BIOASSAYS CONCEPTS, PRINCIPLES AND APPLICATION FOR PESTICIDES - - PowerPoint PPT Presentation

BIOASSAYS

CONCEPTS, PRINCIPLES AND APPLICATION FOR PESTICIDES

10TH ANCAP SUMMER SCHOOL UNZA AUGUST 2013

Robinson H. Mdegela Department of Veterinary Medicine and Public Health

• P. O. Box 3021

Morogoro

SOKOINE UNIVERSITY OF AGRICULTURE FACULTY OF VETERINARY MEDICINE

Who am I?

ENVIRONMENTAL HEALTH WATER SHORTAGE, HYGIENE AND SANITATION

WILDLIFE VETERINARIAN

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WILDLIFE VETERINARIAN

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AQUATIC ECOSYSTEMS

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Course content

 Introduction

 Definition, concepts and practices

 Bioassay  Bio-indicators for pesticide analysis  Concepts and principles of biomarkers  Classification of biomarkers  Potential biomarkers for pesticide studies  Chemical analytical methods and biomarkers for

pesticides

 Biotransformation enzymes and products biomarkers  Biomarkers of Oxidative Stress in Aquatic

Organisms and Risk Assessment

 Advances in Biomarker Working Groups in Africa

Practical

 Practical  Practical exposure on bio-indicators for pesticides  Collection and processing of samples for pesticide

biomarker studies

 Analysis of samples for pesticide biomarkers  Esterase activity biomarkers

Par arace acelsus 1493 lsus 1493 – 1541 1541

All things are poisons (destructive) and nothing is without poison (destruction), only the dose (extent) permits something not to be poisonous (destructive)

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PHD SAMPLE COLLECTION AND ANALYSIS

Gills Liver EROD GST/UGT EROD Bile FACs HM OCs Muscles

Brain, eyes Blood

AChE

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Blood

VTG

LEVELS OF STRUCTURAL AND FUNCTIONAL ORGANIZATIONS

 Subatomic particles  Atoms  Molecules  Complex molecules  Organelles  Single cell organisms  Colonies of single cell organisms  Complex organisms  Populations  Communities  Ecosystems

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Category of biomarkers Biomarkers Biotransformation enzymes Phase I - Cytochrome P4501A (CYP1A) Phase II – Uridine diphosphoglucuronosyl transferase (UGT) Phase II – Glutathione S-transferase (GST) Biotransformation products Polycyclic aromatic hydrocarbons (PAHs) Fluorescent aromatic compounds (FACs) Neuromuscular parameters Acetylcholinesterase (AChE) Stress proteins Metallothionein (MT) Haematological parameters Packed cell volume (PCV) Haemoglobin concentration (Hb) Morphological/gross indices Condition factor (CF) Liver somatic index (LSI) Gonadosomatic index (GSI)

Development and validation of Biomarkers

Molecules Cells Organs Individuals Population Ecosystem

Biological levels

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The principle scheme of responses in organisms towards the detrimental effects of pollutant exposure

increased exposure (dose and time) Early warning signals

• f biomarker responses

Homeostasis normal range

• f biomarkers

Response Observable detrimental effects No

• bservable

detrimental effects impaired reproduction Increased susceptibility to diseases Reduced lifespan

16

Exposure Kinetics in tissues Binding to Receptors (mol) Biochemical Responses (cell) Physiological Alterations (org) Effect on individuals Effect on Pop, comm, & ecosyst

Seconds to minutes Minutes to hours Days to weeks Weeks to months Months to years

Ecological relevance

• f data

Easy of

• btaining

data Time to complete research Present status of knowledge

Ef Effect ect of

• f a

agent gent exposur xposure a e at dif t differ erent le ent levels els

• f
• f biolo

biologica gical l or

• rgan

ganiza ization tion

17

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Integrated study strategy

Field studies Semi- Field studies Laboratory- experiments Evidence control of variables Relevance to natural populations none moderate high Little (extrapolation) moderate high correlation causal

Environmental monitoring methods

 There are five environmental monitoring methods

 Chemical  Bioaccumulation  Biological effect  Health  Ecosystem based monitoring methods

 Biomonitoring - monitoring using living organisms

Bio-monitoring

Biomonitoring Bioindication Bioassay

Why Bioassays?

