Biomarkers of exposure and other things

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Biomarkers of exposure (-and other things)

• Håkan Tinnerberg
• Occupational and environmental medicine
• Lund University Hospital

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Occupational and Environmental Medicine

• Investigate correlations between work-related
  disease and occupational exposure for patients
• Perform research about occupational or
  environmental exposure and disease
• Education/information
• University
• Unions
• Ocuppational health care / primary care

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Occupational Hygiene

• Perform exposure assessment
• Indivdual patients (measurements gt preventive
  measures)
• Epidemiological studies
• Environmental exposures
• Risk assessments
• Consultation
• Research
• Education

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Outline of presentation

• Exposure exposure assessment
• What is a biomarker
• What do you demand from a biomarker
• Instrumentation
• Biomarkers used in practice 5 examples

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Environmental exposure

• can be defined as contact
• between a potential hazardous agent
• in a medium such as air, water or food and
• a surface on a person such as skin, airways or
  gastro-intestinal systemet

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Concentration

• of an agent in a medium means that
• the agent is in the medium with
• a certain concentration
• but there is no exposure
• to the agent if there is not
• a physical contact between the
• agent and the human

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Dose

• is the amount of an agent that really passes the
  border between the medium and the body

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Exposure control

• Method 1 external measurements
• Uptake via inhalation air measurements
• Uptake via gastro-intestinal systemet
  measurement of concentration in food or water
• Method 2 internal measurement
• Biological monitoring measure of some
  biological matrix such as blood or urine

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Exposure assessment
Response/Effect/ Disease
Exposure

• Personal exposure
• Dose-response relationships
• Assessment of risks associated with the exposure

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Exposure-response (antibodies)
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Exposure-response (symptom)
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What is a biomarker?

• A marker that measure a change in a biological
  system that is affected by an external
  environmental exposure (chemical, physical or
  biological)

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Biological monitoring
Exposure
Effect
Effective dose
Internal dose
Susceptibility or sensitivity
Specific methods
Unspecific methods
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Biomarker of exposure I

• Internal dose
• Measurment of a parent compound or its
  metabolites in a biological sample
• Specific method
• Analysis of agent or metabolite
• Most common

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Specific methods

• Examples of analysis
• Lead in blood as a biomarker of exposure to lead
• Mandelic acid in urine as a biomarker of exposure
  to styrene
• Hexahydrophthalic acid in urine as a biomarker of
  exposure to plastic chemicals
• In vivo methods to determine to concentration
• Lead in bone
• Cadmium in kidney

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Urine levels vs. air levels
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Urine levels vs. air levels
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U-2,4-TDA (µg/L)
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10
0
0
5
10
15
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A-2,4-TDI (µg/m3)
Sennbro et al Scand J 2004
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Occupational exposed vs. Controls reference
limit
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Lead in fingerbone X-ray fluorescense
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Biomarker of exposure I

• Internal dose
• Unspecific methods are used as indicators of
  exposure to a group of chemicals
• Thioethers in urine
• a measure of exposure to mutagenic or
  carcinogenic agents
• Diazo positive metabolites in urine
• a measure of exposure to aromatic amines
• Large individual differences due to e.g diet and
  smoking
• Only on group level - not on individuals

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Biomarker of exposure II

• Effective dose
• Methods that show direct or indirect the
  concentration of an agent where the agent is
  acting in the body

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Examples of methods monitoring the effective dose

• Protein-adducts in blood
• Stable
• Show a longer time of exposure
• e.g. exposure to CO or dichloromethane results in
  carboxyhemoglobin
• Integrated time measure since hemoglobin lives
  during 120 days
• HHPA-human serum albumin (allergy)
• DNA-adducts
• Genotoxic agents
• e.g. benso(a)pyren
• Shows the amount of the agent at DNA
• DNA is repaired all the time

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Protein adducts
In vivo
CO

Protein DNA
Conjugate
Hapten
Christian Lindh, Yrkes- och miljömedicin, Lund
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Biomarker of effect

• Measure of endogenous substances or parameters
  that indicate a biological change such as an
  alteration in enzymes in tissues or body fluids
• The effects are reversibel and not dangerous
• Example
• Analysis of delta-aminolaevulinic acid
  dehydratase (ALAD) activity as a measure of lead
  exposure

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ALAD activity and lead in red blood cells (RBC)
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Biomarker of susceptibility or sensitivity

• Markers that show an individual sensitivity to
  exposure to a chemical
• Depends on genetic factors such as the capacity
  of the body to detoxify agents
• CYP450 enzymes
• Capability of acetylation
• amines hydraziner
• Glutationtransferase
• couples a peptid to the chemical to increase the
  excretion
• Ethical problems?

