Document g2mVkLj8o0qvze8jvpjmVndQ
Metabolism and Peripheral Blood Effects in Shanghai Benzene Workers
Benzene 2009 Symposium Munich, Germany September 9, 2009
A. Robert Schnatter ExxonMobil Biomedical Sciences, Inc.
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Background: benzene metabolism
sPMA, ttMA more sensitive, specific vs. phenolic metabolites
Toluene may inhibit (Andrews 1977) or enhance (Wetmore 2008) metabolism, depending on dose
Conflicting findings regarding single gene polymorphisms (SNP's)
Dougherty et al. 2008
CYP2E1 *5 CT or TT I ttMA CYP2E1 *5 CT or TT I Phenol GSTT1 null I sPMA
Positive
1 1 4
Negative
4 1 4
Different proportions I enhanced metabolism for lower exposures
- ttMA favored at lower ambient exposures
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- 9x increase <1 ppm when ambient exposures predicted from urinary benzene
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Benzene metabolism: expected influence of SNP's
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C- N- CH2
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CH3 COOH
S-Phenylmercapturic acid
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Benzene
wild type: increased metabolites
HH
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OHC-CC-CC-CHO II
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Trans, Trans-
Muconaldehyde
INonenzymatic
\aHrearrangement
O c1( l PBenzene
Benzene
HEypdoroxrladese -;:7
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OH
H
Benzene OH
dihydrodiol
Oxide
Oxepin
Nonenzymatic rearrangement
1Drhydrodiol Dehydrogenase
CYP
~H-:?' OH
'- H
0 OH
Diol Epoxide
0 0OH OH
OH
CYP
CYP AoH
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Hydroquinone
~0~wild type:
mutant:
Phenol
Catechol
~~~
lL)wild type:
~)....0 mutant:
reduced CAT
reduced CAT
reduced HQ 1 1 reduced HQ
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Background: benzene and peripheral blood effects
Well established effects for anemia (low RBC), leukopenia (low WBC), and thrombocytopenia (low PLT) early data suggested more
I II
prevalent effects on RBC (Greenberg 1939), or WBC (Aksoy 1971; Kipen 1988)
No consensus on most sensitive effect candidates mentioned: I II - MCV (Yardley-Jones 1988; Collins 1991) - Lymphocytes (Goldstein 1988; Rothman 1996) - Neutrophils (Qu 2002) - Early progenitor cells (Lan 2004)
Early studies showed clear effects >1 0 ppm more recent studies II I show effects at lower levels
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Study design I overview
Cross-sectional study in five factories in and around Shanghai, China
Approved by IRS's at University of Colorado and Fudan University
Consent form, questionnaire, blood sample
Post-shift urine sample in 2of 5factories for metabolite study
Extensive exposure monitoring for benzene, toluene, xylene
Few other chemical exposures
Five metabolites measured via GCMS (CAT, HQ, PH, ttMA, sPMA)
High LOD for sPMA prevented analyses at lower exposures
Five SNP's determined via RFLP in four genes encoding for activating or
detoxification enzymes
NQ01 465C >T, 609C >T
CYP2E11019C >T
MPO 463G >A
GSTT1 null
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Statistical analyses on 4 metabolites and 12 blood indices via correlations,
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general linear models, change-point regressions
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Factories studied
Type Product I Process Workers Males Females
A Rubber
Rubber hoses &belts 352 250 102
B Shoe
Glue soles to uppers 412 113 299
c Sealant Products (rubber) Diluent, adhesive
126 68
58
D Pharmaceutical
Intermediates
128 116
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E Rubber
Rubber hoses
28 16 12
Metabolite Subpopulation
A Rubber
B Shoe
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Same
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Same 47 4 43
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Population description
Administered in-person questionnaire and consent form
95-99/o participation, similar across factories Smoking validated with cotinine for metabolite workers, >98/o agreement
All Workers
Metabolite Subpopulation
Non-Alcohol Us
Alcohol Users
Non-Alcohol
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Alcohol Users
Smokers
Smokers
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Age
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Exposure assessment
1046 workers
133 SEG's
Evaluate homogeneity of SEG via random effects ANOVA; between worker variance 95% confidence limit ratio <4
88/133 (66%) homogeneous SEG's
734workers individually monitored
- 2973 benzene - Weekly average
samples
benzene mg/m3
(x=4/ worker)
Females: median: 10.9 (.07-717)
Males: median: 5.9 (.07-872)
Imputed weekly average from homogeneous SEG's for workers not individually monitored (n=194)
326workers
individually monitored
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Metabolite Subpopulation
Closest benzene reading to blood I urine samples
Females: median: 10.0 (.07-231) Males: median: 4.3 (.07-177)
190 same-day readings
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Benzene I metabolite correlations
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HQ and ttMA particularly highly correlated with one another (r=0.94)
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Prediction models for metabolites
Baseline Model
Benzene~
CATECHOL
0.49***
HYDROQUINONE 0.54***
PHENOL
0.58***
ttMA 0.67*** *p<.05; ** p<.01; *** p<.001
Final Model (p coefficients)
Benzene Benzene2 Toluene Smoking BMI
0.08
0.09**
0.28*** 0.11
0.25**
0.05*
0.10**
0.10 0.04*
0.38***
..
0.27*** 0.21
0.55***
..
