Evaluation of frequencies of HPRT mutant lymphocytes in butadiene polymer workers in a Southeast Texas facility

We examined the frequency of mutant lymphocytes (VFs) in workers (n = 30) occupationally exposed to the petrochemical, 1,3‐butadiene (BD), using the autoradiographic HPRT mutant lymphocyte assay. Current exposures were determined with organic vapor monitors that had a 12‐hr method detection limit (MDL) of 2.5 parts per billion (ppb). HPRT VFs were analyzed with respect to current exposure estimates, age in years, and occupational longevity (OL; defined as years working in the BD industry at this facility). Current exposures were low (mean 93.5 ppb, median 2.5 ppb) with only one individual's estimate (1683.5 ppb) exceeding the Occupational Safety and Health Administration's permissible exposure limit of 1,000 ppb. The majority (>50%) of current exposures were below the MDL. HPRT VFs were not significantly associated with current exposures (n = 29), and they were not significantly associated with age (n = 29). HPRT VFs were, however, significantly associated with OL (n = 29, R² = 0.107, P < 0.046). This result suggests that chronic and/or past, high‐level exposures might leave a mutagenic signature that is revealed by the HPRT assay, possibly through the retention of mutant, long‐term memory T‐cells. While it is encouraging that current occupational exposures to BD in this facility do not appear to be increasing the frequency of mutant T‐lymphocytes, evidence from workers with a lengthy history in the industry (≥30 years in this case) indicates that these individuals likely require additional biomonitoring for possible mutagenic effects resulting from chronic, past exposures. Environ. Mol. Mutagen., 2009. © 2008 Wiley‐Liss, Inc.     

Selective protection of zidovudine‐induced DNA‐damage by the antioxidants WR‐1065 and tempol

The cytokinesis‐block micronucleus cytome (CBMN) assay, introduced by Fenech, was used to demonstrate different types of DNA damage in MOLT‐3 human lymphoblastoid cells exposed to 10 μM zidovudine (AZT). In addition, we explored the cytoprotective potential of two antioxidants, WR‐1065 and Tempol, to decrease AZT‐induced genotoxicity. Binucleated cells, arrested by Cytochalasin B (Cyt B), were evaluated for micronuclei (MN), caused by DNA damage or chromosomal loss, and chromatin nucleoplasmic bridges (NPBs), caused by telomere attrition. Additionally, nuclear buds (NBUDs), caused by amplified DNA, and apoptotic and necrotic (A/N) cells were scored. We hypothesized that AZT exposure would increase the frequency of genotoxic end points, and that the antioxidants Tempol and WR‐1065 would protect against AZT‐induced genotoxicity. MOLT‐3 cells were exposed to 0 or 10 µM AZT for a total of 76 hr. After the first 24 hr, 0 or 5 µM WR‐1065 and/or 0 or 200 µM Tempol were added for the remainder of the experiment. For the last 28 hr (of 76 hr), Cyt B was added to arrest replication after one cell division, leaving a predominance of binucleated cells. The nuclear division index (NDI) was similar for all treatment groups, indicating that the exposures did not alter cell viability. MOLT‐3 cells exposed to AZT alone had significant (P < 0.05) increases in MN and NBs, compared to unexposed cells. Both Tempol and WR‐1065 protected against AZT‐induced MN formation (P < 0.003 for both), and WR‐1065, but not Tempol, reduced the levels of A/N (P = 0.041). In cells exposed to AZT/Tempol there were significantly reduced levels of NBUDs, compared to cells exposed to AZT alone (P = 0.015). Cells exposed to AZT/WR‐1065 showed reduced levels of NPBs, compared to cells exposed to AZT alone (P = 0.037). Thus WR‐1065 and Tempol protected MOLT‐3 cells against specific types of AZT‐induced DNA damage. Environ. Mol. Mutagen. 55:566–572, 2014. © 2014 Wiley Periodicals, Inc.     