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Example

• Acetylation of aromatic amines
• An individual with a slow acetylation
• have a larger risk of getting urinary bladder
  cancer
• but a lower risk of getting colorectal cancer

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Example isocyanate exposed
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Advantages with biological monitoring (BM) I

• Measure of the individual absorption since BM
  includes indiviual properties such as capacity of
  activation detoxification
• Includes uptake from all exposure routes
  inhalation, dermal, oral
• Take into account factors that can affect the
  uptake e.g.
• physical exercise - gives an increased pulmonary
  ventilation and increased uptake of chemicals
• skin damage

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Advantages with BM II

• Exposure is not constant
• BM gives an integrated exposure measure (with
  long half-life)
• Take into account the individual difference in
  handling of chemicals
• It is easy to collect many samples
• Workers often use personal protective equipment
  and their efficiency can be estimated with BM

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Advantages with BM III

• Gives a measure of exposure that is ended (e.g.
  accidents, unexpected exposures)
• Shows also the spare time exposure
• In some situations BM reduces the uncertainties
  produced by day-to-day exposure variability

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Disadvantages with BM

• Only a few occupational exposure limits for
  biomarkers exist in Sweden
• Pb in blood
• Cd in blood
• More knowledge is needed to interpret the results
• Reference limits
• Biological half lifes

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Matrixes that can be used to monitor exposure

• Blood (plasma serum)
• Urine
• Exhaled breath

• Hair
• Saliva
• Milk
• Faeces
• Fat
• Nail
• Sweat
• Semen

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Choise of biological sample depends on

• Point of time for sampling - biological half-life
  of the agent
• How invasive the method is
• The substance of interest and its characteristics
  (e.g. solubility, metabolism, biotransformation
  and excretion)
• Other factors also can influence
• age, sex, food/alcohol, smoking, diseases

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Biological half-lifes

• lt2 h not suitable as a biomarker
• 2-10 h sampling after work or the day after
• 10-100 h sampling at the end of the week
• gt100 h sampling is not critical

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Example of biological half-lives

• Organic solvents in blood minutes - hours
• Metabolites of organic solvents in urin hours
• Hemoglobin adducts 2 months
• Lead, cadmium, PCB months - years
• Lead in skeleton 10 years
• Cadmium in kidney 7 - 30 years

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Ethical aspects on biomarkers

• Sampling involves an invasion of the integrity
• Blood lt Urine lt Saliva lt Exhaled breath
• Sampling can involve a risk of infection
• for both donator and sampler
• How do you communicate the risk if the person is
  exposed?
• High air level Im exposed how do I protect
  myself?
• High biological level I have got it into my
  body how ill will I be?
• Genes?

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Demands on biological monitoring methods

• Specific
• Analytic specificity
• Metabolic specificity
• Source specificity
• Informative
• Give safe and quantitative information about
  exposure, dose or risk
• Better than the alternatives (air samples)
• Cost, information value

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Analytic specificity I

• measurements of the requested agent
• Sensitive method measure low exposures
• Simple, quick and cheap analytical methods
• Simple and safe sampling, no contamination
• Can be stored, stability

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Analytic specificity II

• e.g Hg measured with atomabsorption
  spectrophoto-metry (AAS) includes all Hg-compunds
  (and not only exposure to e.g. Hg-vapour)
• Difficult matrixes or separation problems during
  work-up gt complicated methods, expensive
  analysis. Choice of method is often a compromise
• elements, heavy metalsICP, AAS only minor
  separation problems but at expense on analytic
  specificity
• volatile agents use the simplest matrix
  exhaled air
• polar agentsuse the simplest matrix saliva or
  urine

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Metabolic specificity

• the measured agent or metabolite must originate
  from the agent that is studied
• e.g hippuric acid is not a metabolite specific
  for toluene
• Important with knowledge of the metabolism

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Source specificity

• the measured agent monitor the relevant
  exposure
• Hippuric acid can also indicate exposure to
  benzoic acid through e.g .the food (lingonberry).
  Hippuric acid can be used as a measure of toulene
  exposure in work environments if the exposure is
  high and the intake of benzoic acid through the
  diet is low.
• Hg measured with AAS is a good measure of Hg(0)
  if the exposure is high, but poor if the exposure
  is low due to methyl-Hg from the diet.
• Important with knowledge of exposure sources and
  metabolism

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Validation dilemma

• If there are several exposure routes or if the
  exposure is unclear or intermittent
• biological monitoring is optimal
• hard to establish relationship with exposure
• Uptake only via inhalation, constant exposure
• easy to establish relationship with exposure
• biological monitoring is not so interesting

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Air sampling and biological monitoring are
complements to each other
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Choice of analysis

• Sensitivity/Specificity/Cost
• GC (e.g. FID)
• LC (e.g. UV)
• Mass spectrometry (MS)

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What is a mass spectrometer (MS)?

