Indian Journal of Experimental Biology Vol. 38 August 2000, pp. 753-761

Review Article

Signal transduction by xenobiotics in fish

Shelley Bhattacharya

Environmental Toxicology Laboratory, Department of Zoology, Visva Bharati University, Santiniketan 731235, India

Signal transduction by xenobiotics in fish has recently gained ' much attention. The better known transduction mecha­nisms are those elicited by organochlorines. organophosphates, carbamates and heavy metals. Organochlorines specifically bind to the membrane bound ouabain sensitive Na+-K+-ATPase affecting neural transmission while the organophosphates and carbamates bind specifically to the membrane bound enzyme acetylcholinesterase again affecting neural transmission. Since the nervous system is one of the important integrative and interactive physiological systems in animals, hypofunction of the nervous system leads to secondary effects in the endocrine system including thyroidal, gonadal, interrenal, pituitary and hypothalamic functions . Even low levels of xenobiotics are efficient enough to bring about remarkable changes in the functional physiology of the non target animals. Heavy metals such as cadmium or mercury belonging to the same group 11 B in the periodic table probably have a similar mechanism of action. Avidity of these metals to SH-radicals allow them to bind indiscriminately to SH groups in proteins. One pathway of interaction by inorganic mercury with the membrane bound ouabain sensitive Na+-K+-ATPase has been clearly established in fish liver and ovary. Binding of inorganic mercury to the membrane bound enzyme is through sulfhydryl group which inactivates the sodium pump leading to accumulation of the cation in the cytosol. The inorganic mercury is next conjugated by the cytosolar nucleophile, glutathione, and is transported to the nucleus where dissociation occurs and the free metal binds to the metal regulatory elem~nt to initiate gene expression. The inducible proteins are 3~-hydroxysteroid dehydrogenase in the oocyte and metallothionein and C-reactive protein in the liver. The present review deals with the role of xenobiotic as a stress factor.

The long half life of organochlorines has taken the toll of nature and it is extremely difficult to get rid of these harmful compounds from our environment even after its use was banned in 1970. Considering the higher rate of biodegradation, organophosphate and carbamate compounds were preferred over organochlorines and are in wide use today. Among industrial pollutants, heavy metals such as lead, mercury, cadmium and other metals such as arsenic, copper and zinc are gradually reaching dangerous limits in the soil, water and air. Although much awareness has been generated the scenario is yet to be benign. Like any other stimulus, chronic exposure to xenobiotics also generates stress and the response to such stress is mediated by hormonal, neuronal and other physiological pathways. Interest in xenobiotics as a stress factor is rather recent and an attempt is made in the present review to summarize the various response mechanisms elicited by some candidate xenobiotics.

The elicitation of xenobiotic stress response is basically through two mechanisms of action, intoxication and detoxication. Intoxication by xenobiotics can be expressed in any of the innumerable physiological pathways. Any intoxication process must have a target site of action which is dependent upon the chemical nature of the

xenobiotic, whether it is an organic or an inorganic one and other questions too may be addressed.

Intoxication Signals

Neuronal signal Intoxication signals are directly transduced by the

target cell of the xenobiotic. The neural signalling system is affected by pesticides in general and also by heavy metals. In all cholinergic animal systems the enzyme acetylcholinesterase (AChE) plays the most important role in the transmission of nerve impulse. Organophosphate and carbamate compounds were advocated as pest control agents instead of organochlorines because of their selective inhibitory action on AChE. Interestingly, insects are not the only cholinergic animals, in fact, all vertebrates are cholinergic. Therefore, the selective inhibition of AChE kill the insects and reduce the pest population but the environmental load of pesticides which persists migrates to the non target species 1.2.

