Validation and Evaluation of Biomarkers in Workers Exposed to Benzene in China

EPA Grant Number: R828112C115
Subproject: this is subproject number 115 , established and managed by the Center Director under grant R828112
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Health Effects Institute (2000 — 2005)
Center Director: Greenbaum, Daniel S.
Title: Validation and Evaluation of Biomarkers in Workers Exposed to Benzene in China
Investigators: Qu, Qingshan
Institution: New York University Medical Center , Health Effects Institute
EPA Project Officer: Chung, Serena
Project Period: April 1, 2000 through March 31, 2005
RFA: Health Effects Institute (1996) RFA Text |  Recipients Lists
Research Category: Human Health , Air Quality and Air Toxics , Air


Human exposure to benzene is widespread because it is a component of gasoline and is also used extensively as an industrial solvent. Exposure to high levels of benzene is associated with development of leukemia and other blood disorders, but the risks of exposure to low levels of benzene are not well understood. In the 1990s the Health Effects Institute initiated a research program designed to study the effects of exposure to toxic air pollutants at ambient levels. As one part of this research program, HEI’s Request for Applications (RFA) 93-1 supported studies to develop reliable and sensitive assays for biomarkers of benzene exposure—both recent and longer-term—and of benzene effect. The biomarkers of recent exposure were urinary metabolites (measuring responses up to hours after exposure) and adducts of blood proteins (days to weeks after exposure). The biomarkers of longer-term exposure were chromosomal changes, integrating exposure over months to years. Because chromosomal changes may be determinants of subsequent health effects, they may also be considered early biomarkers of benzene effect. Chromosomal changes may also be due to causes other than exposure to benzene.

To validate the biomarkers characterized in these studies, another part of the research program, Request for Qualifications (RFQ) 95-3, “Transitional Epidemiology Studies for Benzene or 1,3-Butadiene Biomarkers,” solicited applications from investigators with access to suitable human populations exposed to benzene or butadiene. HEI funded a study by Dr Qingshan Qu of New York University School of Medicine to evaluate putative biomarkers in workers occupationally exposed to benzene in China.


Qu and colleagues recruited 181 healthy workers in several factories in the Tianjin region of China. These subjects formed part of a cohort of thousands identified by the US National Cancer Institute (NCI) and the China Academy of Preventive Medicine for a study to evaluate tumor incidence in benzene exposed workers (NCI/China study). In phase 1 of their study, Qu and colleagues evaluated the suitability of using urinary metabolites, blood adducts, or chromosomal aberrations in polymorphonuclear leukocytes and lymphocytes as benzene biomarkers in 25 heavily exposed and 25 unexposed workers. The urinary metabolites measured were phenol, catechol, hydroquinone, benzene triol, S-phenylmercapturic acid (S-PMA), and trans,trans-muconic acid (t,t-MA). The blood adducts measured were benzene oxide and benzoquinone adducts of albumin.

In phase 2, the investigators used biomarkers validated in phase 1 of the study to evaluate relations between benzene exposures and levels of these biomarkers in another 105 benzene-exposed workers and 26 unexposed workers. The investigators focused on obtaining samples from workers whose current day exposures to benzene were no more than 5 ppm, representing the low end of occupational exposure. Qu and colleagues also evaluated whether the number and type of blood cells decreased in the exposed subjects because such decreases may be early indicators of a response to occupational benzene exposure. Some biological samples were analyzed in China and some in the United States.

Expected Results:

This study has made important contributions regarding the utility of biomarkers of benzene exposure in occupational settings. It is the first to evaluate multiple possible biomarkers of benzene across a wide range of exposures and to show effects at the lowest end of the range. In addition to using sensitive assays for urinary metabolites and blood adducts, Qu and colleagues made great efforts to accurately measure and monitor personal exposures to a wide range of benzene levels in the workplace—critical features for assessing the accuracy of biomarker information. The investigators also paid careful attention to quality control issues.

The study’s most novel finding was that benzene exposure was associated with decreases in the numbers of circulating neutrophils and, to a lesser extent, lymphocytes. The decrease in neutrophil numbers is interesting because long-term human exposure to high levels of benzene has been previously associated with the development of cancer in bone marrow precursor cells that give rise to neutrophils. This result—indicating that changes in neutrophil numbers may be a sensitive marker of benzene effects—needs to be corroborated, however, because other studies have found changes in lymphocyte, but not neutrophil, numbers.