 While detection of pollutant by chemical analysis

can suggest toxic potential, chemical analysis alone is insufficient to provide a realist appraisal of actual toxicity

 No instrument has yet been devised to measure

toxicity

 Detection and quantification of chemical can be

measured by instruments

 Only living organisms/materials can be used to

measure toxicity

 Unfortunately, priority chemical lists contain only

those contaminants whose individual toxicity has been well established

 Since most chemicals do not appear singly in the

real world, these lists are very unrealistic

 Chemical analysis alone may be misleading and

may ignore the real toxicants

 Bulk chemical analysis does not account for grain

size, organic carbon content, pH, redox potential, that all have impact on biological effects

 Bioavailability is also difficult to factor in when

chemical analysis is carried out

 Bioassays must be used to determine the actual

adverse impact

 Bioassays have the potential to direct the chemical

analysis to be performed

BIOASSAY

 Assay methods in which the quality of the

environment and the influence of factors (acting alone or in conjunction with others) are judged by the survival and behavior of

• rganisms (test objects) placed in this

environment

 The toxicity of pollutants is most often

evaluated using bioassays under controlled laboratory conditions

Criteria for Bioassays

 Widely accepted by scientific community  Sound statistical basis  Repeatability in different laboratories

(standardized with well defined protocol)

 Realistic range of chemical concentrations

 Realistic durations of exposure  Quantifiable through graphical interpolation or

statistical analysis

 Have field predictive capabilities of similar

• rganisms

 Useful for risk assessment  Economical and easy to conduct  Sensitive enough to detect and measure the effects  Realistic as possible to design

Requirements for methods of bioassay

 Sensitivity  Universality in terms of physical, chemical and

biological effects that are evaluated

 Simplicity  Availability

Examples of application of bioassay

 Establishing regulatory requirements (e.g. water, food,

environmental qualities)

 Ecological monitoring (e.g. sewages discharges,

pesticides etc)

 Environmental monitoring  Environmental impact assessment (new technologies,

treatment facilities, construction and modernization of national economic projects etc)

 Assessment of aquatic ecosystems

Bioassay

 Use living organisms as early warning systems or

sentinels

 Impact does not mean permanent harm but requires

closer inspection

 Must use suite of environmentally related biota  Provide useful insight into potential harm to humans

and to other unintended subjects

Suite of biota

 Use of suite of biota to assess the impact

 Sub-acute, behavioral  Acute, short term  Chronic, long term

 Reproductive  Cancer

REQUIREMENTS FOR BIOASSAYS

Bioassays Bio-indicators Markers (Biomarkers)

Bio-indicators

 A bio-indicator is an organism giving information on the

environmental conditions of its habitat by its presence or absence and its behaviour

 Thus effects at the physiological level are not included in this

definition

 The indicator concept states that the continued presence of

certain species is an indication of the existence of a unique set

• f acceptable environmental conditions, whereas its absence

would indicate the lack of appropriate environmental conditions

 Ecological indicators are parameters describing the

structure and functioning of ecosystems, for example species diversity, population dynamics and nutrient cycling rates

Biomarker

A biomarker is any change that occurs in response to exposure to stressors (xenobiotics, diseases and physical change in the environment including temperature and salinity) that indicates the adaptive responses of an

• rganism beyond the normal range

Enzyme content/activity Specific mRNAs DNA adducts Structural and functional alterations of organelles, proliferation of endoplasmatic reticulum, chromosomal aberrations and micronuclei formation. Histopathological alterations. Liver somatic index. Gonadosomatic index. Immune parameters. Reproductive parameters Physiological parameters. Body condition index. Scope of growth. Fertility. Maturation retardation. Gene frequency. Age structure. Size distribution. Diversity indices. Functional parameters.

Example of biomarkers Molecules Cells Organs Individuals Population Ecosystem Biological level

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Definition of Bioassay

 Bioassay is defined

As the estimation of the potency of an active

principle in a unit quantity of preparation

Detection and measurement of the concentration

• f the substance in a preparation using biological

methods (i.e. observation of pharmacological/toxicological effects on living tissues, microorganisms or immune cells or animals

 Is an assay designed to analyse any compound by

use of a suitable biological system like animals, tissues, microbes etc

 Is an estimation or determination of concentration or

potency of a physical, chemical or biological substance (agent) by means of measuring and comparing the magnitude of the response of the test with that of standard over a suitable biological system under standard set of conditions

 In the analysis the response produced by the test

compound is compared with that of the standard sample using a biological system

Principle of bioassay

 The bioassay compares the test sample with the

same Internationally applicable standard substance

 It determines the quantity of test sample required to

produce an equivalent biological response to that

• f standard substance

 Standard samples are accepted by expert

committee at international level and they represent fixed units of activity

Indications for Bioassay

 Bioassays, as compared to other methods of assays

(e.g. chemical or physical assay) are less accurate, less elaborate, more laborious, more troublesome and more expensive

Indications for Bioassay

 Active principle is unknown  Active principle cannot be isolated  Chemical method is either  Not available  If available, too complex  Insensitive to low doses  Unknown Chemical composition  Chemical composition is different but with the same

action

Principles of Bioassay

 Active principle to be assayed should show the same

measured response in all animal species

 The degree of biological

(pharmacological/toxicological) response produced should be reproducible under identical conditions

