Biomarkers of Exposure to Airborne Criteria and Air Toxic Pollutants

June 10, 2004

California Air Resources Board

Rogene F. Henderson, PhD

Lovelace Respiratory Research Institute Albuquerque, NM

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To wlhat extent dlo,es exposure to· chemical!s •Co,ntrib1ute to diseas.e?

EXPOS,U1 RE

DilSEASE

Use of Biological Markers to Reduce Uncertainties in Risk Assessment

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!Repair IExc1retion

/Excreta . . . Noneffective A / . · Physiological . . . IRepai,r

/ Macromolecular / Resp,onse / . · Adducts

l otall. Dose to Biolog1ica1llly ln_itlial . . . . . . . _. __

-i)lli.... ,Body _,.,.. ..... T~rg:et ► Effectiv·,e , • B1olog•1call • Pre~hm 1cal . ► Clm_1cal Tissue Dose• Changes Les.101ns Lesions

:Burde\. n '-.... . Nontarget

~ CeHs ,01r· ·. · Nonta rget Org1anelles

Tissue

Biological :M,arkers Biologicall Markers

of Do.siimetry of Effect

Biomarkers for Risk Assessment

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Biological Marker of Exposure

An exogenous substance or its metabolite or the product of an interaction between a xenobiotic agent and some target molecule or cell that is measured in a compartment within an organism.

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Ideal Biomarker of Exposure

· Quantitatively relatable to prior exposure to specific chemical

· Quantitatively relatable to, or predictive of, later developing disease

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Sometimes, we only need a qualitative, yes/no answer.

Have you been drinking again?

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Sometimes quantitation is required.

But I only had one beer!

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Desirable Attributes of Biomarkers of Exposure

· Pollutant specific

· Available for analysis by noninvasive techniques

· Sensitive (detectable at trace concentrations)

· Inexpensive

· Integrates exposures from all media

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Exposure? Marker

Strategies to Relate Markers of Exposure to Prior Exposures

What information do we need to relate a biomarker of exposure to prior exposures?

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Exposure? Marker

If we want to be more quantitative, more information is required.

• Rate of formation of biomarker

• Rate of formation of biomarker

From this information, we can predict the steady-state concentrations of the marker following various exposure scenarios.

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Exposure? Marker

Mathematical Models

· If we have information on rate of formation and removal of a biomarker of exposure, and the factors that influence those rates, we can develop a mathematical model that will predict the concentration of the marker following various exposure regimens.

· The concentration of a biomarker cannot be used to indicate a unique exposure scenario, but can indicate the types of exposure regimens that would produce the indicated level of biomarker

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DETAILED I

KINETIC STUD.IES, IN ANIMALS

e PERCENT ABSORBED

I

-MATHEMATICAL

MODELOF MAR.KER

IN ANIM:ALS

e ANIIMAL TOXICOKINETIC DATA

I I

·• T112 ·ro STEADY STATE

e ANIIMAL PHYSIOLOGICAL ,D·ATA

e T1/2 IFOR CLEARAJNCE

e E.FFECTOF ... EXPOSURE CONCENTRATIO·N ... JEXPOSURE RATE - REPEATED EXPOSURES, - ROLJlE OF 1EXPOSURES

-·-MATHEMATICAL

M10 :DELFOR HUMANS

I

eHUMAN PHVSIO,LOGl'CAL D,ATA

e HUMAN METABOLIC DATA IN VITRO

-~

I

VALIDATE HUMAN MO:DEL

e LIMITED, HUMAN DATA

Exposure? Marker

Use of Animal Data on Kinetics of Biomarkers to Predict Kinetics in Humans

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Exposure? Marker

A Second Strategy:

A battery of biomarkers of exposure with differing

half-lives in the body may provide more information

about prior exposures than a single biomarker.

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' ------ ---------

0 1 10 100 1000 TIME AFTER 1EXPOSURE (days)

Henoor.;on, et ,~ .• Crit. Rev .. Toxicol. 20:65-82',. 1989

Exposure? Marker

Hypothetical Relationship Between “Biomarkers” and Time After Exposure

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Marker Marker11, 2 Case I

A minutes +

e. hours +++

C days ++

D weeks +

E m:onths +

Case I - 1R.ecent e,xposure. Case H - Ongoi,ng exposure ..

Case II Case, Ill

+ -+++ -· ++

+++ ++

+++ ++

Case Ill - Exposur·es were severa,11 months ago; no recent exposure.,

Exposure? Marker

Use of a Battery of Biomarkers to Determine Past Exposure

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Marker? Disease

Strategies to Relate Markers of Exposure to Health Outcome

Need to know which markers can be associated with the

disease outcome and the degree of the association. That

is, given a certain level of a biomarker of exposure, what

is the probability of getting the disease?

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Marker? Disease

What information is required to relate markers of exposure to probability of health outcome?

