Environmental diseases

The environment is intimately associated with human health, illness and mortality. Environmental exposures to potentially hazardous agents such as microbes, toxic chemicals and metals, pesticides and ionizing radiations account for many of the diseases of humans, animals and plants. The concept of ‘environmental disease’ is usually applied to illnesses from chemical exposure due to environmental pollution. In other words illnesses and conditions caused by factors in the environment are collectively called environmental diseases. Pesticides, chemicals, radiation, air pollution and water pollution are some of the man-made hazards that are believed to contribute to human illnesses. Human beings made the global commons air, water and land into the global dustbins of human wastes.

Exposures to environmental pollution remain a major source of health risk throughout the world. Globally an estimated 24 percent of the disease burden (loss of healthy life years) and an estimated 23 percent of all deaths (premature mortality) was attributed to environmental factors. Among the children 0-14 years of age, the proportion of deaths attributed to the environment was as high as 36%. Of the 102 major diseases covered by the World Heath Report in 2004, environmental risk factors contributed to disease burden in 85 categories. Poor air quality contributes to cancers, cardiovascular diseases, asthma and other illnesses. Poor water quality can lead to gastrointestinal illness, neurological problems and cancer. Pollution is one of the biggest global killers, affecting over 100 million people.

Globally the two groups of people that are most affected by environmental diseases are the rural poor and the urban poor who live in slums.  About 25-50% of the world’s urban population live in extreme poverty. They lack basic necessesities for a decent life such as adequate housing, drinking water, sanitation or garbage collection. About 75% of the rural households in India burning wood, dung cakes and crop residues for cooking. Epidemiological studies in developing countries have linked exposure to indoor air pollution from biofuels with four major categories of illnesses: acute respiratory infections (ARI) in children; chronic obstructive lung diseases such as asthma and chronic bronchitis; lung cancer and still-births and other problems at birth. Rural coal smoke exposures seem to increase lung cancer risks by a factor of nine or more. Lung cancer in China is attributed to high levels of coal smoke. 

One recent study in Colombia found women exposed to smoke during cooking were more than three times more likely suffer from chronic lung disease. In developed countries, energy efficiency improvements make houses relatively airtight, reducing ventilation and raising indoor pollution levels. Increasing numbers of urban homes and buildings are now ‘air tight’ which can lock in bio-allergens (dust mites, moulds, cockroaches, insect droppings or animal dander) and also irritants (dust, odours from oil paints, room fresheners) and other pollutants. These urban environmental conditions may lead to ‘sick – building syndrome’ (SBS). A number lifestyle changes in living conditions in the indoor environment such as carpeting, upholstered furniture, mattresses, humidifiers and air conditioning make it easier for dust mites and moulds to thrive.

An estimated 500,000 women and children die in India each year due to indoor air pollution (IAP) – related causes, which is 25% of the estimated IAP-related deaths worldwide (World Bank Report). A report of WHO in 2002 showed that 36% of lower respiratory infections were attributable to solid fuel use alone and 1% of all respiratory infections to outdoor pollution.

Causes of environmental diseases

Industrial growth, urbanization and the increasing use of synthetic organic substances have serious and adverse impact on the environment. Population growth indirectly driving climate changes by contributing to deforestation, overgrazing, soil erosion and desertification. Overuse of land may lead to a drop in food production, depletion of natural resources and a rise in pollution.  Chemical fertilization, chemical control of insect pests and weeds, mechanization and irrigation all have an impact on the land environment. The land vegetation and organic matter had been reduced about one-third. ‘Formerly man had been part of nature’ said Lynn White Jr. and ‘now he was the exploiter of nature’. Man dominates all other living creatures and exploits all available natural resources. He has been working as an agent of all environmental degradation and now he becomes its prime victim.

Types of environmental diseases

1.    Vector – borne diseases – transmitted through insects e.g., malaria, dengue fever, encephalitis, schistosomiasis.

2.    Vessel – borne diseases – transmitted through some kind of vessel e.g., a cup, water, food, milk, blood etc. e.g., diarrhoea, dysentery, cholera.

3.    Air – borne diseases – transmitted through air medium e.g., flu, tuberculosis, measles, heart and lung diseases and cancer.

Malaria is strongly connected to environmental factors such as climate, rainfall, irrigation and sanitation. Malaria is caused by the blood parasite plasmodium. This parasite is transmitted by the bite of an infected female anopheles mosquito. An estimated 42% of the global malaria burden could be prevented by environmental management.

World Health organization (2002) estimated that 88% of all causes of diarrhoea globally were attributed to water pollution, sanitation and hygiene. About 18% of the world’s population still lacks access to safe drinking water and nearly 40% have no access to proper sanitation.

Examples of environmental diseases

Air pollutants have both acute and chronic effects on human health. Urban air pollutants can cause or exacerbate cardiovascular disease, cancer, allergies, asthma and lung disease. Benzene, nitrogen dioxide and small particulate matter can cause damage to the bone marrow and the immune system.  Air pollution is shown to be the cause for 1 in 10 deaths due to lung cancer. Air borne particulates increase the severity of asthma attacks, lung disease and chronic obstructive pulmonary disease (COPD). Exposure of humans to air pollutants may be a cause for acute respiratory distress syndrome (ARDS), sick building syndrome (SBS), multiple chemical sensitivities (MCS), and chronic fatigue syndrome (CFS).

Water – and food – borne diseases that result in diarrhoea or dysentery are the leading cause of environment – related health problems in the world. The primary diarrheal diseases include amoebiasis, cholera, giardiasis, and other protozoal diseases; samonellosis, shigellosis, typhoid and paratyphoid fevers and viral diseases. These diseases collectively cause more than 16% of the global environmental disease burden (58 million DALYs per year) and 13% of deaths (1.7 million per year).

