MODELING GENETIC DEFECTS AS A MOLECULAR BIOMARKER IN EVALUATING ENVIRONMENTAL IMPACTS DICHLORVOS ON PULLET CHICKS

ABSTRACT

Environmental pollution and poisoning owing to the widespread use of pesticides in agricultural and domestic pest control may be detrimental to the health of handlers, non target organisms and consumers. In this study, genetic defect was used as a molecular biomarker in evaluating the environmental  impacts  of  dichlorvos,  a  widely  used  pesticide  in  Nigeria,  on  poultry  birds (Gallus domestica). Seven week old pullets with an average weight of 557.5 ± 9.5 g divided into four groups of ten birds each were fed on commercial poultry feed contaminated with 0.01, 0.02 and 0.04% (w/v) dichlorvos. The control group had no pesticide added into their feed. The birds were exposed for ten weeks after which they were sacrificed and the liver taken for analysis. Electrophoresis of isolated liver DNA in 0.8% agarose gels gave variations in band intensity between the control DNA sample and DNA from exposed birds. These variations in band intensity were more pronounced in the RAPD-PCR products amplified with OPE-01 primer, where there is complete disappearance of DNA bands in the birds exposed to 0.04% pesticide. Thermal denaturation of the DNA from the exposed birds resulted in a significant reduction (p< 0.01) in the DNA melting temperature  from  87.2oC  to  81.7oC  while  the  GC/AT ratio  was  also  significantly  reduced (p<0.01) from 0.77 in the control to 0.42 in exposed birds respectively. The percentage weight gain of the birds over the 10 week period was significantly higher (p<0.05) in the control (126.50%) when compared with the birds fed on pesticide contaminated diet (68.75%, 65.10% and 28.10% respectively), but increase in liver weight was not significant (p>0.05). There was also a reduction in feed intake by the birds exposed to pesticides. Egg laying was delayed in the hens exposed to pesticides by as much as eighteen weeks. The ages of the hens at first egg  lay were 18 weeks for the control, 23 weeks for hens fed on 0.01 and 0.02% contaminated diet and 36 weeks for those fed on 0.04% contaminated diet. The average  daily  egg  production  was  reduced  from  5  eggs  in  the  control  group  to  1  egg  in  0.04% contaminated group.  The protein contents of the egg (yolk and albumin) and cholesterol level of the egg yolk were lower in birds exposed to pesticide. There was a general reduction of liver (cytoplasmic and membrane bound) cholesterol, triglycerides and total lipids in the birds fed on pesticide contaminated diet as well as reduction (p<0.05) in GSH levels and GST activity. There was also a significant increase (p<0.05) in lipid peroxidation in the birds exposed to the pesticide. Results of this study suggests that dichlorvos exposure has genotoxic effects in addition to other physiological and biochemical effects on poultry birds.

CHAPTER ONE

INTRODUCTION

1.0 Introduction

The use of pesticides in agriculture has become important considering the huge losses farmers experience due to the ravaging effects of agricultural pests. The large scale use of pesticides in agriculture has proven effective in controlling and minimizing losses due to pests. Experts agree that removal of pesticides from crop protection will result to an immediate drop in food supplies (NAS, 1975). Pesticides have also found application in other areas for example in controlling wood destroying insects like termites and disease control.

Despite the immense benefits that can be derived from the use of pesticides, continuous  and  indiscriminate  use  of  pesticides  has  caused  severe  environmental problems and is a health hazard for humans and animals (Perring and Mellanby, 1975; Partanen et al., 1999). The U.S. Environmental protection Agency (EPA) states that, “By their very nature, most pesticides create some risk or harm to humans or the environment because they are designed to kill or otherwise adversely affect living organisms” (U.S.EPA, 2002). Bioaccumulation of pesticides in the food chain can lead to potentially adverse effects in humans due to their putative toxic effect (Palmeira, 1999).

There is a need to devise an accurate and effective way of monitoring the effects of pesticide exposure to humans and the environment. Until recently, the most common end -point measured when evaluating toxicity of chemicals focuses on mortality values (Otijolu and Onwurah, 2007). Mortality values can only provide a measure of short-term acute toxicity and are not always useful for predicting the ecological consequences of

exposure to a particular chemical (Neuhauser et al., 1984). Exposure to pesticides can be monitored biologically by assessing the absorption of the chemical or its metabolite in body fluids (Kangas and Tuomainen, 1999). This can be done by determining the urinary pesticide levels or measuring the changes in the activity of cholinesterase in the blood of individuals  exposed  tp  organophosphates.  The  determination  of  blood  cholinesterase levels is very important in the monitoring and study of exposure to organophosphorus compounds  and  carbamates.  To this  end  the American  Conference  of  Governmental Industrial Hygienists recommends the use of red blood cell cholinesterase as a biological marker (Partanen et al., 1999). Assessment of exposure to organophosphates and carbamates usually includes baseline cholinesterase determinations before the exposure and subsequent periodic testing during use of these pesticides.