A key positive feature of the study design was Qu’s 2-step approach to validating possible biomarkers in phase 1 before proceeding to the larger study in phase 2. The phase 1 results indicated that S-PMA and t,t-MA, minor metabolites of benzene found in urine, might be the most useful markers of recent benzene exposure. Combined analysis of phase 1 and 2 results confirmed the suitability of S-PMA and t,t-MA as biomarkers for this purpose: both markers had low background levels in unexposed workers and increased levels in exposed workers. S-PMA was found to be the most useful biomarker for recent exposure to benzene because of the extent of the change in its level, its sensitivity in correlating with low occupational benzene exposures, and its specificity for benzene exposure. The urinary metabolites phenol, hydroquinone, and catechol were less sensitive to changes in benzene exposure and had higher background levels than S-PMA and t,t-MA. Therefore, these markers were less suitable for detecting dose- dependent variation across the spectrum of benzene exposures. Benzene triol was found to be unsuitable as a biomarker.

Exposure-dependent changes in blood adduct levels (half-life in blood of approximately 14 days) were found to be suitable measures for evaluating recent exposure although the background levels in unexposed workers were quite high.

Using the fluorescence in situ hybridization (FISH) technique to examine specific chromosomes for effects of longer-term benzene exposure, the investigators did not detect differences between the numbers of chromosomal aberrations in exposed and unexposed workers. In contrast, FISH data in the NCI/China study evaluating the same chromosome (chromosome 7) showed increased numbers of aberrations in exposed workers. However, differences in cell culture conditions, probes evaluated, and scoring criteria make it difficult to compare the FISH results between the 2 studies. In addition, the overall frequencies of numerical aberrations (hyperdiploidy) reported in the unexposed control subjects participating in the NCI/China study were unusually high, which complicates comparisons. Although the median exposures of workers in the NCI/China and HEI studies were similar, workers in the NCI study with above-median exposures were exposed to much higher benzene levels than those participating in the HEI study. These higher exposures may have also contributed to the differences between the FISH results of the 2 studies. Using conventional cytogenetic techniques to evaluate all chromosomes, Qu and colleagues found some increases in aberrations in exposed workers compared with controls. These increases were difficult to interpret because they were not linear with recent changes in benzene exposure. However, a more consistent exposure-response relationship was seen when the aberration frequencies were categorized by cumulative benzene exposures.

The investigators evaluated exposure-response effects in the phase 2 subjects combined with the subjects who had been evaluated in phase 1, which was conducted in the previous year. Combined analysis of phase 1 and phase 2 results may have introduced unmeasured confounding because exposures in the 2 phases were measured in different years and at different sites. Further, they used different subjects with much lower exposure levels—by design—in phase 2 than phase 1. Although Qu and colleagues amply addressed many aspects of this issue in the report, the validity of combining data from phases 1 and 2 of the study remains uncertain.

In conclusion, Qu and colleagues’ study has validated several biomarkers. Urinary levels of S-PMA appear to be the most useful measure of exposure to benzene (detecting changes within a few hours). Blood adducts of benzene and albumin may be useful biomarkers of exposure within days to weeks, but background levels of these adducts are quite high in people not exposed to benzene. Finally, the investigators found that changes in neutrophil levels may be a sensitive and early marker of benzene’s toxicity, but further research is needed to confirm this last finding.

Publications and Presentations:

Publications have been submitted on this subproject: View all 5 publications for this subprojectView all 12 publications for this center

Journal Articles:

Journal Articles have been submitted on this subproject: View all 4 journal articles for this subprojectView all 7 journal articles for this center

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Progress and Final Reports:

  • 2000
  • 2001
  • 2002
  • 2003
  • Final Report

  • Main Center Abstract and Reports:

    R828112    Health Effects Institute (2000 — 2005)