Human health effects of heavy metals in soil include brain and nervous system damage, kidney damage, liver toxicity and birth defects.

Ultraviolet and other ionizing radiations are very damaging to cell components and DNA and they trigger development of cancerous tumours.

Quotes for reflection

“Pollution of the environment  is the root cause of all human health problems”.

"Environmental pollution is an incurable disease. It can only be prevented."
                                                                                 -Barry Commoner.

Environmental Carcinogenesis

Environmental carcinogens include outdoor and indoor air pollutants as well as soil and drinking water contaminants. Epidemiological studies have shown that 70-90% of all cancers are environmental.  Environmental factors such as lifestyle, personal habits, diet, chemicals and radiation and infectious diseases account for about three quarters of all cancers. The China admits that there has been an 80% rise in the mortality rate from cancers over the past 30 years. The United States has one of the world’s highest incidences of cancer associated with environmental pollution. The most recent data suggests there were 223,000 deaths from lung cancer caused by air pollution around the world (International Agency for Research on Cancer, IARC).The IARC classified ambient air pollution as cancer causing agent (carcinogen). Studies also show that nearly 30% of the total mortality in several industrialized countries is due to cancer.

Cancer

A tumour or cancer is an abnormal mass of tissue whose cells undergo rapid and uncontrolled growth at the cost of remaining cells. The tumours are classified as benign or malignant. Benign tumours remain localized in a specific area at the site of origin, forming a single mass enclosed in a capsule. They slow growing and can be removed effectively with surgery. Malignant tumours are cancerous with rapidly growing and actively moving cells.  The cancer cells migrate through the blood and lodge at distant sites which are called metastasis. Cancers or malignant tumours are uncapsulated and invasive.

Carcinogens

Carcinogens are agents that induce cancer. Primary or direct – acting carcinogens are those that do not require metabolic activation e.g., mustard gas. Some carcinogenic chemicals are inactive and require metabolic activation. They are secondary carcinogens e.g., carbon tetrachloride. The parent compound is called a procarcinogen  and is converted to a reactive metabolite called proximate carcinogen and then to a highly reactive species termed as ultimate carcinogen, which are covalently bind to macromolecules like DNA. Co-carcinogens (promoters) are substances that potentiate or promote the effects of carcinogens e.g., cyclopropenoid fatty acids. Carcinogenic agents can further classified as genotoxic or non-genotoxic, based on the ability to alter the genetic systems in cells. Asbestos increase the incidence of cancer, but do not possess genotoxic effects.

 3- stage model of chemical carcinogenesis

 The chemical carcinogenesis comprises 3- sequential and successive steps: initiation, promotion and progression. Tumour initiators can be defined as carcinogens capable to induce a first driver mutation in a dividing cell so that an initial clone of mutated cells emerge. Tumour promoters can be defined as non-genotoxic carcinogens capable of causing clonal expansion of initiated cells i.e., able to induce proliferation of mutated cells. Tumour progressors are carcinogens that advance mutated cells from promotion to progression  and transform a mass of fully malignant cells. So carcinogenesis is a multiple step process.

 Cancer develops over a peroid of several years (latent period) and has many causes. There are more than 100 types of cancers. Scientists have identified more than 300 altered genes called oncogenes that can signal the cell to divide out of control. One of the characteristics of chemical or physical carcinogenesis is the usually long latent periods (years to several decades) between the contact with the carcinogen and appearance of a tumour. The degree of cancer risk from pollutants depends on the concentration, intensity and duration of exposure. E.g., saccharin is carcinogenic only at higher doses.

Environmental carcinogens

Arsenic, asbestos and radon are three prominent human carcinogens strongly associated with lung cancer.  Benzene is known to cause leukemia (blood cancer) in human beings. Benzene has widespread use as solvent in the chemical and drug industries and a gasoline component. Drinking water that is contaminated with a high level of arsenic over a long period of time is known to increase the risk of lung, bladder and certain types of skin cancers. Exposure to arsenic caused 3,700 lung, bladder and skin cancer deaths in Bangladesh alone. An increased risk of stomach cancer has been reported in areas with high nitrate levels in drinking water. There are more than 75,000 chemical compounds in contaminated waters come from industry, agriculture and consumers/homes. Bisphenol A (BPA), a building block of polycarbonate plastic is an endocrine disruptor linked to breast and prostate cancer. Exposure to vinyl chloride (PVC) is linked to the development of liver and brain cancer. Polycyclic aromatic hydrocarbons (PAHs), a product of incomplete combustion of organic compounds is possibly carcinogenic. The chemical compounds with carcinogenic potential include benzopyrene, benzene, organic solvents, pesticides, dioxins, several heavy metals (arsenic, cadmium, lead, mercury) and others.

Cancer is a preventable disease

According to the National Cancer Institute, 80% of the cancers are due to factors that have been identified and can potentially be prevented. About 20 years ago, 1 out of 10 people were diagnosed with cancers and other debilitating diseases.  Now we are faced with 1 out of 2 people being diagnosed with cancer. The environment, which sustains the life of all living organisms, can also be a significant contributor of ill health. The natural environment is crucially a ‘commons’ a public good. Respect the ecosystems and keep them healthy.

Recombinant DNA technology - methods and applications

Recombinant DNA technology is modifying the genetic makeup of an organism either by adding new genes or by changing the existing genes. It is a technique of preparing r DNA in vitro by cutting up DNA molecules and splicing the fragments together from more than one organism. Recombinant DNA is a form of artificial DNA that is made through the combination or insertion of one or more DNA strands.  Recombinant DNA is also referred to as "chimera."

Using recombinant techniques, particular sequences of DNA can be isolated, manipulated, and re-introduced into many different kinds of living things. The desired results are improvement of microorganisms, plants, and animals, for a particular purpose.

