Deliberating Performance Targets: Follow-on workshop discussing PM10, NO2, CO, and SO2 air sensor targets.

The use of air sensor technology is increasing worldwide for a variety of applications, however, with significant variability in data quality. The United States Environmental Protection Agency held a workshop in July 2019 to deliberate possible performance targets for air sensors measuring particles with aerodynamic diameters of 10 µm or less (PM10), nitrogen dioxide (NO2), carbon monoxide (CO), and sulfur dioxide (SO2). These performance targets were discussed from the perspective of non-regulatory applications and with the sensors operating primarily in a stationary mode in outdoor environments. Attendees included representatives from multiple levels of government organizations, sensor developers, environmental nonprofits, international organizations, and academia. The workshop addressed the current lack of sensor technology requirements, discussed fit-for-purpose data quality needs, and debated transparency issues. This paper highlights the purpose and key outcomes of the workshop. While more information on performance and applications of sensors is available than in past years, the performance metrics, or parameters used to describe data quality, vary among the studies reports and there is a need for more clear and consistent approaches for evaluating sensor performance. Organizations worldwide are increasingly considering, or are in the process of developing, sensor performance targets and testing protocols. Workshop participants suggested that these new guidelines are highly desirable, would help improve data quality, and would give users more confidence in their data. Given the wide variety of uses for sensors and user backgrounds, as well as varied sensor design features (e.g., communication approaches, data tools, processing/adjustment algorithms and calibration procedures), the need for transparency was a key workshop theme. Suggestions for increasing transparency included documenting and sharing testing and performance data, detailing best practices, and sharing data processing and correction approaches.

Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes. Involvement of the sphingolipid signaling cascade in cardiac cell death.

In the present study, it was shown that physiologically relevant levels of the proinflammatory cytokine TNFalpha induced apoptosis in rat cardiomyocytes in vitro, as quantified by single cell microgel electrophoresis of nuclei ("cardiac comets") as well as by morphological and biochemical criteria. It was also shown that TNFalpha stimulated production of the endogenous second messenger, sphingosine, suggesting sphingolipid involvement in TNFalpha-mediated cardiomyocyte apoptosis. Consistent with this hypothesis, sphingosine strongly induced cardiomyocyte apoptosis. The ability of the appropriate stimulus to drive cardiomyocytes into apoptosis indicated that these cells were primed for apoptosis and were susceptible to clinically relevant apoptotic triggers, such as TNFalpha. These findings suggest that the elevated TNFalpha levels seen in a variety of clinical conditions, including sepsis and ischemic myocardial disorders, may contribute to TNFalpha-induced cardiac cell death. Cardiomyocyte apoptosis is also discussed in terms of its potential beneficial role in limiting the area of cardiac cell involvement as a consequence of myocardial infarction, viral infection, and primary cardiac tumors.

Interchromosomal gene conversion at an endogenous human cell locus.

To examine the relationship between gene conversion and reciprocal exchange at an endogenous chromosomal locus, we developed a reversion assay in a thymidine kinase deficient mutant, TX545, derived from the human lymphoblastoid cell line TK6. Selectable revertants of TX545 can be generated through interchromosomal gene conversion at the site of inactivating mutations on each tk allele or by reciprocal exchange that alters the linkage relationships of inactivating polymorphisms within the tk locus. Analysis of loss of heterozygosity (LOH) at intragenic polymorphisms and flanking microsatellite markers was used to initially evaluate allelotypes in TK(+) revertants for patterns associated with either gene conversion or crossing over. The linkage pattern in a subset of convertants was then unambiguously established, even in the event of prereplicative recombinational exchanges, by haplotype analysis of flanking microsatellite loci in tk(-/-) LOH mutants collected from the tk(+/-) parental convertant. Some (7/38; 18%) revertants were attributable to easily discriminated nonrecombinational mechanisms, including suppressor mutations within the tk coding sequence. However, all revertants classified as a recombinational event (28/38; 74%) were attributed to localized gene conversion, representing a highly significant preference (P < 0.0001) over gene conversion with associated reciprocal exchange, which was never observed.

