Transcriptome alterations in maternal and fetal cells induced by tobacco smoke.

OBJECTIVES: Maternal smoking has a negative effect on all stages of pregnancy. Tobacco smoke-related defects are well established at the clinical level; however, less is known about molecular mechanisms underlying these pathologic conditions. We thus performed a comprehensive analysis of transcriptome alterations induced by smoking in maternal and fetal cells. STUDY DESIGN: Samples of peripheral blood (PB), placenta (PL), and cord blood (UCB) were obtained from pregnant smokers (n = 20) and gravidas without significant exposure to tobacco smoke (n = 52). Gene expression profiles were assayed by Illumina Expression Beadchip v3 for analysis of 24,526 transcripts. The quantile method was used for normalization. Differentially expressed genes were analyzed in the Limma package and the P-values were corrected for multiple testing. Unsupervised hierarchical clustering was performed using average linkage and Euclidean distance. The enrichment of deregulated genes in biological processes was analyzed in DAVID database. RESULTS: Comparative analyses defined significant deregulation of 193 genes in PB, 329 genes in PL, and 49 genes in UCB of smokers. The deregulated genes were mainly related to xenobiotic metabolism, oxidative stress, inflammation, immunity, hematopoiesis, and vascularization. Notably, functional annotation of the affected genes identified several deregulated pathways associated with autoimmune diseases in the newborns of smokers. CONCLUSIONS: The study demonstrated maternal smoking causes significant changes in transcriptome of placental and fetal cells that deregulate numerous biological processes important for growth and development of the fetus. An activation of fetal CYP genes showed a limited ability of the placenta to modulate toxic effects of maternal tobacco use.

Effect of maternal tobacco smoke exposure on the placental transcriptome.

Smoking in pregnancy increases a woman's risk of preterm delivery resulting in serious neonatal health problems and chronic lifelong disabilities for the children (e.g., mental retardation, learning problems). To study the effects of tobacco smoke on the placental transcriptome, we performed gene expression profiling on placentas from women exposed to tobacco smoke in pregnancy (N = 12) and from those without significant exposure (N = 64). Gene expression profiles were determined by Illumina HumanRef-8 v2 Expression BeadChips with 18,216 gene probes. Microarray data were normalized by quantile method and filtered for a detection P-value <0.01. Differential gene expression was determined by moderated t-statistic. A linear model was fitted for each gene given a series of arrays using lmFit function. Multiple testing correction was performed using the Benjamini and Hochberg method. Abundant levels of transcripts were found for genes encoding placental hormones (CSH1, CSHL1), pregnancy-specific proteins (PSG3, PSG4, PAPPA), and hemoglobins (HBB, HBG, HBA). Comparative analysis of smokers vs nonsmokers revealed the differential expression of 241 genes (P < 0.05). In smoker cohort, we detected high up-regulation of xenobiotic genes (CYP1A1, CYP1B1, CYB5A, COX412), collagen genes (e.g., COL6A3, COL1A1, COL1A2), coagulation genes (F5, F13A1) as well as thrombosis-related genes (CD36, ADAMTS9, GAS6). In smokers, we identified deregulated genes that show tissue non-specific induction and may be considered as general biomarkers of tobacco smoke exposure. Further, we also found genes specifically deregulated in the exposed placentas. Functional annotation analysis suggested processes and pathways affected by tobacco smoke exposure that may represent molecular mechanisms of smoke-induced placental abnormalities.

Bacterial urinary mutagenicity test for monitoring of exposure to genotoxic compounds: a review.

Testing human urine for mutagenic activity towards bacteria has proven to be a useful means for identifying genotoxic exposure. The review documents the utilization of the urinary mutagenicity test using Salmonella typhimurium indicator strains (Ames test) to monitor populations occupationally or environmentally exposed to genotoxic compounds. Confounding factors, mainly smoking and diet, have to be taken into consideration when interpreting the urinary mutagenicity results. Some methodological improvements in the past few years have increased the sensitivity of the urinary mutagenicity test also for identifying environmental exposure to genotoxins. The test appears to be a valid approach for biological monitoring in the field of preventive medicine.

Study of the genotoxicity of toluene.