Objectives of  r DNA Technology

• Artificially synthesize new genes. 
• Altering the genome of an organism.
• Bring about new gene combinations not found in nature. 
• Understanding the hereditary diseases and their cure.
• Improving human genome.

Enzymes are the chemical knives in r DNA technology

DNA or RNA polymerase-replicating or annealing a DNA chain.
Reverse transcriptase – synthesize c DNA from RNA template.
DNA ligase – joining DNA strands together.
Nuclease-breaks phospho-diester bonds within free ends (exonucleases) or in an interior position (Endonucleases ).
Restriction endonuclease – recognizes a specific base sequence and cuts the DNA.

Restriction endonucleases (RE ases)

Restriction endonuclease is a special class of sequence –specific enzymes.It is found in bacteria which protect its genetic material from the invasive attacks of viruses. It is a site-specific enzyme which cleaves DNA molecules only at specific nucleotide sequences. REases recognize DNA base sequences that are palindromes. REases make two single stranded breaks, one in each strand. REases make staggered cuts with complementary base sequences for easy circularization.

Types of Vectors

o   Bacterial plasmid vectors

o   Bacteriophage vectors

o   Cosmid vectors

o   Expression vectors

o   Bacterial Artificial Chromosomes (BAC)

o   Yeast Artificial Chromosomes (YAC)

o   Ti  and Ri vectors

Plasmids

Plasmids are relatively small, self-replicating duplex extra-chromosomal DNAs maintained as independent molecules. Lederberg coined the term ‘plasmid’  in 1952. They are found in bacteria, yeast and streptomyces. Plasmids range in size from < 1 Kb to 500Kb.

Phage Vectors

Two types of phage vectors have been extensively developed-λ and M13. Phage vectors have engineered phage genomes previously genetically modified to include restriction sites. After insertion of foreign DNA, the recombinant phage genome is packaged into the capsid and used to infect host cells

Cosmids

Hybrid vector constructed to contain features from both phages and plasmids. Cosmids  have a selectable marker, multiple cloning sites from plasmids and a cos site from l phage

Artificial Chromosomes

Large fragments of DNA  can  be cloned in artificial chromosomes. Mapping of genes is easier. Artificial chromosomes have played important role in the human genome project. One copy of YAC is present per cell. E.g. yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs)

Host cell types

• Prokaryotic hosts – Bacteria, E . Coli, Bacillus sp., Pseudomonas sp., Streptomyces sp.
• Eukaryotic hosts - Yeast – Saccharomyces, Fungi- Aspergillus, Neurospora, Algae - Chlamydomonas

Two types of host-vectors

• Cloning vector - Propagation of DNA inserts
• Expression vector - Production of proteins

Molecular Cloning / DNA cloning

Molecular cloning refers to the process of making multiple DNA molecules.

Step  1– fragmentation -breaking apart a strand of DNA

Step 2 – ligation-gluing together pieces of DNA in a desired sequence.

Step 3 –Transfection -  inserting the newly formed DNA into cells.

Step 4-Screening / selection – selecting out the cells that were successfully tranfected with the new DNA

Gene transfer technology

• Transduction- Virus mediated gene transfer.
• Tranfection - Chemical or physical tricks to persuade cells to take DNA from the culture medium.
• Direct transfer - Physically inserting the gene e.g. microinjection
• Natural gene transfer - A receptor – mediated lateral binding fusogenic proteins used.

Calcium phosphate –co precipitate  method

This method was described by Graham and Van der Eb in 1973.

It is a process for inserting foreign DNA into bacteria. The bacterial cells are treated with  ice-cold calcium chloride. The plasmid DNA is added  to cells chilled on ice which form calcium phosphate –DNA precipitate. The cell and DNA mixture is heated  to 42^oC. The membrane becomes fluid and plasmid DNA enters bacterial cells and is replicated and expressed

Electroporation

It  involves a brief application of high voltage electric current to the cells resulting in the formation of transient holes in the cell membrane through which plasmid DNA can enter the cell. The transformation efficiency is high. Quick restoration of membrane fluidity and closing of pores is crucial for survival of cell after the pulse.

Selection  techniques for rDNA molecules

•         DNA hybridization assay

•         Colony immunoassay

•         Screening by protein activity

•         Genetic screening methods

Process of selection

Selection is a process designed to facilitate the identification of recombinant bacteria while preventing the growth of non-transformed bacteria. After transformation, the bacteria are challenged with an antibiotic (such as ampicillin). If the E. coli have taken up and expressed an ampicillin resistance gene on a plasmid, they will live - otherwise they will die. This process is called selection because selected bacteria may survive.

DNA hybridization assay

This technique was introduced by Grunstein and Hogness (1978). The target DNA is denatured at 800C and bound to a nitrocellulose filter discs. Such filters are hybridized with radioactive DNA probes. The results are monitored by autoradiography.

Colony immunoassay

The transformed colonies are transferred to a nitrocellulose filter. The colonies are lysed and the released proteins are attached to the matrix. The matrix is treated with a primary antibody which specifically binds to the proteins encoded by the target gene. Then the matrix was washed to remove any unbound antibody. Then the matrix was treated with a second antibody which was an enzyme, alkaline phosphatase. The target protein (antigen) was treated with a colorless substrate. The colorless substrate is hydrolyzed by the alkaline phosphatase into a colored complex.

The Tools of Recombinant DNA Technology

—  Gene Libraries

The collections of cloned DNA fragments from a particular organism contained within bacteria or viruses as the host. The library may contain all genes of a single chromosome. Screening, identification and characterization of cloned fragments are possible with suitable probes.

cDNA Libraries

The library contains only complementary DNA molecules synthesized from mRNA molecules in a cell. mRNA from tissue of interest is isolated and converted into a double-stranded DNA by using the enzyme reverse transcriptase. The newly synthesized molecules are called complementary DNA (cDNA) because it is an exact copy of the mRNA.