Hypermethylation of CpG islands is associated with increasing chromosomal damage in chinese lead‐exposed workers

Lead is a widely existing environmental pollutant with potential carcinogenicity. To investigate the association of blood lead level (B‐Pb) with potential chromosomal damage and cancer, we analyzed micronucleus (MN) frequency of peripheral blood lymphocytes (PBLs) and the methylation status of six human tumor suppressor genes (TSGs) post lead exposure. In the study, 147 lead‐exposed workers were divided into two groups according to their B‐Pb P₅₀ value, with other 50 lead‐unexposed workers as a control group. The cytokinesis‐blocked micronucleus (CBMN) assay was performed to detect chromosomal damage of PBLs of both lead‐exposed and ‐unexposed workers. The methylation‐specific polymerase chain reaction (MSP‐PCR) was further used to examine the methylation status of six TSGs (GSTP1, hMLH1, MGMT, p14, p15, and p16). Results showed that MN frequencies of high B‐Pb workers 8.1 ± 3.1‰ and low B‐Pb workers 5.7 ± 2.3‰ were significantly higher than that of control group 2.8 ± 1.9‰ (P < 0.01), while the MN frequency of high B‐Pb workers was also higher than that of the low B‐Pb workers (P < 0.01). The MN frequency in PBLs of lead‐exposed group with the methylated TSGs was significantly higher than that in PBLs with the unmethylated TSGs (P < 0.05). Notably, the CpG island methylator phenotype (CIMP) correlated with chromosome damage (P < 0.05). Additionally, workers with high B‐Pb had higher chromosome damage than those with low B‐Pb (P < 0.05). Taken altogether, the results suggest that lead‐exposed workers with CIMP positive and high B‐Pb have a higher risk of being vulnerable to tumorigenesis. Environ. Mol. Mutagen. 59:549–556, 2018. © 2018 Wiley Periodicals, Inc.     

Genotoxicity of styrene–acrylonitrile trimer in brain,liver, and blood cells of weanling F344 rats

Styrene–acrylonitrile Trimer (SAN Trimer), a by‐product in production of acrylonitrile styrene plastics, was identified at a Superfund site in Dover Township, NJ, where childhood cancer incidence rates were elevated for a period of several years. SAN Trimer was therefore tested by the National Toxicology Program in a 2‐year perinatal carcinogenicity study in F344/N rats and a bacterial mutagenicity assay; both studies gave negative results. To further characterize its genotoxicity, SAN Trimer was subsequently evaluated in a combined micronucleus (MN)/Comet assay in juvenile male and female F344 rats. SAN Trimer (37.5, 75, 150, or 300 mg/kg/day) was administered by gavage once daily for 4 days. Micronucleated reticulocyte (MN‐RET) frequencies in blood were determined by flow cytometry, and DNA damage in blood, liver, and brain cells was assessed using the Comet assay. Highly significant dose‐related increases (P < 0.0001) in MN‐RET were measured in both male and female rats administered SAN Trimer. The RET population was reduced in high dose male rats, suggesting chemical‐related bone marrow toxicity. Results of the Comet assay showed significant, dose‐related increases in DNA damage in brain cells of male (P < 0.0074) and female (P < 0.0001) rats; increased levels of DNA damage were also measured in liver cells and leukocytes of treated rats. Chemical‐related cytotoxicity was not indicated in any of the tissues examined for DNA damage. The results of this subacute MN/Comet assay indicate induction of significant genetic damage in multiple tissues of weanling F344 male and female rats after oral exposure to SAN Trimer. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Limonene and its ozone-initiated reaction products attenuate allergic lung inflammation in mice

