India Verlecar 2008. Previous study from Hong Kong waters accounted for also seasonal variability in battery of antioxidative biomarkers i.e. antioxidant enzymes
superoxide dismutase SOD, catalase CAT, the lipid peroxidation product malondialdehyde MDA, a phase II detoxification enzyme glutathione-S-
transferase GST and cholinesterase ChE in P. viridis Lau et al 2004. Interestingly, the study revealed that protein content of gill tissues were less
sensitive to seasonal variations compared to protein of whole body which indicated an advantageous of gill tissues as an organ target in using the
biomarkers. Those examples suggested that seasonal variations are important factors and thus should be taken into account when comparing data from different
seasons are conducted. Long term studies on tracing influences of seasonal variations on biomarkers particularly in green mussels in coastal waters of
Indonesia appeared to be necessary for a more accurate application of biomonitoring strategies. It seems that the hot spot strategy which was performed
at certain season is one of cost-effective preferable approaches for conducting effect biomonitoring using biomarkers that introduce general pictures of studied
sites Chapter IV.
6.3.. Organophosphate Pesticides Pollution and Biomarkers
The general formula of OP pesticides is presented in Figure 31 shows the structural basis of the OP pesticides that contain a central pentavalent phosphorus
atom Baird and Cann 2005. The R of most of the OP pesticides are represented by methyl or ethyl groups, while R’ is methyl, ethyl, phenyl, amino, substituted
amino or alkylthio Fukuto 1990. The leaving groups which are sometime symbolized by OX or X define the majority of the structural differences between
individual OP pesticides Cocker et al. 2002. The oxygen atom in the OX group can be substituted by S with some compounds such as the living group of
dimethoate. Likewise, the OP pesticides that contain the thio moiety P=S configuration like dimethoate Figure 32 require metabolic activation to the
corresponding oxon P=O before they can inhibit enzyme activity Galloway and Handy 2003. It is due to the lower electronegativity of sulfur in P=S moiety
compared to oxygen in P=O moiety results in a more electropositive phosphorus atom in P=O moiety, which facilitates attack on phosphorus by nucleophilic
agents such as the serine hydroxyl of AChE Fukuto 1999. On the other hand, trichlorofon Figure 33 is the OP pesticide that has an oxon moiety P=O which
can react to and inhibit acetylcholinesterase AChE directly.
Figure 31. General structure of OP pesticide Connell 2005.
Figure 32. Dimethoate.
Figure 33. Trichlorofon. OP pesticides undergo chemical alteration when they enter into water
system or penetrate to body of living organisms. For instance, trichlorfon is reconfigured in water to be dichlorvos which toxicity is more potent than the
parent compound. Besides, once OP pesticides penetrate the body of living organisms, they are subjected to be metabolized by some enzymes such as
cytochrome P450-dependent monooxigenanses system that lead to mostly enhance the toxicity by forming oxon configurations P=O which so-called
desulfuration Fukuto 1990; Galloway and Handy 2003. Desulfurations of the OP pesticides inside the body of living organisms subsequently bind and inhibit
one of the prominent neurotransmitter enzymes acetylcholinesterase AChE. Inhibition of the enzyme by OP pesticides takes place due to the active site of the
enzyme which is serine-hydroxyl group at catalytic center of the enzyme is phosphorylated Chambers 1992. Accordingly, the prominent function of the
enzyme to breakdown neurotransmitter compound i.e. ACh become choline and acetic acid is unavailable which subsequently cause the accumulation of ACh in
RO R’O
P OX
S or O
CH
3
O P
O S
CH
3
O CH
2
CH
3
O C
S N
O H
CH
3
O P
CH O
CH
3
O Cl
C OH
C
Cl
synaptic cleft. The reactivation of phosphorylated AChE takes place very slow which causes prolonged inhibition. The situation is more complicated when one
alkyl group of the phosphoryl moiety is split non-enzymatically which brings on the formation of negative charge and ultimately sustains it Peña-Llopis 2005.