Cytotoxicity of xanthopterin and isoxanthopterin in MCF-7 cells.

Human mammary carcinoma MCF-7 cell line responsiveness to the pteridines xanthopterin and isoxanthopterin was studied using the MTS assay for measurement of cell viability. The pteridines were tested at concentrations ranging from 7.8 to 500 microM singly and in 11 isoxanthopterin:xanthopterin ratios. IC50s of xanthopterin and isoxanthopterin were 109+/-13 microM (mean+/-SEM of y estimate) and 103+/-9 microM, respectively. The IC50 values for pteridine mixtures were similar although 3:1 and 4:1 isoxanthopterin:xanthopterin ratios seemed slightly more cytotoxic than other mixtures. However, ANOVA revealed no statistical differences in the cytotoxicity of mixtures.

Use of the glycophorin A human mutation assay to study workers exposed to styrene.

The glycophorin A (GPA) assay is a human mutation assay that is potentially useful for large epidemiological studies. The assay is rapid and requires a minimal amount of blood, which can be stored before analysis. The data presented here were collected from workers exposed to styrene in a boat manufacturing plant. This study was the first to apply the GPA assay to an occupationally exposed population. Subjects with a mean styrene exposure of 30 ppm had a higher frequency of GPA N phi variant cells than subjects with mean exposure of 1 ppm, but the subjects differed in respect to smoking and age distribution. Results indicate that the original 1-W-1 version of the assay may not be suitable for studies of small numbers of exposed subjects due to variability and artifacts. The newer BR6 version, however, has much lower variability and shows promise for use in the occupational setting.

An epidemiologic study of early biologic effects of benzene in Chinese workers.

Benzene is a recognized hematotoxin and leukemogen, but its mechanisms of action in humans are still uncertain. To provide insight into these processes, we carried out a cross-sectional study of 44 healthy workers currently exposed to benzene (median 8-hr time-weighted average; 31 ppm), and unexposed controls in Shanghai, China. Here we provide an overview of the study results on peripheral blood cells levels and somatic cell mutation frequency measured by the glycophorin A (GPA) gene loss assay and report on peripheral cytokine levels. All peripheral blood cells levels (i.e., total white blood cells, absolute lymphocyte count, platelets, red blood cells, and hemoglobin) were decreased among exposed workers compared to controls, with the exception of the red blood cell mean corpuscular volume, which was higher among exposed subjects. In contrast, peripheral cytokine levels (interleukin-3, interleukin-6, erythropoietin, granulocyte colony-stimulating factor, tissue necrosis factor-alpha) in a subset of the most highly exposed workers (n = 11) were similar to values in controls (n = 11), suggesting that benzene does not affect these growth factor levels in peripheral blood. The GPA assay measures stem cell or precursor erythroid cell mutations expressed in peripheral red blood cells of MN heterozygous subjects, identifying NN variants, which result from loss of the GPA M allele and duplication of the N allele, and N phi variants, which arise from gene inactivation. The NN (but not N phi) GPA variant cell frequency was elevated in the exposed workers compared with controls (mean +/- SD, 13.9 +/- 8.4 mutants per million cells versus 7.4 +/- 5.2 per million cells, (respectively; p = 0.0002), suggesting that benzene produces gene-duplicating but not gene-inactivating mutations at the GPA locus in bone marrow cells of exposed humans. These findings, combined with ongoing analyses of benzene macromolecular adducts and chromosomal aberrations, will provide an opportunity to comprehensively evaluate a wide range of early biologic effects associated with benzene exposure in humans.

Genetic toxicity of malathion: a review.