Inhalation of indoor air pollutants may cause airway irritation and inflammation and is suspected to worsen allergic reactions. Inflammation may be due to mucosal damage, upper (sensory) and lower (pulmonary) airway irritation due to activation of the trigeminal and vagal nerves, respectively, and to neurogenic inflammation. The terpene, d-limonene, is used as a fragrance in numerous consumer products. When limonene reacts with the pulmonary irritant ozone, a complex mixture of gas and particle phase products is formed, which causes sensory irritation. This study investigated whether limonene, ozone or the reaction mixture can exacerbate allergic lung inflammation and whether airway irritation is enhanced in allergic BALB/cJ mice. Naïve and allergic (ovalbumin sensitized) mice were exposed via inhalation for three consecutive days to clean air, ozone, limonene or an ozone–limonene reaction mixture. Sensory and pulmonary irritation was investigated in addition to ovalbumin-specific antibodies, inflammatory cells, total protein and surfactant protein D in bronchoalveolar lavage fluid and hemeoxygenase-1 and cytokines in lung tissue. Overall, airway allergy was not exacerbated by any of the exposures. In contrast, it was found that limonene and the ozone–limonene reaction mixture reduced allergic inflammation possibly due to antioxidant properties. Ozone induced sensory irritation in both naïve and allergic mice. However, allergic but not naïve mice were protected from pulmonary irritation induced by ozone. This study showed that irritation responses might be modulated by airway allergy. However, aggravation of allergic symptoms was observed by neither exposure to ozone nor exposure to ozone-initiated limonene reaction products. In contrast, anti-inflammatory properties of the tested limonene-containing pollutants might attenuate airway allergy.     

Micronuclei and developmental abnormalities in 4-day mouse embryos after paternal treatment with acrylamide

The developmental consequences of paternal exposure to acrylamide (50 mg/kg i.p. for 5 days) were assessed in preimplantation embryos. There was a significant increase in the proportion of morphologically abnormal embryos after postmeiotic treatment during spermatogenesis (88.7% vs. 14.8% in control). Abnormal embryos had an average of 1.8 ± 3.5 cells and >80% had at least one fragmented nucleus. In addition, morphologically normal embryos were significantly delayed (34.3 ± 12.8 cells per embryo vs. 57.6 ± 15.7 in control, P < 0.001). Acrylamide caused 10- and 20-fold increases in frequencies of cells with micronuclei (MN) in morphologically normal and abnormal embryos, respectively (41 and 93 MN per 1,000 cells). Both centromere-negative (M−) and centromere-positive (M+) were induced. Nuclei of abnormal embryos were significantly larger (900 μm² vs. 250 μm²) than controls. In addition, MN of abnormal embryos were larger than those of normal embryos (21.2 μm² vs. 6.5 μm², P < 0.01). Among control embryos, M+ were significantly larger than M− (P < 0.05). These findings suggest that the preimplantation embryo is a sensitive indicator of paternally transmitted effects on early development. Multiple mechanisms appear to be involved, including cytogenetic damage, proliferation arrest/delay, and fertilization failure. Future studies are needed to establish how induced cytological defects in preimplantation embryos contribute to birth defects and other postimplantation abnormalities. Environ. Mol. Mutagen. 31:206–217, 1998 © 1998 Wiley-Liss, Inc.     

Extracellular amyloid beta 42 causes necrosis,inhibition of nuclear division,and mitotic disruption under both folate deficient and folate replete conditions as measured by the cytokinesis‐block micronucleus cytome assay

