Wednesday, January 27, 2016

Lead: Breaking Down The Bridges Of Life For Centuries

In February of 2015, after months of seeing her children randomly lose hair and complain of stomachaches and other symptoms, LeeAnne Walters finally had the water in her house tested by city officials. As she feared, the results were not good. Lead levels showed a content of 104 parts per billion, nearly 7 times the EPA limit for lead in drinking water. In April she would go on to discover that all four of her children had lead in their blood, and that her son Gavin was actually poisoned by this heavy metal.

The Walters are one of the many families in Flint, Michigan suffering the consequences of the lead poisoning epidemic that resulted from city officials switching the source of city water from Lake Huron to the Flint River. We now know that the corrosive nature of Flint River water leached the lead from aging city pipes; what started as a cost saving measure resulted in nearly tripling the percentage of Flint children having elevated lead blood levels.

Repeated exposure to lead can cause diverse symptoms including abdominal pain, headaches, memory loss, weigh loss, and anemia. Children are particularly susceptible to the ill effects of lead toxicity, which can result in developmental delays and learning disabilities among other long-term, irreversible health issues. Thus, despite the change back to Lake Huron water in October, this man-made disaster will be affecting the residents of Flint for decades to come. Thanks to recent national press attention, the events that led to this water crisis as well as its health-related consequences are well known to most.

The weight equivalent of only ~1/16th teaspoon of salt in lead in the bloodstream of an adult sets off alarms of concern - but what is it about lead that makes it so toxic? What does lead do at the cellular and molecular level that contributes to such widespread and profound effects in the human body?

Mark Nowlin / The Seattle Times
Lead typically enters the body through either ingestion or inhalation of lead particles that contaminate water, food, or the environment. In the United States, up to 20% of the half a million children living with lead poisoning were exposed through the drinking of contaminated water. Once ingested, lead is absorbed through the gastro-intestinal (GI) system, a process that is greatly influenced by age:  while only 10% of lead ingested by adults is absorbed, up to half of that ingested by young children will be absorbed. In addition, diet and nutritional health play a role on absorption levels. Low calorie intake, vitamin, and iron deficiency, as well as a high-fat diet, have all been correlated with enhanced absorption of lead, which contributes to the higher prevalence of lead poisoning among children from economically depressed regions such as Flint, Michigan.

After absorption, lead enters the blood, where it is mostly associated with red blood cells (RBCs). One of the effects of lead is to weaken the membrane of cells leading to their rupture. In the context of RBCs, this results in a process known as hemolysis and contributes to the anemia often associated with lead poisoning. Another key effect of lead that contributes to anemia is the inhibition of enzymes responsible for making heme, a critical component of many proteins including hemoglobin. Unregulated inhibition of enzymes, like that caused by lead, generates an excess of reactive oxygen species (ROS), which in turn can disrupt nearly all components and functions of any cell. Unfortunately, not only does lead produce this so-called “oxidative stress”, but it also inhibits the antioxidant proteins and other molecules our cells would normally use to protect themselves. The net result is a perfect storm of damage and lack of protective mechanisms that spells the ultimate demise of the cell.

Interestingly, while the blood lead level is what health professionals use to monitor exposure, only a small fraction of the total lead burden in the body is found in the blood. A significant amount of lead accumulates in soft tissue organs such as liver, lungs, kidneys, and importantly the brain. Indeed, one of the primary targets of lead toxicity, especially in children, is the central nervous system. The adverse effect of lead in neurons is again a combination of ROS production and inhibition of antioxidants, plus the disruption of proteins responsible for neural functions such as the release of neurotransmitters. Nonetheless, nearly 90% of lead retained by adults and 75% of that retained by children ultimately ends up in the bones and teeth. It actually takes up to 30 years to eliminate half of the lead that enters the bone, and bone-to-blood transfer of lead, which increases during pregnancy, menopause, and aging, can serve as a source of lead toxicity long after initial exposure.

Lead’s uncanny ability to inhibit such a broad range of enzymes is due to its high affinity for sulfhydryl (sulfur and hydrogen) groups, including those attached to carbon, which are known as thiols. A biologically important thiol is the side chain of the amino acid cysteine, one of the 21 building blocks that make up proteins. Because two thiol groups can react with each other to form disulfide bonds, cysteines in different parts of a protein - or even in different proteins altogether - can bind to one another. The ability to form these disulfide bridges makes cysteine a very special amino acid that contributes to a protein’s structure, stability, function, and ability to interact with other proteins.

