THE 4 th INTERNATIONAL CONFERENCE ON ENVIRONMENTAL AND OCCUPATIONAL TOXICOLOGY Kunming, China, October 16-19, 2006

1. On the Neurotoxicity of Carbofuran in Mammalian Systems B. Sharma, Ph.D. Professor of Biochemistry Department of Biochemistry University of Allahabad (A Central University) Allahabad, UP 211002, India E-mail:sharmabi@yahoo.com, Phone:+91-9415715639 THE 4th INTERNATIONAL CONFERENCE ON ENVIRONMENTAL AND OCCUPATIONAL TOXICOLOGY Kunming, China, October 16-19, 2006

2. INTRODUCTION Pesticides are the substances used to prevent, repel, or destroy the pests -the organisms that compete for the food supply, adversely affect comfort, or endanger human health. More than 20000 pesticides with 900 active ingredients, belonging to different classes of compounds serve this purpose. These are insecticides, miticides, herbicide, rodenticide, nematocides, fungicides, fumigants, wood preservatives and plant growth regulators. Pesticides are ubiquitously distributed in environment including food, water, homes, schools, workplaces, lawns, and gardens. Pesticides have significant economic, environmental, and public health impacts rightly evidenced by huge worldwide application, approximately 5684 million pounds annually. Pesticide usage helps improve human nutrition through greater availability, longer storage life, and lower cost of food, reduces labor requirements and attendant risk of injury, and assists in control of food and vector born diseases to the savior of millions of people around the globe.

3. The most commonly used classes of pesticides are composed of different types of chemicals with different mechanisms of action. Most of the insecticides work by interfering with the nervous system functions. Organochlorines interfere with nerve cell membrane cation transport, resulting in neural irritability and excitation of central nervous system. Pyrethrins, which are emitted by plants, are rapidly metabolized by mammals and less neurotoxic used in anti-lice shampoo and as topical treatment for scabs. Repellents such as diethyltoluamide are used in varying proportions to avoid risk of vector stings and vector born diseases such as lyme disease, and rocky Mountain spotted fever. Herbicides such as 2,4-D and 2,4,5-T, are primarily irritative to skin and respiratory tract during acute exposures and work by different mechanisms. Some substances are highly corrosive and can cause multisystem injury and progressive pulmonary failure.

4. Arsenic pesticides, such as copper chromium arsenate are used as wood preservative. These compounds cause CNS depression at sufficient doses. Organophosphates and carbamates, widely used in homes and gardens, inhibit the activity of acetylcholinesterases at nerve endings, resulting in an excessive accumulation of the neurotransmitter acetylcholine and a depolarizing blockade of neural transmission. Currently among all classes of pesticides, carbamates are the most commonly used in agriculture and forestry because among the other alternatives, organochlorines have a long lasting residue persistence problem, and organophosphates (Ops) are extremely toxic and pose a delayed neurotoxicity complications. Among all carbamates carbofuran is the most commonly used because of its broad-spectrum systemic insecticide nematicide and acaricide properties. Here an extensive review of the physical and chemical properties, metabolism, pharmacokinetics, toxicity profile and poisoning incidences due to carbofuran with special reference to biochemical bases of its action in mammalian brain and neurotoxicity in the mammalian systems have been presented. A brief account of the activity of some antidotes to carbofuran toxicity has also been described.

5. POISONING INCIDENCES Tishomingo National Wildlife Refuge, Oklahoma, 1976. Approximately 500 Canada geese died after feeding in a field treated with 0.5 lbs / acre liquid carbofuran. Saskatchewan, Canada, 1986. Forty-five California gulls were found dead after a landowner applied liquid carbofuran to a grain field. Gulls had crops full of grasshoppers; analysis of the grasshoppers showed 4.2-7.2 ppm carbofuran. New Jersey,1990. After carbofuran application to a fruit orchard, approximately 100 carcasses were discovered, including blue jay, American robin, and dark-eyed junco. Laboratory analyses confirmed carbofuran as the cause of death. Linden, California, 1990. Liquid carbofuran was applied by irrigation, with exposure via puddle water. Carcasses of 30 mourning doves, 100 American robins, 200 European starlings, red-winged blackbirds and grackles, and 700-800 goldfinches, sparrows and house finches were recovered. Yountville, California, 1990. Carcasses of one each of acorn woodpecker, bushtit, white-breasted nuthatch, western bluebird, American robin, cedar waxwing, chipping sparrow, white-crowned sparrow and four hermit thrushes, seven yellow-rumped warblers, eleven dark-eyed juncos, nine house sparrows, three house finches, and six lesser goldfinches were reported following drip irrigation of a vineyard with a 6 lb/acre usage rate of flowable carbofuran.

