CYP1A1 and CYP1B1 gene expression and DNA adduct formation in normal human mammary epithelial cells exposed to benzo[a]pyrene in the absence or presence of chlorophyllin.

Benzo[a]pyrene (BP) is a potent pro-carcinogen and ubiquitous environmental pollutant. Here, we examined the induction and modulation of CYP1A1 and CYP1B1 and 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG) adduct formation in DNA from 20 primary normal human mammary epithelial cell (NHMEC) strains exposed to BP (4muM) in the absence or presence of chlorophyllin (5muM). Real-time polymerase chain reaction (RT-PCR) analysis revealed strong induction of both CYP1A1 and CYP1B1 by BP, with high levels of inter-individual variability. Variable BPdG formation was found in all strains by r7, t8-dihydroxy-t-9, 10 epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence assay (CIA). Chlorophyllin mitigated BP-induced CYP1A1 and CYP1B1 gene expression in all 20 strains when administered with BP. Chlorophyllin, administered prior to BP-exposure, mitigated CYP1A1 expression in 18/20 NHMEC strains (p<0.005) and CYP1B1 expression in 17/20 NHMEC strains (p<0.005). Maximum percent reductions of CYP1A1 and CYP1B1 gene expression and BPdG adduct formation were observed when cells were pre-dosed with chlorophyllin followed by administration of the carcinogen with chlorophyllin (p<0.005 for CYP1A1 and CYP1B1 expression and p<0.0005 for BPdG adducts). Therefore, chlorophyllin is likely to be a good chemoprotective agent for a large proportion of the human population.

Coal dust alters beta-naphthoflavone-induced aryl hydrocarbon receptor nuclear translocation in alveolar type II cells.

BACKGROUND: Many polycyclic aromatic hydrocarbons (PAHs) can cause DNA adducts and initiate carcinogenesis. Mixed exposures to coal dust (CD) and PAHs are common in occupational settings. In the CD and PAH-exposed lung, CD increases apoptosis and causes alveolar type II (AT-II) cell hyperplasia but reduces CYP1A1 induction. Inflammation, but not apoptosis, appears etiologically associated with reduced CYP1A1 induction in this mixed exposure model. Many AT-II cells in the CD-exposed lungs have no detectable CYP1A1 induction after PAH exposure. Although AT-II cells are a small subfraction of lung cells, they are believed to be a potential progenitor cell for some lung cancers. Because CYP1A1 is induced via ligand-mediated nuclear translocation of the aryl hydrocarbon receptor (AhR), we investigated the effect of CD on PAH-induced nuclear translocation of AhR in AT-II cells isolated from in vivo-exposed rats. Rats received CD or vehicle (saline) by intratracheal (IT) instillation. Three days before sacrifice, half of the rats in each group started daily intraperitoneal injections of the PAH, beta-naphthoflavone (BNF). RESULTS: Fourteen days after IT CD exposure and 1 day after the last intraperitoneal BNF injection, AhR immunofluorescence indicated that proportional AhR nuclear expression and the percentage of cells with nuclear AhR were significantly increased in rats receiving IT saline and BNF injections compared to vehicle controls. However, in CD-exposed rats, BNF did not significantly alter the nuclear localization or cytosolic expression of AhR compared to rats receiving CD and oil. CONCLUSION: Our findings suggest that during particle and PAH mixed exposures, CD alters the BNF-induced nuclear translocation of AhR in AT-II cells. This provides an explanation for the modification of CYP1A1 induction in these cells. Thus, this study suggests that mechanisms for reduced PAH-induced CYP1A1 activity in the CD exposed lung include not only the effects of inflammation on the lung as a whole, but also reduced PAH-associated nuclear translocation of AhR in an expanded population of AT-II cells.

Immune response signatures of benzo{alpha}pyrene exposure in normal human mammary epithelial cells in the absence or presence of chlorophyllin.