 The reference standard must owe its activity to the

principle for which the sample is being bioassayed

 Activity assayed should be the activity of

interest

 Individual variations must be minimised /

accounted for

 Bioassay might measure a different aspects of

the same substance compared to chemical assay

Types of Bioassays

 Two types of Bioassays  Quantal Assays: Direct endpoint

 Elicits an „All or None‟ response in different animals

 Calculation of LD50 in target test organism (mice, rats,

fish, plants)

 Graded Response Assays [mostly on tissues]

 Graded responses to varying doses  Unknown dose response measured on same tissue

Graded Response Assay

 In these assays, as the dose increases there is an

equivalent rise in response

 The potency is estimated by comparing the Test

sample responses with the standard response curve

 The graded dose response relationship relates the

size of the response to the substance in a single biologic unit

 As the dose administered increases the

pharmacological response also increases and eventually reaches a steady level called the ceiling effect where there will be on further increase in response even with an increase in dose

 The graded dose response curve is obtained by

plotting a graph with dose on the X-axis and response on the Y-axis

 It is usually sigmoid in shape however to be useful in

bio assay the log dose response curve (almost a straight line is used)

 Conc. of unknown = Threshold dose of

standard/threshold dose of test x Conc. of standard

Quantal: End Point Assay

 As the name indicates, the threshold dose of the

sample required to elicit a complete or a particular pharmacological/toxicological effect is determined and compared with standard

 The determination of LD50 (LD=Lethal dose) or

ED50 (ED= effective dose) is done by this method

Methods of Bioassay con1

 Graded Response Assays [ Direct comparison on same

tissues]

Interpolation: Conc. of unknown is read from a

standard plot of a log dose response curve of at least 4 sub maximal concentrations

Matching / Bracketing: Const dose bracketed with

varying doses of standard till exact match is

• btained

Used when test sample is too small Inaccurate & margin of error difficult to estimate

Multiple Point Assays

3 point assay [combines active principles of

matching with interpolation]

4 point assay [combines active principles of

matching with interpolation]

3 point assay [2+1 dose assay]

 Fast & convenient  Procedure [Eg Ach bioassay]

 Log dose response [LDR] curve plotted with

varying conc of std Ach solutions and given test solution

Select two std doses s1& s2 [ in 1:2 dose

ratio] from linear part of LDR [ Let the corresponding response be S1, S2]

Choose a test dose t with a response T

between S1 & S2

 Record 4 sets data [Latin square: Randomisation reduces

error] as follows

 s1

s2 t

 t

s1 s2

 s2

t s1

 s1

s2 t

 Plot mean of S1, S2 and T against dose. Calculate  Log Potency ratio [ M ] = [ (T –S1) / (S2-S1) ] X log d

[d = dose ratio]

4 point assay [2 +2 dose assay]

 Procedure [Eg Ach bioassay]

Log dose response [LDR] curve plotted with

varying conc of std Ach solutions and given test solution

Select two std doses s1& s2 from linear

part of LDR [ Let the corresponding response be S1, S2]

Choose two test doses t1 & t2 with response

T1 &T2 between S1 & S2 ; Also s2/s1 = t2/t1 = 2

Record 4 data sets [Latin square: Randomisation

reduces error]

s1

s2 t1 t2

s2

t1 t2 s1

t1

t2 s1 s2

t2

s1 s2 t1

Plot mean of S1, S2 and T1, T2 against dose.

Calculate

Log Potency ratio [M] = [ (T1 –S1 + T2 –S2) /

(S2-S1 + T2-T1) ] X log d [d = dose ratio]

Measurements

 Dose-response relationships – quantifiable through

graphical interpolation or statistical analysis

 Effective concentrations (50) – useful for risk

assessment

 Biochemical response to stress – Haemoglobin

production

 Daphnia magna  Limnar manda (duck weed)  Chironomus tenstans

Advantages & Uses of bioassay

 Determine the concentration and the potency of the

sample

 Used to standardize drugs, vaccines, toxins or

poisons, disinfectants, antiseptics

 Determine the specificity of a compound  Certain complex compounds like vitamin B-12 which

can't be analysed by simple assay techniques can be effectively estimated by bioassays

 Sometimes the chemical composition of samples are

different but have same biological activity

 For samples where no other methods of assays are

available

Types of Assays

 Chemical Assays

 Spectrophotometry,  Spectrofluorimetry  Chromatography

 Immunoassays  Microbiological assays

Bioassay systems and techniques

 The bioassay systems vary based on the biological

system used

 Animals (mouse, rat, guinea pig, rabbits etc)  Plant bioassay (using plant constituents to evaluate a

sample like(haemolytic activity) microbiological

 Cell based assay (using microbes like bacteria, fungi or

cultured cells for anti biotic compound screening etc)

In vivo techniques

 These techniques employ a living animal

recommended for the purpose of assay

 The techniques aims to study the biological effect or

response of the compound under screening in a living system directly

 Ex: By use of rodents, rabbits etc.

