Time-Course and Sensitivity of Muconic Acid as a Biomarker for Human Environmental Exposure to Benzene

EPA/600/A-92/248
THE TIME-COURSE AND SENSITIVITY OF MUCONIC ACID
AS A BIOMARKER FOR HUMAN ENVIRONMENTAL
EXPOSURE TO BENZENE
Timothy J. Buckley1, Andrew B. Lindstrom1, V. Ross High smith1, William E. Bechtold2, and
Linda S. Sheldon3
'US EPA, Atmospheric Research and Exposure Assessment Laboratory (MD-56) RTP, NC 27711
2Inhalation Toxicology Research Institute, Albuquerque, NM 87185
'Research Triangle Institute, P.O. Box 12194, RTP, NC 27709
Preliminary results; are presented that show the effect of increased benzene exposure on the urinary
elimination of trans, trans-mucomc acid (MA) for an adult male. These results were generated from
a controlled exposure experiment during which an individual was exposed to benzene during a shower
with gasoline-contaminated ground water. Based on measured air and water concentrations, it is
estimated that the 25 minute shower and drying-off exposure period resulted in an inhalation and dermal
absorbed dose of 122 fig and 19 fig, respectively, yielding an average dose rate of 334 fig/h during the
shower period. The measured background dose rate of 1.2 fig/h was exceeded by a factor of 278 during
the shower exposure. The average urinary MA elimination rate increased from 3.7 fig/h during the 30
h period before the exposure to 17.9 fig/h during the 22 h period after the exposure. The post-exposure
profile of muconic acid elimination (jig/h) was characterized by two minor peaks (47 and 35 fig/h)
occurring within 3 h and a major peak (61 fig/h) at approximately 11 h.
Key Words: Muconic Acid, Time Course, Validation, Exposure, Dose, Biomarker, Benzene,
Dermal, Inhalation.
This paper has been reviewed in accordance with the U.S. Environmental Protection Agency's
peer review and administrative review policies and approved for presentation and publication. Mention
of trade names or commercial products does not constitute endorsement or recommendation for use.
This study was reviewed and approved by the Research Triangle Institute Committee for the
Protection of Human Subjects.
Proceedings of the EPA/AWMA International Symposium:
"Measurement of Toxic and Related Air Pollutants"
Durham, North Carolina
May 3-8, 1992
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INTRODUCTION
Human ex posure to benzene in community and occupational environments is common1. This
fact, along with compelling evidence suggesting that benzene exposure causes leukemia in humans2,
gives reason for evaluating and minimizing routes of exposure.
Biomarkers can provide a powerful tool for assessing exposure and risk. The measurement of
a biomarker can provide individual-based evidence that exposure has occurred ex post facto. A
biomarker measurement establishes, in fact, a body burden that can otherwise only be estimated through
external measurements of exposure. Although a biomarker measurement may in theory be a more
valuable means of assessing exposure, its practical value is dependent upon the reliability and validation
of the biomarker.
Various urinary biomarkers of benzene exposure have been investigated including phenol3, S-
phenyl-N-acetylcysteine4,5 and muconic acid6,7,8, trans,trans-Muconic acid (MA) shows particular
promise as a biomarker for human environmental exposure due to its specificity and its presence at
detectable levels in individuals exposed to background benzene levels7. Furthermore, MA provides an
indication of toxicological potential because it is formed from the toxic metabolic intermediate,
muconaldehyde9.
It is the aim of this research to provide further data regarding the validation of muconic acid as
a biomarker of more subtle, non-occupational, benzene exposures. Research to date has generally
involved highly exposed individuals, such as smokers or workers6,7,8. Specific research objectives
include discerning low from relatively high levels of exposure through the urinary elimination of MA
and to characterize the profile of MA elimination following a single acute exposure.
The exposure and muconic acid data presented herein are partial and represent two components
of a multi-faceted study that also included measurements of dosimetry (respiratory and cardiac rate),
blood, and breath benzene. The analysis of the full complement of data will be reported at a later date.
