Exposure Pathways

1
?????????????????
??? ???? ?????????? ?????????? 11-1-2002
? ?????????
2

• ?????????????????????
• ????????????(????????????????????????)???????,????
  ????????????
• ?????????????????????,??????????????
• ???????,??????????????,????????????
• ????????????,???????????????????

3
Exposure Pathways
4
Estimating Exposure UsingBiological Monitoring

• Possible measurements
• Concentration of the chemical itself or its
  metabolites in biological tissues
• Biological effect that occurs as a result of
  exposure
• A sensitive as well as specific analytical tool
  is required to analyze biological samples with
  complicated matrices.

5
Principle of Mass Spectrometry
vacuum
MALDI-TOF ESI-Q-TOF
ESI-TSQ (Sensitivity)-(Specificity)
Mass spectrometry is a sensitive as well as
specific analytical tool
6
Development of Ionization Methods
7
Types of Mass Analyzers

• Magnetic sector
• Time-of-flight (TOF)
• Quadrupole
• Ion trap
• Fourier transform ion cyclotron resonance (FT-ICR)

8
???????????????

• ?????????????
• ??????????????
• DNA????????????

9
Significance of dioxin exposure in Taiwan
About dioxins

• Highly toxic, LD50 KCN in rats, 10 mg/kg
• 2,3,7,8-TCDD in rats 0.03 mg/kg
• Broad effects Imunotoxicity, hepatotoxicity,
  teratogenicity, fetotoxicity, reproductive
  toxicity, etc.
• Carcinogen (2,3,7,8-TCDD, IARC group 1)
• Generated in combustion and certain manufacturing
  processes as by-products
• Persistent in the environment with long
  half-lives
• Many countries had established background
  exposure data, but no data were available in
  Taiwan

10
A Known Mechanism of Dioxin Action Ah Receptor
11
Two major challenges in measuring dioxin exposure

• Ultra-low levels of dioxins in human blood,
• i.e., part-per-trillion (ppt) levels
• -- Established first laboratory (ALTEP) in
  Taiwan to measure human dioxin exposure
• (2) Chinese are not willing to donate blood
  samples.
• -- Reduced serum volume from 60 to 20 mL

12
?????????????????(PCDDs)?????(PCDFs)????????210???
?
13
PCDDs?PCDFs???(congener)???
14
International Toxic Equivalency Factors (I-TEFs)
for PCDD/PCDF (Kutz et al. 1980)
15
Toxic Equivalent (TEQ)

• TEQ Sn1PCDDi x TEFi
• Sn2PCDFi x TEFi

16
??
????
????????
1
???? ????
????
????
????
??
????
17
??????
?????????
???
18
High resolution gas chromatography/high
resolution mass spectrometry (HRGC/HRMS)
????10000??
19
HRMS????????????????????
20
HRMS????????????????????
21
Dioxin concentrations in human blood samples from
across Germany
(from Compilation of EU Dioxin Exposure and
Health Data, 1999)
22
Dioxin exposure levels of different populations
in Taiwan
Tao-Yuan
Tai-Chung
Tai-Nan
Cheng-Si District, Tai-Nan
Yu-Chen Patients
23
Geographical locations of the Tainan City and the
abandoned An-Shun PCP manufacturing plant
24
(No Transcript)
25
???88?6?15???
26
????88?6?16???
27
?????????????????????????????
???88?6?19???
28
???????????????

• ????????????????
• ??????????????
• DNA????????????

29
Significance of benzene exposure in Taiwan

• International Agency for Cancer Research (IARC)
  has classified benzene as a group 1 carcinogen
  leukaemia
• Benzene is a common industrial chemical, a
  component of gasoline, a constituent of engine
  emissions and tabacco smoke, as well as a
  ubiquitous environmental pollutant.
• In Taiwan, gasoline contains 1-3 of benzene.
  However, specific biological monitoring method
  has not been established in Taiwan.

30
Metabolites of benzene
31
The advantages and disadvantages of various
benzene exposure biomarkers
biomarker advantages disadvantages
Benzene in blood Specific, sensitive Invasive sampling
Urinary benzene Specific, sensitive Because benzene is very volatile, great care in analysis is necessary to prevent both contamination by ambient benzene or losses by volatilization
Urinary Phenol Useful for high level exposure High background level
Urinary CAT - Confounding by phenol Limited experience
Urinary HQ - Confounding by phenol Limited experience
Urinary BT - Confounding by phenol Limited experience
Urinary t,t-MA Reasonably specific, sensitive Confounding by sobitol sorbic acid
Urinary S-PMA Specific, sensitive Confounding by carbamazepine Tedious, require sophisticated technique
32
Analytical methods for S-PMAreported in the
literature
Analytical methods Detection Limits Sample preparation Sample preparation References References
Analytical methods Detection Limits Chemical derivation Manual extraction References References
GC-ECD 0.06 µg/L Yes Yes 1996 Einig et al.
GC-MS 0.5 µg/L Yes Yes 2000 Fang et al.
GC-MS/MS 0.6 µg/L Yes Yes 1993 Stanek et al.
HPLC-Flu 1 µg/L yes Yes 1993 Einig et al.
HPLC-UV 20 µg/L No Yes 2000 Inoue et al.
LC-ESI-MS/MS 0.1 µg/L No Yes 1999 Melikian et al.
33
Analytical system requirements for S-PMA

• No chemical derivatization
• No manual liquid/liquid or SPE extraction
• High sensitivity (pre-concentration)
• Detection limit lt 1/5 BEI (BEI25 µg/g
  creatinine)
• Automation
• High-throughput

34
??????????????

