INTRODUCTION
Due to the significant increase in the environmental pollutants and lack of precautionary measures, environmental pollution has become a global problem (BONDARENKO et al., 2004; HELA et al., 2005; ABDEL-HALIM et al., 2006; WILSONT and FOOS, 2006).
The producers have been compelled to intensify their efforts to increase food products to meet the needs of ever-increasing population. The farmers as one of the main components of this chain are using escalating amounts of pesticides to protect their crops (CKAMACA, 2002; STRUGER, 2002). Pesticide residues in food items have been a concern to environmental and consumer groups of their wide spread use (DARKO et al., 2007). Pesticides are widely used to control agricultural pests are also causing threats to the human health through the transfer of dangerous diseases (CKAMACA, 2002; PEDERSEN et al., 2006; RAWN et al., 2006). Although these chemicals are beneficial for the anthropogenic land use but they are dangerous to the biotic species and human living environment (SUDO et al., 2002; NA et al., 2006).
Pesticides are being used in large quantities (IMO et al., 2007) but when applied in any specific area the major part (about 99%) of applied amount remains unused and goes in the environment, where it gets mixed with air, soil, water and plants by several means and causes harmful effects (AGRAWAL et al., 2010). Especially organochlorinat ed and organophosphoru s (OP) pesticides are causing water and soil pollutions, as well as the contamination of vegetables, fruits, milk, food products and other living organisms (DENISTROP, 2000; AHMAD, 2001; ERIN et al., 2001; HAMILTON et al., 2003). Contamination in the rivers is depleting their resources that can put the lives of many people in danger (CHIMWANZA et al., 2006) and can be harmful to the local fauna and flora (FLEEGER et al., 2003; SCOTT and SLOMAN, 2004; LYTLE and LYTLE, 2005).
Besides water river bed sediments also carry significant load of contamination (TSAI et al., 2003; BHATTACHARYA et al., 2003) that enters the rivers from both natural and anthropogenic sources i.e. from agricultural runoff, domestic sewage and industrial effluents (MOKAYA et al., 2004; SINGH et al., 2005; MILOVANOVIC, 2007)
Pesticides can enter surface waters from two sources; as point sources, i.e., at certain locations along the river. The spilling during the filling of the spraying equipment, the cleaning of the equipment and processing of spray waste on paved surfaces are the main causes of point sources (BEERNAERTS et al., 2002; NEUMANN et al., 2002). Or as diffuse sources which are inputs along the whole water course. Run-off is considered to be the most important path way of diffused pollution source. Diffusion through tile drainage is far less important and spray drift is only an issue in specific orchard regions (BATCH et al., 2001; RITTER et al., 2001). From field measurements, inputs by drift, direct spraying or from the atmosphere in precipitation (MAJEWSKI et al., 2000; ROSSI et al., 2003) or dry deposition (FOREMAN et al., 2000) are less important.
The Ravi is the most polluted river in Pakistan. Most wastewater discharge reaches the river in a 60 km stretch between Balloki and Lahore. The Ravi is currently estimated to receive 47 percent of the total municipal and industrial discharge pumped into all rivers of Pakistan. In addition, industrial and agricultural waste from both India and Pakistan pour into the river (JAVED, 2005; RAUF et al., 2009; KUMAR and DUA, 2009).
Traces of pesticides are detected in the surface waters (AHAD et al., 2006) and in shallow drinking water wells of Pakistan nearby the areas where heavily pesticides are being used (AHAD et al., 2000, 2001; TARIQ et al., 2004).
Pesticides in river systems are subjected to different reactions, depending on their physiochemical characteristics and the characteristics of river system itself. This includes biodegradation, bioconcentratio n, hydrolysis, photolysis, redox reaction, and volatilization. The fate process, bioconcentratio n of pesticides is of interest because it helps to predict human exposure to the toxicant in food items, particularly fish (BERMUDEZ-SALDANA et al., 2005; BOSSUYT and JANSSEN, 2005).
Fish serves as a bioindicator species in monitoring the water pollution, because they play an important role in the food web, they are bio-concentrators and are responsive to mutagenic and genotoxic agents at low concentrations such as environmental pollutants (WHITFIELD and ELLIOT, 2002; HARRISON and Whitfield, 2004; ROSET et al., 2007).
Genotoxic agents have the potential to interact with DNA and may cause damage to DNA. Many pesticides have been tested for mutagenicity by a variety of in vitro and in vivo assays, and mutagenic compounds have been found among all major categories of pesticides, including insecticides, fungicides and herbicides.. Several biomarkers have been utilized as tools for detection of exposure to pesticides. Such biomarkers include presence of DNA adducts, chromosomal aberrations, Sister chromatid exchange , DNA strand breaks and micronuclei measurements (GIRI et al., 2002; YADAV and KAUSHIK, 2002; FARAH et al., 2003; BOLOGNESI, 2003; WHITEHEAD et al., 2004; FLORES-MAYA et al., 2005; BULL et al., 2006; ERGENE et al., 2007; BONEY et al., 2008; SOLONESKI and LARRAMENDY, 2010).