The use of biomarkers rather than diseases to access the risk of environmental exposure to pesticides has been on the increase (Albertini, 1976). Biomarkers are characteristics that are objectively measured and evaluated as indicators of normal biological processes, pathogenic processes or pharmacological responses to therapeutic intervention. These are key molecular or cellular events that link a specific environmental exposure to a health outcome. Biomarkers can be classified into three categories: biomarkers  of  exposure  reflecting  the  dose  of  toxic  agents,  biomarkers  of  effect indicating a biological response to exposure, and biomarkers of susceptibility providing information about the inherent sensitivity of individuals to the toxic agents (Groopman et al., 1994). Different types  of molecular  biomarkers  are extensively used worldwide. Among the various types of biomarkers, the following have received special attention: cytochrome    P450    induction,    DNA   integrity,   acetylcholinesterase    activity    and

metallothionein induction (Sarkar et al., 2006). The information obtained from studies of molecular biomarkers in humans and experimental animals can be used for a range of public  health  applications,  from  primary  and  secondary  prevention  to  the  design  of clinical therapies (Groopman et al., 1994).

1.1 Pesticides

The  United  States  Environmental  Protection Agency  has  defined  a  pesticide  as  any substance or mixture of substances used to prevent, destroy, repel or mitigate any pest (US EPA, 2006b). A pesticide may be a  chemical substance, biological agent (such as a virus  or  bacteria),  antimicrobial,  disinfectant  or  device  used  against  any   pest.  Pests include   insects,  plant   pathogens,  weeds,   molluscs,   birds,   mammals,   fish,  nematodes (roundworms),  microbes and people that destroy property, spread or are a  vector for disease or cause a nuisance. Pesticides used to manage insects are called insecticides, those used to manage fungi are fungicides, etc, (Ware and Whitacre, 2004).

Insecticides are agents of chemical or biological origin that control insects (Carpenter and Ware, 2004). There are over 300 commercially available insecticides in hundreds of different formulations (Pedigo, 1991). Most insecticides are natural biochemicals extracted from plants, while others are inorganic chemicals derived from toxic  metals  or  compounds  of  arsenic  (Salgado,  1997).  However,  most  modern insecticides are organic chemicals that have been synthesized by chemists (Aspelin and Grube, 1998). Many of these chemicals are toxic to humans and can be accumulated in the  food  chain  (Tomlin,  2000).  The  insecticides  most  generally  applied  have  broad

spectrum activity and are toxic to a great variety of animals including humans (Pedigo, 1991).

Pesticide formulations may be in either solid or liquid form, or they may be other products, e.g. aerosols. The most common formulations in agriculture are emulsifiable concentrates (EC), soluble concentrates (SC), and wettable powders (WP).

1.1.1 Classification of pesticides

Pesticides can be classified by target  organism, chemical structure, and physical state and mode of action (CSA, 1997; Ware, 2000). Pesticides can also be classed as inorganic, synthetic, or  biologicals (biopesticides), (CSA, 1997), although the distinction can sometimes be blurred. Biopesticides include microbial pesticides and biochemical pesticides (EPA, 2009). Plant-derived pesticides, or “botanicals”, have been developing quickly. These include the  pyrethroids,  rotenoids,  nicotinoids, and a fourth group which includes  strychnine and  scilliroside  (Kamrin, 1997). Pesticides also include plant growth regulators, defoliants, or desiccants (Hagstrum and Subramanyam, 2006). WHO (2001) has classified pesticides according to their toxic effects as class I (extremely hazardous) to class III (slightly hazardous). Pesticides can also be classified based upon their biological mechanism, function or application method.

1.1.2 Classes of Pesticides

Different classes of pesticides exist. The major classes of pesticides are organochlorine pesticides, organophosphates, carbamates and pyrethroids.

1.1.2.1 Organochlorine pesticides

The organochlorine pesticides consist of chlorinated hydrocarbons containing the elements: chlorine, hydrogen and carbon (Thomson, 2001). They are generally synthetic organic insecticides known to be quite toxic to man and animals and very persistent in the environment.   These   groups   of   pesticides   have   the   effect   of   disturbing   normal transmission of nervous signals. Consequently, they are responsible for causing paralysis and nervous dysfunction in exposed agents, e,g, insect pests and other arthropods. The insecticidal  potential  of  chlorinated  hydrocarbons  was  discovered  during  the  second world war (Hassal, 1990). Common examples of organochlorine insectides include dichloro diethylene trichloroethane (DDT), chlordane (benzene hexachloride), aldrin, dieldrin, endrin, toxaphene, endosulphan, etc. The toxicities of this group of pesticides vary greatly, but they have been phased out because of their persistence and potential to bioaccumulate (Kamrin, 1997).