Mammalian in vivo and in vitro studies of technical or commercial grade malathion and its metabolite malaoxon show a pattern of induction of chromosome damage, as measured by chromosome aberrations, sister chromatid exchanges, and micronuclei. Experiments with purified (> 99%) malathion gave weak or negative results. In contrast to the cytogenetic effects of technical grade malathion, responses in gene mutation assays were generally negative except for malaoxon, which was positive for mammalian gene mutations in both tested instances. This result also could be a consequence of chromosome level changes, however. Dermal exposure, a common human route, caused cytogenetic damage in test animals at doses near those producing positive results by intraperitoneal injection. Workers who apply technical grade malathion and other pesticides have higher levels of chromosomal damage than unexposed individuals. Because of the inactivity of malathion mixtures in gene mutation assays, malathion has been thought to be of little genotoxic concern. However, the pattern of chromosome damage in animals and mammalian cells in culture (including human) indicates that technical grade malathion and its components have not been adequately studied for genotoxic potential in humans.

Gossypol-induced DNA breaks in rat lymphocytes are secondary to cytotoxicity.

Gossypol, a male antifertility and potential anticancer agent, was found to induce DNA strand breaks in rat lymphocytes. DNA breaks were measured with the single-cell gel electrophoresis (SCGE) or 'comet' assay. A significant increase in DNA breaks was observed after 1 h incubation at concentrations of 2 microg/ml or greater. The inclusion of 10% fetal bovine serum in the media reduced the toxicity of gossypol, and DNA breaks were only observed at a concentration of 80 microg/ml. However, the increase in DNA strand breaks, for incubations with and without serum, only occurred when cell viability was reduced to less than 70%. Examination of cell morphology and DNA fragmentation at incubations up to 5 h yielded no evidence that DNA strand breaks were occurring due to apoptosis. We conclude that gossypol is not primarily genotoxic in this cell type, and that the DNA breaks observed arose secondary to cytotoxicity.

Quantification of epithelial cell micronuclei by fluorescence in situ hybridization (FISH) in mortuary science students exposed to formaldehyde.

A micronucleus assay employing fluorescence in situ hybridization (FISH) with a centromeric probe was used on specimens of exfoliated buccal and nasal cells collected from mortuary science students exposed to embalming fluid containing formaldehyde. FISH labeling allowed micronuclei (MN) containing a whole chromosome (centromere-positive, MN+) to be differentiated from those containing only chromosomal fragments (centromere-negative, MN-). Each student was sampled before and after the 90 day embalming class. We determined if an increase in MN frequency could be attributed to formaldehyde exposure and was specific to either MN+ or MN-. In buccal cells, total MN frequency was significantly increased from 0.6/1000 to 2/1000 (p = 0.007) following the course, whereas in nasal cells it was not (2 and 2.5/1000, respectively, p = 0.2). Cells with multiple MN were present only in samples taken after exposure to embalming fluid. Although the baseline frequency was higher for MN+ in both buccal (0.4/1000 for MN+ and 0.1/1000 for MN-) and nasal cells (1.2/1000 for MN+ and 0.5/1000 for MN-), the increase in MN frequency was greater for MN-, (9-fold, p = 0.005 for buccal cells; 2-fold, p = 0.03 for nasal cells) than for MN+ (> 2-fold, p = 0.08 for buccal cells; no change, p = 0.31 for nasal cells) in both tissues. Thus, the primary mechanism of micronucleus formation appeared to be chromosome breakage. This finding is consistent with known clastogenic properties of formaldehyde, the component of embalming fluid most likely responsible for micronucleus induction.

Evaluation of a real-time passive personal particle monitor in fixed site residential indoor and ambient measurements.