Carcinogenic polycylic aromatic hydrocarbons can alter immune responses. Changes in immune response gene expression profiles in multiple human mammary cell strains exposed to benzo(alpha)pyrene (BP) (4 microM) in vitro, in the presence or absence of chlorophyllin (5 microM), were observed using Affymetrix gene arrays. Expressions of five immune response genes were altered ~3.0-fold by BP exposure and 24 genes by BP in the presence chlorophyllin. In silico pathway analysis revealed altered immune response genes form interactive gene networks with many cellular processes, suggesting their role in a complex multigenic response to toxins. Additionally, it was suggestive of the possible immunomodulatory potential of chlorophyllin apart from various other well-documented mechanisms of action. Gene expression matrices revealed consistent alteration patterns involving IL1B, SECTM1 and CXCL14 on exposure to BP, and IL1RN, CD86, IF144 and GIP2 in the presence of chlorophyllin and BP, suggesting some of these genes might constitute putative immune response biomarkers of PAH exposure. This study has therefore identified a battery of potential immune response biomarkers of PAH exposure, amidst several genes, for future validation studies.

Transcriptional profiles of benzo(a)pyrene exposure in normal human mammary epithelial cells in the absence or presence of chlorophyllin.

Benzo(a)pyrene (BP) exposure causes alterations in gene expression in normal human mammary epithelial cells (NHMECs). This study used Affymetrix Hu-Gene133A arrays, with 14,500 genes represented, to evaluate modulation of BP-induced gene expression by chlorophyllin in six NHMEC strains derived from different donors. A major goal was to seek potential biomarkers of carcinogen exposure and how they behave in the presence of a chemopreventive agent. NHMECs (passage 6 and 70% confluence) were exposed for 24h to either vehicle control, or BP, or chlorophyllin followed by BP and chlorophyllin together. BP exposure resulted in approximately 3-fold altered expression of 49 genes in at least one of the six NHMEC strains. When cells were exposed to chlorophyllin pre-treatment followed by BP plus chlorophyllin, expression of 125 genes was similarly altered. Genes in the functional categories of xenobiotic metabolism, cell signaling, cell motility, cell proliferation, cellular transcription, metabolism, cell cycle control, apoptosis and DNA repair were identified. Only CYP1B1 and ALDH1A3 were consistently up-regulated by approximately 3-fold in most of the cell strains (at least 4) when exposed to BP. Cluster analysis identified a suite of 13 genes induced by BP where induction was mitigated in the presence of chlorophyllin. Additionally, cluster analysis identified a suite of 16 genes down-regulated by BP where induction was partially restored in the presence of chlorophyllin.

Frequency of human sperm carrying structural aberrations of chromosome 1 increases with advancing age.

OBJECTIVE: To investigate the association between male age and the frequency of sperm with de novo structural chromosomal abnormalities. DESIGN: Semen specimens collected from two groups of 10 healthy, nonsmoking men, aged 22-28 and 65-80 years, were analyzed with the use of a multicolor fluorescence in situ hybridization assay for detecting breaks, segmental duplications and deletions, and aneuploidy and diploidy involving chromosome 1. SETTING: Healthy volunteer workers and retirees from a government research environment. MAIN OUTCOME MEASURE: Sperm carrying numerical and structural chromosomal abnormalities. RESULT(S): We detected significant increases in the frequency of sperm carrying breaks and segmental duplications and deletions of chromosome 1 among older men compared with younger men. Older men carried twice the frequency of sperm with segmental duplications and deletions of chromosome 1. The frequency of sperm carrying breaks within the 1q12 fragile-site region nearly doubled in older men. In contrast to female gametes, there was no effect of age on the frequency of sperm with numerical chromosomal abnormalities. CONCLUSION: Our findings suggest that advancing male age is associated with a gradual and significant increase in the risk of fathering children with various chromosomal defects such as segmental aneusomy syndromes.

Correlation of comparative genomic hybridization results of 100 archival uveal melanomas with patient survival.