1.1.2.2 Organophosphate pesticides

Organophophate (OP) pesticides consist mainly of organophosphorus compounds. They are the most widely used pesticides today (agriculture, industry, home, gardens and in veterinary practice) and the cause of most incidences of pesticide poisoning than any other  class  of  pesticides  (Ames  and  Gold,  2000).  Organophosphate  pesticides  are generally the most toxic of all pesticides and most are chemically unstable and non- persistent (Pedigo, 1991). They are easily broken down a few hours or days after application,  as  against  the  organochlorides  that  take months  or  years  to  disintegrate (Hassal,   1990).   It   was   this   non-persistence   in   the   environment   that   brought

organophosphate pesticides in agricultural use as substitutes to organochlorine pesticides (Charles, 2001). Organophosphates function primarily as cholinesterase inhibitors at the nerve junctions and they disturb nerve transmission impulses in various organs and at the junctions  of  nerves  and  muscle  connections.  Consequently  they  may  account  for paralysis, stupefaction, dizziness and mortality of exposed organisms, especially when applied in high concentration. Common examples of organophosphate pesticides include malathion, fenthione, diazinon, dimethoate, phosdrin (mevinphos), and DDVP (Dichlorvos). Organophosphate pesticides are relatively soluble in water and this offers great advantage for their formulation and application in the field. For example, for spraying on surfaces or vegetations or for dipping of animals in treated chemical troughs as in the case with veterinary handling of livestock and animals for pest control.

In addition to their intended effects like the control of insects or other pests, organophosphates  are sometimes found to affect non-target organisms including humans (Chantelli-Forti et al., 1993; Chandhuri et al., 1999). Exposure to organophosphates is also a potential cause of longer-term damage to the nervous system, with reports of poor mental health and deficits in memory and concentration (Davis, 1991; Mason, 2000; Nigg and Knakk, 2000).

1.1.2.3 Carbamates

The carbamate pesticides consist of carbonic acid esters and they represent a rather popular group of pesticides commonly employed because of their relatively mild nature. Carbamate insecticides are frequently employed to control insects that for some reasons, do  not  readily  respond  to  organophosphates  (Carpenter  and  Ware,  2004).  Like

organophosphates,  carbamates  operate  through  inhibiting  the  enzyme acetylcholinesterase, allowing acetylcholine to transfer nerve impulses indefinitely and causing a variety of symptoms such as weakness and paralysis. Common examples of carbamates include abate (termiphos), carbaryl, pyrolan, propoxur and aprocarb.

1.1.2.4 Pyrethroids

Pyrethroids consist of a particular group of chemical pesticides commonly employed for short term control or suppression of various pest species. There are two kinds of pyrethroids; the natural and synthetic. The natural pyrethroids have long been used as active ingredients in domestic sprays employed against flies and mosquitoes (Ware and Whitacre, 2004). There are four principal active ingredients in pyrethrum flowers known as  pyrethrins  I  and  II  and  cinerins  I  and  II  (Hassal,  1990).  Synthetic  pyrethroid insecticides are derived from natural compounds (the pyrethrins) isolated from the Chrysanthemum  genus  of  plants  (Casida,  1980).  Pyrethroids  are  one  of  the  most important classes of insecticides accounting for 17% of the world insecticide market (Davies et al., 2007). All pyrethroids contain several common features such as presence of an acid moiety, a central ester bond, and an alcohol moiety. The acid moiety contains two  chiral  carbons;  thus,  pyrethroids  typically  exist  as  stereo-isomeric  compounds (Kumar et al., 2010). Common examples of pyrethroids include pyrethrin, alletrin, furethrin,   resmethrin,   bioresmethrin,   cyclethrin,   cypermethrin   and   deltamethrin, Pyrethroid pesticides are used to control a wide range of insects in public and commercial buildings, animal facilities, warehouses, agricultural fields, and greenhouses. They are also applied on livestock to control insects. In agriculture, cypermethrin, cyfluthrin, and deltamethrin have been used frequently on cotton. Pyrethroid insecticides are the most