• Mechanism of disease induction

• Quantitative relationship between marker and probability of progression to adverse health effect

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Marker? Disease

Example:

Predicting cancer induction using DNA adducts as biomarkers

· Identify DNA adducts that are formed by exposure to chemical

· Determine t1/2 of adducts

· Determine mutagenic potential of adducts

· For adducts with long t1/2 and mutagenic potential, determine if mutations induced by adduct are present in tumors induced by chemical

· In animals, develop pharmacodynamic models that describe a quantitative relationship between adduct levels and cancer induction

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Marker? Disease

Example: DNA adducts of vinyl chloride (VC)

VC causes liver cancer in humans and animals. In rats, preweanling rats are more susceptible than adults.

Swenberg et al., Conference of Relevance of Animal Studies to Evaluate Human Cancer Risk, Austin, TX, December 5-8, 1990.

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CYT-P~ , ,ct /c-:c, --------...

0 2• NAO,PH

, ./Cl REAR.RANGEMENT c-c I'/ ' ·O

VINYL CHLORIDE

CHLOROETIHVLENE OXtOE

COVALENT AD1DUCTS WITH NUCLEIC .AcI:o BASES

1/ N

cvl 1,

{d)R

1/' N

N'

~ N

CICH2 -CHO

CHLORO­ACET ALOEIHYDE

7-(2 OXOETHYL) 3,N'4-ETH!ENQ.. 1,.N6-ETHENO- .N•\3.-ETHENO-( DE,OXY} GUANOS.I N'E ,(DEOXY)CVT l'DINE (DEOXY) AD 1ENOSINE DEOXYGUANOSI NE

OEdG EdC EdA EdG

Cirousse1, et al., Biochem. Phannaool. 39·:1109, 1990

Marker? Disease

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Adduct

OEdG

EdC

EdA

EdG

Relative Amount T½ Formed I!!

L.iver :1n

100 62 hr

0.3 >,30 days

0.1 >30 days

1 >30 days

Swenbe1rg et al.., "Prog.ress in Predictive Toxicology" pp. 161-184,, 1:990. 1Elsevier Sci .. Pub.

Marker? Disease

Amount of Each Adduct Formed in Rats

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Rats exp,osed to 6,00p1p,m VC for 5 ,days

Adduct

0 1EdG

EdG

EdC

EdA

1.6 X 1,Q-4

1.8 X 10.6

9 • .'0 X 11 0,-J

1.,8 X. 110 7

Mol1ar Co,ncentration

Day 7

1.2 X 10-5'

8.4 X 10-7

7 .0 X 10-7

'1.3 X 10.7

4.7 X 10-7

6.7x10-1

0.8 X 101

-

7

Swanberg et ,al. • Pro,gress :in Predictive To,xicology"., pp .. 1161 -184, 1990. El1sevier Sc - Pub

Marker? Disease

Persistence of Adducts in Liver

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A,dduct

·OEdG

EdC

Mutation Cau.se,d

none

C-+T C--+A

A-+T Ed'A A-+C

A-+G

EdG G-+.A

Barbin, et al., Cancer Res .. 45: 244,0, 19.85 Barbin, et al., Nucleic Acids ·Res. 9: 375,, 1981 HaU, et al., Ca1rc11no -enes1s 2: 14-11., 1981 Spengler .& Sin:ger, Nucleic Acids Res. 9: 365, 11 981

Marker? Disease

Ability of Adducts to Induce Mutations

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Exposure: 600,ppm VC. 4 hr/day for 5 days.

Concentration (M x 10·01

OEdG, EdG EdA EdC

NeiwbQ:m

Liver 162 1.8 0 .18 ,0.9 0 .. 0 1 ·1

Lung 20 0 .. 25 0 .'11 0 .. 2.5 0.010

Kidney 29 0 .31 ND ND 0 .,011

Brain ND ND o,.oG 0~22.

Spleen ND N1D

Adults

Liver 43 0 .47 0 .19 0 ,.80 0 .. 011

tung 20 0.27 0 .. 014

'Kidney ND ND

Fedtke et al ... Ca,cinogenesi's 111: 1287-1292. 1990

Marker? Disease

Relative Amount of VC Adducts in Tissues of Rats

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AN MAL STU'DIES HUMAN S,TUD_ES SUSPECT ET OLOGl,C AG,ENTS

Identify and Measure Specd1c Biomarkers

Determine Relationship of 1

Biomarker to Exp,osu:re and Adverse Biologic Effect

-

· Model Intervention

Validated osure Ma

1Measure B iomarker 1

in Exposed People

Transitional Epidemiological Study

Cohort Intervention

Validated Risk Marker

John Groopman 1 SOT. Marth 1994

Marker? Disease

The Validation Process: Confirming the Biomarker Disease Link

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Conclusions

· Biomarkers can be valuable for reducing uncertainties in assessing risk for disease from chemical exposures.

· More information is needed on mechanisms of disease induction by chemicals; such studies will suggest the most appropriate biomarkers of the biologically effective dose.

· Much research effort will be required to establish quantitative relationships between the level of markers present and both the degree of prior exposure and the predictability of health outcome.