Comparative genomic hybridization (CGH) was used to elucidate DNA sequence copy number imbalances in 100 archival formalin-fixed, paraffin-embedded (FFPE) uveal melanoma cases. Of these 100 cases, 51 were from patients who survived >or=9 years post diagnosis without evidence of metastasis; the remaining 49 patients died from metastatic disease. Viable probe was generated from 82 of the 100 cases, allowing correlation of CGH findings with survival for all but 18 cases. Copy number imbalances revealed by CGH were tested for univariate prognostic significance. The most powerful predictor of a poor prognosis was gain of 18q11.2, which was subsequently compared with other significant chromosomal regions, as well as histologic and clinical factors, in a multivariate analysis. There was also evidence of differential X chromosome involvement in the survival correlations between male and female cases, which may be of significance to prognosis. This large-scale CGH analysis of archival material is intended to direct further gene-specific study of malignancy in uveal melanoma.

Apoptosis and Bax expression are increased by coal dust in the polycyclic aromatic hydrocarbon-exposed lung.

BACKGROUND: Miners inhaling respirable coal dust (CD) frequently develop coal workers' pneumoconiosis, a dust-associated pneumoconiosis characterized by lung inflammation and variable fibrosis. Many coal miners are also exposed to polycyclic aromatic hydrocarbon (PAH) components of diesel engine exhaust and cigarette smoke, which may contribute to lung disease in these workers. Recently, apoptosis was reported to play a critical role in the development of another pneumoconiosis of miners, silicosis. In addition, CD was reported to suppress cytochrome P450 1A1 (CYP1A1) induction by PAHs. METHODS: We investigated the hypothesis that apoptosis plays a critical role in lung injury and down-regulation of CYP1A1 induction in mixed exposures to CD and PAHs. We exposed rats intratracheally to 0.0, 2.5, 10.0, 20.0, or 40.0 mg/rat CD and, 11 days later, to intraperitoneal beta-naphthoflavone (BNF) , a PAH. In another group of rats exposed to CD and BNF, caspase activity was inhibited by injection of the pan-caspase inhibitor Q-VD-OPH [quinoline-Val-Asp (OMe) -CH2-OPH]. RESULTS: In rats exposed to BNF, CD exposure increased alveolar expression of the proapoptotic mediator Bax but decreased CYP1A1 induction relative to BNF exposure alone. Pan-caspase inhibition decreased CD-associated Bax expression and apoptosis but did not restore CYP1A1 activity. Further, CD-induced lung inflammation and alveolar epithelial cell hypertrophy and hyperplasia were not suppressed by caspase inhibition. CONCLUSIONS: Combined BNF and CD exposure increased Bax expression and apoptosis in the lung, but Bax and apoptosis were not the major determinants of early lung injury in this model.

A father of four consecutive trisomic pregnancies with elevated frequencies of associated aneuploid sperm.

A couple with normal somatic karyotypes had four consecutive trisomic pregnancies, each involving a different chromosome, of which two children were liveborn with confirmed paternal-origin trisomies. The apparently healthy father produced abnormally high frequencies of disomic sperm for each of the four chromosomes involved in the trisomic pregnancies (P< 0.003, by sperm fluorescence in situ hybridization (FISH)). His elevated sperm aneuploidies persisted over a 2-year period and affected all chromosomes evaluated, suggesting that he had a genome-wide defect in meiotic disjunction. He also had the highest frequencies of aneuploid sperm reported for any healthy man to date. His frequencies of aneuploid sperm were comparable to the peak frequencies of the transient responses reported in some cancer patients after receiving aneugenic chemotherapies. These findings indicate that apparently healthy men can produce abnormally high frequencies of sperm aneuploidies that suggest that this condition may contribute to recurrent trisomic pregnancies.

Molecular cytogenetic evaluation of 10 uveal melanoma cell lines.