METHODS
The experimental design consisted of a short-term acute benzene exposure preceded and followed
by periods of low-level background exposure. The short-term acute exposure was generated by an
individual taking a shower using gasoline contaminated ground water. Therefore, a "bolus-like" dose
of benzene was introduced by absorption through the lung and skin after a period of low level
background benzene exposure. The level of contamination, and the likely resulting exposure was
characterized prior to, as well as during the study10,11. The shower dermal and inhalation exposure
was limited to 20 minutes followed by a 5 minute inhalation-only exposure period during drying off.
Background exposures resulted from normal activities within ambient, office, in-transit, and home
microenvironments. The identical shower exposure scenario was conducted three times in 1991 (June
11, 12, and 13) with one shower per day over three consecutive days.
The methods of collecting and analyzing water and microenvironmental air benzene levels are
described in a microenvironmental measurements/intersampler comparison investigation conducted in
conjunction with this biomarker validation experiment12. Integrated and grab samples were collected
throughout the study using Summa™ canisters, Tenax GC™, and glass gas-tight syringes. Low-flow
personal sampling was conducted using the sorbent Tenax GC™ to measure the shower and background
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personal exposures. The pump (DuPont P4000) was operated at 10 cc/minute during the approximately
20 h background j»eriod which preceded and followed each shower exposure. Flow calibration was
conducted at the beginning and end of each sampling period. Personal sampling was delayed for
approximately 2 h following the shower exposure to minimize contamination of the Tenax GC™ with
the elevated levels of exhaled benzene.
All urine passed during each of the three days of the shower plus approximately two days of
background samples was collected. Voids were collected at l-PA and 4 h intervals during day/evening
and night periods, respectively. Each void was collected separately in polypropylene (500 ml) or
polyethylene (100 ml) screw-cap bottle with exact time and date of collection recorded on the bottle
label. Each sample was immediately placed into a freezer or dry-ice cooler. Samples were transferred
to a -20°C laboratory freezer within 1-2 days after collection.
Air samples collected on Tenax GC™ were thermally desorbed and analyzed by gas
chromatography/mass spectroscopy (GC/MS). Water samples were similarly analyzed by GC/MS using
a purge and trap technique. Grab air samples were collected with syringes and analyzed on-site by
GC/PID (photo-ionization detection). Urinary muconic acid was quantified by GC/MS (single ion
monitoring) after the addition of biosynthesized muconic Acid-13C internal standard and liquid extraction
(ethyl ether) according to methods described by Bechtold et al.n
RESULTS
Dose Estimates
MA data are currently available only for the June 13th shower exposure. Air and water benzene
concentrations and quality assurance results are reported by Lindstrom et al.n The relevant data
required for the biomarker assessment are specified here.
Personal sampling yielded air concentrations of 1-2 fig/m3 during the background periods. The
benzene air concentration during the 20 minute shower and 5 minute dry-off period was 525 and 398
Hg/m1 as determined from the integrated Tenax GC™ and grab syringe samples (20 and 25.5 minute),
respectively. From these measurements, dose was estimated using equation 1.
A-w = £
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Where: Dd^ is the dose absorbed through the skin (/xg); CW is the concentration of benzene in water
Oxg/L); SA is the surface area of a 180 lb, 6'4" male (2090 cm2); Kp is the dermal permeability constant
for benzene in an aqueous solution (0.111 cm/h)16; t is the duration of the exposure (0.33 h); and U
is the units conversion factor (1 L/1000 cm3).
Therefore, it is estimated that a total benzene dose of 141 ftg (13% dermal absorption, 87%
inhalation) resulted from the 25 minute exposure on June 13th giving an hourly dose rate of 334 jtg/h.
This dose rate exceeded the background dose rate of approximately 1.2 /tg/h by a factor of 278.