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
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    R828112C047 Effect of Nitrogen Dioxide on Bacterial Respiratory infection in Mice
    R828112C048 Effects of Ozone Exposure on Airway Epithelium
    R828112C049 Inhalation of Aldehydes and Effects on Breathing
    R828112C050 Does Ozone Cause Precancerous Changes in Cells?
    R828112C051 Effects of Formaldehyde on Human Airway Epithelial Cells Exposed in a Novel Culture System
    R828112C052 Carbon Monoxide and Cardiac Arrhythmias
    R828112C053 Effects of Formaldehyde and Particle-Bound Formaldehyde on Lung Macrophage Functions
    R828112C054 Mechanisms for Protecting Lung Epithelial Cells Against Oxidant Injury
    R828112C055 Relationship of Nitropyrene-Derived DNA Adducts to Carcinogenesis
    R828112C056 Particle Trap Effects on Heavy-Duty Diesel Engine Emissions
    R828112C057 Carbon Monoxide and Atherosclerosis
    R828112C058 Nitrogen Dioxide and Respiratory Illness in Children
    R828112C059 Noninvasive Methods for Measuring Ventilation in Mobile Subjects
    R828112C060 Oxidant Air Pollutants and Lung Cancer: An Animal Model
    R828112C061 Detection of Carcinogen-DNA Adducts: Development of New Methods
    R828112C062 Effects of Carbon Monoxide on Heart Muscle Cells
    R828112C063 Development of Personal Ozone Samplers: Three Approaches
    R828112C064 Development of Biomarkers to Monitor Carcinogen Exposure
    R828112C065 Effects of Prolonged Ozone Inhalation on Collagen Structure and Content in Rat Lungs
    R828112C065II Prolonged Ozone Exposure and the Contractile Properties of Isolated Rat Airways
    R828112C065III Changes in Complex Carbohydrate Content and Structure in Rat Lungs Caused by Prolonged Ozone Inhalation
    R828112C065IV Genetic Control of Connective Tissue Protein Synthesis After Prolonged Ozone Inhalation
    R828112C065V Pulmonary Function Alterations in Rats After Chronic Ozone Inhalation
    R828112C065VII Prolonged Ozone Exposure Leads to Functional and Structural Changes in the Rat Nose
    R828112C065VIII - IX Studies of Changes in Lung Structure and Enzyme Activitiesin Rats After Prolonged Exposure to Ozone
    R828112C065X An Innovative Approach to Analyzing Multiple Experimental Outcomes: A Case Study of Rats Exposed to Ozone
    R828112C065XI The Consequences of Prolonged Inhalation of Ozone on Rats: An Integrative Summary of the Results of Eight Collaborative Studies
    R828112C066 Interactive Effects of Nitropyrenes in Diesel Exhaust
    R828112C067 Detection of Formaldehyde–DNA Adducts: Development of New Methods
    R828112C068I Comparison of the Carcinogenicity of Diesel Exhaust and Carbon Black in Rat Lungs
    R828112C068II An Investigation of DNA Damage in the Lungs of Rats Exposed to Diesel Exhaust
    R828112C068III No Evidence For Genetic Mutations Found In Lung Tumors From Rats Exposed To Diesel Exhaust or Carbon Black
    R828112C069 Noninvasive Determination of Respiratory Ozone Absorption: The Bolus-Response Method
    R828112C070 The Effects of Inhaled Oxidants and Acid Aerosols on Pulmonary Function
    R828112C071 Biochemical Consequences of Ozone Reacting with Membrane Fatty Acids
    R828112C072 DNA Mutations in Rats Treated with a Carcinogen Present in Diesel Exhaust
    R828112C073 Developmental Neurotoxicity of Inhaled Methanol in Rats
    R828112C074 Methanol Distribution in Non Pregnant and Pregnant Rodents
    R828112C075 Is Increased Mortality Associated with Ozone Exposure in Mexico City?
    R828112C076 Effects of Fuel Modification and Emission Control Devices on Heavy-Duty Diesel Engine Emissions
    R828112C077 Metabolic Studies in Monkeys Exposed to Methanol Vapors
    R828112C078 Effects of Ozone on Pulmonary Function and Airway Inflammation in Normal and Potentially Sensitive Human Subjects
    R828112C079 Improvement of a Respiratory Ozone Analyzer
    R828112C080 Mechanism of Oxidative Stress from Low Levels of Carbon Monoxide
    R828112C081 Long-Term Exposure to Ozone: Development of Methods to Estimate Past Exposures and Health Outcomes
    R828112C082 Effects of Ambient Ozone on Healthy, Wheezy, and Asthmatic Children
    R828112C083 Daily Changes in Oxygen Saturation and Pulse Rate Associated with Particulate Air Pollution and Barometric Pressure