Basic mechanism of AChE inhibition In the schematic events of neural transmission in

cholinergic nerves, acetylcholine (ACh) an excitatory neurotransmitter, is released from the synaptic vesicle of the nerve terminal in the pre synaptic region. ACh binds to its receptor in the post synaptic membrane, opens cation channels and causes the influx of Na+

754 INDIAN J EXP BIOL, AUGUST 2000

ions thereby depolarizing the membrane. Until and unless the ACh is removed the state of depolarization of the nerve continues and "the stimulation persists . The activity of AChE is vital at this stage initiating hydrolysis of ACh and the ACh receptor reverts back to its initial state. AChE is a membrane bound enzyme which is specifically inhibited following a bimolecular rate kinetics by pesticides such as organophosphates and carbamates. The selectivity of these pesticides towards AChE is owing to the specific binding of the phosphoric acid or carbamic acid moieties to the active site of the enzyme, namely serine. The process of phosphorylation and carbamylation leads to the formation of phosphorylated or carbamylated AChE which are more stable intermediate reaction products than the acetylated AChE formed during ACh hydrolysis. The target action of all organophosphate and carbamate pesticides is mediated via this pathway.

In the next phase of anticholinesterase poisoning by organophosphates and carbamates, ACh accumulates in the synapse' . Thus inhibition of AChE is considered as a significant parameter to assess complex toxicogenetic effecrs4

• Carbofuran exposure to Channa punctatus and Anabas testudineus effected dose dependent decline of brain AChE activit/ accompanied by ACh accumulation in the brain6

.

Phenthoate, an organophosphate was also found to elicit similar responses in the fish7

• It has been reported that pesticide treated fish 5.7,8 have a higher level of ACh in the brain than that in the untreated fish. Interestingly, greater accumulation of brain ACh was found to initiate a faster rate of recovery of the inhibited enzyme in the treated fish8

• Sublethal carbofuran exposure led to a series of events in the brain of Channa punctatus6

• The authors demonstrated that dose dependent inhibition of AChE occurs during the exposure; and faster rate of recovery is observed in the fish with maximally inhibited AChE. Moreover, low doses of the pesticide elicited a delayed toxic response in two teleosts, Channa punctatus and Anabas testudineus and a higher dose of pesticide exposure resulting in ACh accumulation definitely protects the enzyme being further inhibited by the circulating inhibitors5

.

Ghosh and Bhattacharya2 demonstrated in vivo and in vitro effects of Metacid - 50, an organophosphate and Carbaryl, a carbamate, on brain AChE of C punctatus under natural field conditions which clearly suggests a specific mode of intoxicating signals in terms of neurotoxic responses.

Interestingly, the rate of in vitro inhibition of AChE from pretreated fish always recorded a faster rate of inhibition9 which demonstrates a preformed effect of pesticides during in vivo exposures. AChE is an

. enzyme which shows distinct product inhibition, hence the role of ACh in the recovery of the inhibited AChE remained enigmatic, because ACh combines with the alcohol binding site of the acetylated enzyme retarding deacetylation lO

. The signal of recovery was undoubtedly mediated through ACh and it was elegantly deciphered by Ghosh et alII . Two experiments were designed, i) in vivo brain AChE activity was determined in fish administered intramuscularly with lomustine, an inhibitor of protein synthesis, which crosses the blood brain barrier and ii) in vitro experiment with fish brain slice to study the inhibitory effect of Actinomycin D on protein synthesis . The in vivo administration of Lomustine inhibited brain AChE activity by 22% and 35% at 24 and 48hr after Injection respectively against the control. Interestingly exogenous administration of ACh caused a reversal of inhibition and significant enhancement of AChE activity. In the in vitro experiment it was proved beyond reasonable doubt that increasing concentrations of ACh (0.25 to I ~mole) in the system augmented the Actinomycin­D inhibited AChE activity highly significantly.