Alzheimer's disease is associated with accumulation of extracellular beta amyloid peptide 42 (Aβ42) which may induce DNA damage and reduce cellular regenerative potential. These effects may be exacerbated under conditions of folate deficiency. The aim of this study was to investigate whether extracellular Aβ42 induces DNA damage and cell death in human peripheral lymphocytes and whether there is an interactive effect between extracellular Aβ42 and folic acid status. Peripheral blood lymphocytes were cultured in medium under conditions of both low and high folate (20 and 200 nM, respectively) and challenged with either Aβ42 or the physiologically normal form Aβ40 (both at 5, 10, 15 µM). Genome stability and cytotoxicity events were investigated using the cytokinesis‐block micronucleus cytome (CBMN‐cyt) assay. Outcome measures scored included the nuclear division index (NDI), necrosis, apoptosis, binucleated cells with micronuclei (MN), nucleoplasmic bridges (NPB), and nuclear buds (NBUD) and abnormally shaped nuclei (circular, (CIR) and horse‐shoe, (HS) that may be indicative of mitotic disruption. Folic acid deficiency significantly reduced NDI (P < 0.001) and increased all the DNA damage biomarkers (MN, NPB, NBUD, HS, CIR), (P < 0.001). In contrast, exposure to Aβ40 had no impact on CBMN cytome biomarkers but Aβ42 significantly reduced NDI (P < 0.01), increased necrosis (P < 0.05) and frequency of cells with circular nuclei (P < 0.01). There was no evidence of an interaction between Aβ42 and folic acid with respect to CBMN cytome biomarkers. Extracellular Aβ42 appears to have cytotoxic and cytostatic effects but its effect on chromosomal instability appears to be small relative to folate deficiency. Environ. Mol. Mutagen. 55:1–14, 2014. © 2013 Wiley Periodicals, Inc.     

Genotoxicity of doxorubicin in F344 rats by combining the comet assay,flow‐cytometric peripheral blood micronucleus test,and pathway‐focused gene expression profiling

Doxorubicin (DOX) is an antineoplastic drug effective against many human malignancies. DOX's clinical efficacy is greatly limited because of severe cardiotoxicity. To evaluate if DOX is genotoxic in the heart, ~7‐week‐old, male F344 rats were administered intravenously 1, 2, and 3 mg/kg bw DOX at 0, 24, 48, and 69 hr and the Comet assays in heart, liver, kidney, and testis and micronucleus (MN) assay in the peripheral blood (PB) erythrocytes using flow cytometry were conducted. Rats were euthanized at 72 hr and PB was removed for the MN assay and single cells were isolated from multiple tissues for the Comet assays. None of the doses of DOX induced a significant DNA damage in any of the tissues examined by the alkaline Comet assay. Contrastingly, the glycosylase enzymes‐modified Comet assay showed a significant dose dependent increase in the oxidative DNA damage in the cardiac tissue (P ≤ 0.05). In the liver, only the top dose induced significant increase in the oxidative DNA damage (P ≤ 0.05). The histopathology showed no severe cardiotoxicity but non‐neoplastic lesions were present in both untreated and treated samples. A severe toxicity likely occurred in the bone marrow because no viable reticulocytes could be screened for the MN assay. Gene expression profiling of the heart tissues showed a significant alteration in the expression of 11 DNA damage and repair genes. These results suggest that DOX is genotoxic in the heart and the DNA damage may be induced primarily via the production of reactive oxygen species. Environ. Mol. Mutagen. 55:24–34, 2014. © 2013 Wiley Periodicals, Inc.^?     

Carbon nanotubes induce oxidative DNA damage in RAW 264.7 cells

The induction of DNA and chromosome damage following in vitro exposure to carbon nanotubes (CNT) was assessed on the murine macrophage cell line RAW 264.7 by means of the micronucleus (MN) and the comet assays. Exposures to two CNT preparations (single‐walled CNT (SWCNT > 90%) and multiwalled CNT (MWCNT > 90%) were performed in increasing mass concentrations (0.01–100 μg/ml). The frequency of micronuclei was significantly increased in cells treated with SWCNT (at doses above 0.1 μg/ml), whereas MWCNT had the same effect at higher concentrations (1 μg/ml) (P < 0.05). The results of the comet assay revealed that the effects of treatment with SWCNT were detectable at all concentrations tested (1–100 μg/ml); oxidized purines increased significantly, whereas pyrimidines showed a significant increase (P < 0.001) only at the highest concentration (100 μg/ml). In cells treated with MWCNT, an increase in DNA migration due to the oxidative damage to purines was observed at a concentration of 1 and 10 μg/ml, whereas pyrimidines showed a significant increase only at the highest mass concentration tested. However, both SWCNT and MWCNT induced a statistically significant cytotoxic effect at the highest concentrations tested (P < 0.001). These findings suggest that both the MN and comet assays can reliably detect small amount of damaged DNA at both chromosome and nuclear levels in RAW 264.7 cells. Moreover, the modified version of the comet assay allows the specific detection of the induction of oxidative damage to DNA, which may be the underlying mechanism involved in the CNT‐associated genotoxicity. Environ. Mol. Mutagen., 2010. © 2010 Wiley‐Liss, Inc.     