Disrupting and remaking disulfide bridges between proteins is the basis of chemicals used by hairdressers to straighten and curl up hair.
By strongly interacting with thiols, lead can break disulfide bridges and ruin the structure and function of enzymes, sometimes permanently. The ubiquitous presence of thiols in proteins and their importance to function is what allows lead to affect such a broad range of cells, organs, and processes. Another chemical property of lead that contributes to its toxicity is its divalency (ability to make two bonds). Once inside of cells, lead can take the place of biologically important divalent ions such as calcium and magnesium with dire consequences. Calcium, in particular, plays an important role as a signaling molecule and therefore its level inside of the cell is tightly regulated. When lead levels are high, it can activate proteins and processes, such as neurotransmitter and hormonal release, which are normally controlled by calcium fluxes.

Lead has been intrinsically interconnected with human history. Ancient civilizations considered it the father of metals and the Romans laced their wine and aqueducts with it. Soft, highly malleable, and with a low melting point, lead is in many ways an ideal material. But its more pernicious character as a poison has also been recognized for centuries. There is no known safe level of lead and its effects are widespread, long lasting, and unpredictable. While the pharmacokinetics, mechanism of toxicity, and chemical properties of lead are fascinating, they are insignificant and almost irrelevant in the context of the human cost lead poisoning has inflicted throughout history and continues to inflict. LeeAnne Walters’ son Gavin and the many other afflicted children from Flint, Michigan are unfortunately the latest to pay the price.

Contributed by:  Gustavo Arrizabalaga, Ph.D.

References and resources:

Flora G, Gupta D, & Tiwari A (2012). Toxicity of lead: A review with recent updates. Interdisciplinary toxicology, 5 (2), 47-58 PMID: 23118587

Thursday, January 21, 2016

A 5,300 Year Old Stomach Ache

In 1991, one of the best preserved mummies was found frozen in the Ötztal Alps, a mountain range near the border of Austria and Italy. This 5,300 year old man was named Ötzi and has been fascinating nosey scientists ever since. So far, scientists have learned about Neolithic fashions and dietary habits, and even identified his cause of death...solving one of the oldest “cold cases” in CSI history. (For the curious, poor Ötzi appears to have been attacked. He took a blow to the head and an arrow to his shoulder).

The well-preserved remains of the “iceman” named Ötzi.

Remarkably, genetic studies of folks living in Austria today reveal that Ötzi has some living relatives. Researchers found that at least 19 people may share a common ancestor with Ötzi, but the odds of one being a direct descendent of Ötzi himself are very remote. Some of these living relatives can be seen in a series of commercials for the auto insurance company Geico:


Ötzi provides a window into our past - a glimpse of what life might have been like 5,300 years ago. It turns out that Ötzi suffered from many of the same problems that we still have to contend with today. His body shows signs of heart disease, tooth decay, and joint pain, possibly caused by Borrelia borgdorferi, the bacteria that causes Lyme disease. The latest secret that scientists Frank Maixner and Albert Zink have coaxed out of Ötzi is that he was infected with another bacterial species that still causes grief in millions of people here and now:  Helicobacter pylori.

This is an artist’s rendition of Ötzi. But scientists didn’t just find a 5,300 year old human ancestor that day. They also found 5,300 year old bacteria.
Thanks to a landmark study in the 1980s, we now know that Helicobacter pylori is a causative agent of gastritis or stomach ulcers. Previously, doctors believed that stomach ulcers were simply caused by environmental factors - too much stress, spicy food, or smoking. But Drs. Barry Marshall and Robin Warren had a “gut feeling” that this model was wrong after they repeatedly found bacteria in the peptic ulcer biopsies from patients.

For decades, doctors believed that gastric ulcers were caused by stress. The idea that they were caused by a bacterial infection was a profound discovery and revolutionized treatment of this common ailment.
A lot of medical professionals had a hard time swallowing the idea that gastric ulcers were really an infection. Marshall and Warren faced great difficulty getting their results published in scientific journals. Many other doctors at the time mentioned how controlling the acid in the stomach usually helped patients feel better, and they balked at the idea that bacteria could survive in the acidic milieu of the stomach. Today, we now know that some bacteria can thrive in even the most inhospitable of places, including areas of high acidity. Helicobacter pylori is one such “acidophile” – it grows best under acidic conditions. This also explains why some ulcer patients recover after taking medication that reduces stomach acid; acid reducers cause a gastric climate change that is less favorable to the growth of Helicobacter pylori.

