Definition of a Pesticide Any substance intended for preventing, destroying, repelling, or mitigating any pest Insecticides Herbicides Fungicides

Pesticides are Not New Fumigants Insecticides Rodenticides Sulfur (1000 BC) Bordeaux mixture (CuSO4, lime (Ca(OH)2, H2O)- 1882) Insecticides Arsenic (16th Century) Tobacco leaves (Nicotiana tabacum - 1690) Rotenone (Derris eliptica – 1800s) Pyrethrum (Chrysanthemum cinerriafolum- 1800s) Paris Green (copper arsenite 1800s) Rodenticides Nux vomica (Strychnos nux-vomica)

There is No Such Thing as a Completely SAFE Pesticide But Pesticides can be USED safely. All Pesticides have some type of biological activity The trick is to balance efficacy and safety.

Pesticide Regulations Wiley or Sherman Act (1906) Federal Food, Drug, and Cosmetic Act (1938) Pesticide tolerances set in 1954 and 1958 1958: Delaney Act No additive is deemed safe if found to cause cancer Pesticides were one type of additive Federal Insecticide, Fungicide, and Rodenticide Act 1947 (initially administered by USDA) 1972 (switch to EPA) Defines registration and labeling requirements Can cancel registration Set tolerances FDA monitors residue levels Food Quality Protection Act 1996 Special considerations for children Higher susceptibility Consider child consumption patterns

Development of a New Pesticide is a Lengthy and Costly Process FIFRA Product and residue chemistry Environmental fate Toxicology Acute toxicity Oral, dermal, inhalation, irritation, ocular, delayed neurotox, dermal sensitization Subchronic Rat, mouse, dog (dermal, inhalation, neurotox) Chronic Rodent, dog, carcinogenicity Reproductive Fertility, teratology, in vitro mutagenicity Biotransformation Occupational exposure Spray drift Environmental impact on non-target species Efficacy Costs: $30 to 80M (full development and testing of a new pesticide may take 10 years)

The Majority of Insecticides are Neurotoxic

Toxicological Syndromes Associated with Insecticides Acute syndromes Cholinesterase inhibitors Arsenic Pyrethrins and pyrethroids Chronic toxicosis Delayed neuropathy (OPIDN) Residues Organochlorine insecticides DDT Wild-life problem

Anticholinesterase Insecticides Organophosphorus Insecticides Carbamate Insecticides

Acetylcholinesterase Inhibitors Organophosphates (OP) Phosphoric acid derivatives 30 in use First synthesized in 1937 (Bayer) Tetraethyl pyrophosphate (TEPP) Chemical warfare agents Tabun (GA) Sarin (GB) – Japanese subway attacks Soman (GD) VX CMPF (GF) Parathion (O,O-diethyl O p-nitrophenyl phosphate) Replaced DDT in 1950s Number of acute poisoning cases resulted Chlorpyrifos Parathion

Acetylcholinesterase Inhibitors Carbamates Carbamic acid derivatives ~25 in use Originally developed as fungicides (1930s) Carbaryl Physostigmine (alkaloid from Calabar bean)

Interference With Neurotransmitter (Acetylcholine) Function Synthesized in neuron cell body Release triggered by an action potential Sudden influx Ca2+ ---> ACh release Broken down by acetylcholinesterase Primary neurotransmitter in PNS Smooth and skeletal muscle CNS (distributed throughout) In the developing brain, every neuron expresses cholinesterase activity even if it isn’t cholinergic in adulthood. 4

Effector organs: Smooth muscle Mode of Action Cholinergic neuron Acetylcholine Effector organs: Smooth muscle Skeletal muscle CNS

Mode of Action Acetylcholinesterase inhibitors Carbamates Carbamylation of enzyme occurs Reversible Organophosphates Enzyme phosphorylation occurs “Aging” Irreversible Aging occurs over a 12-24 hr time period

Mode of Action C Carbamate Organophosphate Op Choline + Acetylcholine Treatment: Atropine 2-PAM Choline + Acetylcholine Acetate Acetycholinesterase inhibitors C Op Acetylcholinesterase Synaptic cleft Carbamate Organophosphate Muscarinic Nicotinic

Clinical Signs Clinical signs related to excessive stimulation of nicotinic and muscarinic receptors Muscle tremors CNS effects Respiratory paralysis (this is what causes death) Salivation, Lacrimation, Urination, and Defecation CNS signs Bronchospasm Bronchial secretions Miosis Bradycardia