Target action of organochlorine pesticides Davis and Wedemeyer l2 reported the inhibition of

Na+- K+ - ATPase in a heavy microsomal membrane preparation from the brain of rainbow trout. Interestingly, this ubiquitous membrane bound enzyme is inhibited by organochlorines not only in the neuronal but also the non-neuronal cells. Leadem et aID recorded the inhibition of both Na+ - K+ - and Mg + - ATPases in the liver of rainbow trout exposed to DDT concentrations of 0.3 and 1.0 ng/I irrespective of the salinity of the water. Several cyclodiene insecticides l4

, I5 caused significant inhibition of brain Na+ - K+ - ATPase in bluegill in an in vitro system. The effects of DDT on various membrane related functions were demonstrable in vitro in the salmon id fish where both Na+ - K+ - and Mg+ - ATPases were found to be significantly inhibited l6

, although the in vitro effect was not comparable to the in vivo toxicity of these compounds. It was further revealed l7 that DDT and other organochlorines, in general, have the potential to bind allosterically to the membrane bound enzymes, either in the plasma membrane or the

SHELLEY BHATTACHARYA : SIGNAL TRANSDUCTION BY XENOBIOTICS IN FISH 755

mitochondrial membrane, by being incorporated in the lipid structure of the membranes.

Other metal ATPases such as Cd2+ - and Zn2+ -ATPases were also inhibited in the liver mitochondria isolated from the bluegill by chlordane, dieldrin, heptachlor, kepone, lindane, methoxychlor and toxaphene ls. Aldrin inhibited the Zn2+ - but not the Ca2+ - ATPase while aldrin, kepone and heptachlor inhibited both the enzymes. Mg2+ - ATPase was found to be stimulated by dieldrin, lindane, toxaphene and diazinon and inhibited by aldrin, endrin, heptachlor and kepone. Finally DOE, toxaphene and diazinon increased but chlordane and endrin inhibited Ca2+ -A~ase acivity.

Non target actions of organochlorines Few studies were conducted prior to 1970 on the

non target actions of the organochlorines to assess the metabolism of these toxic agents in the fish system. It has been reported that chronic low doses of chlorinated hydrocarbons can stimulate and near lethal doses suppress thyroid function while the intermediate doses were stimulatory at first and inhibitory in the later phase of the exposurel9. Moffet and Yarbrough20 concluded from their studies on hepatic and brain mitochondrial SUCCinIC dehydrogenase in insecticide resistant mosquito fish that the mitochondrial membranes being less permeable to the organochlorines the fish could withstand the inhibitory effects of dieldrin and DDT.

Elevated levels of serum transaminases (GOT and GPT) were described in carp exposed to dichlorvos and trichlorfon21 . These level,S were concomitant with changes in the serum glucose triglyceride and total cholesterol content. These evidences encouraged the author to propose that biochemical lesions produced by the organochlorine compounds are impaired glycogen metabolism and increased muscle catabolism. Mixed function oxidase (MFO) activity played a significant role in large mouth bass and blue gill in metabolizing aldrin and chlordane, while endrin strongly inhibited aldrin epoxidation in vitro22. The relative toxicity of endosulfan was also found to be mediated by the MFO pathway 23.

Non target actions of organophosphate (OP) and carbamate (CA) compounds

Sakaguchi21 was one of the earliest authors to report the non target action of OP and CA agents. Malathion, dipterex, and DDVP significantly elevated serum transaminase activity accompanied by similar changes in serum glucose, triglycerides and

cholesterol. It may be opined that insecticides in general interact with various enzyme systems and cannot be considered as either activators or inhibitors of these enzymes. Aldolase was attacked by 4.8 to 6.22 ppm of malathion and 2.5 to 6 ppm of dimecron in the liver, brain and gills of Clarias batrachus. Labelled amino acid incorporation study in malathion exposed tilapia revealed a general increase in the protein synthesis in various tissues such as liver, muscle and gill22.

Low levels of malathion (0.5,0.7.0.9 and 1.1 ppm for 7 days) caused significant decrease in the hepatic nucleic acid and protein content24. It was suggested

. that malathion interferes with or blocks key steps of RNA synthesis, because on depuration of the fish the inhibitory effect of malathion was quickly reversed. However, malathion can affect the protein profile in three ways: 1) it can inhibit RNA synthesis; 2) it can affect the uptake of amino acids in the polypeptide chain and 3) it increase the catabolism of proteins. Cashman et aes conducted elegant investigations with eptam to reveal the mechanism of action of the OP compound in fresh and salt water striped bass. In the fresh water fish hepatic microsome eptam is S­oxygenated by monooxygenase and in the salt water variety both eptam S-oxide and eptam sulfone are formed independent of monooxygenase activity. Thus the bioactivation of an OP agent and rendering it more toxic than the parent compound was amply clear in the fresh water fish.

