented products and alcoholic beverages. The genotoxicity of urethane requires metabolic activation. Metabolism of urethane is mediated by multiple pathways, and ethanol is known to inhibit the esterase hydrolysis pathway of urethane, which accounts for over 95% of urethane metabolism. This report shows that ethanol also inhibits the induction of micronuclei by urethane in mouse bone marrow erythrocytes, presumably by inhibiting the minor pathway that generates genotoxic metabolite(s). In this study, male CD-1 mice were administered urethane, ethanol, or urethane co-administered with increasing amounts of ethanol in single intraperitoneal injections. Bone marrow polychromatic erythrocytes (PCE) obtained 24 h after injection were scored for micronuclei. The dose of urethane was 1000 mg/kg, and the doses of ethanol were 0, 625, 1250, 2000, 2250, 3000 and 3500 mg/kg. The blood ethanol level at each dose was determined. Two pharmacokinetic parameters, C max and AUC, were estimated for each dose. The observed C max of ethanol at doses of 1250, 2000, 2250, 2500, 3000 and 3500 mg/kg were 1.39, 2.84, 3.15, 3.69, 4.13 and 4.76 mg/ml, with AUCs of 1.37, 4.84, 5.88, 7.28, 10.76 and 13.51 mg·h/ml, respectively. Urethane treatment alone markedly increased the micronucleus frequency from 0.1% in the vehicle control to 2.47%. This magnitude of increase was suppressed when urethane was co-administered with ethanol at ethanol doses of 2500 mg/kg and above. At 2500, 3000 and 3500 mg/kg, the micronucleus frequencies reduced from 2.47% to 0.9, 0.44 and 0.28%, respectively. This study shows that ethanol inhibits the induction of micronuclei by urethane.

In vivo genotoxic interactions among three phenolic benzene metabolites

Three benzene metabolites, hydroquinone (HQ), cathecol (CAT) and phenol (PHE) were studied to define their possible interaction in inducing micronuclei (Mn) in mouse bone marrow polychromatic erythrocytes (PCEs). HQ and CAT, administered separately, induced Mn while PHE showed no genotoxic effects. Binary and ternary mixtures of two or three metabolites gave different results, causing considerable increase or decrease in Mn induction. HQ and PHE, in binary mixtures, as well as PHE and CAT, increased Mn synergistically, while HQ and CAT interacted negatively. The genotoxicity of ternary mixtures was mainly the consequence of two metabolites: HQ and CAT. The maximal effect obtained is far below the induction of Mn consequent to benzene treatment. These data suggest that toxic and genotoxic effects of benzene alone could be the result of more complex interactions among these and other metabolites.

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.

The comparative clastogenic activity of mainstream tobacco smoke from cigarettes widely consumed in Armenia

Whole-body exposure of male albino Swiss mice to the mainstream smoke produced by 10 types of cigarettes widely consumed in Armenia resulted in a significant increase (2.4–5.6-fold) of the number of micronucleated bone marrow polychromatic erythrocytes. The smoke produced by cigarettes manufactured in Armenia, Russia and Bulgaria was more clastogenic than the smoke produced by cigarettes manufactured in the USA. A high direct correlation was observed between the number of micronucleated polychromatic erythrocytes and the content of tar and nicotine.

An assessment of the in vivo clastogenicity of erythrosine

In an investigation of the in vivo clastogenic potential of the food colouring erythrosine (ER), male B6C3F 1 mice were treated by ip injection at doses of 50, 100 and 200 mg/kg, repeated 24 hr apart. Signs of toxicity were observed at the highest dose of ER administered. The three cytogenetic endpoints analysed were sister chromatid exchanges (SCEs) in peripheral blood lymphocytes (PBLs), micronuclei in bone marrow polychromatic erythrocytes (PCEs), and micronuclei in peripheral blood reticulocytes (PBRs). SCE frequencies in PBLs were 4.13, 4.58, 4.33 and 4.60 SCE/cell at 0, 50, 100 and 200 mg ER/kg, respectively. At the same doses, the frequencies of micronucleated PCEs were 3.5, 3.2, 2.0 and 2.5/1000 PCEs. Micronuclei in PBRs ranged from 1.2 to 3.6 and from 1.4 to 3.0/1000 PBRs in control and treated mice, respectively. These results indicate that ER is inactive as a clastogen in mouse blood and marrow cells. This result supports the hypothesis of a non-genotoxic mechanism for ER carcinogenicity.

