Interaction between the aryl hydrocarbon receptor (AhR) and tryptophan on the hepatic and microbial toxicity of polychlorinated biphenyl 126 (PCB126)

We are exposed to persistent organic, man-made pollutants, also called forever chemicals, in our daily lives. Many have a structure that allows them to bind with a cellular receptor, known as the aryl hydrocarbon receptor (AhR). Ligands for this receptor include dioxins, polychlorinated biphenyls (PCBs), polybrominated biphenyls, and pesticides. AhR ligands are compounds that may bind and activate the AhR. After binding with foreign contaminants, the AhR moves from the cytoplasm of the cell to the nucleus where it interacts with the cellular genome and changes the expression levels of many different genes. Gene expression can be altered in this way by many AhR ligands, including some PCBs, mixtures of chlorinated biphenyls, and dietary factors such as tryptophan (Trp).

PCBs were produced from 1929 to 1977 in the US. After their ban by the Environmental Protection Agency in 1979, PCBs remain a problem still today due to their persistence in the environment and their continued inadvertent production. PCB126 is the most acutely toxic PCB and leads to many different dysfunctions throughout the body, primarily mediated by the activation of the AhR. Changes by PCB126 in gene expression can lead to alterations in energy metabolism and in the balance of glucose, fatty acids, and carbohydrates. Consequences of this toxicity can manifest as disturbed energy metabolism, non-alcoholic fatty liver disease, immune dysfunction, and wasting disease.

Trp is an essential amino acid found in many food sources that acts as a nutrient enhancer. Unlike the activation of the AhR by PCB126, Trp and/or Trp metabolites (such as serotonin, tryptamine, indole, etc.) can activate the AhR beneficially. Activation of the AhR by Trp can also alter gene expression and regulate energy metabolism. Studies have shown that Trp can strengthen immune function and reduce prevalence of diseases like diabetes and non-alcoholic fatty liver disease. Therefore, a co-exposure to both a beneficial and toxic AhR ligand may reduce toxicity.

While PCB126 causes detrimental effects to bacteria in the gut, known as the colon microbiome, Trp is a key player in balancing intestinal immune tolerance and gut microbiota maintenance. The gut microbiome consists of thousands of different species of bacteria and is sensitive to diet and environmental exposures. For instance, exposure to PCB126 and Trp can directly impact the gut microbiome. Alternatively, changes in the colon microbiome can lead to changes in the host. Adverse relationship between the microbiome and the host can result in compromised immune function and diseases such as non-alcoholic fatty liver disease, inflammatory bowel disease, type 2 diabetes, or colorectal cancers.

The first two studies of this dissertation focused on PCB126 alone and the effect on both liver gene expression and the colon microbiome. We hypothesized that the toxic effects caused by PCB126 to the major energy metabolism pathways, as well as the colon microbiome, would be mediated by the AhR. These studies aimed to determine 1) the effects of PCB126 on liver energy metabolism, 2) the effects of PCB126 on the colon microbiome, and 3) if these alterations could be linked with the AhR.

The following two studies introduced Trp as a potential supplementation to reduce the toxic effects of PCB126 on liver and microbial systems. Therefore, we hypothesized that supplementation of excess Trp would decrease the toxicity of PCB126. Overall, these studies aimed to determine, 1) whether an oral exposure to PCB126, with or without a co-exposure to Trp, would significantly a) alter the liver energy metabolism or b) alter the gut microbiome, and 2) if any or all of the alterations could also be linked to the AhR.

In order to test our hypotheses, we utilized two animal studies. In the first study, male and female WT (wild-type, normal) and KO (AhR knockout, no functional AhR) rats received an injection of either PCB126 in corn-oil (5 μmol/kg) or corn-oil (control). Four weeks later rats were sacrificed, livers removed to measure changes in gene expression, and fecal pellets from the colon were removed to measure any gut microbial changes. The second study used the same AhR KO rat model: male and female WT and KO rats were put on either a 2% Trp or a control diet (containing 0.2% Trp). Then after one week, the rats were given a weekly oral exposure of either PCB126 in corn-oil (1.25 μmol/kg) or corn-oil (control) for 4 weeks. Rats were then sacrificed, livers removed once again to measure changes in gene expression, and fecal pellets from the colon were removed to measure any gut microbial changes. To determine the impact of PCB126 and/or Trp, statistical analyses were conducted to detect differences in liver gene expression and colon microbes.

In the first study, knocking out AhR resulted in changes in expression of genes involved in energy metabolism without PCB126 exposure, notably in males. In contrast, PCB126 caused a larger number of changes in gene expression and alterations in associated sub-pathways in WT females than in any other genotype. Glucose homeostasis and fatty acid oxidation after PCB126 exposure had significantly fewer changes in activation states in KO rats than WT rats, both in males and females. Carbohydrate metabolism displayed a similar number of changes in both WT and KO rats exposed to PCB126. Additionally, KO rats exposed to PCB126 inhibited many carbohydrate metabolism and fatty acid oxidation pathways while WT rats exposed to PCB126 activated many of the same pathways. Overall, KO rats had very low numbers of changes in gene expression and energy metabolism sub-pathways.

In the second study, overall diversity of the microbiomes and Firmicutes to Bacteroidetes ratios were not altered after PCB126 exposure. This means that the microbiomes found across and within treatments were not different from one another, and none of the treatment groups showed signs of obesity or irritable bowel disease based on their microbial communities. However, PCB126 and knocking out the AhR can lead to specific taxon alterations after a one-time injection. These alterations can all cause changes in the host energy metabolism.

The third study showed that PCB126 altered many genes in both WT males and females. However, WT females had more changes in gene expression after PCB126 exposure than males. Trp decreased the number of genes altered by PCB126 in WT females but not in WT males while Trp caused more changes in gene expression than PCB126 in males. Additionally, Trp reduced the PCB126 effect on energy metabolism even though the co-exposure to Trp and PCB126 caused more overall genes to be altered in WT males. Overall, Trp did not significantly alter the diversity of genes in WT rats exposed to PCB126. However, Trp reduced the effects of PCB126 on glucose homeostasis and fatty acid oxidation but not carbohydrate metabolism in WT rats while KO rats showed very few changes in comparison across all analyses.

In the final study, overall diversity of the microbiomes and Firmicutes to Bacteroidetes ratios were once again not altered after oral exposure to PCB126 and/or Trp. However, oral exposure to PCB126, alone or with Trp, and knocking out the AhR, led to specific bacterial phyla and genera alterations. Interestingly, these alterations have been reported to cause changes in energy metabolism of the host.

The results of these studies demonstrate the essential role of AhR in energy metabolism pathways even during normal conditions and the significant disturbance by PCB126. Also, males may be at higher risk of adverse effects on energy metabolism while females may respond in a broader, more adaptive way. The second study with an injection implies that the circulation of PCB126 throughout the body can lead to changes in the gut even when the route of exposure is not through ingestion. The third study demonstrates the effects of PCB126 and Trp through the AhR in energy metabolism pathways; Trp reduced the effects of PCB126 in females but did not benefit males. Finally, the last study’s results demonstrate the effects of PCB126 and Trp on the colon microbiota through an oral exposure and how Trp can help mediate some of the responses. Therefore, the link of AhR ligands, such as PCB126 and Trp, to metabolic diseases and microbial alterations, and the possibility of additive or ameliorating effects should be further explored.