Supporting techniques of Recombinant DNA Technology

      Multiplying DNA in vitro by Polymerase Chain Reaction (PCR)

It was developed in the 1980s by Kary Mullis. This technique is used for making copies, or amplifying, a specific sequence of DNA in a short period of time. It is a repetitive process consisting of three steps: Denaturation, Priming and Extension.It can be automated using a thermocycler. At the end of one cycle, the amount of DNA has doubled. Cycles are repeated 20–30 times

      Separating DNA Molecules by Gel Electrophoresis

The molecules are separated on the basis of electrical charge, size, and shape. It allows to isolate DNA of interest. Negatively charged DNA drawn toward positive electrode. Agarose makes up gel; acts as molecular sieve. Smaller fragments migrate faster than larger ones.Size is determined by comparing distance migrated to standards.

Applications of rDNA technology

Production of transgenic organisms - Recombinant plants and animals altered by addition of genes from other organisms. Transgenic plants that can produce their own insecticides e.g.Better Crops (drought , heat and salt resistance)
Recombinant DNA technology has been used for creating new animal species using the cloning technology. rDNA technology is used to elucidate molecular events in biological processes like cell differentiation, aging etc.
Recombinant Vaccines (ie. Hepatitis B
Gene therapy - Missing or defective genes replaced with normal copies e.g. sickle cell anaemia and severe combined immuno-deficiency (SCID).
Large-scale production of human proteins by genetically engineered bacteria such as : insulin, Growth hormone, Interferons and blood clotting factors (VIII & IX)

Disadvantages of Recombinant technology:

Recombinant technology  can be commercialized and became big source of income for businessmen. The products have effects on the natural immune system of the body. The transgenic organisms can destroy natural ecosystem that relies on organic cycle. They are prone to undergo mutation that could have harmful effects. There is possibility of manufacturing of biological weapons such as botulism & anthrax to target humans with specific genotype. There is a concern of creating super‐human race.

Environmental hormones

Daily use of chemicals is an essential habit of modern society. Large scale production and repeated use of synthetic chemicals introduce many toxic and persistent chemical residues into the environment. As a result, humans and wildlife are constantly exposed to the chemical residues through air, food and water. Some of these chemicals in the environment interfere with, block or mimic the effects of natural hormones. Such environmental contaminants which modulate the activities of natural hormones are called environmental hormones, endocrine disrupting chemicals (EDCs), or environmental estrogens (EE) or xenoestrogens. It has been scientifically shown that environmental hormones may elicit a variety of adverse health effects in both humans and wildlife including promotion of hormone- dependent cancers, reproductive organ disorders and reduction in reproductive fitness.

Kinds of hormone mimicking chemicals

The term ‘environmental estrogen’ is currently used to denote both plant – derived estrogens (phytoestrogens) and the anthropogenic (synthetic) estrogens (xenoestrogens). The naturally occurring estrogens in either humans or animals are called endogenous estrogens and all compounds with estrogenic properties entering the body from an outside source as exogenous estrogens.

Phyto-xenoestrogens

Phytoestrogens (phyto meaning plant) are naturally occurring estrogenic compounds that are found in a variety of plant foods such as beans, seeds and grains. These compounds are generally weak estrogens when compared to xenoestrogens. Many phytoestrogens belong to the large group of plant phenolics. Caffeic acid is one of the most common plant phenolics found in chicory coffee, artichoke, olive oil and red wine. The other major phytoestrogens consumed in excess quantities by humans are  isoflavonoids and lignans. Strong phytoestrogens are present in soy, red clover, caffeine and Chester berry.

Synthetic xenoestrogens

The word xenoestrogens is derived from the Greek words xeno, meaning foreign, estrus, meaning sexual desire and gene, meaning to generate and literally means ‘foreign estrogen’. The synthetic xenoestrogens include both chlorinated and non-chlorinated compounds. The synthetic xenoestrogens are found in plastics (BPA), pasticizers (phthalates), industrial chemicals (polychlorinated biphenyls,PCBs; dioxins), pesticides(DDT, methoxychlor),fungicides(vinclozolin), pharmaceuticals(diethylstilbestrol) and heavy metals(arsenic, lead, chromium, cadmium).

Mode of action

Environmental estrogens have been shown to directly bind to the estrogen receptor(ER) and function as either agonists or antagonists. The estrogen agonists are compounds that mimic the effects of natural estrogen. But estrogen antagonists block the action of estrogens by interfering with the normal functions of the estrogen receptor. In general environmental estrogens have the ability to mimic/ antagonize the effects of natural hormones. They also modify hormone receptor levels and the pattern of synthesis and metabolism of natural hormones.

Sources of synthetic xenoestrogens

Plastic additives- phthalates and bisphenol A(BPA) are plastic additives. Phthalates are used extensively in industry, plastic packaging, inks, paints, and vinyl products. Bisphenol A is used in the production of epoxy resins and polycarbonate plastics. They are lipophilic and accumulate in the fat. They cause human breast cancers.

Brominated flame retardants(BFRs) – are a group of industrial chemicals mainly used in electric devices, textiles and cars. They are highly lipophilic and bioaccumulate in adipose tissues. They have been reported to disrupt thyroid, androgen and estrogen signalling.

Perfluorinated chemicals(PFCs) – are used to make non-stick cookware. They are completely resistant to biodegradation. PFOA decreases sperm quality and causes kidney disease or thyroid disease.

Organotins,TBT (tributlytin) is an active ingredient in antifouling paints. Exposure to TBT causes obesity and metabolic disorders.

Dioxins – are the most deadly organochlorine chemicals found in pesticides, plastics, solvents, detergents, and cosmetics.  More than 90% of human exposure is through food, mainly meat and dairy products, fish and shellfish. They are highly toxic and bio- accumulate through food chain.