Bone marrow micronucleus assay: a review of the mouse stocks used and their published mean spontaneous micronucleus frequencies.

We have examined published negative control data from 581 papers on micronucleated bone marrow polychromatic erythrocytes (mnPCE) for differences in mean frequency and the frequency distribution profile among the mouse stocks used with the bone marrow micronucleus assay. For the 55 mouse stocks with published micronucleus assay data, the overall mean frequency is 1.95 mnPCE/1,000 PCE (1.95 mnPCE/1,000); for the 13 stocks most commonly used in the assay, it is 1.88 mnPCE/1,000. During the last 5 years, the mnPCE rate for these 13 major stocks has been 1.74 mnPCE/1,000. This current mean frequency is a substantial decrease from the mean of 3.07 mnPCE/1,000 observed for these 13 stocks for data published prior to 1981. Of the major stocks, the highest mean mnPCE negative control frequencies were observed for MS/Ae > BALB/c > C57Bl/6, and the lowest for CD-1 < Swiss Webster. We note that hybrid mouse stocks appear to have lower and less variable negative control frequencies than either of their parent strains and that the negative control frequency for some progeny stocks have diverged significantly from that of the parent stocks. Overall mean negative control frequencies appear to be correlated with breadth of the frequency distribution profile of published mean negative control values. Furthermore, a possible correlation between negative control frequency in the micronucleus assay and sensitivity to clastogens of different mouse strains may be indicated. The databases generated here allow us to define a range of norms for both the historical mean frequency and individual experimental mean frequencies for most stocks, but in particular, for the more commonly used mouse stocks. Our analysis, for the most part, bears out the recommendation of the first Gene-Tox Report on the micronucleus assay that the historical negative control frequency for a mouse stock should fall between 1 and 3 mnPCE/1,000. Eighty-six percent of the most commonly used mouse stocks have historical mean frequencies within this range. Though individual experimental mean values would not necessarily be expected to fall within the 1-3.00 mnPCE/1,000 range, 65.3% of the 2,327 published negative control values do, and 83.5% are < 3 mnPCE/1,000. The frequency with which an individual experimental mean value lies outside the 1.00 to 3.00 mnPCE/1,000 range differs among stocks and appears related to the mouse mean frequency. We suggest that the recommended range for historical mean frequency be extended slightly, to approximately 3.4 mnPCE/1,000, to accommodate some commonly used strains with overall mean negative control frequencies just above 3.00 mnPCE/1,000.(ABSTRACT TRUNCATED AT 400 WORDS)

Dominant-lethal mutations and micronucleus induction in male BALB/c, BDF1 and H mice by tobacco smoke

The mutagenicity of whole tobacco smoke (TS) was examined in male BALB/c, BDF1 and H mice using the dominant lethal and micronucleus tests in bone marrow polychromatic erythrocytes. Male mice (about 80 days old) from the three strains were treated with TS on 5 days/week for 8 weeks. The animals were divided into three groups for every strain: control and two experimental groups. Two doses - low (1h treatment/day) and high (2 h treatment/day) - were used. In the first case 8 exposures of 7.5 min each with 1-min intervals were given and to realize the high dose this was repeated 4 h later. It was found that TS induces significant dominant-lethal mutations in both experimental groups of BALB/c, BDF1 and H mice ( p < 0.001), but some strain differences existed. The data obtained suggest that in BALB/c and BDF1 mice TS induces dominant-lethal mutations mainly in spermatocytes, spermatogonia and gonial stem cells, while in H mice only spermatids and spermatocytes are affected. The bone marrow from each strain and each dose group was investigated for the presence of micronucleated polychromatic erythrocytes (PCE) at 5, 19, 38, 54, and 63 days after beginning of the treatment with TS, using the same exposure regimen. Exposure of male mice to TS caused an up to 2–3-fold increase in the number of PCE in the bone marrow of BALB/c and BDF1 mice in both dose groups. In H mice this effect is observed only on days 19 and 38 of sampling. No cumulative or dose-dependent effects were detected. Increased formation of micronuclei within PCE of femoral bone marrow after passive smoking was regarded as being due to the effect of TS.