What effect does rotenone have on the electron transport chain and the formation of ATP?

What is Rotenone?

Rotenone is a naturally occurring compound found in the roots of several plant species of the pea (Leguminosae) family. Being the first member of the rotenoid family, its use includes:

• Insecticide (insect control) in home gardening
• Piscicide (fish eradication) for freshwater management
• Acaricide (lice, scabies, mites and tick control) for pets

It is generally classified as a botanical insecticide and it is a commonly used pesticide.

Rotenone is used alone or in combination with pyrethrin, pyrethrum, and piperonyl butoxide to control a variety of insects on food crops (5). Rotenone is a moderately hazardous Class II pesticide. In the U.S. and Canada, rotenone is mainly used as a piscicide.

Biotransformation

Please see the metabolic scheme of the major biotransformation pathways of rotenone by hepatic microsomes

• Metabolites: hydroxyrotenone, rotenolone I and II, dihydroxyrotenone
• Physicochemical Properties:
  • High melting point
  • Insoluble in water
  • Soluble in organic solvents
  • Highly lipophilic
• Stability:
  • Decomposes rapidly when exposed to light and air
    • Loses toxicity within days
• Formulations:
  • Dust – to control beetles and aphids on produce
  • Wettable powder – to control parasitic mites, lice, and ticks
  • Emulsion – used for undesirable fish in water management
  • Formulated with other pesticides for wide-ranging effects

Infographic by Oluwatobi Clement via Canva

Infographic by Oluwatobi Clement via Canva

Mechanism of Action

Remember that electron transfer is needed in order to pump protons (H+ ions) across the gradient from the mitochondrial matrix into the cell. The resulting protocol gradient triggers the molecular rotation of the ATP synthase, producing ATP. Need a refresher? Let’s revisit the electron transport chain (ETC):

https://u.osu.edu/clement.103/files/2020/05/electron-transport-chain-2.mp4

(Video Source)

Rotenone interferes with the electron transport chain by inhibiting the transfer of electrons from iron-sulfur centers in Complex I to ubiquinone. This inhibition interferes with NADH during ATP synthesis. Complex I is then unable to pass off its electron to Coenzyme Q (CoQ), creating a back-up of electrons within the mitochondrial matrix. In mammals and fish, rotenone inhibits the oxidation of NAD and substrates glutamate, alpha-ketoglutarate and pyruvate.

In addition to its effects on the ETC, rotenone interferes with reactive oxygen species (ROS) by inducing apoptosis. Rotenone inhibits the ATP production and increased generation of ROS causing the inhibition of neuronal activity (2). The increased ROS results in increased calcium and activation of calcium-sensitive potassium channels. This is called nitrosative stress.

Other actions of rotenone include:

• Increased nitric oxide and malondialdehyde levels
• Global cellular impairment
• Aggregation of alpha-synuclein and polyubiquitin
• Activation of astrocytes and microglial cells
• Neuroinflammatory reaction
• Glutamate excitotoxicity
• Oxidative stress
• Microtubule assembly and spindle formation in mitosis (4) resulting in chromosomal aberrations

Which organs does it target?

• Cutaneous membrane (skin)
• Mucous membranes:
  • Respiratory tract (inhalation)
  • Digestive tract (digestion)
  • Eye irritant (eye contact)
• Central Nervous System
• Peripheral Nervous System: causes anesthetic effect when in contact with nerve axons
• Skeletal and Smooth muscle

Signs and symptoms of toxicity

Parkinson’s Disease

In addition to the effects noted earlier, the following activities have been observed when rotenone is administered experimentally:

• Degeneration of striatal-nigral dopaminergic neurons similar to neurotoxin MPP (a well-known Parkinsonism-causing chemical and Complex I inhibitor)
• Degeneration of dopaminergic neurons in rotenone-evoked Parkinsonism
• Production of protein inclusions, similar to Lewy bodies
  • Stain positive for ubiquitin and alpha-synuclein

Ultimately, no strong evidence of Parkinson disease-like signs or neurodegenerative pathologies have been found with rotenone exposure. However, a strong association between rotenone and increased risk of PD has been noted.

Click (1), (2) or (3) for more information.

NIH News Release (here)

Biomarkers

Rotenone is 100x more toxic when administered IV than oral due to poor absorption from GI tract.

