**3. Phase I**

and surrounding environments were contaminated with different insecticides (Arjmandi et al., 2010; Bagheri et al., 2000; Ghassempour et al., 2002; Rahiminezhhad et al., 2009; Tarahi Tabrizi, 2001). The continuous presences of insecticides are the consequence of application (timing, rate, frequency) and the rainfall during the application period (Lydy and Austin, 2004; U.S.A. EPA, 2005; Bouldin et al., 2007; Mast et al., 2007; Echols et al., 2008; Vryzas et al., 2009; Werimo et al., 2009; Ding et al., 2011; Hope, 2012). Although monitoring the presence of insecticides in surface water and ground water are generally poor in much of the world and especially in developing countries, the effect of these pollutants on aquatic animals' health frequently was investigated (Chambers et al., 2002; Richards and Kendall, 2002;Lam and Wu, 2003; Scott and Sloman, 2004; Cengiz, 2006; Box et al., 2007; Sun and Chen, 2008; Banaee et al.,

Since fishes are important sources of proteins and lipids for humans and domestic animals, so health of fishes is very important for human beings. Fish like other aquatic organisms may be exposed to a great range of insecticides during the course of their life cycle. In fish, different insecticides can be absorbed through gills, skin or alimentary ducts (Schlenk, 2005; Banaee et al., 2011; Banaee, 2012). Fishes are particularly sensitive to environmental contamination of water. Hence, pollutants such as insecticides may significantly damage certain physiological and biochemical processes when they enter into the organs of fishes (Banaee et al., 2011). So,

Recently, many studies have been conducted to determine the mechanisms of insecticides' damage in fishes, with the ultimate goal of monitoring, controlling and possibly intervening in xenobiotics exposure and its effects on the aquatic ecosystem. The main mechanism of action of organophosphate and carbamate insecticides is block of enzyme acetylcholinesterase action that results in signs and symptoms of intensive cholinergic stimulation. Organochlorines are neurotoxins which effect on sodium and potassium channels in neurons. Decrease of potassium permeability and inhibition of cadmodulin, Na/K and Ca-ATPase activity occur by organochlorine insecticides. Pyrethroids can block Na channel and effect on the function of GABA-receptors in nerve fiber. Oxidative stress is another mechanism for toxicity of insectisides resulting in cell death includescellular necrosis and apoptosis and dysfunction in cellular physiology include alterations in metabolic and vital functions of the cells.Hence, fish should be able of managing environmental exposure by detoxifying these xenobiotic. In order to do this task, fish like aerobic animals have evolved complex of detoxification system, composed of two main parts including enzymatic and non-enzymatic components. Theroles of enzymatic and non-enzymatic detoxification system of animal's body lessen the potential damages caused by the toxicity of environmental pollutants. Although it is possible that reactive oxygen species (ROS) produced during the insecticides detoxification process in liver tissue may react with vital macromolecules such as lipid, protein, carbohydrate and nucleic acid and result in oxidative damage to aquatic organisms (Üner et al., 2006). ROS derived damage to natural and structure cellular components are generally considered a serious mechanism involved in the physiological and pathological disorders (Sepici-Dinçel et al., 2009).So, much literature suggests an association between impaired detoxification and disease

2008; Banaee et al., 2011; Banaee and Ahmadi, 2011).

104 Insecticides - Development of Safer and More Effective Technologies

the effects of insecticides on fishes are of great concern.

The phase I detoxification system, composed over 10 families of enzymes which played important role in the metabolism of various xenobiotic. The phase I detoxification system, composed mainly of the cytochrome P450 supergene family of enzyme, that are present in all eukaryotes and some prokaryotes and is generally the first enzymatic defense system against xenobiotic.A great diversity of cytochrome P450 enzymes in fish has been recognized (Stegeman and Hahn, 1994), and CYP1A, CYP2B, CYP2E1, CYP2K1 and CYP3A have been recently identified in liver of some freshwater fish (Nabb et al., 2006) which play an important role in the detoxification of organophosphate and carbamate insecticides (Ferrari et al., 2007). The common pathways of biotransformation of different kinds of insecticides include three cytochrome P450 (CYP) mediated reactions: *O*-dealkylation, hydroxylation, and epoxidation of insecticides (Keizer et al., 1995; Kitamura et al., 2000; Straus et al., 2000; Behrens & Segner, 2001; Nebbia, 2001). Most insecticides are metabolized through phase I biotransformation. In general, CYP450 enzymes mediated reactions by using oxygen and NADH, as a cofactor, lead to detoxification and subsequent excretion of xenobiotic. The CYP450 enzymes can also facilitate dealkylation, dearylation, aromatic ring hydroxylation, thioether oxidation, and deamination (Table 1.). However, CYP450 enzymes mediated metabolism can also cause formation of reactive metabolites which is far more dangerous than parental compounds. For example, oxidative group transfer of certain organophosphorous insecticides to the toxicorganophosphate, e.g. conversion of parathion to paraoxon,oxidative dechlorination of chloroform to phosgene,activation of ethyl carbamate to urethan. However, many of these same chemicals are also detoxified by cytochrome P450 by conversion to less toxic metabolites. In some cases, the same enzyme may catalyze activation and detoxification reactions for a given chemical. The resulting toxic effect of a xenobiotic chemical is thus due to a balance between metabolic activation and deactivation (Casarett and Doull, 1996).