• It is an instrument that can separate charged
  molecules/atoms after their mass-to-charge ratio
  (m/z)
• Example of instrument
• GC-MS, GC-MS/MS
• LC-MS, LC-MS/MS
• ICP-MS

Ion source
Detector
Separator (e.g. Quadrupole)
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How are the molecules charged?

• To separate the molecules after their mass they
  must be charged. This process takes place in the
  ion source. Each molecule can take up several
  charges, both positive and negative.

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Exampel of interface and ion source of LC-MS/MS

• Ion spray Electrospray Ionisation (ESI)
• Used for polar agents

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What happens with the charged molecular ions?

• The formed molecular ions are accelerated with an
  electric field to the mass analyser. You can
  select to detect either positive or negative
  ions. The molecules that are not ionised are
  evacuated.

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Separation in Quadrupole

• Combined radio frequency field and voltage field
• Ions in a field bend depending on the strength of
  the field and the ion mass-to-charge ratio
• Works as a mass filter
• When the ions reaches the detector (e.g. electron
  multiplikator) you get a mass spectra

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LC-MS/MS
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ICP-MS
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How is the agent quantified?

• Choice of suitable internal standard (usually
  deuterium labelled)
• Properties of the molecule
• Optimising of GC- or LC-method
• Optimising of MS-method
• Standard curve used for quantification

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Routine analysis performed at Occupational and
Environmental Medicine

• Bly i blod Cyklofosfamid Ifosfamid i urin
• Kadmium i blod Mandelsyra och fenylglyoxylsyra
• Kadmium i urin i urin
• Kvicksilver i blod och plasma Metaboliter av
  isocyanater och
• Kvicksilver i urin aminer i plasma eller urin
• Mangan i urin Pesticider i urin
• Nickel i urin TTCA i urin
• Syraanhydridmetaboliter i urin
• PCB CB-153 och /eller p,p-DDE i serum
• HCB i serum eller plasma
• Kotinin i urin
• 1-Hydroxypyren i urin
• Specifika antikroppar, IgE mot isocyanater i
  serum
• Specifika antikroppar, IgE mot organiska
  syraanhydrider i serum

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Biomarkers used in practice I

• Styrene is an organic hydrocarbon that is used as
  a solvent in the manufacturing of fiber
  reinforced polyester plastic

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• In a plastic boat plant workers are exposed to
  styrene when they spray, laminate and paint.
• Inhalation
• Skin exposure
• To asses the exposure of the workers urine is
  collected during the last 3 hours of the work
  shift

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• Styrene is metabolised in the body and its
  metabolites is excreted in the urine
• No endogen formation of the metabolites
• Exposure to ethylbensen also involves excretion
  of mandelic acid, therefore is both mandelic and
  phenylglyoxylic acid measured
• LC-UV method
• The values should not exceed 320 mmol/mol creatine

Mandelic acid
Phenylglyoxylic acid
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Biomarkers used in practice II

• Anticancer drugs are common drugs used in the
  health care. These drugs can be toxic and
  carcinogenic. Health care workers handle these
  drugs during drug preparation, during
  administration to patients and during care taking
  of treated patients.

Skin inhalation
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• How do we assess the exposure to antineoplastic
  drugs?
• Half-life is ?4 h
• Urine is sampled during the 4 last h of the work
  shift
• Cyclophosphamide in urine is used as a biomarker.
• LC-MS/MS method
• No background, all values above the LOD is
  exposure

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Biomarkers used in practice III
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Hg in blood nmol/ml
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Levels of Hg in blood (above) and urine (below)
for one subject. The yellow part is suspended
time.
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When starting up a new facility

• How should you survey the exposure during
  start-up?
• How should you survey exposure when production is
  under control?

