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New Understanding of How PCBs, a Class of Long-Banned Chemicals, Release Their Toxic Effects Inside the Body

Plastics, paints, electronic equipment, insulating fluids, and other industrial and commercial products all included polychlorinated biphenyls (PCBs). Due to their toxicity, they were widely prohibited from production starting in the late 1970s; yet, significant amounts of them are still present in our environment and accumulate inside the bodies of animals.

Chiral PCBs are PCBs having two mirror-image isomers that are exact reflections of one another and share the same chemical structure. Because chiral PCBs include more chlorine atoms, which are difficult for the body to break down, they can build in the body quickly. Additionally, because their isomers are metabolized differently, they can induce toxicity (particularly neurodevelopmental issues) that is specific to each isomer.

However, the process behind this selective metabolism was not known. A study team has shed light on how body-produced enzymes unevenly digest the mirror-image isomers to remedy this. These findings will enable the estimation of animal PCB metabolism and detoxification mechanisms. In order to gain a better knowledge of the potential toxicity in humans and other mammals, they will also aid in the creation of technologies that can anticipate the mirror isomers of chiral PCBs.

These findings were made by a multi-institutional research collaboration, which included Associate Professor INUI Hideyuki (Kobe University Biosignal Research Center), Lead Researcher MATSUMURA Chisato (Hyogo Prefectural Institute of Environmental Sciences), Professor YAMAMOTO Keiko and Professor ITOH Toshimasa (Showa Pharmaceutical University), Associate Professor MORI Tadashi (Osaka University Graduate School of Engineering), and Visiting Professor NAKANO Takeshi (Osaka University Research Center for Environmental Preservation).

These research results were published online in the international academic journals Environmental Science & Technology on July 8, and Chemosphere on September 6, 2022.

Main points

  • PCBs were once found in a huge variety of industrial and consumer goods. These extremely cancer-causing chemical substances permeate our environment and build up inside living things.
  • PCBs have a dioxin-like toxicity and research into PCB metabolism is advancing.
  • However, research had yet to uncover how chiral PCBs’ mirror-image isomers are metabolized.
  • The researchers split the two atropisomers (mirror-image isomers) found in each type of chiral PCB and used them as substrates for CYP enzymes.
  • Even though an atropisomer pair is physically and chemically similar, their levels of metabolization differ significantly.
  • The atropisomers are metabolized differently because of variations in the binding inhibition of each atropisomer by the amino acids of CYP.
  • These results will be helpful for measuring chiral PCB atropisomers, which are known to readily accumulate inside the bodies of animals.

Research Background

Despite the fact that the production and use of PCBs were outlawed around 50 years ago, they are still present in the environment. It has been found that eating food causes PCBs to accumulate in both human and animal bodies.

Particularly water resistant and difficult to degrade are PCBs with many chlorine linkages. This makes it possible for huge amounts of these PCBs to collect inside the bodies of animals, which is harmful to their health. The aryl hydrocarbon receptor (AhR) is responsible for PCB toxicity, which has detrimental effects similar to dioxin poisoning, including cancer, teratogenesis, and immune system impairment.

Research is being done on specific PCB types, such as dioxin-like PCBs with one ortho chlorine substitution in the biphenyl ring or PCBs with no substitutions, which are well known to have these effects.

However, if a PCB has more than 3 chlorine substitutions at the ortho position of the biphenyl ring, it becomes a mirror-image isomer called chiral PCB. These chiral PCBs do not demonstrate dioxin-like toxicity but are far more dangerous, binding with the ryanodine receptors (RyR) in organisms to become neurotoxic. The two mirror-image isomers (called atropisomers) in chiral PCB have identical physical and chemical properties and exist at a 1:1 ratio in commercial chiral PCB.

However, biased ratios are often observed in the environment and in animals such as earthworms and whales, as well as humans. It is believed that this unbalanced ratio is mainly caused by metabolism and that one of chiral PCB’s atropisomers is more effected by the metabolic reaction thus reducing its concentration.

However, very little research has been carried out into differences in how these atropisomers are metabolized nor the structural arrangement of the metabolic enzymes.

Research Methodology

To address this knowledge gap, the team conducted research focusing on the metabolic enzyme cytochrome P450 (CYP enzyme). The CYP enzyme reacts with foreign compounds that enter an animal’s body (for example, chemicals or pollutants in food, or medicines). CYP can convert them into water-soluble compounds and promote their expulsion from the body.

Previous research by this group has shown that CYP enzymes hydroxylate and dechlorinate dioxin-like PCBs. This decreases PCB’s binding with AhR and increases its water solubility, promoting expulsion from the body and therefore counteracting its toxicity. In other words, CYP is an important enzyme that determines whether or not PCBs are treated as toxic compounds by the body.