Kupffer cell activation by ambient air particulate matter exposure may exacerbate non-alcoholic fatty liver disease

Owing to increased obesity, non-alcoholic fatty liver disease (NAFLD) is now the most prevalent liver disease in the United States. NAFLD is considered a component of metabolic syndrome, a cluster of disorders that also includes diabetes mellitus, dyslipidemia, arteriosclerosis, and hypertension. Exposure to ambient air particulate matter with aerodynamic diameters?<?2.5?μm (PM_2.5) is a risk factor for arteriosclerosis and lung disease, but its effect on NAFLD is unknown. PM_2.5 induces pulmonary dysfunction via Toll-like receptor (TLR) activation on alveolar macrophages. TLR activation of Kupffer cells, resident hepatic macrophages, and subsequent pro-inflammatory cytokine production have been shown to play a key role in NAFLD progression. We hypothesized that PM_2.5 exposure is a significant risk factor for the progression of NAFLD. Thus, following exposure of male C57BL/6 mice fed high fat chow (HFC) to concentrated air particulate matter (CAPs) or filtered air for 6 weeks, progression of NAFLD was evaluated by standardized histological assessment of hepatic inflammation and fibrosis. In mice fed HFC, the hepatic inflammatory grade (3.00?±?0.00 vs. 1.50?±?0.71, P?<?0.001) and fibrosis stage (1.00?±?0.00 vs. 0.60?±?0.52, P?=?0.023) were both significantly higher in mice exposed to CAPs versus filtered air, respectively. Increased numbers of Kupffer cells contained PM in CAPs-exposed mice scores of (2.00?±?0.94 vs. 0.20?±?0.42, respectively, P?<?0.001). PM exposure increased IL-6 secretion up to seven-fold in a dose-dependent manner by isolated wild-type but not TLR4^?/? Kupffer cells (P?<?0.050). In conclusion, ambient PM_2.5 exposure may be a significant risk factor for NAFLD progression.     

DNA damage in Pakistani agricultural workers exposed to mixture of pesticides

A cross‐sectional study was designed to determine whether occupational exposure to a complex mixture of pesticides results in a significant increase of DNA damage in farmers chronically exposed to pesticides in open fields. Leukocytes from 47 agriculture workers exposed to pesticides and 50 controls were evaluated with comet assay. Workers recruitment was based on their exposure to pesticides during the spraying season on cotton crop. Serum from these individuals was also analyzed for pesticides presence using high performance liquid chromatography. Statistically significant difference (P < 0.001) in DNA damage of exposed individuals (mean ± S.D 14.80 ± 3.04 μm) was observed when compared with control group (6.54 ± 1.73 μm) as studied on the basis of comet tail length. Smokers had significantly higher mean comet tail length than nonsmokers and ex‐smokers in both workers (20.26 ± 3.53 vs. 14.19 ± 4.25, P < 0.001) and controls (7.86 ± 1.09 vs. 5.80 ± 1.59, P < 0.001), whereas age had a minimal effect on DNA damage (P < 0.05). The length of pesticide exposure is positively associated with DNA damage in exposed individuals (P < 0.001). Our study shows that chronic exposure to pesticides produces DNA damage in pesticide sprayers and suggests that this type of monitoring is recommended in preventive policies for pesticide sprayers. Environ. Mol. Mutagen., 2009. © 2008 Wiley‐Liss, Inc.     