Hormonal signals Organochlorines in general were found to interfere

. h h I h . I· . I 26 27 wIt t e regu atory pat ways InVO vlng COrtI so . which led to immune suppression2s and synthesis of a vitellogenin like protein which was capable of binding and transporting insecticides in the non reproductive season29. Aroclor 1254 was found to interfere with steroid biosynthesis and metabolism in the testis and head kidneys of male Gadus morhus. At 5 and 10Ilgig feeding levels of polychlorobiphenyl the synthesis of cortisol, cortisone and corticosterone was stimulated significantly while at 50Ilg/g feeding level conversion of progesterone was grossly hindered30. Thomas31 .32 opined that Aroclor 1254 disrupts the fish reproductive functions by interfering with the activity of hypothalamus-pituitary-gonadal-liver axis. Chaudhury et al.33 demonstrated depression in both progesterone and estradiol synthesis by Metacid - 50, an organophosphate in the perch, Anabas testudineus. On the other hand, Mondal34 reported elevation of

756 INDIAN J EXP BIOL, AUGUST 2000

both these steroids in Cd-treated Channa punctatus as recorded earlier by Thomas35. Investigations by Mondal34 further revealed the delayed effect of xenobiotics (heavy metals, Cd and Hg; pesticides Metacid-50 and endosulfan) in the fresh water murrel. Interestingly the level of cortisol does not always increase under stressful condition and cannot be taken as an indicator.

Thyrotoxicity, in terms of iodide peroxidase inhibition and plasma thyroxine profiles, by

"d 3637 d h I 383940 h b pestlcl es' " an eavy meta s' " , ave een abundantly clarified. Besides it was evidenced that the thyrotoxicity was mediated via the cholinergic pathways56,57. Mukherjee et al.41 demonstrated inhibition of gonadal steroidogenesis in Cyprinus carpio at very low levels of phenol and sulfide present in the pulp mill effluent. In India the field application schedule . of pesticides usually coincides with reproductive season of the fish. It was interesting to note in both laboratory and field studies depression of GtH, GnRH42 and estradiol profiles33 , Information on xenobiotic action on steroidogenesis from endogenous precursors is rather meager43 and although cortisol is a major corticosteroid in many teleosts there is only one report on its profile under xenobiotic stress44

•

Xenobiotic action on steroid hormones Seasonal reproductive cycles in teleosts,often

regulated by photoperiod and temperature, are mediated by the hormones of the hypothalamus and the pituitary which in turn act on the gonads to stimulate the synthesis of steroid hormones, Thus fish reproduction is dependent on the precise orchestration and sequencing of a number of interrelated events. Xenobiotic action can be attributed to the disruption of the endocrine system, inhibition of hormone production leading to loss of fecundity, breeding or development.

Decreasesd plasma estrogen has been shown to result from exposure to Aroclor in trout and carp45, gamma-BHC and malathion in C. batrachus46

,

Aroclor 1254, lead and benzo[a]pyrene in Atlantic croaker3 I ,32,47, cadmium, malathion and 3-metyl­cholanthrene in Monopterus albus48

, bleached kraft mill effluents in white sucker49 and gamma-BHC in H. !ossilis5

o,51. However, these measures of plasma concentration of steroids reflect the final outcome of xenobiotic action and not the action itself.Sangalang and O'Halloran52 had demonstrated the role of in vitro Cd (1O-IO,OOOfJ.g/g tissue from normal brook trout testis in steroidogenesis with 14C-pregnenolone as the

precursor inhibiting the synthesis of 11-ketotestosterone. Contrastingly, Kime53 reported the stimulatory effect of Cd on steroid synthesis from endogenous precursor, specially of testosterone and 11 ~-hydroxytestosterone. Concentration of the xenobiotic has an important role in the expression of the steroidogenetic effect. Low concentrations can stimulate while higher ones become inhibitor/3.5

4. Stress as mediated by adrenal, has well known

ffi h d · 55 56 57 d . h e ects on t e repro uctlve system ,-, an mlg t account for significant indirect effects leading to decline in hormone synthesis. Under stress, high blood level of cortisol for extended periods of time have deleterious effects on growth, reproduction and the immune system58. Habituation of the stress response, evidenced by a decline of the previously elevated plasma cortisol level, has been reported in fish under continuous exposure to Cd59. Only few reports are available either on the cortisol output from interenals60, or the mechanism of action of xenobiotics6l ,62,63.