6. PHYSICAL & CHEMICAL PROPERTIES OF CARBOFURAN Carbofuran is a white crystalline solid with slight phenolic odor. Its melting point is 153-1540C, vapor pressure 3.4x10-6 mm Hg at 250C, specific gravity 1.18 at 200C, water solubility 0.7 g/L of water at 250C. Its chemical name is 2, 3-Dihydro-2, 2-dimethyl-7-benzofuranyl-N-methyl carbamate and its chemical structure is shown in Fig.1. Chemical formula of carbofuran is C12H15NO3. Its CAS number is 1563-66-2. It is sold under different trade names such as Furadan, Bay 70143, curaterr (Bayer AG), D1221, ENT 27164, FMC 10242, NIA 10242, Pillarfuran and Yeltox etc.

7. METABOLISM & PHARMACOKINETICS Carbofuran is rapidly absorbed by digestive tract upon ingestion. Carbofuran is metabolized through hydroxylation or oxidative mechanisms resulting in to formation of carbofuran phenol, 3-hydroxy carbofuran, 3-hydroxy carbofuran phenol, 3-keto carbofuran and 3-keto carbofuran–7- phenol. In the oxidative pathway carbofuran is oxidized at benzylic carbon to yield 3-hydroxy carbofuran, which can be hydrolyzed to 3-hydroxy carbofuran phenol and 3-keto carbofuran-7-phenol. In hydrolytic pathway which is more common in mammals than in insects and plants, it is hydrolyzed directly to carbofuran phenol. Hepatic cytochrome P450 (CYP450) families of enzymes are responsible for oxidative metabolism of carbofuran. In humans the major isoform is CYP3A4 which is associated with CYP1A2 and CYP2C19, significant contributors in this process. In rodents it is CYP2C family. The oxidized metabolites are conjugated to glutathione and glucoronide and excreted in feces and urine mainly.

8. OXIDATION & HYDROLYSIS OF CARBOFURAN

9. NEUROTOXICITY Carbofuran (CF), an anticholinesterase compound, is commonly used for a variety of purposes such as in agriculture and in human and veterinary medicines. It exerts toxicity in mammalian system primarily by virtue of acetylcholinesterase (AChE) inhibition at the synapses and neuromuscular junctions, leading into the signs of hypercholinergic preponderance. The mechanism of action of carbofuran is mediated via carbamylation of hydroxyl group of serine residue present at the active site of AChE. This inhibition is reversible in nature.

10. Reaction Scheme showing carbamylation and decarbamylation of AChE by carbofuran. Here EH, CX and COH represent AChE enzyme, carbofuran with carbamylating radical C and carbamic acid, respectively; whereas k+1, k-1, k2, ki and k3 denote binding constants for forward reaction, binding constant for backward reaction, carbamylation rate constant, the bimolecular rate constant governing the overall rate of inhibition and the decarbamylation constant which controls the enzyme recovery, respectively.

11. When carbofuran serves as an alternate substrate, the alcohol moiety is cleaved, giving rise to the carbamylated AChE. In contrast to acetylated AChE, carbamylated AChE is more stable. Thus the hydrolysis of acetylcholine (ACh) precludes due to the sequestration of AChE, which leads to accumulation of ACh and finally to neurotoxicity. Rapid carbamylation of AChE with a potential inhibitor results into higher toxicity because such inhibitors posses low ki values. The IC50 value for AChE (isolated from rat blood) by carbofuran has been determined to be 33 nM. IC50 values for different carbamates decrease as the side chain becomes larger and bulkier. The variations in the IC50 values (33 nM to 307 µM) for different carbamate inhibitors could be partly due to different tissues and experimental conditions employed such as temperature, pH and incubation time etc. The decarbamylation of AChE resulting in recovery of carbofuran induced inhibition of this enzyme is quite rapid as recovery merely requires dissociation of the methylcarbamyl moiety from the enzyme. The recovery of AChE activity in a very short period can be partly explained by kinetics of the reversible AChE-carbofuran complex .