Uveal melanoma is the most common intraocular tumor in adults and often results in unilateral blindness and/or death. Previous cytogenetic characterizations of this tumor consistently revealed chromosomal abnormalities involving chromosomes 3, 6, and 8; reports of other abnormalities vary in frequency. We defined cytogenetic abnormalities of this tumor using complementary in situ hybridization techniques on 10 uveal melanoma cell lines. Synthesis of comparative genomic hybridization (CGH) and spectral karyotyping (SKY) results revealed that chromosomal rearrangement is involved in DNA sequence copy number abnormalities throughout the genome, but monosomy 3 was not found. Monosomy 3 is thought to be a significant prognostic indicator, so its absence was investigated further. Fluorescence in situ hybridization (FISH) for chromosome 3 revealed approximately 1 centromere signal per cell, but probes for 3p and 3q revealed multiple telomere signals per cell, suggesting chromosomal rearrangement without whole-chromosome loss. Based on combined CGH, SKY, and FISH data, we propose that chromosome 3 is more frequently involved in chromosomal rearrangements than whole-chromosome loss in uveal melanoma. Future approaches should be designed to confirm and enhance the resolution of regions of imbalance in primary tumors. Once identified, conserved chromosomal alterations that contribute to uveal melanoma may reveal the underlying aspects of uveal melanoma onset, metastasis and resistance to current treatment modalities.

Characterization of calpastatin gene in fish: its potential role in muscle growth and fillet quality.

Calpastatin (CAST), the specific inhibitor of the calpain proteases, plays a role in muscle growth and meat quality. In rainbow trout (RBT), we identified cDNAs coding for two CAST isoforms, a long (CAST-L) and a short isoform (CAST-S), apparently derived from two different genes. Zebrafish and pufferfish CAST cDNA and genomic sequences were retrieved from GenBank and their exon/intron structures were characterized. Fish CASTs are novel in that they have fewer repetitive inhibitory domains as compared to their mammalian counterparts (one or two vs. four). The expressions of CAST mRNAs were measured in three RBT strains with different growth rates and fillet firmness that were fed either high energy or control diets. CAST-L and S expressions were significantly lower (p<0.01) in the strain that has the slowest growth rate and yielded the softest fillet. Strain or diet did not affect level of calpain mRNAs. However, the decrease in the CAST/calpain ratio at the mRNA level did not lead to a corresponding change in the calpain catalytic activity. Further investigation should reveal a potential use of the CAST gene as a tool to monitor fish muscle growth and fillet firmness.

Identification and molecular characterization of the rainbow trout calpains (Capn1 and Capn2): their expression in muscle wasting during starvation.

Calpains are calcium regulated proteases involved in cellular functions that include muscle proteolysis both ante- and postmortem. Here, we describe the molecular characterization of the rainbow trout catalytic subunits of the mu- and m-calpains, respectively. The cDNA sequence for Capn1 encodes a protein of 704 amino acids with a calculated molecular mass of 79.9 kDa. The amino acid sequence shows 66% and 86% identity with the mouse and zebrafish Capn1, respectively. The Capn2 cDNA codes for a protein consisting of 701 amino acid residues with a calculated molecular mass of 78.2 kDa. The protein shows 65% amino acid sequence identity with the mouse and chicken Capn2. The two isozymes of rainbow trout have the characteristic domains: I (propeptide), II (cysteine catalytic site), III (electrostatic switch), and IV (contains five EF-hands). Because starvation induces muscle wasting, the hypothesis of this study was that starvation could affect regulation of the calpain system in muscle. Starvation of rainbow trout fingerlings (15-20 g) for 35 days stimulated the expression of Capn1 (2.2-fold increase, P < 0.01), Capn2 (6.0-fold increase, P < 0.01), and calpastatins (1.6-fold increase, P < 0.05) as measured by quantitative real-time RT-PCR. The mRNA changes led to a 1.23-fold increase in the calpain catalytic activity. The results suggest a potential role of calpains in protein mobilization as a source of energy under fasting condition.