Xenobiotic interactions with steroidogenesis Only very few reports are available which

demonstrate the xenobiotic action on steroid synthesis such as those of Civen et al.64 on sterol-ester hydrolase, Donovan et at. 65 and Clement66 on testosterone hydroxylase. 3~-hydroxysteroid dehydrogenase (HSD) has been shown to be one of h 'f' f b" 6768 Th t e target sItes 0 actIOn 0 xeno IOtlCS '. e

response to stressors have molecular or biochemical basis the studies on which are still in its infancy and xenobiotics can bind directly to proteins at sites such as steroid hormone receptors that either initiate or promote the transcription of genes55

• There are reports that OP poisoning stimulates hormone mediated protein synthesis although Cisson and Wilson69

opined that direct action ofaxenobiotic on protein synthesis cannot be ruled out.

Detoxication signals Resilience of the fish system to the hazardous

xenobiotics is on record which points towards the existence of an efficient detoxication sytem in fish liver. One of the best known mechanisms of detoxication is the glutathione (GSH)- GSH-S­transferase system, It was earlier contended that the nucleophilic GSH reacts with electrophilic carbon atoms70 But Fuhr and Rabenstein71 had demonstrated the conjugation of GSH with non carbon atoms such as Cd, Zn, Pb and Hg and Chatterjee and Bhattacharya72 with ammonia. The enzyme GSH-S-

SHELLEY BHATTACHARYA : SIGNAL TRANSDUCTION BY XENOBIOTICS IN FISH 757

transferase (GST) was first reported in fish by Nimmo et al.73 and then by Chatterjee and Bhattacharya74. This finding clearly indicates the involvement of the GST in the detoxication of heavy metals in fish. GSTs are capable of binding the xenobiotic through covalent bond on the enzyme surface which may lead to the inactivation of the enzyme but prevent xenobiotic action. This pathway is termed as "suicide inactivation" of GST where the cell allows this protein to encounter and "disarm" the xenobiotic75.

Other detoxication mechanisms which provide adequate protection to the host is the xenobiotic­induced synthesis of stress proteins such as metallothioneins (MT) and C-reactive protein (CRP). MT is a single polypeptide chain with a molecular . weight ranging between 6000-7000 Da, high cysteine content (one third of the toial residues), serine and glycine but devoid of any aromatic amino acids. The arrangement of cysteine is highly organized, the 61 residue chain is interspersed with a series of C-X-C, C-X-X-C and C-X-X-X-C units (C=cysteine; X= other amino acids). Due to high cysteine content the protein binds avidly with a large number of metal atoms (7 in case of Cd and Zn, 12 in case of Cu/mole of the protein) . MT is found in very high concentrations in the liver of mature fish exposed to metals76 or metals and non metals77

. The most striking feature is that MT not only binds heavy metals but it is also induced by them in mammals78 and fish79. It has been proposed that MT protects the animal from metal toxicity by sequestration and detoxication80. CRP is an inducible plasma acute phase protein increasing by 100-1000 fold in mammals81 while in fish it is a normal component of the plasma increasing significantly on exposure to xenobiotics such as pesticides and heavy metals82.

Mechanism of signal transduction by a candidate xenobiotic .