12. There are two basic types of cholinesterase measurements. (1) Blood cholinesterase levels which consist of plasma cholinesterase or RBC (true) AChE are the easiest to measure, as they require only blood samples and do not necessitate the termination of the subject. But blood cholinesterase serves best as markers of exposure to such chemicals, as they are not directly linked to AChE in central or peripheral nerves. (2) Evaluation of functional level of AChE activity requires taking out tissue samples, most commonly of brain or muscles. Depression of nerve or neuromuscular AChE is most indicative of adverse effects of toxicants, while depression of blood cholinesterase provides a useful indicator of potential impairment. Clinical signs of cholinesterase poisoning consisting of tremors, salivation, miosis, dyspnea, and piloerection appeared at the highest dose within five min, with high mortality. However, AChE assays for carbofuran and other carbamates must be done quickly and carefully because of the reversible nature of the cholinesterase inhibition by carbamates.

13. The binding of carbofuran with carboxylesterases (as well as blood cholinesterase) may prevent the direct action of carbofuran on AChE activity. Pretreatment of rats with nonspecific esterase inhibitor, iso-OMPA (1mg/kg, subcutaneous) one hr prior to carbofuran administration of 0.5mg/kg potentiated carbofuran toxicity by more than three-fold. The severe signs of cholinergic depression has been reported in rats. With each drug given alone (iso-OMPA at 1mg/kg or carbofuran at 0.5 mg/kg) significant depression of carboxylesterase activity was reported in brain structures, while AChE activity was unaffected. Carbofuran induced characteristic alterations in different brain regions; AChE activity being maximally inactivated in cortex with carbofuran. Unlike AChE, carboxylesterase (CarbE) did not show brain regional variability in controls, and its activity was uniformly inhibited in all brain regions by carbofuran.

14. OXIDATIVE STRESS & NEUROTOXICITY The mechanism(s) involved in brain/muscle damage appear also to be linked with alteration in antioxidant and the scavenging system leading to free radical-mediated injury. Carbofuran causes excessive formation of F2–isoprostanes and F4 neuroprostanes, in vivo biomarkers of lipid peroxidation and generation of reactive oxygen species (ROS), and of citrulline, a marker of NO / NOS and reactive nitrogen species (RNS) generation. In addition, during the course of these excitatory processes and inhibition of AChE, a high rate of ATP consumption, coupled with the inhibition of oxidative phosphorylation, compromise the cell’s ability to maintain its energy levels and excessive amounts of ROS and RNS may be generated. Pretreatment with N-methyl D-aspartate (NMDA) receptor antagonist memantine, in combination with atropine sulfate, provides significant protection against inhibition of AChE, increases of ROS/RNS, and depletion of high-energy phosphates induced by DFP/carbofuran. Similar antioxidative effects are observed with a spin trapping agent, phenyl-N-tert-butylnitrone (PBN) or chain breaking antioxidant vitamin E.

15. All aerobic organisms are susceptible to oxidative stress simply because semireduced oxygen species, superoxide and hydrogen peroxide, are produced by mitochondria during respiration. The exact amount of ROS produced is considered to be about 2% of the total oxygen consumed during respiration, but it may vary depending on several parameters. Brain is considered abnormally sensitive to oxidative damage. Brain is enriched in the more easily peroxidizable fatty acids (20:4 and 22:6), consumes an inordinate fraction (20%) of the total oxygen consumption for its relatively small weight (2%), and is not particularly enriched in antioxidant defenses. In fact, brain is lower in catalase activity, about 10% of liver. Additionally, human brain has higher levels of iron (Fe) in certain regions and in general has high levels of ascorbate. Thus, if tissue organizational disruption occurs, the Fe/ascorbate mixture is expected to be an abnormally potent pro-oxidant for brain membranes.