Biomarkers of rotenone exposure include:

• Residue detection of rotenone and/or its metabolites in blood, urine, feces, or liver
  • Determined using HPLC with a fluorescence detector or Liquid Chromatograph-Mass Spectrometer (LC-MS-MS)
• Metabolites (in decreasing order of toxicity):
  • Hydroxyrotenone: have reduced inhibitory activity in insect and rat liver mitochondria
  • Rotenolone I and II
  • Dihydroxyrotenone
• Characteristic toxicological symptoms
• Histopathological changes

Infographic by Oluwatobi Clement via Canva Source

Let’s Test Your Knowledge!

1. Which of the following metabolites causes the most systemic toxicities?
   1. Hydroxyrotenone
   2. Rotenolone I
   3. Dihydroxyrotenone
   4. Rotenolone II
2. Where is rotenone mainly metabolized?
   1. Dermally (Sweat)
   2. Feces
   3. Exhalation
   4. Urine
3. Which of the following vitamins is an experimental therapy for acute rotenoid exposure?
   1. Vitamin D
   2. Vitamin B12
   3. Vitamin E
   4. Vitamin K
4. Which of the following techniques are used to quantitatively detect rotenone biomarkers in the blood?
   1. GC and MS
   2. FPLC and ELISA
   3. HPLC
   4. HPLC and LC-MS-MS
5. For an accidental ingestion, one should induce vomiting within 1 hour of exposure.
   1. True
   2. False
6. Which of the following is not a rotenoid metabolite?
   1. Dihydroxyrotenone
   2. Trimethylrotenone II
   3. Rotenolone I
   4. Hydroxyrotenone
7. Which of the following cardiac toxicities are observed in chronic exposures?
   1. Arrythmia
   2. Prolonged QT interval
   3. Tachycardia
   4. None of the Above

Answers:

1. A
2. B
3. D
4. D
5. B
6. C
7. D

Resources

1. Costa LG. Toxic Effects of Pesticides. In: Klaassen CD. eds. Casarett and Doull’s Toxicology: The Basic Science of Poisons, Eighth Edition New York, NY: McGraw-Hill; 2013. http://accesspharmacy.mhmedical.com.proxy.lib.ohio-state.edu/content.aspx?bookid=958&sectionid=53483747. Accessed May 25, 2020.
2. Yee AG, Freestone PS, Bai J, Lipski J. Paradoxical lower sensitivity of locus coeruleus than substantia nigra pars compacta neurons to acute actions of rotenone. Experimental Neurology. 2017;287:34-43. http://www.sciencedirect.com.proxy.lib.ohio-state.edu/science/article/pii/S0014488616303338. doi: https://doi-org.proxy.lib.ohio-state.edu/10.1016/j.expneurol.2016.10.010.
3. Newhouse K, Shih-Ling Hsuan, Chang SH, Beibei Cai, Yupeng Wang, Zhengui Xia. Rotenone-Induced Apoptosis Is Mediated By p38 And JNK MAP Kinases In Human Dopaminergic SH-SY5Y Cells. Toxicological Sciences. 2004;79(1):137-146. doi:10.1093/toxsci/kfh089.
4. Krieger, R. (ed.). Handbook of Pesticide Toxicology. Volume 2, 2nd ed. 2001. Academic Press, San Diego, California., p. 1186
5. Bavli D, S., Ezra E, Levy G., Cohen M.,Vinken M.,Vanfleteren J., Jaeger M., Nahmias Y., Proceedings of the National Academy of Sciences Apr 2016, 201522556; DOI: 10.1073/pnas.1522556113
6. U.S. Department of Health and Human Services. Toxicology and carcinogenesis studies of rotenone. 1988
7. Gupta R. Biomarkers in toxicology. Second ed. Academic Press; 2019:455-475.
8. International Programme on Chemical Safety, (IPCS). Rotenone. http://www.inchem.org.proxy.lib.ohio-state.edu/documents/pims/chemical/pim474.htm#DivisionTitle:9.4.3.1%20%20Central%20Nervous%20System%20(CNS). Updated 1989. Accessed May 26, 2020.
9. International Labour Organization, ROTENONE International Chemical safety cards (ICSCs) 2000.
10. National Center for Biotechnology Information. PubChem Database. Rotenone, CID=6758, https://pubchem.ncbi.nlm.nih.gov/compound/Rotenone (accessed on May 27, 2020)
11. Gosalvez, M. (1983). Carcinogenesis with the insecticide rotenone. Life Sci. 12, 809-816
12. Greenman, D., Allaben, W., Burger, G., Kodell, R. Bioassay for carcinogenicity of rotenone in female wistar rats. Fundamental and Applied Toxicology. 1993;20:383-390.