Chromosome analysis was conducted for peripheral lymphocytes of 23 printers exposed to toluene concentrations of 590 mg/m3 in a rotary machine workshop and to rotogravure printing inks. The percentages of aberrant cells were 2.30 in the printers and 1.46 in the control group (n = 22) (p < .05). The concentration of hippuric acid in printers was significantly higher than in the control group (p < .01), and the level of blood toluene at the end of the workshift was 0.500 mg/l. The authors also examined rotogravure printing inks-considered a potential source of genotoxic polycyclic aromatic hydrocarbons because they contained carbon black-their use in printing plants, and previous documentation of increased chromosomal aberrations in rotogravure printers. Only milligrams of fluorene and phenanthrene per gram of the printing inks were found; no polycyclic aromatic hydrocarbons with carcinogenic properties were discovered in the inks. The authors used Salmonella typhimurium indicator strains TA 98, TA 100, TA 1537, and YG 1041 in spot tests and indicator strains TA 98 and TA 100 in plate-incorporation assays to determine that there was no bacterial mutagenicity of all four colors of rotogravure inks. Urinary mutagenicity, which was evaluated with a microsuspension assay containing YG 1041 indicator strain both in the presence and absence of metabolic activation, was also studied. No significant difference in bacterial mutagenicity was found between the exposed and control groups. The increased percentage of aberrant cells in printers can be explained by exposure to genotoxicants that are not excreted in urine. Toluene was the most likely cause of the aberration.

Genotoxicity of urban air pollutants in the Czech Republic. Part I. Bacterial mutagenic potencies of organic compounds adsorbed on PM10 particulates.

As part of a long-term program to investigate the impact of air pollution on the health of a population in a polluted region in Northern Bohemia, mutagenicity of extractable organic matter (EOM) from air particles PM10 was investigated by the means of Salmonella typhimurium indicator strains TA98 and YG1041 using the Ames plate incorporation assay. The air samples were collected in both the polluted and the control districts during the summers and winters of 1993-1994. In the polluted district, the collection was repeated during the winter of 1996-1997. The crude extracts from filters pooled according to the locality and the season were fractionated by acid-base partitioning into acid, base, and neutral fractions. The neutral fractions were further fractionated by silica gel column chromatography into five subfractions. The induction of revertants with the crude extracts was higher in winter samples than in summer samples. Both indirect-acting and direct-acting mutagenicity were observed. The indirect mutagenic potency of aromatic subfractions containing polycyclic aromatic hydrocarbons (PAHs) was generally low. The mutagenic potency detected with TA98 was more distinct only in the winter sample 1993-1994 from the polluted area, where the aromatic subfraction accounted for 23% of total mutagenicity. In both strains, the highest direct-acting mutagenicity was found in slightly polar fractions containing nitro-PAHs. The mutagenic potency detected with YG1041 was about two orders of magnitude higher than that detected with TA98. No substantial locational- or time-related variances in the mutagenic potencies of EOM, or in the spectrum of chemical components identified in individual fractions were found. The polluted district, in comparison to the control district, was found to have higher amounts of EOM, carcinogenic PAHs and mutagenicity of air particles (rev/m(3)). The fractionating process, combined with the bacterial mutagenicity test, confirmed that nitro-derivatives are the most important contributors to the bacterial mutagenicity of air particles. However, this study did not fulfill the expectancy to bring substantially new, clear-cut information on the composition and the biological activity of air pollution in both districts.

Mutagenicity monitoring of airborne particulate matter (PM10) in the Czech Republic.

The mutagenic activities associated with inhalable airborne particulate matter (PM10) collected over a year in four towns (Czech Republic) have been determined. The dichloromethane extracts were tested for mutagenicity using the Ames plate incorporation test and the Kado microsuspension test both with Salmonella typhimurium TA98 and its derivative YG1041 tester strains in the presence and absence of S9 mixture. The aim of this study was to assess the suitability of both bacterial mutagenicity tests and to choose the appropriate indicator strain for monitoring purposes. To elucidate the correlation between mutagenicity and polycyclic aromatic hydrocarbons (PAHs), the concentration of PAHs in the air samples were determined by GC/MS. In general, the significant mutagenicity was obtained in organic extracts of all samples, but differences according to the method and tester strain used were observed. In both mutagenicity tests, the extractable organic mass (EOM) exhibited higher mutagenicity in the YG1041 strain (up to 97 rev/microg in the plate incorporation and 568 rev/microg in the microsuspension tests) than those in TA98 (up to 2.2 rev/microg in the plate incorporation and 14.5 rev/microg in the microsuspension tests). In the plate incorporation test, the direct mutagenic activity in YG1041 was on average 60-fold higher and in microsuspension assay 45-fold higher with respect to strain TA98. In the presence of S9 mix, the mutagenic potency in YG1041 declined (P<0.001) in summer, but increased in TA98 (P<0.05) in samples collected during the winter season. The microsuspension assay provided higher mutagenic responses in both tester strains, but in both strains a significant decrease of mutagenic potency was observed in the presence of S9 mix (P<0.001 for YG1041, P<0.05 for TA98 in winter). The mutagenic potencies detected with both indicator strains correlated well (r=0.54 to 0.87) within each mutagenicity test used but not (for TA98) or moderately (r=0.44 to 0. 66 for YG1041) between both of the tests. The mutagenic activity (in rev/m(3)) likewise the concentration of benzo[a]pyrene and sum of carcinogenic PAHs showed seasonal variation with distinctly higher values during winter season. A correlation between the PAH concentrations and the mutagenicity results for the plate incorporation, but not for the microsuspension tests was found. In samples from higher industrial areas, the higher mutagenicity values were obtained in plate incorporation test with TA98 and in both tests with YG1041 in summer season (P<0.05). According to our results, plate incorporation test seems to be more informative than microsuspension assay. For routine ambient air mutagenicity monitoring, the use of YG1041 tester strain without metabolic activation and the plate incorporation test are to be recommended.