The signal transduction mechanism adopted by a xenobiotic cannot be generalized because the nature of response is dictated by various factors. The organochlorine pesticides are seen to bind to ACh receptors or Na+-K+-ATPase and the OP and CA agents to AChE. Thus their action is mediated by inhibtion of neurotransmission . Since the nervous system has integrative and coordinating function to maintain the hormonal homeostasis dysfunction of the nervous system leads to a cascade of inhibitory events in the endocrine system. Heavy metals may be neurotoxic83 but they are more potent in the induction

of stress proteins such as MT and CRP. Investigations in this area is rather scanty although mercurials have been studied in some detail owing to their avid binding relationship to SH-radicals84. However, the target site of inorganic mercury retention in fish is liver and kidney85.86.87. 203Hg distribution profile in liver demonstrated a clear excretion of Hg after 6h of administration with a second rise in its level at 48h of treatment. While retention of Hg is drastically reduced in the kidney of the same fish. Interestingly, cytosolar concentration of GSH and MT being higher in the liver than in the kidney accumulation of Hg would also be higher in the liver. GSH has a major role to play in the clearance of Hg in fish as evidenced by rapid decline in the Hg level after exogenous administration of GSH88.

Increased MT concentration in response to metals is a general phenomenon of induction in majority of the animals. Induction of MT occurs at the transcriptional level by various cis-acting DNA sequences in the promoter region of the MT gene. Depending on the inducibility characteristics these sequences are classified as (1) metal regulatory elements (MREs) and (2) glucocorticoid regulatiory elements (GREs) which are capable of inducing the MT gene on interaction with glucocorticoids89.90

• Two separate genes, MT-I and MT-II have also been identified91 .92.George et al. 93 have demonstrated CD­induced synthesis of MT-I mRNA which was subsequently followed by increased MT level in the liver and kidney of a marine flat fish. Thus the binding of inorganic mercury to the MRE and subsequent induction of MT mRNA and MT has been clearly established. It has also been demonstrated that inorganic Hg partitions to the nucleus within a very short span of time of administration in both fish and rat liver which could be correlated with MT synthesis94. Bhattacharya et al.95 further clarified the mechanism of signal transduction of inorganic Hg to be mediated via its specific binding to the plasma membrane Na+-K+-ATPase in rat liver. It can be concluded from these evidences that inorganic mercury has specific binding sites in the plasma membrane, namely the ouabain sensitive Na+-K+­ATPase as reported earlier95.96.97.98 and the increase in cytosolar Na+ 78 in Cd-treated rat liver appears to be due to inhibition of Na+-K+-ATPase. Interestingly, Hg bound to the plasma membrane enzyme dissociates to be conjugated to cytosolar GSH. The Hg-GSH conjugation is effected by the enzyme GST which is

758 INDIAN J EXP BIOL, AUGUST 2000

highly stimulated by the increased Na+ concentration 95.

The disruptive effect of Cd and Hg has revealed the unusual build up of progesterone in the fish ovary which suggested the increased synthesis of the enzyme responsible for the conversion of pregnenolone to progesterone, 3-B hydroxysteroid dehydrogenase (HSD). It has been demonstrated that in an in vitro cell free translation system synthesis of both specific RNA and the polypeptide (HSD) is significantly stimulated in the ovary of Cd-treated fish99

• The signal transduction mechanism by inorganic mercury is thus found to be universal as revealed in rat liver95

• fish liverS?, fish oocyte99 and rat platelet (unpublished observation of Kumar and Bhattacharya).

Conclusion Xenobiotic physiology is a vast virgin area of

research . Information available are clearly not sufficient enough to propose a universal mechanism of signal transduction by xenobiotics in fish. Basically the signals are of two types, intoxication and detoxication . Intoxication signals have been discussed here as neuronal and hormonal only considering the limitation of space and also to avoid the multifarious responses of each category of xenobiotic. The mechanism of signal transduction by organochlorines, organophosphates and carbamates and heavy metals is better understood which has been discussed in the present review.

Acknowledgement The author is grateful to her students, Ravi Kumar

and Vinaya Kumar, for their painstaking effort in the preparation of the manuscript. Financial assistance from DBT, DST, ICAR, CSJR was of great help in elucidating some of the phenomenal events in signal transduction. UGC is acknowledged for the DSA support to this department which accelerated research programmes in environmental toxicology.

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