Repeated ozone exposures enhance bronchial allergen responses in subjects with rhinitis or asthma

BACKGROUND: Single exposures to > 200 p.p.b. of ozone are capable of enhancing the early-phase lung function response to allergen. OBJECTIVE: The aim of the present study was to compare the effect of single vs. repeated exposures to ozone on early and late-phase allergen responses. METHODS: Eleven subjects with allergic asthma and 22 subjects with allergic rhinitis underwent single exposures to filtered air, 125 p.p.b. and 250 p.p.b. ozone, as well as repeated exposures to 125 p.p.b. ozone on four consecutive days. Twenty hours after the (final) exposure, subjects inhaled a single dose of allergen and a sputum induction was performed 6-7 h later. RESULTS: In the subjects with rhinitis, the mean early-phase response of FEV1 and the number of > or = 20% reductions were significantly greater after exposure to 250 or 4 x 125 p.p.b. ozone compared with filtered air. In addition, most of the > or = 15% late-phase responses in FEV1 occurred after exposure to 4 x 125 p.p.b., as well as the strongest effects on sputum parameters. The rise in the number of eosinophils was statistically significant in both groups. Regarding the number of lymphocytes and the concentrations of mast cell tryptase, histamine or LDH, significance was, however, only reached in the asthma group. CONCLUSION: Our data suggest that repeated exposure to ozone, at a peak ambient air level, can enhance both functional and inflammatory responses to inhaled allergen in subjects with pre-existing allergic airway diseases, and that these effects might reach a clinically relevant magnitude.     

γH2AX formation kinetics in PBMCs of rabbits exposed to acute and fractionated radiation and attenuation of focus frequency through preadministration of a combination of podophyllotoxin and rutin hydrate

DNA damage can be assessed by the quantitation of γH2AX foci that form at DSB sites. This study examines the generation and persistence of γH2AX foci, variability in foci size after acute and fractionated radiation exposure, and the effect of pretreatment with a safe radioprotective formulation termed G‐003M on foci generation and persistence. G‐003M contains a combination of podophyllotoxin and rutin hydrate, and was administered intramuscularly to rabbits 1 hr prior to Co⁶⁰ gamma irradiation. Rabbits were assigned to one of the following treatment groups: untreated, G‐003M alone, irradiated (single dose 8 Gy, fractionated 2 Gy/day for 4 days or single dose 2 Gy) or G‐003M preadministration followed by radiation exposure. Foci continuously persisted for a week in peripheral blood mononuclear cells of rabbits exposed to a single 8 Gy dose. However, the number of foci gradually decreased after reaching a maximum at 1 h. In rabbits exposed to fractionated radiation, foci detected 1 hr after the final exposure were significantly larger (P < 0.001) than in rabbits exposed to a single 8 Gy dose, but disappeared completely after 24 h. In both groups, foci reappeared on days 11‐15 in terminally ill animals. G‐003M pretreatment significantly (P < 0.05) attenuated the formation of γH2AX foci in all irradiated rabbits. This study reveals that γH2AX focus assessment could be used to confirm radiation exposure, that focus size reflects the type of radiation exposure (acute or fractionated), that the re‐appearance of foci is a strong indicator of imminent death in animals, and that G‐003M provides protection against radiation. Environ. Mol. Mutagen. 57:455–468, 2016. © 2016 Wiley Periodicals, Inc.     