Urinary Muconic Acid Elimination
Muconic acid elimination results are shown in Figure 1 as a frequency distribution of elimination
rates for the background and post-shower exposure periods. Muconic acid elimination is reported as
a rate (jig/h) based on the assay results (ng/ml), the void volume (ml), and the time between collections
(h). The post-shower mean elimination of 17.9 fig/h exceeded the mean background elimination rate
of 3.7 figfh by a fictor of 4.8. Duplicate analysis (n=21) yielded an average deviation from the mean
of 8% (range: 1-53%).
The time course of muconic acid elimination relative to the background and shower exposure
periods is illustrated in Figure 2. Relative to the background period, the shower exposure appears to
be associated with two minor peaks in MA elimination at post-shower times of 1.3 h and 2.8 h with the
most substantial psak at 11 h. Since the subject was exposed to two showers, one and two days prior
to the day for which these results are reported, this time course may in part reflect more than one
exposure.
CONCLUSIONS
Urinary MA elimination resulting from a relatively high short-term dermal and respiratory
exposure shows two minor peaks occurring within the first three hours and a dominant peak
approximately 11 hours following the exposure. Interpretations of this time course will be made based
on these results, and the confirming results from the two previous exposures when they become
available.
These data demonstrate the relationship between benzene exposure and MA elimination for a
single individual during one of three repeated controlled exposure experiments. Data from the two
unreported experiments will be used to confirm these findings and to further investigate the validity of
MA as an exposure biomarker.
The increased rate of MA elimination corresponded to an increased benzene exposure suggesting
that MA has some capacity as a biomarker for non-occupational exposures. Although the time metric
by which dose and MA elimination are reported are not directly comparable, it is noted that the benzene
dose increased 280 fold while MA elimination increased four-fold. This suggests that large changes in
exposure are reflected by relatively small changes in MA elimination. Additional studies characterizing
MA response to viirying levels of benzene exposures are required to more fully assess this relationship
and the sensitivity of MA resulting from benzene exposures.
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REFERENCES
1. L.A. Wallace 'The exposure of the general population to benzene," Cell Biol. Toxicol.. 5(3):297-314
(1989).
2. International Agency for Research on Cancer, Evaluation of the carcinogenic risk of chemicals to humans.
IARC monograph no. 29,1ARC, Lyon France (1982).
3. L. Drummond, R. Luck, A.S. Afacan, H.K. Wilson, "Biological monitoring of workers exposed to
benzene :in the coke oven industry," Br. J. Ind. Med.. 45:256-261 (1988).
4. F.J. Jonjeneelen, H.A.A.M. Dirven, C.-M. Leijdekkers, P.T. Henderson, R.M.E. Brouns, and K. Halm,
"S-pheny 1-N-Acetylcysteine in urine of rats and workers after exposure to benzene," J. Anal. Toxicol..
11:100-104(1987).
5. P. Stomrael, G. Muller, W. Stacker, C. Verkoyen, S. Schobel and K. Norpoth, "Determination of S-
phenylmisrcaptaric acid in the urine-an improvement in the biological monitoring of benzene exposure,"
Carcinogenesis. 10(2):279-282 (1989).
6. W.E. Be;htold, G. Lucier, L.S. Birabaum, S.N. Yin, G.L. Li, and R.F. Henderson, "Muconic acid
determinations in urine as a biological exposure index for workers occupationally exposed to benzene,"
Am. Ind. Hvg. Assoc. I.. 52(ll):473-478 (1991).
7. P. Ducos, R. Gaudin, A. Robert, J.M. Francin, and C. Maire, "Improvement in HPLC analysis of urinary
trans,fra,u-muconic acid, a promising substitute for phenol in the assessment of benzene exposure," Int.
Arch. Occur). Environ. Health. 62:529-534 (1990).