DNA and RNA, the nucleic acids, are the molecules responsible for the hereditary information that commands the protein synthesis in living beings. The nucleic acids derived indices, especially muscle ribonucleic acid (RNA) to deoxyribonuclei c acid (DNA) ratio provides an estimate of short term (hours to days) growth rate and better understanding of physiology, trophic interaction and changes in composition and structure in aquatic ecosystems. While DNA per cell remains relatively constant, the amount of RNA will vary with physiological status, requirements for protein synthesis and growth (BUCKLEY et al., 1999).
Molecular methods based on nucleic acid derived indices (BERGERON et al., 1997) and the polymerase chain reaction has recently become an important tool in marine ecology (NEJSTGAARD et al., 2003). One of the most widely used nucleic acid derived indices in marine ecology is the RNA:DNA ratio. This index gives a measure of the synthetic capacity of the cell and usually correlates with nutritional status (BUCKLEY et al., 1999). In fact RNA:DNA ratios have been used on a wide range of marine organisms, mainly plankton, (IKEDA et al., 2007), fish (CALDARONE et al., 2003; GARCIA et al., 2007), bivalves (CHICHARO et al., 2001), cephalopods (SYKES et al., 2004) and crustaceans (LEMOS et al., 2002; CHICHARO et al., 2007). RNA:DNA ratios have often been used in marine fish. Few studies have measured RNA:DNA ratios in fresh water fish under natural conditions (AUDET and COUTURE, 2003; TARDIF et al., 2005).
RNA:DNA ratio for selected body tissues has frequently been used as an indicator of nutritional status and growth rate in fish (CHICHARO, 1998; HEYER et al., 2001; WEBER et al., 2003; OLIVER et al., 2009). Ratios were stated to be positively correlated with the growth of some fish (FAKUDA et al., 2001) or feeding conditions of juvenile Atlantic code (SMITH and BUCKLEY, 2003). There is evidence from laboratory and field studies that reduction in average nucleic acid ratios are associated with reduced survival rates (CLEMMENSEN et al., 1997; PEPIN et al., 1999).
Effects of environmental factors such as food availability, temperature and environmental toxins on growth and condition of fishes have been successfully evaluated with RNA:DNA ratios (DAHLHOFF, 2004). In a variety of taxa, fish being fed a high ration had a higher RNA:DNA ratio than fish maintained on low rations or deprived of food for a few days. (ADAY et al., 2000; TANAKA et al., 2008).The ratio between these two nucleic acids increased with rise in temperature from 9.3°C to 22.3°C in juvenile signal crayfish but were reduced when the fishes were exposed to high temperatures or heavy metal salts. Likewise, there was also a significant decrease in RNA:DNA ratio of pre spawing of Labeo rohita after 9 and 30 days of mercury and methyl parathion (ADITYA et al., 2002).
RNA:DNA ratios vary in response to many environmental and biological conditions, but typically minimum values are greater than 1 when the ratio falls below 1 it is usually attributed to physiological stress , such as food limitation (KONO et al., 2003) or hypoxia (ADAY et al., 2000). The ratio can also be used as a bioindicator of contaminant stress, as growth rates can change with contaminant exposure.
Dimethoate is an organophosphate (OP) pesticide. Because of its electrophilic nature, it is possible that the enzyme necessary for DNA synthesis might have been inhibited by this insecticide. Inhibition of DNA synthesis, thus, might affect both protein as well as amino acid levels by decreasing the level of RNA in protein synthesis machinery. (TRIPATHI et al., 2003).
Endosulfan is an organochlorine pesticide, that has been found to be generally more toxic to fish than invertebrates. Endosulfan did not produce any significant effect on DNA content and RNA:DNA ratio. However, the RNA and protein contents of brain, liver and skeletal muscle decreased significantly in tissues. The maximum decrease in RNA and protein was approximately 30% to 37%. Withdrawal of endosulfan from the medium for 21 days restored the RNA and protein contents nearly to their control levels (TRIPATHI and VERMA, 2004).
People generally show an increasing interest in healthy diets, in which fish has a prominent place for its energy and bio-nutritive values. The n-3 polyunsaturated fatty acids in fish, eicosapentaenoi c acid (EPA) and docosahexaenoic acid (DHA), are likely to prevent sudden cardiac death, manifested as fatal ischemic heart disease or arrhythmic death (LEMAITRE et al., 2003; MOZAFFARIAN et al., 2006) and possibly as ischemic stroke (MOZAFFARIAN et al., 2005). Fish consumption also prevents asthma and airway abnormalities through their anti inflammatory effects in the airway wall (SCHWARTZ et al., 2000; WOOD et al., 2001, 2002) and is associated with a modestly decreased risk of developing rheumatoid arthritis (MAGDALENA et al., 2009) and may also have various effects on cancer risk (MACLEAN et al., 2006).
The aquatic environment can be polluted by contaminants that have been bio accumulated by fresh water fish. Concern has been raised that contaminants in some fish may lead to increase in total cancer risk because of pesticide contaminants (HUANG et al., 2006). It is evident that the consumption of fish is beneficial to humans, but if these fish are contaminated, they pose mutagenic, teratogenic and carcinogenic health risks to consumers. So the present study aimed to asses the effect of pesticide residues in muscles of major carps (Labeo rohita, Catla catla, Cirrhinus mrigala) inhabiting the river Ravi, using nucleic acid index (RNA:DNA ratio) as bioindicator for pollution.