    R828112C084 Evaluation of The Potential Health Effects of the Atmospheric Reaction Products of Polycyclic Aromatic Hydrocarbons
    R828112C085 Mechanisms of Response to Ozone Exposure: The Role of Mast Cells in Mice
    R828112C086 Statistical Methods for Epidemiologic Studies of the Health Effects of Air Pollution
    R828112C087 Development of New Methods to Measure Benzene Biomarkers
    R828112C088 Alveolar Changes in Rat Lungs After Long-Term Exposure to Nitric Oxide
    R828112C089 Effects of Prenatal Exposure to Inhaled Methanol on Nonhuman Primates and Their Infant Offspring
    R828112C090 A Pilot Study of Potential Biomarkers of Ozone Exposure
    R828112C091 Effects of Concentrated Ambient Particles on the Cardiac and Pulmonary Systems of Dogs
    R828112C092 Cancer, Mutations, and Adducts in Rats and Mice Exposed to Butadiene and Its Metabolites
    R828112C093 Effects of Concentrated Ambient Particles in Rats and Hamsters: An Exploratory Study
    R828112C094I The National Morbidity, Mortality, and Air Pollution Study: Methods and Methodologic Issues
    R828112C094II The National Morbidity, Mortality, and Air Pollution Study: Morbidity and Mortality from Air Pollution in the United States
    R828112C095 Association of Particulate Matter Components with Daily Mortality and Morbidity in Urban Populations
    R828112C096 Acute Pulmonary Effects of Ultrafine Particles in Rats and Mice
    R828112C097 Identifying Subgroups of the General Population That May Be Susceptible to Short-Term Increases in Particulate Air Pollution
    R828112C098 Daily Mortality and Fine and Ultrafine Particles in Erfurt, Germany
    R828112C099 A Case-Crossover Analysis of Fine Particulate Matter Air Pollution and Out-of-Hospital Sudden Cardiac Arrest
    R828112C100 Effects of Mexico City Air on Rat Nose
    R828112C101 Penetration of Lung Lining and Clearance of Particles Containing Benzo[a]pyrene
    R828112C102 Metabolism of Ether Oxygenates Added to Gasoline
    R828112C103 Characterization and Mechanisms of Chromosomal Alterations Induced by Benzene in Mice and Humans
    R828112C104 Acute Cardiovascular Effects in Rats from Exposure to Urban Ambient Particles
    R828112C105 Genetic Differences in Induction of Acute Lung Injury and Inflammation in Mice
    R828112C106 Effects on Mice of Exposure to Ozone and Ambient Particle Pollution
    R828112C107 Emissions from Diesel and Gasoline Engines Measured in Highway Tunnels
    R828112C108 Case-Cohort Study of Styrene Exposure and Ischemic Heart Disease Investigators
    R828112C110 Effects of Metals Bound to Particulate Matter on Human Lung Epithelial Cells
    R828112C111 Effect of Concentrated Ambient Particulate Matter on Blood Coagulation Parameters in Rats
    R828112C112 Health Effects of Acute Exposure to Air Pollution
    R828112C113 Benzene Metabolism in Rodents at Doses Relevant to Human Exposure from Urban Air
    R828112C114 A Personal Particle Speciation Sampler
    R828112C115 Validation and Evaluation of Biomarkers in Workers Exposed to Benzene in China
    R828112C116 Biomarkers in Czech Workers Exposed to 1,3-Butadiene: A Transitional Epidemiologic Study
    R828112C117 Peroxides and Macrophages in the Toxicity of Fine Particulate Matter in Rats
    R828112C118 Controlled Exposures of Healthy and Asthmatic Volunteers to Concentrated Ambient Particles in Metropolitan Los Angeles
    R828112C119 Manganese Toxicokinetics at the Blood-Brain Barrier
    R828112C120 Effects of Exposure to Concentrated Ambient Particles from Detroit Air on Healthy Rats and Rats with Features of Asthma or Mild Bronchitis
    R828112C121 Field Evaluation of Nanofilm Detectors for Measuring Acidic Particles in Indoor and Outdoor Air
    R828112C123 Time-Series Analysis of Air Pollution and Mortality: A Statistical Review
    R828112C126 Effects of Exposure to Ultrafine Carbon Particles in Healthy Subjects and Subjects with Asthma
    R828112C128 Neurogenic Responses of Rat Lung to Diesel Exhaust
    R828112C130-I Relationships of Indoor, Outdoor, and Personal Air (RIOPA). Part I. Collection Methods and Descriptive Analyses
    R828112C132 An Updated Study of Mortality Among North American Synthetic Rubber Industry Workers