Human health effects

Synthetic xenoestrogens may act as false messengers and disrupt the process of reproduction.  Xenoestrogens induce precocious puberty in girls with premature secondary sexual characters.

They may be associated with the development of learning disorders, severe attention deficit disorders and developmental defects.

TBT may act as androgens and fungicide vinclozolin act as anti-androgens.

In males, exposure to xenoestrogens decreases sperm quality and counts, incidence of testicular cancers and crytorchidism (undescended testis). In females exposure causes endometriosis and endometrial cancer.

Exposure to dioxins can cause reproductive and developmental problems, damage the immune system, interfere with hormones and also cause cancers.

Environmental xenobiotics

A xenobiotic is a compound which is foreign to a particular organism. The term ‘xenobiotic’ is a combination of the Greek words ‘xenos’ meaning strange or foreign and ‘bios’ meaning life. They are mostly synthetic substances used as agrochemicals, pharmaceuticals, petrochemicals, colorants, adhesives, preservatives and certain chemicals in plastics.

 Kinds

The xenobiotics may be naturally occurring as well as man-made (anthropogenic). The man-made chemicals are synthetic substances like pesticides, organic solvents, medicaments, ethanol etc. The naturally occurring chemicals are produced by plants , microorganisms or animals as ‘chemical warfare agents’. E.g. pyrethrins, nicotine, mycotoxins, tetrodotoxin (newt) and antibiotics.

    Xenobiotics can be exogenous to living organisms, which include drugs, food additives, pollutants, insecticides, chemical carcinogens etc. They are not normally ingested or utilized by the organisms.

Endogenous xenobiotics are not foreign substances but are synthesized in the body or produced as metabolites of various processes in the body e.g. bilirubin, bile acids, steroids, eicosanoids and certain fatty acids.

Sources

 The major sources of xenobiotic compounds are from the chemical and pharmaceutical industries, mining operations, fossil fuels and intensive agriculture.  Food additives are xenobiotics which have no nutritional value, are of no use in the body and can be harmful, if consumed in excessive amounts. Human beings are increasingly exposed to the kinds and amounts of xenobiotic agents from industrial, agricultural, pharmacological and lifestyle applications.

Sites of action

 The xenobiotic agents target the active sites of enzymes, DNA (genetic material) and lipid membranes.

Mechanism of toxicity

The exposure to xenobiotic agents disrupts normal cell functions. They easily bind and damage structural and dynamic proteins e.g. enzymes. They also bind and damage DNA and induce mutations (nucleophilic).  They bind and damage lipid membranes (lipophilic).

They react in the cell with oxygen to form ‘free radicals ‘which damage lipid, protein and DNA.

Effects of xenobiotics

The metabolism of xenobiotics can result in cell injury/cell death by cytotoxicity, immunologic damage (altering its antigenicity) or cancer (disorder in cell growth).

The xenobiotics may directly bind to a cellular component and inhibit its normal function. For example carbon monoxide binds to haemoglobin in the red blood cells and prevents the haemoglobin from binding with oxygen.

Cadmium binds with a transporting blood protein metallothionein which accumulates in the kidney and damage the filtering function (tubular cells).

Metabolism of xenobiotics

The main organ involved in xenobiotic metabolism is liver. The xenobiotic transforming enzymes are present in the cytosol and endoplasmic reticulum of hepatocytes.

The biotransformation involves phase I and phase II reactions.

The main purpose of the reactions is converting the xenobiotic lipophilic (lipid-soluble) agents into hydrophilic compounds and facilitates excretion. First phase of reactions are performed by liver or gut enzymes before the compounds reaches the systemic circulation and limits its bioavailability. Several enzyme systems participate in phase one metabolism of xenobiotics. The cytochrome P450s (CYPs:450s) detoxify and / or bioactivate a vast number of xenobiotic chemicals. Phase I involves the addition of reactive functional groups by oxidation, reduction or hydrolysis.  Phase I reactions convert xenobiotics into more reactive metabolites (metabolic activation). Phase II biotransformation is catalysed often by the ‘transferase’ enzymes that perform conjugating reactions. Phase II reactions include glucuronidation, sulfation, methylation, acetylation, glutathione conjugation and amino acid conjugation.  Phase II reactions cause the xenobiotic metabolites into more hydrophilic and readily excretable compounds.

‘Poisons are xenobiotics, but not all xenobiotics are poisonous’

Polymerase Chain Reaction (PCR) - methods and applications

Polymerase chain reaction is a biochemical technique used in Experimental Biotechnology to amplify a specific fragment of target DNA. PCR is a novel molecular procedure based on thermal cycling which consist of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of defined DNA sequences.
PCR was discovered by Kary B. Mulis in 1983 of Cetus Corporation, a Biotech company in California, USA. He won the Nobel Prize for Chemistry in 1993 for ‘contributions to the developments of methods within DNA-based chemistry’. ‘Taq polymerase’ an enzyme used in PCR was described as ‘molecule of the year’ 1989. PCR is now an indispensable technique used in medical and biological research labs for a variety of applications.

A copying machine for DNA molecules

PCR multiplies a single, microscopic strand of the DNA molecule into billions of times within hours. PCR has a major impact on recombinant DNA technology. PCR has multiple applications in medicine, genetics, biotechnology, and forensics.
PCR-A DNA multiplication protocol
PCR is a powerful technique, in which from a single copy of a DNA molecule, millions of copies can be obtained with high accuracy, specificity and in a very short time. DNA amplification process in PCR is cyclical and the concentration of DNA doubles at each cycle. The total amount of DNA concentration increases exponentially during the cyclical process of PCR machine.

The ‘master mix’ components for PCR machine

· A thermostable DNA polymerase: tag polymerase
· A template DNA
· A complete set of deoxynucleotide triphosphates e.g. dATP, dCTP, dGTP and dTTP
· Tris buffer of pH 8.8
· A pair of oligonucleotide primers
· Mg 2+ and detergents
· 2-mercaptoethanol to stabilize proteins during thermal cycle.