common active ingredient in commercially available insect sprays and are also used as structural termiticides. Certain pyrethroid insecticides (such as permethrin, resmethrin, and sumithrin) are also registered for use in mosquito-control programs in the United States. Outside the U.S., deltamethrin has been used for indoor protection against mosquitoes that carry malaria, in some situations replacing the use of DDT. About two million pounds of permethrin and one million pounds of cypermethrin have been applied annually (U.S. EPA, 2006a, 2006b). Permethrin is also used in skin lotions and shampoos as medical treatments for lice and scabies. Pyrethroid pesticides are generally formulated as complex mixtures of different chemical isomers, solvent oils, and synergists, such as piperonyl butoxide.  Pyrethroid pesticides have low volatility, bind to soils, and are rarely detected in ground waters (USGS, 2006). Generally, they are not persistent in the environment due to their rapid degradation within days to several months. This class of pesticides has low toxicity in birds and mammals, but pyrethroids are highly toxic to fish and some aquatic invertebrates, so usage is restricted near water (U.S. EPA, 2002).

1.2 Environmental effects of pesticides

Pesticides are used in every realm of the environment to control undesired pests, such as insects, weeds, fungi and rodents. Although there are benefits to the use of pesticides, there  are  also  drawbacks,  such  as  potential  toxicity  to  humans  and  other  animals. Pesticide use raises a number of environmental concerns. Pesticides are one of the most potentially harmful chemicals introduced into the environment. Though they have contributed  considerably  to  human  welfare,  their  adverse  impacts  on  non-target

organisms are significant (Hazarika and Das, 1998; John, 2007). Over 98% of sprayed insecticides and 95% of herbicides reach a destination other than their target species, including non-target species, air, water and soil (Repetto and Baliga, 1996;, Miller, 2004). Pesticide drift occurs when pesticides suspended in the air as particles are carried by wind

to other areas, potentially contaminating them. Pesticides are one of the causes of  water

pollution, and some pesticides are  persistent organic pollutants and contribute to  soil contamination.

In  addition,  pesticide  use also  reduces  biodiversity and  results  in  lower soil quality

(Johnston,  1986),  reduce   nitrogen fixation  (Rockets,  2007),  contribute  to   pollinator decline (Wells, 2007), can reduce habitat, especially for birds (Palmer et al., 2007), and can threaten  endangered species (Muller, 2004).

Pesticides can be dangerous to consumers, workers and close bystanders during manufacture, transport, or during and after use (U.S. EPA, 2007).  The World Health Organization and the  UN Environment Programme estimate that each year, 3 million workers  in  agriculture  in  the  developing  world  experience  severe   poisoning from pesticides, about 18,000 of them die (Muller, 2004). According to one study, as many as 25 million workers in developing countries may suffer mild pesticide poisoning yearly (Jeyaratnam,1990).   Organophosphate pesticides have increased in use, because they are less damaging to the environment and they are less persistent than organochlorine pesticides (Jaga and Dharmani, 2003). These are associated with acute health problems for workers that handle the chemicals, such as abdominal pain, dizziness, headaches, nausea, vomiting, as well as skin and eye problems (Ecobichon, 1996). Additionally,

many studies have indicated that pesticide exposure is associated with long-term health problems  such  as  respiratory  problems,  memory  disorders,   dermatologic  conditions (Arcury et al., 2003; O’Malley, 1997), cancer (Daniels et al., 1997),  depression (Beseler et al., 2008),  neurological deficits (Kamel, 2003),  miscarriages, and  birth defects (Cordes and Foster, 1988; Eskenazi et al., 1999; Engel et al., 2000;; Das et al., 2001). Summaries of peer-reviewed research have examined the link between pesticide exposure and neurologic outcomes and  cancer, perhaps the two most significant things resulting in organophosphate-exposed workers (Alavanja et al., 2004; Kamel and Hoppin, 2004).

According to researchers from the  National Institutes of Health (NIH), licensed pesticide applicators who used chlorinated pesticides on more than 100 days in their lifetime were at greater risk of  diabetes. One study found that associations between specific pesticides and incident diabetes ranged from a 20 percent to a 200 percent increase in risk. New cases of diabetes  were reported by 3.4 percent of those in the lowest pesticide use category compared with 4.6 percent of those in the highest category. Risks were greater when users of specific pesticides were compared with applicators who never applied that chemical (Montgomery et al., 2008).

There are concerns that pesticides used to control pests on food crops are dangerous to people who consume those foods. These concerns are one reason for the  organic food

movement. Many food crops, including fruits and vegetables, contain  pesticide residues

after being washed or peeled. Chemicals that are no longer used but which are resistant to breakdown for long periods may remain in soil and water and thus in food (Cornell University, 1999).

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