Using tiny engineered liver organs derived from human donor cells, researchers found that many genes involved in drug metabolism are under circadian control. These circadian fluctuations affect how much of a drug is available and how effectively the body can break it down. For example, they found that enzymes that break down Tylenol and other drugs are more abundant at certain times of the day. Administering drugs at different times of the day could have a significant impact on how they are metabolized in the liver. In total, the researchers identified more than 300 liver genes that follow a circadian clock, including many involved in drug metabolism as well as other functions such as inflammation.

Analyzing these rhythms could help researchers develop better dosing schedules for existing drugs. One of the first applications of this method could be the fine-tuning of already approved drugs to maximize their efficacy and minimize their toxicity. The study also showed that the liver is more susceptible to infections such as malaria at certain times in the circadian cycle when fewer inflammatory proteins are produced. Bhatia is the lead author of the new study, which was published in Science Advances. The lead author of the study is Sandra March, research associate at IMES.

How Circadian Cycles Affect Liver Function

It is estimated that about 50 percent of human genes follow a circadian cycle, and many of these genes are active in the liver. However, studying the effects of these cycles on liver function has been difficult because many of these genes are not identical in mice and humans, so mouse models cannot be used to study them. Bhatia’s lab has already developed a method to grow miniaturized livers from liver cells (hepatocytes) from human donors. In this study, she and her colleagues wanted to investigate whether these artificially produced livers have their own 24-hour clocks.

In collaboration with Charles Rice’s group at Rockefeller University, they identified culture conditions that promote the circadian expression of a clock gene called Bmal1. This gene, which controls the cyclical expression of a variety of genes, enabled the liver cells to develop synchronized circadian oscillations. The researchers then measured gene expression in these cells every three hours for 48 hours and were able to identify more than 300 genes that were expressed in waves.

Most of these genes clustered into two groups – about 70 percent of the genes peaked together, while the remaining 30 percent peaked when the others peaked. These included genes involved in a variety of functions, including drug metabolism, glucose and fat metabolism, and various immune processes. Once the engineered livers had established these circadian cycles, the researchers were able to use them to study how circadian cycles affect liver function. First, they examined how the time of day affects drug metabolism by studying two different drugs – acetaminophen (Tylenol) and atorvastatin, a drug used to treat high cholesterol.

When Tylenol is broken down in the liver, a small amount of the drug is converted into a toxic byproduct known as NAPQI. The researchers found that the amount of NAPQI produced can vary by up to 50 percent depending on what time of day the drug is administered. They also found that atorvastatin has higher toxicity at certain times of the day. Both drugs are metabolized in part by an enzyme called CYP3A4, which has a circadian cycle. CYP3A4 is involved in the processing of about 50 percent of all drugs, so the researchers now plan to test more of these drugs with their liver models.

The MIT researchers are now working with colleagues to analyze a cancer drug that they suspect is affected by circadian cycles, and they hope to investigate whether the same is true for drugs used to treat pain.

Susceptibility to Infections

Many of the liver genes that exhibit circadian behavior are involved in immune responses such as inflammation, so the researchers wondered whether this variation could affect susceptibility to infection. To answer this question, they exposed the manipulated livers to Plasmodium falciparum, a parasite that causes malaria, at different points in the circadian cycle. These studies showed that the livers were more likely to be infected after exposure at different times of the day. This is due to differences in the expression of genes called interferon-stimulated genes, which contribute to the suppression of infections.

The researchers hypothesize that these cyclical fluctuations are due to the liver dampening its response to pathogens after meals, when it is normally exposed to an influx of microorganisms that could trigger inflammation, even if they are not actually harmful. Bhatia’s lab is now taking advantage of these cycles to study infections that are normally difficult to establish in engineered livers, including malaria infections caused by parasites other than Plasmodium falciparum.

This is very important for research, because simply by setting up the system and choosing the right time of infection, the experts can increase the infection rate of their culture by 25 percent and thus enable drug studies that would otherwise not be feasible.