Organophosphate Insecticides Toxicology Delayed effects (OPIDN) Ginger Jake paralysis Tri-o-tolyl phosphate (TOTP) Leptophos, mipafox, chlorpyrifos, DFP All organophosphates are required to be tested for their ability to produce OPIDN before they are marketed (Hen test) 5

OPIDN Organophosphate Induced Delayed Neuropathy Clinical signs Ataxia and paralysis Develop 10 to 14 days after exposure Neuropathology Wallerian-type degeneration Mode of Action Inhibition of neurotoxic esterase (NTE) is generally predictive 10

As Animals Mature, They Become Less Sensitive to Chlorpyrifos Toxicity (Moser et al, Toxicol. Sci, 1998)

Generic OP Pathway Oxon Parent Pesticide Bind to CaEs Inhibit AChE Hepatic Activation Bind to CaEs Hydroylzed by A-Esterases Inhibit AChE

Acetylcholinesterase in the Young Brain is NOT more Sensitive to Organophosphorus Pesticide Inhibition Mortensen et al., Toxicol. 1998

Developmental Profiles of Carboxylesterase and A-Esterase in Rats (Mortensen et al., J. Biochem. Toxicol. 1996; Moser et al, Toxicol. Sci, 1998)

Generic OP Pathway Adult vs Young Rat Oxon Hepatic Activation and probably Deactivation Parent Pesticide Bind to CaEs Hydroylzed by A-Esterases Inhibit AChE

Developmental Profiles of Arylesterase (U/mL) Arylestease (U/mL) Age (months) A B C Developmental Profiles of A-Esterase in Humans Cole et al, 2003

Pyrethrin and Pyrethroid Insecticides

Pyrethrin and Pyrethroid Insecticides Pyrethrins Naturally derived insecticide Chrysathemum Natural pyrethrins include pyrethrin I, pyrethrin II, jasmolin I, jasmolin II, cinerin I and cinerin II.3 Pyrethroids Synthetic insecticides Slightly more persistent Fenvalerate Deltamethrin

Pyrethroids Natural pyrethrins are light sensitive and undergo rapid photodegradation Pyrethroids that contain a cyano substituent at the alpha-carbon of the phenoxy-benzyl moiety have been classified as type II; pyrethroids which lack this alpha cyano moiety as type I Type I (T syndrome): Tremors, tachypnea, "running fits," hyperthermia, and salivation within 1-2 hours of injection Allethrin Pyrethrin I Resmethrin Tetramethrin Type II (CS syndrome): Whole body tremors, hypersensitivity, occasional running fits, choreoathetosis (sinuous writhing), hypothermia, and generalized seizures Cypermethrin Deltamethrin Fenvalerate

Pyrethrin and Pyrethroid Insecticides Toxicity Low oral toxicity LD50 quite variable 25 to 10000 mg/kg Rapid hydrolysis in the gastrointestinal tract Liver metabolism Synergists Microsomal enzyme inhibitors Piperonyl butoxide

Mode of Action (Overstimulation of the Nervous System) Interference with voltage gated sodium channels. Type I keep channels open for shorter period vs. Type II Enhanced sodium ion conductance

Clinical Signs Muscle tremors Mixed CNS effects Increased salivation CNS depression CNS excitation Seizures Increased salivation Vomiting Ataxia Hypo and hyperthermia Paresthesia

Generic Pyrethroid Pathway Keep Na Channels Open Hydrolyzed by P450s Hydroylzed by CAEs

Time After Dosing (minutes) Choreoathetosis Salivation 50 100 150 200 250 300 350 400 Choreoathetosis Score 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Weanling, 4 mg/kg DLT Adult, 30 mg/kg DLT Choreoathetosis Salivation Score Weanling, 4 mg/kg DLT Adult, 30 mg/kg DLT Salivation

Newer Pesticides: “Mectins” (Nicotinic receptor agonists, specific for non-mammalian receptors) Avermectin Ivermectin Anthelmentic, insecticide Management of river blindness (Onchocerca) Low dermal absorption Minimal biotransformation

Final Thoughts Most of what has been presented relates to acute toxicology. What about long term effects of low level exposure to pesticides? What about effects on the developing nervous system?