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Criteria Air Pollutants

Two oxidizing agents: Ozone and Nitrogen Dioxide

Ozone

– 8-oxo-7,8-dihydroguanine (8-oxodG)

Found in oral swabs, white blood cells and urine

Urinary 8-oxodG results from DNA repair processes

Nonspecific marker of oxidative stress

– Clara cell protein in serum

Reported to be sensitive to as low as 0.060-0.084 ppm ozone

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Criteria Air Pollutants (Con’t)

Two oxidizing agents: Ozone and Nitrogen Dioxide

Nitrogen Dioxide

– Can form 8′-nitroguanosine in RNA and 8-nitro-2′-deoxyguanosine in DNA

The latter is unstable

The 8-nitroguaniosine has been used as a marker of endogenous reactive nitrogen species

– Urinary nitrate does not correlate with NO2 exposure

– NO-heme (cannot distinguish between endogenous and exogenous reactive nitrogen)

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NO2 Disproportionates in Water

2 NO2 + H2O HNO2 + HNO3

2 HNO2 NO + NO2 + H2O

NO + heme protein

... 7

... 7

.... 7 NO/heme protein

NO/heme complex is detectable by its distinctive ESR signal

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►

►

►

A Biological Marker of Exposure to SO2:

S-Sulfonates in Nasal Lavage Fluid

H+ –SO2 + H2O + HSO3

– –HSO3 + –S –S –S–SO3

– ––S–SO3 CN– HSO3

OH

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1

V

CO

Carb

oxy h

emo

glo

bin

LeadB

loo

d lead

so

ft tissue lead

Urin

ary Pb

reflect recen

t lead exp

osu

resP

lasma P

b

Bo

ne P

b

Hair P

b

reflect lon

g term

Pb

expo

sures

Teeth

Pb

H2 SN

o b

iom

arker of exp

osu

re fou

nd

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Particulate Matter

Carbon elemental organic (including PAH)

Metals

Markers

8-oxod G (in WBC)

1-hydroxy pyrene

Fibrinogen

Platelet counts

Total WBC

Particles in sputum macrophages

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Air Toxics

VOCs

· Offer many opportunities for chemical-specific biomarkers of exposures:

– Parent compound or metabolites in blood, urine or exhaled breath

– DNA or protein adducts

· Studies by Albertini on 1,3-butadiene illustrate good transitional study

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I

BIOMARKER RES,PONSES IN BUTADIENE- EXPOSED CZECH W·O'RKERS,:

EXPOSURE

EXTENSIVE EXTERNAL, EXPOSURE

ASSESSMENT

A TRANSITIONAL E.PIDEMIOLOGICAL STUDY

t+ ,,

INTERNAL DOSE

I

URJN - M1 & M2 MET ABOl!.lliES

____., BIOLOGICALLY EfFECTNE DOSE

l HSIOGLOBIN HBVall & THBVal .ADDUCTS

1---tJ,

,, EA!RLY' I

BIOLOG1CAL

i---+ EFFECTS,

HP.RT MUTATIONS .. Autar.adlo,graphy • Cloning

I

MUTATIONAL SPECTRA CYTOGENETICS

- AbenatilOfla: MSCE - FISH

~LTERED STRUCTIJRE

FUNCTION

DIRECTl:ON' OF VALl1:DATIOiN

M1 :• 1,2:-DlhydrOK~N-acetylc:ysteln,t)-ButeM Ma • 1.ffydro:llCy.2---(N-A.c.tytcysite: n)4)-3-Butane HBVal = N--(2~ydrm:y-3-Butenyl)V lne THBVal • N-(2,311-'.Trlhyd'roxybulyl)Vallne FISH = FluorescenQ In· sit!:, Hybridl'ailon

CYP2E1 = A P450 Enf)TI'le GST 111 Glutatlt ~T,ran1feraae EH • Epoxlde HydroliH ADH • Af.cohol DehydrogeHH

& rt, CILINICAL DISEASE

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@ GLUTADHONE CONJUGATES

' IIUCONALDEHYDE

H

◄ [~ --..... _ .. 0

BENZOQU NONE

...

BENZENE OXIDE

OH

... H

HYDROQIHNONE

~

' OH

© PHENOL

I

OH

@(OH

"' CATECHOL

/ GWCURONIDE AND SULFAT!

CONJUGATES

OH

OH

0

H IIUCONIC A.CID

OH

TRIHY:DROXY BENZEN1E

Metabolic Pathway for Benzene

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· Metals

– Markers of exposure are the metals in blood, urine, hair and toe or fingernails.

· PAHs

– Urinary 1-hydroxypyrene

– Total bulks DNA adducts by 32P-postlabeling technique

· Dioxin-like compounds

– Compounds in fat

· Mixtures

– Need signature markers for specific sources of pollutants

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· Proteomics and Genomics

– Exciting new tools that may provide excellent biomarkers in the future

– Currently we are still learning how to interpret our findings

· Sensitive Subpopulations

– Polymorphisms can be valuable biomarkers

– Examples:

· NA D(P)H: quinone oxidoreductase: enzyme responsible for reducing reactive quinones to less reactive hydroquinones

· Glutathione-S-transferase M1: enzyme required for detoxication of many electrophilic metabolites

· DNA repairs enzyme