Recent experimental findings in animals and humans indicate adverse respiratory effects from short-term exposures to particulate air pollutants, especially in sensitive subpopulations such as asthmatics. The relationship between air pollution and asthma has mainly been determined using particulate matter (PM) measurements from central sites. Validated tools are needed to assess exposures most relevant to health effects. Recently, a personal passive particulate sampler (personal Data-RAM, pDR, MIE Inc., Bedford, MA) has become available for studying personal exposures to PM with time resolution at 1 min. The pDR measures light scatter from PM in the 0.1-10 microM range, the significant range for health effects. In order to assess the ability of the pDR in predicting gravimetric mass, pDRs were collocated with PM2.5 and PM10 Harvard Impactors (HI) inside and outside nine homes of asthmatic children and at an outdoor central Air Pollution Control District site. Results are presented of comparisons between the HI samplers and the pDR in various modes of operation: passive, active, and active with a heated inlet. When used outdoors at fixed sites the pDR readings exhibit interference from high relative humidity (RH) unless operated with a method for drying inlet air such as a heater, or if readings at times of high RH are adjusted. The pDR correlates more highly with the HI PM2.5 than with the HI PM10 (r2 = 0.66 vs. 0.13 for outdoors, r2 = 0.42 vs. 0.20 for indoors). The pDR appears to be a useful tool for an epidemiologic study that aims to examine the relationship between health outcomes and personal exposure to peaks in PM.

Households contaminated by environmental tobacco smoke: sources of infant exposures.

OBJECTIVES: To examine (1) whether dust and surfaces in households of smokers are contaminated with environmental tobacco smoke (ETS); (2) whether smoking parents can protect their infants by smoking outside and away from the infant; and (3) whether contaminated dust, surfaces, and air contribute to ETS exposure in infants. DESIGN: Quasi-experiment comparing three types of households with infants: (1) non-smokers who believe they have protected their children from ETS; (2) smokers who believe they have protected their children from ETS; (3) smokers who expose their children to ETS. SETTING: Homes of smokers and non-smokers. PARTICIPANTS: Smoking and non-smoking mothers and their infants < or = 1 year. MAIN OUTCOME MEASURES: ETS contamination as measured by nicotine in household dust, indoor air, and household surfaces. ETS exposure as measured by cotinine levels in infant urine. RESULTS: ETS contamination and ETS exposure were 5-7 times higher in households of smokers trying to protect their infants by smoking outdoors than in households of non-smokers. ETS contamination and exposure were 3-8 times higher in households of smokers who exposed their infants to ETS by smoking indoors than in households of smokers trying to protect their children by smoking outdoors. CONCLUSIONS: Dust and surfaces in homes of smokers are contaminated with ETS. Infants of smokers are at risk of ETS exposure in their homes through dust, surfaces, and air. Smoking outside the home and away from the infant reduces but does not completely protect a smoker's home from ETS contamination and a smoker's infant from ETS exposure.

Pharmacokinetic model of dioxin and furan levels in adipose tissue from sawmill work involving chlorophenate fungicides.

Sawmill workers in British Columbia (B.C.), Canada, have been exposed to chlorophenate fungicides which are known to be contaminated with polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Due to concern about the potential of these workers to have significant body burdens of PCDD/Fs, and the absence of measurements in these workers, a single-compartment pharmacokinetic model was developed to estimate the concentration of PCDD/Fs in the fat tissue of the sawmill workers. Data from a large cohort of B.C. sawmill workers and literature-based data on chlorophenate exposures and PCDD/F concentrations in chlorophenates were used in Monte Carlo simulations to predict a PCDD/F body burden distribution. The median concentrations of HxCDF and HpCDF predicted using the model for the B.C. sawmill worker population exceeded the range measured in unexposed populations. PeCDF and OCDF concentrations exceeded the range measured in unexposed populations at the 70th percentile of the model-predicted distribution, and PeCDD at the 90th percentile. The primary limitation of the model was the scarcity of input data about actual dermal and inhalation exposures to chlorophenates.

Evaluation of urinary trans-3'-hydroxycotinine as a biomarker of children's environmental tobacco smoke exposure.