Differential basal expression of genes associated with stress response, damage control, and DNA repair among mouse tissues.

Efficient recognition and repair of DNA damage is essential for maintaining genomic integrity. Tissues and cell types within tissues appear to vary in both DNA damage susceptibilities and cancer incidences, yet the molecular mechanisms underlying these differences are not well understood. The purpose of this study was to characterize the baseline transcription profiles of selected genes involved in DNA damage recognition and repair processes among several tissues of healthy adult B6C3F1 mice (testis, brain, liver, spleen and heart), which are routinely used by the National Toxicology Program (NTP) to conduct long-term chemical carcinogenicity studies. Stress response, damage control and DNA repair-associated genes were differentially expressed among the tissues examined. Overall, stress response genes exhibited the greatest variation among tissues with the highest expression in liver and heart while DNA repair genes exhibited the least variation. Damage control genes associated with cell cycle regulation and DNA repair genes generally had the highest expression in testis. The expression levels of several genes were rank correlated with the spontaneous cancer incidences among these tissues. Variations in basal expression of DNA damage recognition and repair-associated genes among healthy tissues may contribute to their differential response to genotoxic agents and susceptibility to genetic disease.

Respirable coal dust particles modify cytochrome P4501A1 (CYP1A1) expression in rat alveolar cells.

Cytochrome P4501A1 (CYP1A1) metabolizes polycyclic aromatic hydrocarbons in cigarette smoke to DNA-binding reactive intermediates associated with carcinogenesis. Epidemiologic studies indicate that the majority of coal miners are smokers but have a lower risk of lung cancer than other smokers. We hypothesized that coal dust (CD) exposure modifies pulmonary carcinogenesis by altering CYP1A1 induction. Therefore, male Sprague Dawley rats were intratracheally instilled with 2.5, 10, 20, or 40 mg CD/rat or vehicle (saline); and 11 d later, pulmonary CYP1A1 was induced by intraperitoneal injection of beta-naphthoflavone (BNF; 50 mg/kg). Fourteen days after CD exposure, CYP1A1 protein and activity were measured by Western blot and 7-ethoxyresorufin-O-deethylase activity, respectively. CYP1A1 and the alveolar type II markers, cytokeratins 8/18, were localized and quantified in lung sections by dual immunofluorescence with morphometry. The area of CYP1A1 expression in alveolar septa and alveolar type II cells in response to BNF was reduced by exposure to 20 or 40 mg CD compared with BNF alone. CD exposure significantly inhibited BNF-induced 7-ethoxyresorufin-O-deethylase activity in a dose-responsive manner. By Western blot, induction of CYP1A1 protein by BNF was significantly reduced by 40 mg CD compared with BNF alone. These findings indicate that CD decreases BNF-induced CYP1A1 protein expression and activity in the lung.

Effects of male age on the frequencies of germinal and heritable chromosomal abnormalities in humans and rodents.

OBJECTIVE: To review evidence regarding the effects of male age on germinal and heritable chromosomal abnormalities using available human and rodent studies and to evaluate possible underlying mechanisms. DESIGN: Review of English language-published research using MEDLINE database, excluding case reports and anecdotal data. RESULT(S): There was little evidence from offspring or germ cell studies for a generalized male age effect on autosomal aneuploidy, except in rodents. Sex chromosomal nondisjunction increased with age in both human and rodent male germ cells. Both human and rodent data showed age-related increases in the number of sperm with chromosomal breaks and fragments and suggest that postmeiotic cells are particularly vulnerable to the effects of aging. Translocation frequencies increased with age in murine spermatocytes, at rates comparable to mouse and human somatic cells. Age-related mechanisms of induction may include accumulation of environmental damage, reduced efficiency of DNA repair, increased genomic instability, genetic factors, hormonal influences, suppressed apoptosis, or decreased effectiveness of antioxidants and micronutrients. CONCLUSION(S): The weight of evidence suggests that the increasing trend toward fathering at older ages may have significant effects on the viability and genetic health of human pregnancies and offspring, primarily as a result of structural chromosomal aberrations in sperm.