This reaction is called aging or dealkylation which then makes the reactivation of the enzyme is completely unavailable. The deleterious effects of OP pesticides
inhibition become more fatal when the accumulation of ACh occurs at very high concentrations in the synaptic cleft which cause what so-called desensitization
that blockage some cholinergic transmissions Chambers 1992. Synaptic blockage causes to tetanus, paralysis of the diaphragm muscles and respiratory
failure which ultimately followed quickly by the death of organisms Walkers et al. 2001.
Mechanisms of OP pesticides actions to AChE activity through activation of biotransformation enzymes provide rationale basis for the use of the alteration
of the enzymes as biomarkers in laboratory test Sturm and Hansen 1999; Frasco and Guilhermino
2002 and field biomonitoring programs Triebskorn et al. 2001; Binelli et al. 2005; Schiedek et al. 2006. However, the current dissertation
focus only on the enzyme that shows a principal mode of action of the OP pesticides on nervous system which is AChE activity from two mussels species
i.e. green mussel Perna viridis and blue mussel Mytilus edulis as a biomarker. It is due to the fact that the OP pesticides are fabricated determinedly to be
effective inhibitors of AChE by binding the nucleophilic active site serine of the enzyme Galloway and Handy 2003. Moreover, the disruption of AChE activity
is considered as much reasonable explanation of the nervous system failure that will be manifested on higher levels of biological organization such as feeding
activity and changes in inter-individual relationship Lagadig 2001. Interestingly, the current research observed that reduction of siphoning rate which
is the behavioral biomarker exhibits correlations with inhibition of ChE activity from mantle, gill and posterior adductor muscle of M. edulis after exposed by
trichlorfon Chapter II. Observable correlation between ChE activity of hemolymph and physiological biomarker i.e. cardiac activity from crab Carcinus
maenas has been measured by Lundebye et al 1997. Therefore, measured
activity of this enzyme provides a quantification of disability induced by the pesticides that might be reflected in population or in higher level.
There is emerging evidence that OP pesticides not only inhibit AChE activity but also disrupt immune system of aquatic organisms De Guise et al.
2004; Gagnaire et al. 2006: Chang et al. 2006; Ivan et al 2007; Canty et al. 2007. Accordingly, it has been postulated that pesticides disrupt immune-
competence of organisms through direct and indirect mechanisms by phosphorylating and inhibiting critical protein involved in immune function
Pruett 1992; Galloway and Handy 2003. Directly the pesticides impair the immunity via inhibition of serine hydralase class of enzymes which play key roles
in immune function, hampering membrane associated esterase of lymphocytes and monocytes which alter structural and functional immunocytes population,
generating oxidative damage to immune organs, modulation of signal transduction pathway controlling cell proliferation and differentiation Rickwood and
Galloway 2004; Galloway and Handy 2003. Indirect mechanisms of the pesticides to induce immune-competence of living organism are by alteration of
cholinergic tone to lymphoid organs, influencing on GABA gamma amine butyric acid-mediated transmission, and alteration of metabolism Galloway and
Handy 2003. In invertebrates, phagocytosis is a principal mechanisms of cellular defense which was performed by circulating hemocytes Galloway and Depledge
2001. The disruption effects of OP pesticides to phagocytic activity will be reflected in the impairment of the immune function of living organism.
Therefore, phagocytosis which operates through well-characterized phases, including recognition of non-self material, chemotaxis, adhesion to cell membrane
receptors, ingestion, and destruction of the foreign agent Russo and Lagadic 2004 has been used as biomarker to detect the impact OP pesticides and other
xenobiotic compounds Galloway and Depledge 2001; Blaise et al. 2002. The applicability of the phagocytosis activity from blue mussels M.edulis
as biomarker has been studied in the current studies by exposing the blue mussels to OP pesticide, dimethoate. The study revealed intriguing results by emerging
non-dose dependent response pattern of the phagocytosis activity that so called U- Shape hormetic-like response Figure 5. In contrast, inverted U- Shape hormetic-
like responses of phagocytic activity have been observed in M. edulis when the animals were exposed to OP pesticide, chlorfenvinphos Rickwood and Galloway
2004. Hence, to take into account the hormetic-like response as one of phenomena that likely appeared in vivo study concerning phagocytosis activity of
blue mussels is necessary.
6.4.. Cholinesterase Activity: An Enzymatic Biomarker