Requirements for PCR

·        DNA template – DNA segment to be amplified.

·    Two primers- a short segment of DNA (forward and reverse primers) about 20–25 bases long.

·    Taq polymerase – an enzyme to synthesize DNA copies.

·     Deoxynucleotide triphosphates – the building blocks for new DNA strand.

·     Buffer solution – a suitable chemical environment.

·     Divalent cations – Mg ²⁺ ions

·     Monovalent  ions – Potassium ions

·     PCR machine – a thermal  cycler

Thermostable DNA polymerase

The thermophilic DNA polymerases catalyze template-directed synthesis of DNA from nucleotide triphosphates.

Several thermostable polymerase enzymes are used in PCR

•         Pfu DNA polymerase obtained from - Pyrococcus furiosus

•         Vent polymerase obtained from- Thermococcus litoralis

•         Taq polymerase  obtained from - Thermus aquaticus

Oligonucleotide primers

They are synthesized chemically to be complementary to sequences which flank the region of DNA to be amplified. They are usually about 20-25 nucleotides in length. The primers are designed to anneal specifically to the opposite strands of the template molecule. It is the specificity of the primer annealing reaction which ensures that the PCR amplifies the appropriate region of the template DNA.

Critical steps in PCR

1.     Sample Preparation

2.     Target selection

3.      Primer selection

3 – Temperature cycle in PCR

•         The temperature - 90-98⁰C separates two strands of target DNA.

•         The temperature– 40-60⁰C anneals two complementary primers to the ends of separated single strands of target DNA. 

•         The temperature 72⁰ C allows taq polymerase to use ss target DNA and primers to synthesize new strands.

PCR protocol

1. Denaturation of ds DNA template –melting of target DNA-it is the thermal denaturation of the dsDNA molecules at 950C for 1 min.
2. Annealing of two oligonucleotide primers at 680C for 60 sec. The annealing temperature is dependent on the length and G+C content of the primer sequences.
3. Extension of dsDNA molecules – temperature is raised to 750C for about 30 sec.
The step cycle programme makes the instrument to heat and cool to the set temperatures due to solid state Peltier-effect device, which actively modulates the desired temperature. There may be as many as 30-35 cycles.

Variants of PCR

·    In standard PCR, the sequences of both ends of target DNA have to be known. Two primers define the ends of target DNA and only that part is amplified.

·   In single sided PCR, the DNA is rearranged before amplification so that only one primer is needed. This is also called Anchored PCR.

·   In inverse PCR, the DNA at primer sites rather than between two primers is amplified because primer sites which are bracketing may have important sequence like promoter for triggering target gene into action.

Characterization of PCR product

•        Contamination of the reagents by foreign DNA or annealing of primers to alternative sites in the template DNA may produce unwanted DNA molecules.

•        Multiple bands in the electropherogram suggest primers annealing to multiple sites.

•        Smear of DNA suggests presence of excess template DNA.

Problems and limitations

• Contamination of reaction mixture by bacteria, viruses, and our own DNA presents a real problem.
• PCR cannot substitute for cell- based gene cloning, when large amounts of a gene are desired.
• Taq polymerase used in PCR often lack 3' to 5' exonuclease activity. This enzyme lacks the ability to correct mis-incorporated nucleotides.
• PCRs of longer products are less efficient due to enzyme activity loss. PCR can be applied only to short DNA fragments.

Applications of PCR

• Detection of pathogens in food, water and tissue specimens.
• Detection of tuberculosis, AIDS and other microbial diseases.
• Diagnosis of genetic diseases-e.g. sickle cell anemia, β-thalasemia, hemophilia
• Identification of criminals, disputed parentage
• Monitor gene expression in genetic engineering or gene therapy experiments.
• To study genetic profile of animals and to trace evolutionary and cultural lineage of human beings.
• To study DNA polymorphism.
• To determine orientation and location of restriction fragments relative to one another.
• To conduct microbial surveillance of the environment.

References

•        Saiki, R., Scharf, S., Faloona, F., Mullis, K., Horn, G., and Erlich, H. (1985). Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 230: 1350-54

•        Mullis, K. and Faloona, F. (1987). Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155: 335-350.

•        Mullis, K. (1990). The unusual origin of the polymerase chain reaction. Scientific American April 56-65

•        Rabinow, P. (1996). Making PCR: A story of biotechnology. University of Chicago Press.

Biological monitoring of water quality

Water quality can be described in terms of physical, chemical and biological characteristics. Bioindicator organisms are those that can be used to identify and quantify the effects of pollutants on the environment. The presence, condition and numbers of certain species of fishes, insects, algae and plants can provide accurate information about the health of a specific river, stream, lake, wetland or estuary. Biological methods are inexpensive and data can be obtained by periodic (e.g.monthly or yearly) visits to the sites. The data is unambiguously related to pollutional impact and scientifically credible. Since pollution is a threat to the biological components of the ecosystem, the assessment of biological variables is the most appropriate. Biological monitoring can indicate past and ongoing pollution events. The reactions of individual organisms, such as behavioural, physiological or morphological changes, can also be studied as responses to pollutant stress. Certain contaminants, particularly metals and organic compounds, may be accumulated in the tissues of organisms. The chemical analysis of the appropriate biological tissues can be used to show that the organism has been exposed to contaminants. The biological approaches can be cheaper than chemical methods in terms of manpower and equipment.

Pros and cons of biomonitoring

Monitoring of biological variables can be cheaper, precise, rapid, easy to perform, require less sophisticated instruments and reflect the integrated expression of pollution load. Biological indicators could identify the possible environmental problems before the health of the aquatic system is seriously altered. Since there is no instrument devised by man to measure toxicity of chemical pollutants, a living organism is a most ‘sensitive sensor’ to evaluate the relative toxicity of pollutants. However the biological approach suffers from the problems posed by the extreme complexity of organisms and ecosystems, endless form of interactions and the non-specific nature of population and community responses.