Application of a geographic information system to explore associations between air pollution and micronucleus frequencies in African American children and adults

Exposure to air pollution has been associated with adverse respiratory and cardiovascular health outcomes in both children and adults. In this study, we used geographic information systems (GISs) to explore possible associations between chromosomal damage in 65 African American children and their mothers from Oakland, California, and both proximity to traffic and regional ozone levels. Study participants were interviewed at the Healthy Child Clinic of Children's Hospital, Oakland, and their blood and buccal cells were collected for assessment of chromosomal damage by the micronucleus (MN) assay. Regional ozone levels, which decreased from April to November with a secondary peak in late summer, were highly correlated with season by month (r=-0.84, P=0.02) and strongly associated with MN frequency (frequency ratio (FR): 3.37, 95% confidence interval (CI): 1.30-8.72) in both cell types of children and adults. Additionally, MN frequencies were modestly associated with individual measures of traffic density in children (FR=2.45, 95% CI=0.86-7.10), but not in adults; this suggests a greater vulnerability to traffic-related air pollution in children. Smoking in the household also increased MN frequency in the lymphocytes of children (FR: 1.13, 95%CI: 1.01-1.24) and adults (FR: 1.06, 95%CI: 0.99-1.13), whereas vitamin use in adults decreased MN frequency in both lymphocytes and buccal cells (FR: 0.17, 95%CI: 0.02-1.31; FR: 0.18, 95%CI: 0.03-1.18, respectively). Our data indicate that GIS-generated measures of traffic density for individual households augment regional ozone monitoring data used to assess effects of air pollution. This approach helped to demonstrate elevated cytogenetic damage in exposed minority children.     

Effects of the DNA topoisomerase II inhibitor merbarone in male mouse meiotic divisions in vivo: Cell cycle arrest and induction of aneuploidy

In order to clarify possible risks of aneuploidy induction in germ cells by cancer chemotherapy we studied effects of a non complex-stabilizing DNA topoisomerase II (topo II) inhibitor merbarone in male mouse meiotic divisions in vivo. Two cytogenetic approaches were used: (1) C-banding on meiotic chromosome preparations and (2) analysis of spermatid micronuclei (MN) combined with immunocytochemical staining of kinetochore proteins using CREST serum. For comparison, another topo II inhibitor, VP·16, known to form cleavable complexes, was studied. The microdissection technique of mouse seminiferous tubules enabled us to carefully examine effects at specific phases of meiosis. Merbarone injections increased percentages of polyploid and hypoploid metaphase II spermatocytes at time intervals corresponding to the treatment of the first meiotic division and diplotene-diakinesis. The highest level of MN induction (5.8 MN/1000 spermatids, P < 0.001) was observed in animals injected 48 hours before the harvest, corresponding to the treatment of diplotene-diakinesis spermatocytes. Most of the induced MN (80%) contained kinetochore signals, indicating that they resulted from detachment of a whole bivalent or chromosome from the meiotic spindle. The high frequency of MN with two kinetochore signals at opposite sides (33%) most likely denotes lagging of whole bivalents during MI. Inhibition of cell proliferation was determined by scoring cells arrested at different phases of MI and MII. All groups of treated animals showed a clear increase in the frequency of arrested divisions compared to controls (P < 0.001). Thus, merbarone was shown to severely damage normal meiotic processes. Environ. Mol. Mutagen. 29:16–27, 1997 © 1997 Wiley-Liss, Inc.     

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. © 1994 Wiley-Liss, Inc.     

Cytogenetic biomonitoring of Indian women cooking with biofuels: micronucleus and chromosomal aberration tests in peripheral blood lymphocytes

India currently has the largest number of indoor air pollution-related health problems in the world, with three-quarters of its households burning wood, cowdung, or crop residues ("traditional" biomass fuels) for cooking, and the remainder using kerosene and relatively clean-burning liquefied petroleum gas (LPG). Combustion of these fuels produces various pollutants that may cause serious health effects in exposed populations. In this study, the micronucleus (MN) and chromosomal aberration (CA) assays were used to evaluate the relative amounts of DNA damage produced by the use of these cooking fuels. Cytogenetic evaluation of 179 female subjects showed a significant increase in both MN and CA frequency in blood lymphocytes from users of biomass-fuels in comparison to lymphocytes from LPG users (used as a reference population). The relative MN and CA frequencies for the users of the various fuels decreased in the order cowdung > cowdung/wood >/= wood > kerosene >/= LPG. Further, the results indicated an effect of subject age, and the duration of exposure on the MN and CA frequencies in biomass fuel users. Age had no significant effects on the genotoxicity responses in subjects with 10 years, CA and MN frequencies were higher in older individuals (>30 years of age) than younger subjects. Regardless of age, subjects burning biomass fuels had higher MN and CA frequencies than LPG users only when exposures were of at least 5 years duration. These results indicate that burning biomass-based fuels increases the frequency of cytogenetic alterations in blood lymphocytes of exposed populations, possibly because of exposure to the various noxious gases and toxic substances present in biomass fuels. These cytogenetic markers could be used in the field to assess the genotoxic consequences of burning various cooking fuels and for early detection of genetic abnormalities in people exposed to various pollutants and toxicants.     

