Clock Gene Can Make High Drug Doses More Toxic to the Liver

Higher doses of drugs that yield toxic byproducts when broken down by the liver do the most damage to the organ's cells when its circadian clock genes are more active, according to a new study in Science Advances involving an experimental human liver system.

Like their name suggests, circadian clock genes maintain cells' circadian rhythms, or 24-hour cycles dictated by organisms' biological clocks. This new study details the ways in which clock genes regulate drug metabolism and immune responses in liver cells.

"By understanding when our liver cells are most active, either in how they interact with drugs, or how they react to invading infections, we can design new medicines to take advantage of these pathways," said Heather Fleming, immunologist and research director at the Laboratory for Multiscale Regenerative Technologies (LMRT). "Perhaps most excitingly, we can make better use of medicines that we already have, by learning the best time to deliver them to patients to maximize their function and minimize their negative side effects."

The investigators focused on the pain-relieving drug acetaminophen and the cholesterol management drug atorvastatin, both of which produce metabolites that can cause liver failure at higher concentrations. Their analyses spotlighted a clock gene called CYP3A4. When this gene peaked in activity, the drugs' toxicity increased. Understanding how CYP3A4's circadian activity affects drug metabolism and leads to heightened toxicity could enable new ways to ensure drug safety and efficacy, such as the development of time-centric dosage guidance.

"This approach could make a big difference," explained Sandra March, first and corresponding author of the paper and a research scientist at LMRT specializing in liver pathophysiology. "For drugs that are changed by the liver to make a toxic side effect, we can plan to give that drug when the liver is less active to reduce any dangerous risks of the medicine."

Moreover, March and her colleagues found that liver immune responses against bacteria and malaria may be similarly beholden to circadian gene activity.

"The phase of the circadian cycle can have a potential impact on how well a person's liver can fight against an invading parasite, like the one that causes malaria. Our cells have internal defenses, but we've found that these tools are also influenced by the time of day," said March.

On The Clock

Colloquially, people think of circadian rhythms in the context of sleeping and disruptions to sleep cycles. Jet lag is a classic case of circadian disruption, where the body must catch up and adjust over a few days to get back on track with its normal sleep and wake cycles. But circadian rhythms exist on a cellular and molecular level too.

"All the cells of our body have their own circadian 'clock.' This can influence many aspects of life including the effectiveness of medicines and vaccines," said Sangeeta Bhatia, corresponding author on the paper, director of LMRT and professor at the Massachusetts Institute of Technology (MIT).

Cells are controlled by clock genes, which encode proteins that uphold time-dependent functions. For example, the protein created by the Bmal1 gene, called BMAL1, bonds with another protein CLOCK. In mammals, the BMAL1-CLOCK protein helps control 24-hour cyclical patterns of expression in around 50% of genes.

"Many of these fluctuating genes are active in the human liver and have important jobs in managing how our bodies produce energy, how our immune system works to fight infections, and how our body uses the medicines that we take," noted Fleming.

Partners In Time

To understand the relationship between circadian-enforcing Bmal1 and another clock gene known as CYP3A4 that aids in the liver's drug metabolization, the scientists used their previously designed system involving human liver cell cultures.

"Human liver is difficult to study in the lab, since its cells are challenging to grow outside the body. Our group has established methods to grow human liver cells in dishes, and that have been shown to preserve many of their functions for weeks at a time," Bhatia said.

When they knocked out Bmal1 expression in liver cells, they found that CYP3A4 activity decreased. This result confirmed CYP3A4 's status as a clock gene and revealed its dependency on the famous time-keeper gene Bmal1.

After establishing the Bmal1and CYP3A4 partnership, the team profiled CYP3A4 expression in cell cultures at 16 points over 48 hours, or two 24-hour cycles. At the same time, they administered gradually increasing doses of acetaminophen and atorvastatin to the cell cultures. They found that CYP3A4 enzymatic activity correlated with drug toxicity. For example, when the gene's expression peaked at 36 hours, drug toxicity for higher doses also peaked.

Since there are at least 189 other drugs metabolized by CYP3A4, determining the optimal times to take these drugs could help ensure safety.

"By finding that toxicity of established drugs varies throughout the day in a pattern that matches the activity of CYP3A4, it may help make better recommendations about what time of day people should take or avoid taking already-approved drugs that are known to carry some risk of side effects," said Bhatia.

Turn Of Phase

After exploring the intersection between drug toxicity and circadian gene expression, Bhatia and her co-authors investigated how circadian genes affect immunity in the liver.

"If we understand more about when our body is naturally more resistant to infection, and what pathways it uses naturally, we can use this knowledge to design more weapons to fight back, either to reduce how many cells become infected, or to limit the spread of the infection," said March.

In their analyses, they identified many immune genes operated along circadian Bmal1-related pathways. Next, they exposed the liver cells to a bacterial cell wall lipid commonly used to trigger immune reactions in laboratory experiments. They administered this lipid to liver cells at 24 hours, 36 hours or 48 hours and saw that 36 hours yielded the most robust immune response.

When they infected liver cells with the malaria-causing parasite Plasmodium falciparum at 24 hours and at 36 hours, they again saw that immunity was strongest at 36 hours. These findings reinforced the idea that inherent circadian cellular patterns dictate liver immunity.

Moving forward, the group plans to use their approach to untangle how circadian genes can influence the processing of medicine related to cancer and pain.

"This information may become very valuable in the design of clinical trials. There may be new medicines that haven't worked well enough or that cause too many side effects to be approved, but if they are given to patients at a different time of day, they may be able to pass their clinical trials - which increases the number of medicines that doctors have at their disposal," said March.