Biological monitoring is an important tool for water quality management.

Biological monitoring measures the cumulative effect of all the pollutants and overall health of the aquatic ecosystems. Biological can integrate all environmental variables over long periods of time. Generally biological effects occur at concentrations below the analytical capabilities. Toxicity is a property that can be measured only by an organism’s response(Mount,1980).

Definition of biological monitoring

Biomonitoring is the introduction of biological variables for the assessment of the structural and functional aspects of ecosystems. Biological variables are most sensitive to stress which can be easily measured and quantified.A bioindicator is an organism (or community of organisms) that contains information on the quality of the environment. Organisms which are used as indicators of water quality are called sentinel organisms or biological litmus paper or pollution thermometer. The indicator organisms are either plants or animals which show clear symptoms of the possible presence of pollutants. e.g. macroinvertebrates in general( caddis fly larva, may fly larva, stone fly larva etc.), bivalve molluscs (clams, mussels, oysters) and microcrustaceans.

Bioindicators

Indicator organisms may be true indicators or scale indicators.
In true indicators, the degree of pollution- induced damage is related to morphological and/or physical symptoms in one single species.
In scale indicators, the degree of pollutional stress is related to the presence or absence of a sensitive species in a community.
Active bioindication is meant when bioindicators bred in laboratories are exposed in a standardized form in the field for a defined period of time. At the end of the exposure, the reactions provoked in the organisms are analysed.
Passive bioindication organisms already occurring naturally in the ecosystem are examined for their actions.

Biomonitors

A biomonitor is an organism (or community of organisms) that contains information on the quantitative aspects of the quality of the environment.
Accumulation indicators / monitors are organisms that accumulate one or more elements or compounds from their environment. e.g. fresh water mussels.
Effect or impact indicators / monitors are organisms that demonstrate specific or unspecific effects in response to exposure to a certain element or compound or a number of substances.

Criteria for a bioindicator species

1. Highly susceptible to pollutant stress.
2. Widely distributed in many habitats.
3. Taxonomically stable and well known.
4. Low genetic and biological variability
5. Well known natural history with abundant ecological and physiological data .
6. Ready and easy to be sampled, surveyed and manipulated.
7. Economically/ biologically important species.
8. Easily held or cultured in the laboratory for experimental ecotoxicological procedures.

Criteria for selecting a bioindicator species

1.       Relevance – causal relationship to ecologically significant endpoints

2.       Sensitivity – dose responsiveness to specific stressors.

3.       Specificity – responds to specific stressors.

4.       Broad applicability – over temporal and spatial scales.

5.       Representatives – role as surrogate for other responses.

6.       Variability – low variability relative to noise in a ecosystem.

7.       Cost – reasonable for available resources and scope of study.

Benthic macroinvertebrates as good bioindicators

Benthic macroinvertebrates are small animals that live on the bottom of a pond, lake, stream or river for at least part of their lives. E.g. aquatic insects – may flies, damsel flies, dragon flies, stone flies, caddies flies, Dobson flies, true flies and beetles.EPT index is a measure of total number 3 aquatic insect orders such as Ephemeroptera, Placoptera and Trichoptera.

Properties of benthic macroinvertebrates

1.       Live in water for all or most of their life with limited mobility.

2.       Stay in areas suitable for their survival.

3.       Easy to identify, sample or survey

4.       Differ in the range of tolerance to amount and types of pollution.

5.       Are integrators of environmental conditions.

Biomarkers

Biomarkers reflect pollution – induced effects at several levels of biological organization. Biomarkers are measurable biological parameters at the genetic, enzymatic, physiological and morphological levels which indicate qualitative and quantitative aspects of environmental pollution.

Biomarkers can be divided in to markers of exposure and toxic effects.

Biomarkers of exposure represent responses such as induction or inhibition of specific enzymes involved in biotransformation and detoxification as a result of chemical exposure. These biomarkers show early response at the molecular or cellular level and specific in their reaction.

Biomarkers of toxic effects reflect pathological endpoints and are determined at each level of biological organization. These biomarkers serve as integrative markers of complex toxicities and ecologically relevant indicating environmental health at higher levels of biological organization( individual, population, and community level).

Molecular level biomarkers – The activity of cytochrome p 450-dependent monooxygenase system can be analysed as a biomarker of organic chemical pollutant exposure e.g. PCBs, PAHs, dioxins, furans.

Subcellular level biomarkers – the integrity of lysosomal system is analysed to find out the influence of PAHs, heavy metals and organochlorines.

Cellular level biomarkers – the accumulation of neutral lipids in fish hepatocytes is analysed for toxicity induced liver fat metabolism.

Individual level biomarkers – the macrophage aggregate activity is analysed as a marker of pollution induced changes of cellular immune responses.

A biosensor is a measuring device that produces a signal in proportion to the concentration of a xenobiotic substance in a biological system. E.g enzyme, antibody, membrane, organelle, cell, or tissue.

Biological indicators of water quality

Saprobic index(S.I) – a measure of level of organic pollution(Pantle and Buck 1955).

Nyggard’s algal index (Nyggard 1949) and Palmer’s algal index(Palmer 1969) – pollution index using algal species.
Biological index of pollution – BIP
Shannon – Weiner index (Shannon – Weiner 1949)

Potential uses of bioindicators

1. Indicate pollutant exposure

2. Help identify mechanisms of toxicity
3. Provide early warning of impending ecological damage
4. Reveal early indication of environmental recovery or remediation.
5. Important in linking cause/ effect relationship.
6. Can be included in ecological risk assessment.