Increased expired NO and roles of CO2 and endogenous NO after venous gas embolism in rabbits

Venous gas embolism (VGE) is a feared complication in diving, aviation, surgery and trauma. We hypothesized that air emboli in the lung circulation might change expired nitric oxide (FeNO). A single intravenous infusion of air was given (100 μl kg⁻¹) to three groups of anaesthetized mechanically ventilated rabbits: (A) one with intact NO production, (B) one with intact NO production and where end-tidal CO₂ was controlled, and (C) one with endogenous NO synthesis blockade (l-NAME, 30 mg kg⁻¹). Air infusions resulted in increased FeNO of the control group from 20 (4) [mean (SD)] ppb to a peak value of 39 (4) ppb within 5 min (P < 0.05), and FeNO was still significantly elevated [27 (2) ppb] after 20 min (P < 0.05). Parallel to the NO increase there were significant decreases in end-tidal CO₂ (ETCO₂) and mean arterial pressure and an increase in insufflation pressure. In group B, when CO₂ was supplemented after air infusion, NO was suppressed (P = 0.033), but was still significantly elevated compared with pre-infusion control (P < 0.05). In group C, all animals died within 40 min of air infusion whereas all animals in the other groups were still alive at this time point. We conclude that venous air embolization increases FeNO, and that a part of this effect is due to the concomitant decrease in ETCO₂. Furthermore, an intact NO production may be critical for the tolerance to VGE. Finally, FeNO might have a potential in the diagnosis and monitoring of pulmonary gas embolism.     

Studies on the genotoxicity of molybdenum salts in human cells in vitro and in mice in vivo

Molybdenum is an essential element in plants and animals as a cofactor for enzymes. Molybdenum trioxide is used in metallurgical processes, in cosmetics as a pigment, and in contact lens solution, yet limited information is available on molybdenum genotoxicity. In the present study the micronucleus (MN) assay in human lymphocytes and mouse bone marrow and the dominant lethal assay in mice were used to assess the genotoxic effects of molybdenum salts in vitro and in vivo. Two salts of molybdenum were tested in whole blood cultures. Ammonium molybdate was more potent than sodium molybdate in causing a dose-dependent decrease in viability and replicative index and an increase in MN formation in binucleated lymphocytes (P < 0.001). A dose–response in both kinetochore-positive MN (caused by chromosome lagging) and kinetochore-negative MN (associated with chromosome breakage) was observed. Based on the results of a toxicity study of sodium molybdate, two doses, 200 and 400 mg/kg, were assessed in the bone marrow MN assay in mice (two i.p. injections 24 and 48 hr prior to euthanasia). A modest but statistically significant increase in MN frequency in polychromatic erythrocytes was observed (P < 0.05). The same treatment protocol was used to analyze dominant lethality. A dose-dependent increase in postimplantation loss represented mostly by early resorptions was observed the first week after treatment (P = 0.003). These preliminary data suggest that sodium molybdate induces dominant lethality at the postmeiotic stage of spermatogenesis. Overall, molybdenum salts produced moderately positive results both in vitro in human cells and in vivo in mice. Environ. Mol. Mutagen. 32:251–259, 1998 © 1998 Wiley-Liss, Inc.