**Metabolism Category Reaction pattern**

Dehalogenation R1 RX→R-H+X- X = halogen

Dehydrohalogenation R2 CH - CX→C = C + HX X = halogen

Sulfone, sulfoxide R5 -S(O2) - → - S(O) - → - S - Hydrolysis

Multiple bond R4 -N=N - → - NH - NH - , - C≡C - → -C=C- → - CH - CH -

X, Y = O, S ROS(= O)OR'

[X=halogen]

Nitro group R3 -NO2→ - NO →NHOH→NH2

Carboxyl ester H1 -C(= O)OR→COOH + R - OH

Amide H3 -C(= O)NR - → - COOH + NHR

Urea H5 -NHC(= O)NR - → - NH2 + RNH -

Carbamate H4 -NC(= O)O(or S)R - → - NH + RO(or S)H

Glucuronidation C1 R - XH→R - X - Gla X = O, COO, S, NH

Sulfation C3 R-X-H→R - X - SO3H X = O, NH

*N-*acylation C5 -NH2→ - NHCHO, - NHC(= O)CH3

Methylation C7 R-O(or NH)H→R-O(or NH)CH3

Amino acid conjugation C6 R - COOH→RC(= O)NHC(R'

Miscellaneous M Isomerization, rearrangement, etc.

**Table 1.** Classic detoxification pathways of insecticides in aquatic organisms (adapted from Katagi, 2010)

C=C→epoxide, ketone, NH2→NHOH

Physiological Dysfunction in Fish After Insecticides Exposure

http://dx.doi.org/10.5772/54742

107

→NO, R - X→R - OH X = NO2


→ROH + R'SO3H


R - XH→R - X - Glu→R-X- (6*O* - R*'*

R - X + GSH→R - SG→R - Cys - Gly →R - Cys→R - (N - acetyl - Cys)

X = O, COO, S, NH; R*'*

)Glu

)COOH

= acetyl, malonyl, pentosyl

Others O8

Phosphoryl (sulfonyl) ester H2

Others H6

Glucosidation C2

Glutathione conjugation C4

**Reduction**

**Conjugation**

### **4. Phase II**

In phase II reactions, metabolites produced in phase I detoxification often conjugate with glutathione, uridyl-diphosphate glucose (UDPG), uridyl-diphosphate-glucuronic acid (UDPGA), amino acid derivatives and sulfate derivatives and can readily excrete from the fish body (Keizer et al., 1995; Kitamura et al., 2000; Straus et al., 2000; Behrens & Segner, 2001; Nebbia, 2001). In fact, this results from enzymatic oxidation and hydrolysis that produce metabolites with OH, COOH and NH2, SH functional groups. These functional groups are then subject to conjugation with carbohydrates, glutathione, sulfate, andamino acids, and then, the final metabolites may also be excreted from the body of fish through the skin, gills, genital products, urine as sulphated and glucuronidated metabolites and stool as glutathione conjugated metabolites (Kitamura et al., 2000; Straus et al., 2000; Behrens & Segner, 2001; McKim & Lein, 2001; Nebbia, 2001).



2001; Nebbia, 2001). Most insecticides are metabolized through phase I biotransformation. In general, CYP450 enzymes mediated reactions by using oxygen and NADH, as a cofactor, lead to detoxification and subsequent excretion of xenobiotic. The CYP450 enzymes can also facilitate dealkylation, dearylation, aromatic ring hydroxylation, thioether oxidation, and deamination (Table 1.). However, CYP450 enzymes mediated metabolism can also cause formation of reactive metabolites which is far more dangerous than parental compounds. For example, oxidative group transfer of certain organophosphorous insecticides to the toxicorganophosphate, e.g. conversion of parathion to paraoxon,oxidative dechlorination of chloroform to phosgene,activation of ethyl carbamate to urethan. However, many of these same chemicals are also detoxified by cytochrome P450 by conversion to less toxic metabolites. In some cases, the same enzyme may catalyze activation and detoxification reactions for a given chemical. The resulting toxic effect of a xenobiotic chemical is thus due to a balance

In phase II reactions, metabolites produced in phase I detoxification often conjugate with glutathione, uridyl-diphosphate glucose (UDPG), uridyl-diphosphate-glucuronic acid (UDPGA), amino acid derivatives and sulfate derivatives and can readily excrete from the fish body (Keizer et al., 1995; Kitamura et al., 2000; Straus et al., 2000; Behrens & Segner, 2001; Nebbia, 2001). In fact, this results from enzymatic oxidation and hydrolysis that produce metabolites with OH, COOH and NH2, SH functional groups. These functional groups are then subject to conjugation with carbohydrates, glutathione, sulfate, andamino acids, and then, the final metabolites may also be excreted from the body of fish through the skin, gills, genital products, urine as sulphated and glucuronidated metabolites and stool as glutathione conjugated metabolites (Kitamura et al., 2000; Straus et al., 2000; Behrens & Segner, 2001;

**Metabolism Category Reaction pattern** Oxidation Alkyl oxidation O1 R - CH3→R - CH2OH→R - CHO→RCOOH

Ring hydroxylation O3 Ar - H→Ar - OH

*S*-oxidation O6 -S - → - S(O) - → - S(O2)-

Desulfuration O7 P=S→P = O, C - SO3H→C - OH

*O* (N)-dealkylation O2 -O{N} - CH3→ -O{N} - CH2OH → - O{N}H

O5 Quinone

O4 Ar - H→Ar - (OH)

2

between metabolic activation and deactivation (Casarett and Doull, 1996).

106 Insecticides - Development of Safer and More Effective Technologies

**4. Phase II**

McKim & Lein, 2001; Nebbia, 2001).

**Table 1.** Classic detoxification pathways of insecticides in aquatic organisms (adapted from Katagi, 2010)