Ex vivo techniques

 These techniques employ a tissue or cells of

recommended living system to study the effect of compound under test in suitable conditions within the stipulated time of organ survival outside the body

 Eg The methods described in the videos employ a

living tissue of an animal in an apparatus to study the contractile effect of drugs

In vitro techniques

 These techniques employ a cell culture of

recommended biological system to study the effect

• f compound under standard condition not similar to

that of living environment.

 The cell culture survives by utilization of the nutrition

in the media

 Ex: use of stem cells, cell culture, microbes (bacteria) etc

Key points for bioassay

 Do thorough literature search  Utilize, build on existing information  Identify the gaps  Use living biota to assess the impact  Use suite of ecologically relevant biota  Know the details of biota of your choice  Multi-disciplinary experts is essential

The purpose of bioassay

 The purpose of bioassay is to ascertain the potency

• f a drug and hence it serves as the quantitative

part of any screening procedure (Research).

 Other purpose of bioassay is to standardize the

preparation so that each contains the uniform specified pharmacological/toxicological activity

 In this way, it serves as a pointer in the Commercial

Production of drugs when chemical assays are not available or do not suffice.

 From the clinical point of view, bioassay may help in

the diagnosis of various conditions

Biomonitoring Bioindication Bioassay

Bio-indicators

 A bio-indicator is an organism giving information on the

environmental conditions of its habitat by its presence or absence and its behaviour

 Thus effects at the physiological level are not included in this

definition??

 The indicator concept states that the continued presence of

certain species is an indication of the existence of a unique set

• f acceptable environmental conditions, whereas its absence

would indicate the lack of appropriate environmental conditions

 Ecological indicators are parameters describing the

structure and functioning of ecosystems, for example species diversity, population dynamics and nutrient cycling rates

Bio-indicators

 Organisms, chemical markers or biological

processes whose change indicates the altered environmental conditions

 While direct sampling provides information about

the conditions at the time of sampling only, bio- indicators provide a time-integrated estimate of past environmental conditions

 Time scales can vary depending on the actual

indicator chosen

 They therefore serve to detect changes in the

environment even when measurements are not available or are too variable

 For example, a reduced abundance of large

foraminifera (marine micro-organisms) or the darkening of coral pigmentation may indicate that a reef has been exposed to poor water quality for several weeks or months

 Bio-indicators can also provide information on the

harmful effects of contaminants at biochemical, molecular and cellular levels and can act as an early warning system for larger-scale effects

 For example, reduced photosynthesis in a plant or a

coral may indicate stress from exposure to herbicides

BIOMARKERS

Analytical approach

 Chemical analysis  Biomarkers

Definition

 Biomarker

 Any change that occurs in response to exposure to stressors

(xenobiotics, disease causing agents and physical change in the environment including temperature and salinity) that indicates the adaptive responses of an organism beyond the normal state

 Examples

 Induction of heat shock proteins which are triggered in

response to raised temperatures

 Reduction in fecundity – due to environmental pollutants  Plasma protein differentiating live and degenerated

Taenia solium cysts

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Biomarker concept

 Biomarker  biological response measured to indicate exposure,

effect/response or susceptibility

 Biomarkers  Allow the identification of pollutants and their potential risks to the

environment

 Give additional information that cannot be obtained from

chemical analysis

 May show integrated effects of chemical mixtures

Attributes of Biomarkers

 Biomarkers are used to identify biological changes due to toxic

chemicals and as part of integrated approach in the assessment of environmental health

 They increase the ability to identify the long-term risks due to toxicant

exposure in particular due to risks of developing cancer

 The field of biomarkers identifies early markers of toxicity in the field of

environmental toxicology or Ecotoxicology

 The ultimate aim of using biomarkers is to identify problems as early as

possible, thus avoiding adverse effects on whole populations and communities

 The goal of using biomarkers is to identify the adverse effects of

chemical contaminants at the lowest level of biological organization so as to avoid toxicological problems at later stages which are both more difficult to identify and to correct

 It is possible to identify biomarkers at all levels of biological

• rganization extending from molecular of an individual to the ecosystem

The principle scheme of responses in organisms towards the detrimental effects of pollutant exposure

increased exposure (dose and time) Early warning signals

• f biomarker responses

Homeostasis normal range

• f biomarkers

Response Observable detrimental effects No

• bservable

detrimental effects impaired reproduction Increased susceptibility to diseases Reduced lifespan

81

Exposure Kinetics in tissues Binding to Receptors (mol) Biochemical Responses (cell) Physiological Alterations (org) Effect on individuals Effect on Pop, comm, & ecosyst

Seconds to minutes Minutes to hours Days to weeks Weeks to months Months to years

Ecological relevance

• f data

Easy of

• btaining

data Time to complete research Present status of knowledge

Ef Effect ect of

• f a

agent gent exposur xposure a e at dif t differ erent le ent levels els

• f
• f biolo

biologica gical l or

• rgan

ganiza ization tion

82

Important criteria for biomarkers

 Ideally, biomarkers should be:  The assay to quantify the biomarker should be sensitive, reliable

and relatively cheap and easy to perform

 The baseline data (concentration or activity) of the biomarker

should be well defined in order to distinguish between natural variability (noise) and contaminant-induced stress (signal)

 The basic biology and physiology of the organism should be

known so that sources of uncontrolled variation (growth and development, reproduction, food sources) can be minimized.