Metallothionein isoform 1 and 2 gene expression in a human urothelial cell line (UROtsa) exposed to CdCl2 and NaAsO2.

Studies have shown that metallothionein (MT) is overexpressed in some human bladder cancers and that overexpression can predict treatment response to neoadjuvant cisplatin, methotrexate, and vinblastine chemotherapy. In the present study the UROtsa cell line, a model of normal human urothelium, was used to determine the expression of the human MT-1 and MT-2 genes and MT protein following exposure to CdCl(2) or NaAsO(2) at lethal and sublethal levels. Acute exposure was modeled by treating confluent cultures with 100 microM NaAsO(2) or 53.4 microM CdCl(2) for 4 h followed by a 48-h recovery period. Extended exposure was modeled by treating confluent cells with 1, 4, and 8 microM As(3+) or 1, 5, and 9 microM Cd(2+) for 16 d, with the highest concentrations producing cell lethality. The expression of MT mRNAs and protein were determined by reverse-transcription polymerase chain reaction (RT-PCR) and immunoblot analysis. Cell viability was determined by the MTT assay. It was shown that acute exposure to either As(3+) or Cd(2+) increased the levels of mRNAs for the MT-1E, MT-1X, and MT-2A genes, whereas extended exposure only increased these mRNAs following exposure to Cd(2+). It was shown that both acute and extended exposure to either As(3+) or Cd(2+) increased the levels of MT protein, reaching a maximal value of 8 ng MT protein/microg total protein for acute exposure to Cd(2+). This is in contrast to previous studies using cultured human proximal tubule cells, where similar extended treatment with Cd(2+) resulted in over 20-fold higher MT protein levels. These studies demonstrate that human urothelial cells accumulate only modest amounts of MT protein when exposed to either Cd(2+) or As(2+) for a 16-d time period.

Stable transfection and overexpression of metallothionein isoform 3 inhibits the growth of MCF-7 and Hs578T cells but not that of T-47D or MDA-MB-231 cells.

The third isoform of metallothionein (MT-3) is overexpressed in some breast cancers and its expression is associated with a poor disease outcome. In the PC-3 prostate cancer cell line, MT-3 expression has been shown to inhibit cell growth and increase drug resistance. The goal of the present study was to determine if MT-3 overexpression would influence the growth of human breast cancer cell lines. To determine this, the coding sequence of the MT-3 gene was stably transfected into two estrogen receptor positive (MCF-7 and T-47D) and two estrogen receptor negative cell lines (Hs578T and MDA-MB-231) having no basal expression of MT-3. Cell growth was determined by counting DAPI-stained nuclei, cadmium resistance by the colony formation assay, MT mRNA expression by RT-PCR, and MT protein by immuno-blot. It was demonstrated that MCF-7 and Hs578T cells that overexpress the MT-3 gene were growth inhibited compared to untransfected cells. In contrast, T-47D and MDA-MB-231 cells that overexpress MT-3 were not growth inhibited. Stable transfection of the MT-1E gene had no effect on the growth of any of the four cell lines. It was also demonstrated that the overexpression of both MT-3 and MT-1E only increased the resistance of MCF-7 cells to Cd(+2). In all instances, stable transfection of the MT-3 or MT-1E gene had no effect on the expression of the other MT isoforms. The study shows that MT-3 can influence the growth of some breast cancer cell lines.

Expression of hsp 27, hsp 60, hsc 70, and hsp 70 stress response genes in cultured human urothelial cells (UROtsa) exposed to lethal and sublethal concentrations of sodium arsenite.