Monitoring of human exposure to occupational genotoxicants.

The human exposure to genotoxic agents can be detected by using genetic monitoring procedures which is mainly concerned with markers of exposure and effect. Cytogenetic analysis of human peripheral lymphocytes and urine mutagenicity are routinely used in Hygiene Service of the Czech Republic. The review demonstrated the activity of National Reference laboratory and other laboratories in Hygiene Service of the Czech Republic in the problem dealing with monitoring of population exposure to genotoxic substances. Altogether, 7 regional and 15 district laboratories have been in function. Several thousands of occupationally exposed and control persons have been examined by now. The most followed population at risk were those exposed to cytostatics, polycyclic aromatic hydrocarbons, complex mixtures of chemicals, metals and others. The system of genetic monitoring helped to detect the exposure of population at risk to genotoxic contaminants, to use the obtained data for quantification of exposure and for preventive measures application and to control the efficacy of applied regulatory action.

Urinary cyclophosphamide excretion and chromosomal aberrations in peripheral blood lymphocytes after occupational exposure to antineoplastic agents.

In this study we have compared the results of a method for the detection of cyclophosphamide in urine and the results of analysis of chromosomal aberrations in peripheral blood lymphocytes of four groups of subjects with various exposure statuses. These groups are 17 Dutch and 11 Czech exposed workers (mainly hospital nurses and pharmacy technicians) handling antineoplastic agents and 35 Dutch and 23 Czech controls (nurses, medical doctors, pharmacy and lab technicians) not handling these drugs. The groups were subdivided into smokers and non-smokers because of a confounding effect of smoking. Within the Dutch groups, the percentage of aberrant cells and the number of breaks per cell were increased for smokers compared to non-smokers. The percentage of aberrant cells was increased in Dutch exposed workers in comparison with Dutch control workers. Within the Czech groups the percentage of aberrant cells and the number of breaks per cell were increased in exposed workers in comparison with control workers. However, both Dutch and Czech smokers mainly contributed to the increase. The results suggest an additive effect of exposure and smoking in the Dutch subjects and a more than additive effect in the Czech subjects. In urine samples of three out of 11 Dutch exposed workers cyclophosphamide was found in a range of 0.1-0.5 micrograms/24 h. Higher levels were detected in the urine of eight out of 11 Czech exposed workers, a range of 0.1-2.9 micrograms/24 h. No correlation was observed between the amounts of cyclophosphamide excreted in urine on the one hand and the percentage of aberrant cells and the number of breaks per cell on the other hand. The present study is the first study showing that hospital workers having an increase in chromosome aberrations related to their work are exposed to at least one antineoplastic agent.

Effect of ascorbic acid prophylaxis on the frequency of chromosome aberrations in the peripheral lymphocytes of coal-tar workers.

The possible anti-mutagenic potential of prophylactically administered ascorbic acid (AA) preparations was studied in a group of 35 coal-tar workers occupationally exposed to PAH and benzene. The effectiveness of AA prophylaxis was assessed by differences in the frequency of chromosome aberrations in peripheral blood lymphocytes of the exposed workers examined before and after a 3-month treatment with AA at the daily doses of 1.0 g for 5 days a week. In the exposed group the cytogenetic analysis of peripheral blood lymphocytes revealed a significant drop in the frequency of aberrant cells (AB.C.) from the initial 5.07% AB.C. (0.0657 B/C, breaks per cell) to 1.77% AB.C. (0.0197 B/C). In a group of matching controls the frequency of AB.C. was 1.50% (0.0170 B/C) and 1.45% (0.0180 B/C), respectively. The study showed that the risk of genetic injury assessed by the frequency of chromosome aberrations in peripheral blood lymphocytes was substantially reduced after AA prophylaxis.