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IV. Pesticides in the general Swedish population

• The aim was to investigate the Swedish
  populations exposure to some pesticides
• 100 persons participated
• Collected 24 h urine during spring or autumn
• Questinnaire about diet and lifestyle
• Analysis of 2,4-dichlorophenoxy acetic acid
  (2,4-D) 2,4,5-trichlorophenoxy acetic acid
  (2,4,5-T)

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• ()
• Urine collected during
• spring 54
• Women 57
• Age gt40 years 65
• Comsume organic 45
• Consume exotic fruit 54

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Results

• Low amounts of pesticides were found in the urine
  (ng/ml)
• mean median range
• 2,4-D 0,17 0,10 lt0,05-3,0
• 2,4,5-T lt0,1 lt0,1 lt0,1-0,3

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Biomarkers used in practice V

• In our environment there are several man-made
  persistent organochlorine compounds such as
• DDT (dichlorodiphenyltrichloroethane) was used
  as an insecticide for several decades. Was banned
  in Sweden in the 1970s.

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• PCBs (polychlorinated biphenyls) were used as
  coolants and insulating fluids for transformers
  and capacitors, pesticide extenders, cutting
  oils, flame retardants, hydraulic fluids, paints
  etc
• In Sweden PCB was banned in 1972 due to the high
  toxicity and due to bioaccumulation in animals.

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Exposure to PCB DDT

• Exposure to PCB is measured with the biomarker
  CB-153
• Exposure to DDT is measured with the biomarker
  p,p-DDE
• Serum
• GC-MS/MS
• Concentrations in the general population
• CB-153 up to 1,9 ng/ml
• DDE up to 4,2 ng/ml

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Has a high intake of fish from the Baltic sea an
effect on mens and womens health?
Lars Rylander, Yrkes- och miljömedicin, Lund
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What is in fish?
-

Proteins Polyunsaturated fatty acids Trace
compounds (selen) . . .
Methyl mercury Persistent organochlorine pollut
ants . . .
-

-

-

-
-


Lars Rylander, Yrkes- och miljömedicin, Lund
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Persistent organochlorine compunds (POC)

• PCB
• DDT
• Unwanted by-products in industrial
  processes
• PCDD (dioxines)
• PCDF (furans)

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Important properties for POC
High fat-solubility and persistent Long
half-lives (5-10 years) Easily taken up in the
body by the gastro-intestinal system Mainly
accumulated in the liver and adipose tissue
Cross easily the placental-barrier and the
blood-brain barrier and is excreted in the breast
milk Hormone disruptive properties
Lars Rylander, Yrkes- och miljömedicin, Lund
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Which concentration is present in the fatty fish
from the Baltic sea?
7.8
Dioxine levels in herring (pg/g weight)
9.0
2.6
7.7
The Baltic sea is one of the most POC
contaminated area in Sweden
3.4
From Bergqvist m fl 1989
8.1
Lars Rylander, Yrkes- och miljömedicin, Lund
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Relationship between intake of fish from the
Baltic sea and dioxine in blood
Dioxine TEQ (pg/g fat)
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27
19
From Svensson et al. 1991
0
Daily
1 times/week
Lars Rylander, Yrkes- och miljömedicin, Lund
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Fish intake (meal/month) among the Swedish
fishermen and among men from the general
population
From Svensson et al 1995
Lars Rylander, Yrkes- och miljömedicin, Lund
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Concentrations of dioxine in blood from Swedish
fishermen and from men in the general population
pg/g fat
pg/g fat
From Svensson et al 1995
Lars Rylander, Yrkes- och miljömedicin, Lund
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Suitable groups to study health effects on!
Fishermen and their families from the Swedish
WEST COAST.
Fishermen and their families from the Swedish
EAST COAST.
Different exposure to persistent organochlorine
compounds. Socioeconomic status of the groups
is similar
Lars Rylander, Yrkes- och miljömedicin, Lund
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Studied health effects in the fishermen studies

• - MORTALITY
• CANCER
• REPRODUCTION
• Time to pregnancy
• Miscarriages
• Reproductive outcome
• Growth
• Male fertility
• OSTEOPOROSIS
• DIABETES

Lars Rylander, Yrkes- och miljömedicin, Lund
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Studied health effects in the fishermen studies

• - MORTALITY
• CANCER ()
• REPRODUCTION
• Time to graviditet
• Miscarriages
• Reproductive outcome
• Growth
• Male fertility ()
• OSTEOPOROSIS
• DIABETES

Lars Rylander, Yrkes- och miljömedicin, Lund
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Diabetes
Fraction with diabetes ()
Men
Women
Women
Men
(From Rylander et al 2005)
p,p-DDE
CB-153
Lars Rylander, Yrkes- och miljömedicin, Lund
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