The stress response is one mechanism that the bladder urothelium could potentially employ to protect itself from cellular damage after exposure to arsenic and, in so doing, influence the shape of the dose-response curve at low concentrations of exposure to this environmental pollutant. In the present study, we used the cultured human urothelial cell line UROtsa, a model of human urothelium, to determine the expression of heat shock proteins hsp 27, hsp 60, hsc 70, and hsp 70 after acute and extended exposure of the cells to lethal and sublethal levels of sodium arsenite (NaAsO2). Acute exposure was modeled by exposing confluent cultures of UROtsa cells to 100 micro M NaAsO2 for 4 hr followed by a 48-hr recovery period. Extended exposure was modeled by exposing confluent UROtsa cells to 1, 4, and 8 micro M NaAsO2 for 16 days, with the highest concentration producing cell death by 4 days of exposure. The expression of hsp 27, hsp 60, hsc 70, and hsp 70 mRNA and protein was determined by reverse-transcription polymerase chain reaction and Western analysis. Cell viability was determined by the MTT [(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The results demonstrated that the expression of hsp 27, hsp 60, and hsc 70 mRNA and protein were not consistently increased by either acute or extended exposure to NaAsO2. In contrast, hsp 70 expression was induced by NaAsO2 after both acute and extended exposure. The degree and duration of the induction of the hsp 70 protein in the extended time course of exposure to NaAsO2 correlated directly with UROtsa cell cytotoxicity. The substantial level of basal expression of hsp 27, hsp 60, and hsc 70 shown previously in human bladder urothelium, coupled with the inducible expression of hsp 70, could provide the human urothelium with a mechanism to withstand and recover from a low level of arsenite exposure.

DNA damage assessment by comet assay of human lymphocytes exposed to jet propulsion fuels.

Exposure to jet fuel damages DNA and results in a number of physiological changes in liver, lung, immune, and neurological tissue. In this study the single-cell gel electrophoresis assay or comet assay was used to compare the DNA damage in human peripheral lymphocytes produced by three jet propulsion fuels: JP-8, JP-5, and JP-8+100. These fuels consist of complex mixtures of aliphatic, aromatic, and substituted naphthalene hydrocarbons. Two exposure times were investigated which correspond to estimated occupational exposure times and concentrations of fuels were used that were based on previous fuel toxicity studies. Analysis of samples for the extent of DNA damage as determined by tail moment and percent tail DNA was performed on exposed cells following a brief recovery time. All fuels produced significant increases in DNA damage; however, only JP-8+100 was genotoxic at the lowest exposure concentration (1:500). At the highest exposure concentration (1:75), the mean tail moments for JP-8 and JP-8+100 (32.041 +/- 2.599 and 45.774 +/- 4.743, respectively) were significantly greater than for JP-5 (1.314 +/- 0.474). These results indicate that JP-8+100 is the most potent inducer of DNA damage in human peripheral lymphocytes and that both JP-8+100 and JP-8 are capable of damaging lymphocyte DNA to a greater extent than JP-5.

CENP-B is not critical for meiotic chromosome segregation in male mice.

Centromere protein B (CENP-B) is a constitutive protein that binds to a highly conserved 17bp motif located at most mammalian centromeres. To determine whether disruption of this gene affects chromosome segregation in male germ cells, we evaluated the frequencies of disomic and diploid sperm in CENP-B heterozygous and homozygous null mice using the mouse epididymal sperm aneuploidy (m-ESA) assay, a multicolor FISH method with probes for chromosomes X, Y and 8. The specificity and sensitivity of the m-ESA assay was demonstrated using Robertsonian (2.8) translocation heterozygotes as positive controls for sperm aneuploidy. Our results show that the frequencies of disomic and diploid sperm did not differ significantly between CENP-B heterozygous and homozygous null mice (P> or = 0.5) or from 129/Swiss isogenic mice (P> or = 0.5) and B6C3F1 mice (P> or = 0.2). These findings indicate that CENP-B does not have an essential role during chromosome segregation in male meiosis.