 The confounding factors (intrinsic and extrinsic) to the

biomarker response should be well established

 It should be established whether changes in biomarker

concentration are due to physiological acclimation or to genetic adaptation

 The biomarker response should correlate with the „health‟ or

„fitness‟ of the organism

 It should preferably be non-invasive or non-destructive to

allow or facilitate monitoring the effects of environmental pollution in protected or endangered species

Use of Biomarkers

 The use of biomarkers measured at the molecular or cellular level has

been proved to be of great value as a sensitive early warning tool for measuring biological effects in environmental quality assessments

 The most compelling reason for using biomarkers is that they can give

information on the biological effects of pollutants, rather than a mere quantification of environmental levels

 Biomarkers applied both in laboratory and field studies can provide an

important linkage between the laboratory toxicity and field-based assessments

 Using field samples, biomarker data may provide an important index of

the total external load that is biologically available in the exposure environment

 The merits of using biomarkers are summarized as follows:  They can demonstrate the interactions that have taken place

between contaminants even at sub-lethal concentrations and effects in the organisms

 They can detect the presence and/or effects of both known and

unknown contaminants

 They have the ability to allow early detection of effects from

contaminants, thus providing an opportunity for remedial or preventive action to be taken, before irreversible environmental damage with ecological consequences occurs

 They provide a temporal and spatial integrative measure of

bioavailable pollutants

 They can help to establish the important routes of exposure if

applied to species from different trophic levels and thus can aid in prioritizing monitoring schemes and strategies for intervention or remediation

 They can detect exposure to, and the toxic effects of parent

compounds and metabolites of readily metabolized and eliminated contaminants such as PAHs and organophosphates

 They can integrate the toxicological interactions of mixtures of

various pollutants and give an expression of cumulative effect at molecular, cellular or tissue targets

Classification

 Three major groups that include:

 Biomarkers of exposure  Biomarkers of effect or response  Biomarkers of susceptibility

Biomarkers of exposure

 A biomarker of exposure refers to an exogenous substance or its

metabolite, or the product of an interaction between a xenobiotic agent and some target molecule or cell, which is measured in a compartment within an organism

 Biomarkers of exposure can be used to confirm and assess the exposure

• f an individual or population to a particular substance (group), by

providing a link between external exposure and internal dosimetry

 Biomarkers of exposure are divided into markers of internal dose and

makers of effective dose

Markers of internal dose

 Give an indication of the occurrence and extent of

exposure of the organism and thus the likely concentration

• f the present compound or the metabolite at the target

site

 Markers of internal dose are useful in establishing the

dose of a compound which has been absorbed in ecological studies and in human studies when they provide information about long-term carcinogen exposure

Markers of effective dose

 Gives an indication of the true extent of the exposure of

what is believed to be the target molecule, structure or cell

 Chemicals can bind covalently to cellular macromolecules

such as nucleic acids and proteins which may be the target molecule for the compound

 These are called adducts and can be measured in tissues

• r body fluids

 Both markers are preferable to measuring external levels of

the compound in question such as in workplace since they take into account the biological variations in absorption, metabolism and distribution of the compound in an individual

 Due to many inter-individual differences in the rate and route

• f metabolism of a compound and also the accessibility of the

target, any measurement of the internal dose and effective dose will be different

 Hence, the effective dose at the target site is the preferred

measurement to internal dose

Biomarkers of effect or response

 These are biomarkers that have been used most widely and routinely  A biomarker of effect refers to a measurable biochemical, physiological

• r other alterations within an organism that, depending upon the

magnitude, can be recognized to be associated with an established or possible health impairment or disease

 Biomarkers of effect can be used to demonstrate either preclinical

alterations or adverse health effects due to external exposure and absorption of a chemical

 They can be grouped into several categorises such as metabolic,

pathological, clinical, behavioural etc.