The utility of urinary trans-3'-hydroxy cotinine (3HC) as a biomarker of environmental tobacco smoke (ETS) exposure was investigated in comparison with urinary cotinine (COT), the sum (3HC + COT), and ratio of the two nicotine metabolites (3HC/COT). Participants were 150 ETS exposed children (aged 1-44 months) and their parents. Child urine samples were collected during 3weekly baseline assessments and at interviews administered 3, 6, 12, and 18 months after baseline. Findings indicate that 3HC and COT can be measured reliably (rho = 0.96, 0.88) and show equivalent levels of repeated measures stability (rho = 0.71, 0.75). COT, 3HC, and 3HC + COT showed equally strong associations with air nicotine levels, reported ETS contamination, and reported ETS exposure (r=0.60-0.70). The intraclass correlations of 3HC/COT were lower than those for COT or 3HC. Older children had a higher 3HC/COT ratio than younger children (3.5 versus 2.2), and non-Hispanic White children had a higher ratio than African-American children (3.2 versus 1.9). These findings suggest that COT, 3HC, and 3HC + COT are approximately equivalent and equally strong biomarkers of ETS exposure in children. Moreover, 3HC/COT may provide a useful indicator to investigate age- and race-related differences in the metabolism of COT and 3HC.

Novel biomarkers of genetic damage in humans: use of fluorescence in situ hybridization to detect aneuploidy and micronuclei in exfoliated cells.

Here we describe new techniques that employ fluorescence in situ hybridization (FISH) with centromeric and chromosome-specific DNA probes to detect aneuploidy and micronucleus formation in exfoliated human epithelial cells. Micronuclei arise from chromosome breakage or lagging, which results in chromosome fragments and whole chromosomes being left outside of the main nucleus at telophase. By using a centromeric DNA probe to detect the presence of whole chromosomes in micronuclei and propidium iodide as a general DNA stain in exfoliated nasal, buccal, and bladder cells, we have developed a new fluorescent method that can detect micronuclei and determine the mechanism of formation. The new fluorescent technique gave results that were very similar to those obtained with the standard Feulgen-fast green method. The spontaneous levels of micronuclei in healthy volunteers were buccal, 0.13%, nasal, 0.21%, and urothelial, 0.07%, in approximately 1500 cells per data point. These values are lower than that found in cultured lymphocytes, 0.4-0.8%. Approximately 50% of the exfoliated cell micronuclei contained whole chromosomes (centromeric DNA). FISH was also used to detect aneuploidy in exfoliated buccal and bladder cells. A DNA probe specific for chromosome 9 was used. Average frequencies for 0, 1, 2, 3, and 4 hybridization regions were 4.8, 9.3, 84.8, 0.8, and 0.3% for urothelial cells and 8.2, 9.9, 80.1, 1.4, and 0.4% for buccal cells. The estimated frequency of aneuploidy in exfoliated cells is similar to that found in human lymphocytes analyzed by FISH with the same probe for chromosome 9. These techniques are potentially useful for epidemiological studies of exposed populations and are currently being applied in our laboratory for studies of arsenic- and formaldehyde-exposed populations.

Distribution of DRB1 and DQB1 HLA class II alleles in occupational asthma due to western red cedar.

Occupational asthma caused by western red cedar is a common problem in sawmill industries. The objective of this study was to examine a possible association of human leukocyte antigen (HLA) class II genetic markers with susceptibility or resistance to western red cedar induced asthma. The distribution of DRB1 and DQB1 HLA class II alleles and DRB1-DQB1 haplotypes was studied in 56 Caucasian patients with proven red cedar asthma and 63 healthy Caucasian control subjects exposed to red cedar dust. DRB1 and DQB1 high resolution typing was performed by the polymerase chain reaction-based method. Patients with red cedar asthma had a higher frequency of HLA DQB1*0603 and DQB1*0302 alleles compared to a group of healthy exposed control subjects and a reduced frequency of DQB1*0501 allele. The frequency of the DRB1*0401-DQB1* 0302 haplotype was increased and the DRB1*0101-DQB1*0501 haplotype was reduced. These findings suggest that genetic factors such as human leukocyte antigen class II antigens may be associated with susceptibility or resistance to development of red cedar asthma.

Monitoring of 1-min personal particulate matter exposures in relation to voice-recorded time-activity data.