8. E.S. Johiison and G. Lucier, "Perspectives on risk assessment impact of recent reports on benzene," Am.
J. Ind. Med.. 21:749-757 (1992).
9. G. Witz, L. Latriano, and B.D. Goldstein, "Metabolism and toxicity of frans,rra/ii-muconaldehyde, an
open-ring microsomal metabolite of benzene," Environ. Hlth. Perspect.. 82:19-22 (1989).
10. A.B. Lindstrom, Unpublished data.
11. W.J. Pats, NC Department of Environmental Health and Natural Resources, Unpublished data.
12. A.B. Lindstrom, V.R. Highsmith, TJ. Buckley, W.J. Pate, L.C. Michael, and R.M. Johnson, "Household
exposures to benzene from showering with gasoline-contaminated ground water," in Proceedings of the
1992 EPA/AWMA Symposium on Measurement of Toxic and Related Air Pollutants. AWMA, Pittsburgh,
In Press.
13. W.E. Be;htold, G. Lucier, L.S. Birabaum, S.N. Yin, G.L. Li, and R.F. Henderson, "Muconic acid
determinations in urine as a biological exposure index for workers occupationally exposed to benzene,"
Am. Ind. Hvg. Assoc. J.. 52(11):473-478 (1991).
14. US EPA;, Water Quality Criteria Documents. Federal Register 45(231):79318-79379.
15. A.C. Guyton, Textbook of Medical Physiology. W.B. Saunders co., Philadelphia, (1971).
16. I.H. Blank and D.J. McAuliffe, "Penetration of benzene through human skin," J. Invest. Dermatol..
85:522-526 (1985).
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20
Figure 1.
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0 3 6 9 1215182124273033363942454851545760
MUCONIC ACID ELIMINATION RATE (ug/h)
H Background S Post Shower
Frequency distribution of MA elimination rate during background and post-exposure
periods.
MUCONIC ACID ELIMINATION RATE (ug/h)
¦
Snowar
Background
Expotur*
2 2.5 3
TIME (days)
Poat-
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Figure 2. Time: course of MA elimination during background and exposure periods.
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TECHNICAL REPORT DATA
(Heme read Instructions on the reverse before comple
1. REPORT NO. J.
EPA/600/A-92/248
3. -
4. title and subtitle
The Time-course iiad Sensitivity of Muconic Acid as a
Biomarker for Human Environmental Exposure to Benzene
6. REPORT DATE
S. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
T.J. Buckley, A.B. Lindstrom, V.R. Highsmith,
W.E. Becbtold and L.S. Sheldon
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Atmospheric Rssearch & Exposure Assessment Laboratory
Human Exposure & Field Research Division
Human Exposure Research Branch (MD-56)
RTP, NC 27711
10 PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Office of Research and Development
Atmospheric Research & Exposure Assessment Laboratory
RTP, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Preliminary results are presented that show the effect of ^increased benzene exposure on the urinary elimination
of trans,trans-muconi c acid (MA) for an adult male. These results were generated from a controlled exposure
experiment whereby an individual was exposed to benzene during a shower with gasoline-contaminated ground
water. Based on measured air and water concentrations, it is estimated that the 25 minute shower and drying-off
exposure period resulted in an inhalation and dermal absorbed dose of 122 /*g and 19 fig, respectively, yielding an
average dose rate of 534 ^g/h during the shower period. The measured background dose rate of 1.2 ng/h was
exceeded by a factor cf 278 during the shower exposure. The average urinary MA elimination rate increased from
3.7 figfh during the 30 h period before the exposure to 17.9 iigfh during the 22 h period after the exposure. The
post-exposure profile of muconic acid elimination (jxg/h) was characterized by two minor peaks (47 and 35 pg/h)
occurring within 3 h and a major peak (61 figfh) at approximately 11 h.
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b. IOENTIF IE RS/OPEN ENDED TERMS
c. COSATi Field.Croup
Muconic Acid, Time Course, Validation, Exposure,
Dose, Biomarker, Benzene, Dermal, Inhalation.

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