 A metabolic lesion may or may not be the result of

altered pathology, rather it may predict or precipitate a pathological lesion, making them potential early warning markers such as elevated glucose levels in diabetic patients

 There is also a growing interest in the use of and

identification of non-invasive biomarkers rather than invasive biomarkers

 Non-invasive biomarkers allow routine sampling and

also overcome the ethical issues

 Thus biomarkers identified in urine, breath or saliva

are more useful/preferred than those measured in blood

Behaviour and clinical markers

 They are simplest biomarkers which are sometimes referred as

gross indices

 They include changes in body weight, urinary output, food

consumption, population size and general behaviour

 These changes may signify a change in the biochemistry or

pathology of an individual

 These changes may be the first indication that there is a problem in

the environment

 In toxicology trials, body weight can be very sensitive of adverse

effects of a compound

Pathology

 Invasive markers of tissue damage that cover an array of pathological

techniques including gross pathology, organ weight and histology using light and electron microscopy

 Measurement of enzyme change using immunohistochemistry

Clinical chemistry/pathology

 Traditionally, fluids have been a source of biochemical

markers which are able to identify both site and severity

• f a lesion within the organism

 Elevate serum levels of enzyme which have leaked from

the damaged tissue

 Biochemical changes such as elevated bilirubin  Changes in biochemistry of urine and cerebrospinal fluid

Enzymatic changes (Induction /inhibition)

 Changes in enzyme activity can be used as

biomarkers of specific chemical exposure

 Organophosphate exposure – inhibition of blood

acetylcholinesterase

 Lead exposure – inhibition of serum aminoaevulinic

acid dehydrase (ALAD)

 As these biomarkers are believed to bespecific, the

degree of enzyme inhibition has also been used as biomarkers of “effective dose”

 Induction of specific enzymes e.g. cytochrome P450 isoenzymes

is an adaptive response to challenges from a wide variety of compounds including organochlorines, polycyclic aromatic hydrocarbons

 Direct measurements require tissue be sampled, although

urinary markers of CYP P450 activity such as excretion of D- glucaric acid in urine can be used as a non-invasive marker

 In the recent years it has been shown that the constituents of

breath are also potential source of CYP P450 generated metabolites which should also be used as biomarkers

Protein synthesis

 Some cellular proteins increase in response to external stressors  Heat shock proteins which were first identified as proteins which are

rapidly synthesized within minutes to hours following slight rise to temperature

 They include hsp 90, hsp 70 and hsp 60 (also called chaperonin and

ubiquitin)

 Metallothionein – increases in organisms exposed to heavy metals such

as cadmium

 Antibody poteins (IgG, IgM and IgA serum levels) are biomarkers of

exposure to antigens

Others

 Excretory products  DNA damage and gene expression  Tumour genes and tumour markers.

Biomarkers of susceptibility

 A biomarker of susceptibility refers to an indicator of an inherent or

acquired ability of an organism to respond to the challenge of exposure to a specific xenobiotic substance

 This includes genetic factors and changes in receptors which alter the

susceptibility of an organism to that exposure

 Biomarkers of susceptibility help to elucidate variations in the degree of

response to toxicant exposure observed between different individuals

 Any variation in the response of an individual to identical exposures may

represent some difference in susceptibility due to either the genetic makeup of the individual or variables and environmental influences such as diet or the uptake and absorption of the xenobiotic

 Metabolism

 The most important source of variability is often the

metabolism of the compound by the organism which may be genetically determined

 Genotype

 In several familial cancers, the presence of specific

genes has been identified and can be considered as markers of susceptibility

Type of biomarker Biomarker Specific example Stressor Exposure DNA adducts Styrene oxide-O6 guanine Styrene exposure Protein adduct N7-Guanyl-aflatoxin B1 Dietary aflatoxin B1 Exposure and effect (response) Enzyme inhibition Acetylcholinesterase inhibition Organophosphates Urinary metabolites Mercapturic acids Buta-1,3 diene,allyl chloride Effect (response) Plasma/serum enzymes Aspartate aminotransferase (AST) Xenobiotics causing necrosis Enzyme induction CYP P450 PAHs Stress proteins Hsp 90, hsp 70, hsp 60 Cd and heat Protective proteins MT Heavy metals Antibodies Antigens Population changes Breeding patterns, migrations Climate change Susceptibility Phenotype Acetylator phenotype (NAT 2)

• Oncogenes

Dominant oncogenes (ras, mic)

• Cancer genes

Breast-ovary cancer gene (BRCA 1)

• Examples of different biomarkers illustrated with specific examples and

examples of stressors which may result in the biomarker changes

Justification for Biomarker studies in Africa

• Limited use of biomarkers in developing countries
• Need to develop and validate biomarkers for

monitoring environmental pollution and changes

• Cheap, sensitive, robust
• Universal applications

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Clarias gariepinus

• Important tropical

freshwater fish

• Natural distribution 52oN –

28oS

• Clean & polluted

waterbodies

• Lifespan – 8 years
• Benthic & omnivorous

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108

Sex dependent studies

Experimental studies at SUA

Acclimatization

 Concrete tanks  2 months  Fed on locally

formulated food

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Exposure of fish in glass tanks

Kept in glass tanks 24 hours before exposure

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Selecte Selected d biomar biomarker ers