To measure the metabolic action of CYP on chiral PCB, the researchers set up a CYP enzyme and PCB docking model. They used this to estimate the structure of PCB metabolites and the structure of the CYP that decides to metabolize each of the PCB atropisomers differently.

For the experiment, the group selected three types of chiral PCB, each with a different number of substituted chlorine atoms; CB45 (4 chlorine substitutions), CB91 (5 chlorine substitutions), and CB183 (7 chlorine substitutions). They separated the atropisomers for each type of chiral PCB using chromatography and let them react with a human CYP enzyme.

It is thought that research on separating the atropisomers and letting them react has not been done before now. The results revealed big differences in how each atropisomer is metabolized. This revealed that even though the two atropisomers in one PCB have the same physical and chemical composition, they are biologically different.

The researchers found that one of the chiral PCB atropisomers was metabolized more than the other one, disrupting the 1:1 ratio. In addition, it is thought that the amount of (aS)-CB183 atropisomer decreases because it is metabolized more than the other atropisomer, and this is supported by the reports of low accumulation of (aS)-CB183 in humans.

But why are these physically and chemically identical atropisomers metabolized differently by the CYP enzyme? To solve this mystery, the researchers used a computer model to investigate how easily each chiral PCB atropisomer binds to the chemical structure of CYP. They found that when an atropisomer fills up the substrate-binding cavity inside the CYP enzyme, CYP’s amino acids (that form the cavity) interfere with the binding between CYP and the atropisomer.

Therefore, the atropisomer that isn’t interfered with by CYP’s amino acids becomes easy to metabolize (atropisomer (aR)-CB45 in CB45, and (aS)-CB183 in CB183), resulting in alterations to the original 1:1 ratio of atropisomers found in chiral PCB.

Further Research

The results of this research will be useful for making predictions about the atropisomers of chiral PCBs, which accumulate easily inside animals’ bodies. In other words, it will be possible to work out which atropisomer is reduced by the metabolic reaction with CYP enzymes and which atropisomer remains inside the body.

Chiral PCB’s toxicity is activated by binding with RyR, however, the ability to bind with RyR differs between the atropisomers. Therefore, this research will make it possible to estimate the toxicity of chiral PCBs.

Glossary

  • Polychlorinated biphenyl (PCB): A chemical compound with between 1 to 8 chlorine atoms bound to its biphenyl ring. There are 209 types of PCB, each with different structures. Until the late 1970s, PCBs were manufactured for use as insulating oil and were also found in a variety of products, however their manufacture and use were banned worldwide after it was discovered that they are highly toxic. PCBs with many chlorine bonds do not break down easily so large quantities remain in the environment and inside organisms even almost 50 years after they were banned.
  • Chiral PCB: This is a PCB that has 3 or more chlorine substitutions at the ortho position. This structure inhibits the rotation of the bond connecting the 2 benzene rings. It also has 2 mirror isomers (a pair of atropisomers). There are 19 types of chiral PCB and according to the Cahn-Ingold-Prelog priority rules, their atropisomers are referred to as ‘aS’ and ‘aR’.
  • Mirror-image isomer (enantiomer): A pair of isomers that are non-superimposable mirror-images of each other, slightly similar to your left and right hands. Asymmetrical carbon compounds have mirror-image isomers. These isomers have the same physical and chemical properties. In chemical synthesis, a racemic mixture consists of an equal amount of each isomer in a 1:1 ratio.
  • Cytochrome P450 monooxygenase (CYP enzyme): This metabolic enzyme is found in most organisms- it is responsible for the oxygenation reaction in the biosynthesis pathway for various chemical compounds in the body. In addition to this it has a detoxifying function; it oxygenates foreign compounds in the body, which makes them water soluble and easier to excrete.
  • Receptor: These are proteins found in organism cells that bind to chemical substances and promote or control the expression of the gene required for the organism to respond to the chemical substance. Aside from the estrogen receptor (ER) that binds to the female hormone and the aryl hydrocarbon receptor (AhR) that binds to dioxins, there are many other known receptors for various hormones.
  • Dioxin: Dioxins are generated by incinerating trash at low temperatures. They are difficult to break down and persist in the environment and inside organisms for a long period of time. When dioxins enter an animal’s body via food, they bind to AhR (aryl hydrocarbon receptors) inside the cells and the gene transcription for the enzyme that detoxifies dioxins is activated.

This study was supported by a Grant-in-Aid for Challenging Exploratory Research (grant number 25550064) from the Japan Society for the Promotion of Science and the Japan Science and Technology Agency’s CREST program (grant number JPMJCR2001).

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