Recent studies on the association between exposures to airborne particulate matter (PM) and disease have identified short-term peaks in PM exposures as posing especial health threats. Lightweight personal instruments are needed to characterize short-term exposures to PM and to identify the most important sources of high PM excursions. In this study, we measured exposure to fine PM using a small personal nephelometer (pDR; MIE, Inc) to investigate the utility of this instrument in identifying activities and microenvironments most associated with high PM exposures and the magnitude and duration of peaks in PM exposures. Ten adult volunteers wore a pDR recording PM concentrations at 1-min time intervals for 1 week each. PM concentrations were measured by the pDR in units of microg/m(3) based on light scatter. The use of a time-stamped voice recorder enabled activity and location to be continuously documented in real time. In addition, a small, inexpensive light intensity logger was affixed to the pDR to evaluate the potential of this instrument to assist in verifying wearer- recorded data. For each person, patterns of PM exposure were remarkably consistent over daily activities and showed large excursions associated with specific indoor and outdoor microenvironments and activities, such as cooking. When the magnitude and duration of excursions in PM were analyzed, we found that high PM levels occurred in relatively few of the minutes measured but comprised a substantial fraction of the total exposure to PM. Fifteen-minute averaged PM levels were found to be as much as 10 times the daily average. When the data were analyzed with a generalized estimating equation model to account for effects of autocorrelation and clustering, PM exposure was significantly higher during subject-reported events including barbeque, yard work, being near pets or construction activities, cooking, and environmental tobacco smoke exposure, as compared with periods with no pollution events. When light intensity data were explored to determine whether these loggers could be of potential use in establishing or verifying indoor vs outdoor location for future PM studies, we found that personal light intensity measurements differed among indoor, outdoor, and in-car environments (P<0.001). Overlap between measured values implies that light intensity cannot be used to absolutely predict location; however, a sudden increase or decrease in light intensity was highly associated with participant report of location change between indoors and outdoors. This study demonstrates the utility of the pDR in identifying patterns of personal exposures to particulate matter and especially in registering the magnitude and duration of excursions in PM in relation to location and activity.

Sensitivity analysis of a new model of carcinogenesis.

A new simulation model of carcinogenesis is described which, in addition to the features of a standard clonal two-stage model (loss of both copies of a tumor suppressor gene by point mutations, cell division and cell death), includes a quantitative description of mitotic recombination, DNA repair, and cell to cell interactions in all stages. The model is implemented as a discrete event process. The results of a sensitivity analysis of the model are presented. The most sensitive parameters were found to be: the number of normal cells at risk, and the division rate, death rate and DNA repair efficiency for the intermediate stage cells. Accurate information about these parameters is important for a quantitative understanding of carcinogenesis. The sensitivity of the model to the number of normal cells indicates the importance of understanding the nature of the cells at risk, for example, stem cells vs. differentiated cells. The model can be used to assess the importance of chromosomal damage such as mitotic recombination and epigenetic mechanisms such as hyperplasia and cytotoxicity in the onset of malignant tumors.

Compliance with federal and state legislation by indoor tanning facilities in San Diego.

BACKGROUND: The prevalence rates of both skin cancers and indoor tanning among the US population are high and have increased substantially in recent years. Low compliance by indoor tanning facilities with safety regulations may place consumers at greater risk of skin and ocular damage. OBJECTIVE: This study quantified the level of compliance by indoor tanning facilities with selected federal and state regulations and recommendations. METHODS: Tanning facilities (N = 54) in San Diego County, California, were visited by a confederate posing as a prospective customer. Compliance with 13 regulations/recommendations were assessed by either direct query or observation of the presence/absence of signs and warning labels. Operators' responses to 5 risk-based questions also were noted. RESULTS: No facility complied with all 13 regulations/recommendations. Compliance with 3 protective eyewear regulations was high (89%-100%). In contrast, compliance with maximum tanning frequency recommendations (approximately 6%) and parental consent regulations (approximately 43%) was disturbingly low. CONCLUSION: The investigators recommend instituting mandatory, comprehensive training for operators, as well as systematic compliance monitoring with enforcement of penalties for noncompliance.

Benzene induces gene-duplicating but not gene-inactivating mutations at the glycophorin A locus in exposed humans.