• Biotransformation enzymes
• Phase I

– CYP450 1A

• Phase II

– GST – UGT

• Biotransformation products (FACs)
• Neuromuscular parameters (Esterase)
• Stress proteins (MT)
• Haematological parameters (PCV, Hb)
• Gross indices (CF, LSI & GSI)
• Oestrogenic pollutants (VTG)

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Sample collection and analysis

Gills Liver EROD GST/UGT EROD Bile FACs HM OCs Muscles

Brain, eyes Blood

AChE

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Blood

VTG

Biotransformation pathway

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2 4 6 8 CF CM EF EM EROD activity (pmole min-1 mg P-1

a a a a

0.1 0.2 0.3 CF CM EF EM EROD activity (pmole min-1 filament -1

a b b a

Gill EROD

Liver EROD

2 4 6 8 10 CF CM EF EM µg B[a]P equivalents mL bile

a b b a

Bile FACs Biomarkers

• Gill EROD and FACs
• Simple, sensitive, robust
• First pass metabolism
• GST not sensitive

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A B BC C

0.05 0.10 0.15 0.20 0.25 0.30 0.35 1 3 6 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.05 0.10 0.15 0.20 0.25 0.30 0.35 1 3 6 B[a]P+EE2 EE2 B[a]P Control B[a]P+EE2 EE2 B[a]P Control B[a]P Control

Days pmol min-1 filament tip-1

A

A AB B

A

B B B A B BC C A B BC C

0.05 0.10 0.15 0.20 0.25 0.30 0.35 1 3 6 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.05 0.10 0.15 0.20 0.25 0.30 0.35 1 3 6 B[a]P+EE2 EE2 B[a]P Control B[a]P+EE2 EE2 B[a]P Control B[a]P Control

Days pmol min-1 filament tip-1

A

A AB B

A

A AB B

A

B B B

A

B B B

40 20 60 80 100 140 120 0.5

Bile FACs (µg mL-1

1 3 6

Days

1 3 6 1 3 6

Days

A A B C B B A A A A B B

1 3 6

Days

1 3 6 1 3 6

Days

10 20 30 40 50

nmol min-1 mg protein-1

A AB B B A A AB B A A A A

Gill EROD Liver EROD FACs

Interactions of chemicals

• First pass metabolism
• Gill EROD sensitive to inhibitors
• Higher chemicals reaching liver

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117

Background cont‟

Organophosphates and Carbamites studies

Actellic super – Pirimiphosmethyl Diazinon – Diazinon Rogor – Dimethoate Selecron – Profenofos Sumithion – Fenitrothion Steladone – Chlorfenvinphos Carbaryl - sevin dudu dust

OP C

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http://www.franklincoll.edu

Inhibition of Acetylcholinesterase activity In fish

AChE - largely found in plasma, eyes & brain

• BChE – largely found in serum,

liver & adipose tissues

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Methodology cont‟

 Experiment 1  Determination of baseline activities

 6 males and 7 female fish were used

 Experiment 2

 Determination of IC50 of Chlorfenvinphos

 7 female fish & different concentration of Chlorfenvinphos were used

 Experiment 3

 Dose dependent study of Chlorfenvinphos

 24 female fish & different concentration of Chlorfenvinphos were used

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Methodology cont‟

 Cholinesterase activity determined by Ellman‟s colorimetric

method

 Acetylthiocholine

Thiocholine + Acetate

 Thiocholine + DTNB

Yellow colour

Enzyme

Pesticide Dose (µM) Plasma (% inhibition) Brain (% inhibition) Eyes (% inhibition) Chlorfenvinphos 0.0003 12.5 23

• 2.5

0.003 24.5 20

• 8

0.03 83.5 50 49 0.06 81 49 42 Carbaryl 0.0005

• 3

20 46 0.005 6 25 31 0.05 19.5 28 50

• Clinical signs only in Chlorfenvinphos exposed fish at 0.03 & 0.06 µM
• Restlessness
• Reduced opercula movement
• Erratic swimming
• Loss of equilibrium
• Lethargy

In vivo inhibition of AChE activity in plasma, brain & eyes in C. gariepinus following 24 hours of chlorfenvinphos & carbaryl exposure

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Main findings

 Experimental studies demonstrated sensitive and robust

biomarkers

 Gill filament EROD and FACs

 Gills are sensitive to inducers and inhibitors

 Inhibition of AChE activity  GST – not sensitive

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123

27.01.2014 124

Integrated study strategy

Field studies Semi- Field studies Laboratory- experiments Evidence control of variables Relevance to natural populations none moderate high Little (extrapolation) moderate high correlation causal

Mindu dam catchment area in Morogoro urban and peri-urban areas, Tanzania

Studies carried out

• Hotspot characterization
• Chemical analysis of fish, prawn,

water and sediments

• Biomarker responses

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0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Mindu Mazimbu Mafisa Mzumbe Somatic index (%)