0.16** 0.11
BZ*TOL term did not enter final models (BZ, toluene correlation= 0.70) No SNP terms were important metabolite predictors
Additional models run for BZ, TOL, BZ*TOL only:
Metabolite
p-value for interaction term
CAT <0.0001
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0.37 0.21 0.72
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Change-point regression results
Change-point regressions determine response that is different than background response Lowest change point found for ttMA and HQ
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Benzene metabolite proportions across benzene concentrations
Metabolite production rate across benzene concentrations
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Uncorrected
Background metabolite values identified in 27 controls Different percentiles of control distribution used for correction factor Correction factor subtracted from metabolite concentration Corrected metabolite concentrations converted to micromoles
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Mean
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P75 factor: stable metabolism Median factor: 3-4X
Mean correction factor:
P25 factor: 5-6X
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2-3X enhanced metab <1 ppm
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Benzene effects on peripheral blood elements
Twelve indices examined (6 WBC, 4 RBC, 2 PLT parameters) Continuous outcomes modeled via GLM, out-of-normal range defined using
Chinese National norms and examined via logistic regression Covariates: smoking, age, BMI, alcohol, SNP's, gender
Parameter
Crude B(Benzene)
Adjusted B(Benzene)
WBC -0.06*
Lymphocytes
-0.01
Monocytes
-0.00
Neutrophils
-0.05**
In (Eosinophils)
0.01
Basophils
-0.00*
RBC -0.06***
HGB -0.14*
MCV 0.26***
RDW 0.03
Platelets
-1.64
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-0.18***
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-0.06** -0.02 -0.00* -0.01* 0.01 0.00 -0.04*** -0.07*** 0.36*** 0.02 -2.32** -0.18***
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Effect of toluene exposure
Toluene confounded several blood index I benzene relationships
Toluene I benzene exposure correlation =0.66, thus collinearity may explain
this observation
Toluene effect significant I benzene effect non-significant
Neutrophils Monocytes
RBC MCV Hemoglobin Platelets
Toluene increased I significant benzene effect
Lymphocytes Eosinophils
No toluene effect I significant benzene effect
WBC MPV
No toluene or benzene effect
Basophils ROW
Future analysis will assess toluene exposure and potential synergistic and antagonistic effects
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Present analyses is analogous to several studies in the literature where
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Change-point regressions
Examined whether background blood elements could be distinguished from benzene-induced effect
Lower change points => more sensitive effect
Still may not represent clinically relevant effect
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WBC
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Neutrophil change-point regression results
Neutrophils
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Benzene concentrations affecting different blood elements
Neutrophils and MPV showed lowest change points Lymphocytes showed highest change point
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Benzene exposure (ppm)
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Logistic regression results for clinically relevant blood elements
Parameter
WBC Lymphocytes Neutrophils Eosinophils RBC MCV HGB Platelets
OR
1.18 1.04 1.05 1.07 1.28 1.68 1.11 1.28
95/o Cl
1.00-1.39 0.91-1.19 0.92-1.20 0.88-1.30 1.12-1.47 1.35-2.10 0.99-1.24 1.01-1.63
Significant effects for: leukopenia, anemia, high MCV, and thrombocytopenia Strongest effects for MCV and RBC
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MCV: out-of-range values by benzene exposure
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Logistic regression results by exposure category (OR, 95/o Cl):
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1-<10ppm
10+ ppm
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5.65 (0.62, 51.1) 5.91 (0.75, 46.5) 17.7 (2.35, 134.1)
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RBC: out-of-range values by benzene exposure
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Logistic regression results by exposure category (OR, 95/o Cl):
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10.5 (1.39, 79.7) 4.68 (0.61, 36.1) 14.7 (1.97, 110)
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Summary I Conclusions
Metabolism
Higher metabolic levels: males, smokers, alcohol use, toluene co-exposure No strong effects for: age, SNP's Benzene levels of 0.5 ppm can distinguish metabolites (HQ, ttMA) from
background levels Metabolism rate slightly enhanced (2-3x) for lower (<1 ppm) exposures,
although correction factor value is critical
Blood effects
Benzene affects most blood indices, however, toluene co-exposure and
collinearity under further investigation
Most sensitive parameter for continuous indices is neutrophils (7.8 ppm) ...
appears to be a mild effect
When clinically relevant effects are examined, benzene has an effect on
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Stronger effects for RBC and MCV, albeit at higher concentrations
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Collaborators
EMBSI and former EMBSI Yimei Zhou, Min Chen, Mark Nicolich, Tom Armstrong
Ginpathogen I former Joint Molecular and Clinical Laboratory Patrick Kerzic, Rich Irons
Fudan University Fu Hua, Lu Lin
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Acknowledgements
1078 study participants from Shanghai, China
SHS consortium (BP, Chevron, Conoco, ExxonMobil, Shell Chemical)
Science Review Panel
Ethics Review Panel
Shanghai Municipal IPHS and CDC (Zhu Surong)
Fudan University (and former) Miao Liuzhong
Data Processing I QA- Susan Marcella (EMBSI), Gail Jorgensen
(former EMBSI)
University of Colorado IRB
Fudan University Ethics Panel
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Lymphocyte change-point regression results
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Effect of NQ01*2 on hydroquinone levels
Analysis of Covariance for logHQ_ug_ml
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