• An automatic on-line sample clean-up and
  electrospray ionization tandem mass spectrometry
  system is used for the quantitative detection of
  benzene exposure biomarker S-phenylmercapturic
  acid (SPMA) in urine.
• The required sensitivity is provided by
  electrospray ionization in negative mode
• The required specificity is provided by tandem
  mass spectrometry
• Sample clean-up is done by a on-line column
  switching device

35
PE Sciex API 365 Triple-stage-quadrupole Mass
Spectrometer
36
API 365 Electrospray Ionization Source
37
(No Transcript)
38
API 365 Triple-stage-quadrupole Tandem Mass
Spectrometer
Q1
Q2
Q3
39
Tandem Mass Spectrometry (MS/MS) Single Reaction
Monitoring (SRM)
Q1
Q2
Q3
Collision Induced Dissociation (CID)
Product Ion
Precursor Ion
m/z 109
m/z 238
40
??????????????

• An automatic on-line sample clean-up and
  electrospray ionization tandem mass spectrometry
  system is used for the quantitative detection of
  benzene exposure biomarker S-phenylmercapturic
  acid (SPMA) in urine.
• The required sensitivity is provided by
  electrospray ionization in negative mode
• The required specificity is provided by tandem
  mass spectrometry
• Sample clean-up is done by a on-line column
  switching device

41
???????????????????????????????
42
LC-MS/MS???????

• ???????
• (Peak Width vs. Flow Rate)
• ????????
• (Peak Intensity vs. Wash Time)
• ???????(Concentration vs. Mass Dependent)

43
????????????????
???????
??????
1.03e5
5.63e5
44
The advantages of flow splitting before ESI
1/20
600 mL/min
30 mL/min

• No decrease in signal intensity
• No heating necessary
• Less drying gas (Turbo gas)
• Much less sample matrix deposition at the orifice
  to the mass spectrometer ( n gt 2000 runs without
  clogging at the orifice)

45
?????????????????

• ??????????????????????????????????????????????,???
  ????
• (1)????????
• (R2 0.998, 0.45112.5 µg/g creatinine)
• (2)????????? (LOD0.35 µg/L)
• (3)???????(11.3 45 µg/g creatinine)
• (4)???????(11.3 45 µg/g creatinine)
• (5)??????
• (6)??????????

46
???
??13C6 S-PMA?????
47
???????
48
??????????
Concentration µg/g creatinine number ???? ???? ????? ?????
Concentration µg/g creatinine number CV ( ) RE( ) 13C6 S-PMA CV ( ) RE( ) 13C6 S-PMA
11.25 ( ½ BEI ) 6 4.8 -4.7 2.0 10.4
22.5 ( BEI ) 6 5.6 -2.4 4.1 9.9
49
Summary of the methods
Analytical methods Methods reported in literature Methods reported in literature Methods reported in literature This method
Analytical methods GC HPLC HPLC On-line SPE/HPLC
Analytical methods ECD MS FLU ESI-MS/MS ESI-MS/MS
Chemical Deriv. Yes Yes No No
Manual extraction Yes Yes Yes No
Analysis time 3 hr 30 min 3 hr 30 min 1 hr 10 min 0.5 min 10 min
Detection limits 0.06 µg/L 0.5 µg/L 1 µg/L 0.1 µg/L 0.35 µg/L
Urine volume used 5 mL 110 mL 0.55 mL 1 mL 0.05 mL
50
Neat 0.02 ppb SPMA
Detection of Urinary SPMA by on-line sample
clean-up and ESI-MS/MS
Newborn babys urine
unexposed individual in Tainan city
51
Benzene exposure levels revealed by urinary
biomarker S-PMA levels
Residents of Tainan City
Neonates
Factory workers
52
???????????????

• ????????????????
• ??????????????
• DNA????????????

53
DNA????????????

• Many genotoxic cacinogens and mutagens are
  electrophilic chemical compounds and can react
  with nucleophilic sites in DNA
• The introduction of DNA adducts may be
  responsible for the subsequent development of
  mutation and cancer in animals and humans
• The detection of DNA adducts in biological
  specimen provides a biomarker for adduct
  formation resulted from chemical exposure
• The major challenge for the detection of DNA
  adducts in biological samples is that the
  concentration of adducts in DNA can be very low

54
DNA???,N7-HEG,?????????

• Ethylene oxide has been widely used as a
  industrial chemical and a disinfectant in
  hospital.
• Exposure to ethylene oxide has been associated
  with lymphatic and haematopoietic cancer
• The International Agency for Research on Cancer
  (IARC) has classified ethylene oxide as a known
  carcinogen in humans (Group 1)
• The major product of ethylene oxide-DNA adduct
  formation is N7-(2-hydroxyethyl)-guanine (N7-HEG)
  (82)

55
Fragment Ions Derived from N7-HEG by CID-MS/MS
experiment
Product Ion
Precursor Ion
56
High Sensitivity and Specificity Provided by
LC-MS/MS for N7-HEG Detection
Matrix effect considered
Without matrix
4 fmol
16 fmol
LC-MS/MS
128 fmol
1.02 pmol
LC-MS
57
High Sensitivity and Specificity Provided by
LC-MS/MS for N7-HEG Detection
Matrix effect considered
Without matrix
128 fmol
1.02 pmol
LC-MS/MS
128 fmol
1.02 pmol
LC-MS
58
Logarithmic dose-response curve showing N7-HEG
formation in Hep G2 cells after 8-hr incubation
with ethylene oxide-containing medium
59
???????????????

• ????????????????
• ??????????????
• DNA????????????

60
??
??
???
??
??
??
??
??
??
??
??
???
??
??
??