Liver Gonad

b b a d d d b c

A

0.002 0.004 0.006 0.008 Mindu Mazimbu Mafisa Mzumbe (p.mole/min/gill filament) a a b a

C

20 40 60 80 100 120 Mindu Mazimbu Mafisa Mzumbe (p.mole/min/mgP) a b b a

D

10 20 30 40 50 60 70 80 90 Mindu Mazimbu Mafisa Mzumbe Percentage PCV or Hb

% HB % PCV

a a ab b

B

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Mindu Mazimbu Mafisa Mzumbe Somatic index (%)

Liver Gonad

b b a d d d b c

A

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Mindu Mazimbu Mafisa Mzumbe Somatic index (%)

Liver Gonad

b b a d d d b c 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Mindu Mazimbu Mafisa Mzumbe Somatic index (%)

Liver Gonad Liver Gonad

b b a d d d b c

A

0.002 0.004 0.006 0.008 Mindu Mazimbu Mafisa Mzumbe (p.mole/min/gill filament) a a b a

C

0.002 0.004 0.006 0.008 Mindu Mazimbu Mafisa Mzumbe (p.mole/min/gill filament) a a b a 0.002 0.004 0.006 0.008 0.002 0.004 0.006 0.008 Mindu Mazimbu Mafisa Mzumbe (p.mole/min/gill filament) a a b a

C

20 40 60 80 100 120 Mindu Mazimbu Mafisa Mzumbe (p.mole/min/mgP) a b b a

D

20 40 60 80 100 120 20 40 60 80 100 120 Mindu Mazimbu Mafisa Mzumbe (p.mole/min/mgP) a b b a

D

10 20 30 40 50 60 70 80 90 Mindu Mazimbu Mafisa Mzumbe Percentage PCV or Hb

% HB % PCV

a a ab b

B

10 20 30 40 50 60 70 80 90 Mindu Mazimbu Mafisa Mzumbe Percentage PCV or Hb

% HB % PCV

a a ab b 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 Mindu Mazimbu Mafisa Mzumbe Percentage PCV or Hb

% HB % PCV % HB % PCV

a a ab b

B

Use of suite of biomarkers

126

Phase II enzyme activities

0,2 0,4 0,6 0,8 1 1,2 1,4 Mindu dam Ponds nmoles min-1 mg protein Mafisa Mazimbu Mzumbe a b b b 0,2 0,4 0,6 0,8 1 1,2 1,4 Mindu dam Ponds nmoles min-1 mg protein Mafisa Mazimbu Mzumbe a b b b

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UGT activities in hepatic microsomes

Use of suite of biomarkers

Parameter Mazimbu Mafisa Mzumbe LSI + + + GSI + + + Hb

• +

+ Gill EROD

• +

Liver EROD

• +

+ Liver UGT + + + MT

• AChE

GST

128

Main findings and Conclusion

 In the field study, the following biomarkers showed graded

responses under conditions where levels of pollutants were unknown

 EROD activities in gills and liver  FACs in bile  Liver uridine diphosphate glucuronosyl transferase (UGT)  Stress protein metallothionein  Liver somatic index  Gonadosomatic index  Haemoglobin concentration  The results also demonstrated that C. gariepinus is suitable for

experimental and monitoring studies of environmental pollution in tropical regions

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Further studies

 Development and validation of additional biomarkers  Investigations on the influence of abiotic and biotic factors on

biomarker responses in C. gariepinus

 Studies on the effects of long-term, low-level exposure

situations where acute responses are not observed

 Development and validation of in-vitro models  Cell cultures  Development and validation of DNA based techniques

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130

Subatomic particles

Ecs Comm‟

Popln‟

Individuals Colonies Microorganisms Organelles Complex molecules Molecules

Atoms

Ecological relevance Ability to solve challenges Time to develop competence

Status of OH and EC knowledge

The principle scheme of responses in organisms towards the detrimental effects caused by agent exposure

increased exposure (dose and time) Early warning signs Exposure responses Homeostasis normal range Response Observable detrimental effects No

• bservable

detrimental effects Disease outbreaks Increased susceptibility to diseases Reduced lifespan

132

Description of the elephant by blind people

ECOSYSTEM HEALTH

27-Jan-14

• Prof. Robinson H. Mdegela

Sokoine University of Agriculture Department of Veterinary Medicine and Public Health; P. O. Box 3021; Morogoro Tanzania Tel: +255 754 371 628 Fax: +255 23 260 46 47 E-mail: mdegela@suanet.ac.tz rmdegela2012@gmail.com rmdegela@yahoo.com Web: www.suanet.ac.tz

Thank you very much

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135

27.01.2014 136

e GR

CYS CYS GLU GLU GLY GLY

S S HS SH

Acknowledgement to you all