Occupational exposure to benzene is known to cause leukemia, but the mechanism remains unclear. Unlike most other carcinogens, benzene and its metabolites are weakly or nonmutagenic in most simple gene mutation assays. Benzene and its metabolites do, however, produce chromosomal damage in a variety of systems. Here, we have used the glycophorin A (GPA) gene loss mutation assay to evaluate the nature of DNA damage produced by benzene in 24 workers heavily exposed to benzene and 23 matched control individuals in Shanghai, China. The GPA assay identifies stem cell or precursor erythroid cell mutations expressed in peripheral erythrocytes of MN-heterozygous subjects, distinguishing the NN and N phi mutant variants. A significant increase in the NN GPA variant cell frequency (Vf) was found in benzene-exposed workers as compared with unexposed control individuals (mean +/- SEM, 13.9 +/- 1.7 per million cells vs. 7.4 +/- 1.1 per million cells in control individuals; P = 0.0002). In contrast, no significant difference existed between the two groups for the N phi Vf (9.1 +/- 0.9 vs. 8.8 +/- 1.8 per million cells; P = 0.21). Further, lifetime cumulative occupational exposure to benzene was associated with the NN Vf (P = 0.005) but not with the N phi Vf (P = 0.31), suggesting that NN mutations occur in longer-lived bone marrow stem cells. NN variants result from loss of the GPA M allele and duplication of the N allele, presumably through recombination mechanisms, whereas NO variants arise from gene inactivation, presumably due to point mutations and deletions. Thus, these results suggest that benzene produces gene-duplicating mutations but does not produce gene-inactivating mutations at the GPA locus in bone marrow cells of humans exposed to high benzene levels. This finding is consistent with data on the genetic toxicology of benzene and its metabolites and adds further weight to the hypothesis that chromosome damage and mitotic recombination are important in benzene-induced leukemia.

LPS-induced TNF-alpha release from and apoptosis in rat cardiomyocytes: obligatory role for CD14 in mediating the LPS response.

The bacterial endotoxin lipopolysaccharide (LPS) contributes to the cardiovascular collapse and death observed in patients with sepsis. Because LPS has such profound effects on cardiac performance, we speculate that direct effects of LPS could be demonstrated on cardiomyocytes in culture, and that these direct effects are mediated by the LPS receptor, CD14. Accordingly, in this study, we provide evidence for CD14-dependent cardiotoxic effects of LPS including the LPS-stimulated secretion of tumor necrosis factor alpha (TNF-alpha) from cardiomyocytes. TNF-alpha is an inflammatory cytokine which is known for its negative inotropic effects on cardiac performance, but has not until recently been shown to be produced by cardiac cells. In this study, LPS was found to stimulate strongly in a dose-dependent manner the secretion of TNF-alpha from cultured adult rat cardiomyocytes. Further, LPS-induced TNF-alpha secretion was blocked by an inhibitor of TNF-alpha processing, metallomatrix protease inhibitor (TAPI). Molecular and immunological evidence demonstrated the presence of LPS receptors (CD14) on cardiomyocytes. Attenuated TNF-alpha secretion following PI-PLC treatment confirmed the functional importance of CD14 for LPS-mediated myocardial effects. Importantly, LPS also triggered apoptosis in cultured cardiomyocytes as quantified by single-cell gel electrophoresis of nuclei exhibiting DNA fragmentation patterns characteristic of apoptosis (i.e. cardiac comets). Apoptotic cell death was blocked by pre-incubation with the soluble TNF-alpha receptor fragment (TNFRII:Fc), suggesting that LPS-induced apoptosis was TNF-alpha-dependent and probably involved an autocrine function for the TNF-alpha whose secretion was under LPS control. The results of this study suggest that the cardiodepressant effects of LPS are dependent on CD14 signaling and may not only be due to acute negative inotropic effects of TNF-alpha but also may be complicated by TNF-alpha-induced apoptotic cell death which effectively reduces the number of working myocardial cells.