16. ANTICHOLINESTERASE TOXICITY & OXIDATIVE STRESS Carbofuran exposure has been reported to cause a significant increase in LPO observed in cerebral cortex, cerebellum and brain stem following carbofuran exposure. A strong correlation between the accumulation of acetylcholine and the extent of LPO has been reported. End products of LPO are believed to be largely responsible for the cytotoxic effects observed in various neurodegenerative conditions. The mechanism of increased production of reactive oxygen species involves inhibition of cytochrome c oxidase. It is also reported that carbofuran induces nitric oxide synthase, which is implicated in the overproduction of superoxide anions. Glutathione (GSH)-dependent defense against xenobiotics is a multifaceted phenomenon that has been well characterized in mammals. Carbofuran treatment has been reported to cause significant decrease in the GSH; maximum reduction in GSH being in cerebellum followed by brain stem and cerebral cortex.

17. The mechanism involved in GSH depletion after carbamate exposure involves carbomylation of –SH groups. The decrease in GSH levels might diminish the overall antioxidant potential of the brain resulting in increased LPO following carbofuran. GSH depletion may also effect the activation and translocation of transcription factors like NF-jB and c-Jun/activating transcription factor. Antioxidant enzymes have evolved with primary function to keep the amount of free radicals in the body under control. The enzymatic antioxidants studied include SOD, CAT, GSH–Px and GR. A significant decrease in the activity of SOD in different brain regions of rat such as cerebral cortex, cerebellum and brain stem of the carbofuran treated group has been reported. Antioxidant enzymes, SOD and CAT are the first line of defense against oxidative stress. SOD offers protection from highly reactive superoxide anions (O2-) and converts them to H2O2. The decreased CAT activity in response to carbofuran might reduce the protection against free radicals.

18. Reduction in the activity of both SOD and CAT is reported to make the brain more vulnerable to carbofuran-induced oxidative stress. Activity of GSH–Px and GR were decreased in the different regions of brain after carbofuran exposure. GSH–Px protects cells from H2O2 and lipid peroxides. Glutathione reductase is an important enzyme required for maintaining high GSH/GSSG ratios. The enzyme has ten cysteine residues per monomer that participate in catalysis. It is speculated here that carbofuran might be inhibiting GR activity by blocking the –SH groups or by carbomylating free –SH groups at the active siterequired in the catalytic process. The diminished activity of SOD after carbofuran exposure has been shown leading to accumulate superoxide anions, which in turn inactivates selenium-dependent GSH–Px by its reaction with selenium at the enzyme active site. Carbamates, like other anti-cholinesterases have been shown to interfere with the process of learning and memory.

19. Antidotes Complete success in developing antidotal treatment against anticholinesterase poisoning has never been achieved, since there is no single therapeutic agent yet available that can antagonize the toxic effects, central as well as peripheral evoked by overstimulation of both muscarinic as well as nicotinic Ach receptors. Carbofuran acute toxicity treatment so far rests with Atropine sulphate, which readily the muscarinic–associated effects. A new cholinesterase reactivator alloxime, shows a marked therapeutical benefits in association with atropine in acute intoxication rats with carbamate pesticides such as carbofuran, pirimor and elocron. The mechanism of alloxime's therapeutical action is due to its capacity to restore cholinesterase activity in the central nervous system, normalizing neuromuscular transmission and hepatic and renal cytochrome P-450 levels. Atropine works through competitive antagonism of ACh at the muscarinic receptor. A combined antidotal treatment with memantine HCI and atropine sulfate provided complete protection against acute carbofuran toxicity in rats by multiple mechanisms. Carbofuran also perturbed the activities of mitochondrial/cytoplasmic biomarker enzymes (creatine kinase, CK andLDH) in diaphragm muscle

20. Any substance with antimuscarinic properties is an effective antidote. The substances with greater CNS penetration i.e. scopolamine or 3-quinuclidinyl benzilate (BZ) offer theoretical advantages for reversal of CNS effects. However, the inherent toxicity of these “antidotes” when carbofuran is not present led to their rejection in favor of atropine.

21. ACKNOWLEDGEMENTS • Devendra K. Rai, Research Fellow, NET-CSIR • Department of Biochemistry, University of Allahabad, India • Prashant K Rai, Department of Chemistry, University of Allahabad,India • & • Council of Scientific and Industrial Research, New Delhi, India

22. THANKS