Ecotoxicological concepts and applications

 The combination of the disciplines ecology and toxicology evolved into another integrative discipline known as ecotoxicology. The science of ecotoxicology is an outgrowth of the link between toxicology, ecology and chemistry. Environmental toxicological studies focus on the nature, properties, effects and detection of toxic substances in the environment and in any environmentally exposed species. The term ecotoxicology was first introduced by Truhaut in 1969 as a natural extension of toxicology. The major difference between toxicology and ecotoxicology is that toxicology deals with the effects of poisons on individual organisms whereas ecotoxicology deals with the effects on population of individuals.

 Aim of ecotoxicology

The aim of ecotoxicology centered on determining the effects of pollutants on the structure and functions of intact ecosystems, communities or assemblages. Ecotoxicologist is one who uses ecological parameters to assess the effects of toxic substances on ecosystems.

Scope of ecotoxicology

- to generate data that will be useful for risk assessment and environmental management.

-to meet legal requirements for regulating the development, manufacture or release of potentially dangerous substances.

- develop empirical or theoretical principles to further understanding of the behavior and effects of chemicals on living systems.

Toxic agent + Environment + host = toxic effects

       Objectives of ecotoxicology

The objective of ecotoxicology is to understand the mechanisms and processes whereby the environmental chemicals exert their effects on ecosystems and their impact on the populations or communities. The purpose of ecotoxicological tests is to predict the response of natural systems using tests in laboratories or model ecosystems. It is therefore an essential tool in the prevention of pollution by supporting environmental policies, laws, standards and control measures.

      

Definitions of ecotoxicology

 Ecotoxicology is a branch of toxicology concerned with the study of toxic effects, caused by natural and synthetic pollutants, to the constituents of ecosystems, animals (including human), vegetables and microbes in an integrated context (Truhaut,1977).

Ecotoxicology is the study of the effects of toxic substances occurring in both natural and man-made environments (Duffus 1980).

Ecotoxicology is the study of the impacts of pollutants upon the structure and function of ecological systems(from molecular to ecosystem) (Landis and Yu1995)

Ecotoxicological tools

Toxicological impacts in the ecosystem can be elucidated through a combination of bioassays using environment/ animal models and long-term field observations. Laboratory bioassays are performed to study feeding, growth, respiration, reproduction, histology, enzyme assays and mortality. Field observations such as tissue concentrations of toxins, species number, species density and population dynamics are crucial. Field experiments like the containment of test organisms at contaminated sites and environmental simulations (microcosms and mesocosms) aid in constructing theoretical models. Modeling throws more insights into our understandings of mechanisms of chemical movements in the environmental compartments.

Toxicological impact on the environment is a four-part process

The release of a chemical into the environment.
The transport of the chemical into biota, with or without chemical/bio-transformation.
The exposure of the chemical to one or more target organisms and
The response of the constituents or whole of the biosphere to the chemical exposure.

The paradigm shift from toxicology to ecotoxicology: Assumptions:

The range of variables that affect population responses is greater than the range that affects individual responses to pollutants.The sublethal effects on individuals may be as important as lethal effects.
 Different individuals of a given species or different populations of the same species may not respond in an identical manner to a pollutant.

Toxic substances have a strong influence both on the ecosystems and on the organisms in the ecosystems. These interactions may be complex and involve a number of parameters or organisms. This may also involve food chains and complex food webs. So there is a great deal of complexity exists because of the great variety of environmental factors and their interactions. The science, techniques and applications of ecotoxicology are evolving rapidly and will continue to contribute more on the understanding of pollution problems.

    Toxicologic principles and laws

Paracelsus (1567) stated “all substances are poisons; there is none which is not a poison. The proper dose separates a poison from a remedy”. Paracelsus is often referred to as the father of toxicology. According to him, the dose makes the poison and the sublethal dose determines the magnitude of the response. There is a threshold concentration for every toxin, which begins to produce effects on organisms; concentrations below this threshold will not have effect.

In order to understand the effects of toxins, Paracelsus believed  the following:

1. It was necessary to use experiments to identify and understand responses to chemicals.
2. There is a difference between toxic and therapeutic properties of chemicals.
3. The dose of a chemical is important in making the distinction between therapeutic and harmful effects and
4. It is possible to identify to some extent, the degree of chemical specificity.

M.J.B.Orfila (1815) described the harmful effects of chemicals on organism. He is often referred to as the founder of toxicology.

  Terminology
A toxic agent is anything that can produce an adverse biological effect. It may be chemical, physical or biological in form.

Chemical agent-e.g. cyanide

Physical agent-e.g. radiation

Biological agent – e.g. snake venom

A toxic substance is simply a material which has toxic properties. It may be a individual toxic chemical or a mixture of chemicals e.g. lead chromate.

It may be organic toxins or inorganic toxins. Organic toxins contain carbon and are man-made large molecules. Inorganic toxins are specific chemicals that are derived from minerals.

Toxic substances may be systemic toxins or organ toxins. A systemic toxin is one that affects the entire body. e.g. potassium cyanide. It affects every cell and organ in the body.

A organ toxin may affect only specific tissues or organs. Lead is a specific organ toxin. It affects 3 target organs namely CNS, kidney and hematopoietic system.

Concept of toxicity – toxicity is usually defined as the inherent property of a chemical to produce adverse biological effects. Toxicity is a function of  concentration (dose) and the duration of exposure. It is characterized in terms of acute or chronic effects and local or systemic effects. Analytical instruments cannot measure toxicity. They can measure the concentration of a chemical in the environment or in organism.

Application of knowledge and techniques of ecotoxicology

1. Determining contaminants leaching from wastes, their critical ecological thresholds and breakpoints.
2. Developing bio-marker –based monitoring systems.
3. Establishing protocols for the protection of natural environment.
4. Developing guidelines for risk assessment of anthropogenic wastes.
5. Contributing remedial ecological restoration measures.