On June 12, 1984, Barry Marshall drank a culture of live Helicobacter pylori. He developed an ulcer, which he successfully treated with antibiotics. A tabloid newspaper first covered “the guinea-pig doctor” story, alongside stories of alien abductions and celebrity gossip. Ultimately, the stunt finally convinced the skeptics and won Marshall and Warren the Nobel Prize in Physiology or Medicine for 2005.
How do we get infected with Helicobacter pylori? The bacteria can be contracted through saliva or accidental ingestion of material coming out of the other end of a person. Somewhere along the way, a little poo from an infected person got into your food/water (or on your hands) and found its way into your gut. As gross as that sounds, it must happen a lot because Helicobacter pylori is present in the gut of billions of people. However, it causes ulcers in only 10% of them. 

Scientists don’t currently know why the bacteria attack the stomach lining of some people but not others. But if you are one of those unlucky few, the damage caused by the bacteria allows stomach acid to pass through the protective lining, which can cause bleeding and digestive problems, and obviously a lot of pain and discomfort.

But thanks to the renegade efforts of Barry Marshall, doctors now know how to treat gastric ulcers more effectively with a simple course of antibiotics. It is perhaps no surprise to you now that our old friend Ötzi had Helicobacter pylori in his tummy. The bug is very common and easily contracted, especially in his day when hand sanitizers were not easily accessible. Whether Ötzi’s Helicobacter pylori actually caused a gastric ulcer is hard to say, as his stomach lining was not preserved well enough to draw firm conclusions. One day we may find a specific type of genetic mutation in people prone to stomach ulcers, which would allow us to revisit the question.

If you want to learn more about the cracking of the stomach ulcer mystery, check out the following video.


Contributed by:  Bill Sullivan, Ph.D.

Follow Bill on Twitter.

Williams AC, Edwards HG, & Barry BW (1995). The 'Iceman': molecular structure of 5200-year-old skin characterised by Raman spectroscopy and electron microscopy. Biochimica et biophysica acta, 1246 (1), 98-105 PMID: 7811737

Tito RY, Knights D, Metcalf J, Obregon-Tito AJ, Cleeland L, Najar F, Roe B, Reinhard K, Sobolik K, Belknap S, Foster M, Spicer P, Knight R, & Lewis CM Jr (2012). Insights from characterizing extinct human gut microbiomes. PloS one, 7 (12) PMID: 23251439

Maixner, F., Krause-Kyora, B., Turaev, D., Herbig, A., Hoopmann, M., Hallows, J., Kusebauch, U., Vigl, E., Malfertheiner, P., Megraud, F., OSullivan, N., Cipollini, G., Coia, V., Samadelli, M., Engstrand, L., Linz, B., Moritz, R., Grimm, R., Krause, J., Nebel, A., Moodley, Y., Rattei, T., & Zink, A. (2016). The 5300-year-old Helicobacter pylori genome of the Iceman Science, 351 (6269), 162-165 DOI: 10.1126/science.aad2545

Thursday, January 7, 2016

Daraprim: The 62 Year Old $750 Pill

In September 2015, the CEO of a new company called Turing Pharmaceuticals gained instant notoriety when he jacked up the price of a little known drug called Daraprim from $13.50/pill to $750/pill. Turing is not the first company to raise drug prices to astronomical levels, but the Daraprim situation is unique for a number of reasons. First, Turing inflated the cost of Daraprim over 50-fold overnight, making it one of the largest jumps in a drug’s price ever. Second, the company did not invent or improve upon Daraprim; this drug is 62 years old and all Turing did was pony up $55 million to acquire the rights to it.

Martin Shkreli, who unapologetically increased the cost of Daraprim by 5,500%, has been dubbed “Pharma Bro” and “the most hated man in America” by various media outlets.
Third, the company justified the huge price increase by stating that it will use a portion of the profits to invest in generating better drugs for toxoplasmosis, the neglected disease that Daraprim treats. This reverses the polarity of drug development:  most pharmaceutical companies use money they’ve already raised or earned to develop a new drug. But Turing already has the drug and claims that it will develop a better drug with the profit made from overcharging current patients. Many would argue that Turing should have used the $55 million to develop a new toxoplasmosis drug in the first place, rather than gouge current patients and further strain the US health care system. Moreover, the patients who are forced to pay for this research will not get their money back if Turing fails to deliver, which is often the case in drug research.

The world had Daraprim before Elvis had a song played on the radio!
In addition to highlighting problems with the nation’s policy on drug pricing, the situation has also taken toxoplasmosis from the shadows and into the limelight. This disease is caused by a single-celled parasite called Toxoplasma gondii. The parasite infects nearly anything with a backbone, but animals (including people) that have a healthy immune system keep the parasite in check, locking it into a latent, encysted form. If immunity deteriorates due to disease, such as HIV/AIDS, or chemotherapy, such as that given to fight cancer, the latent Toxoplasma parasites will start replicating again, leaving massive tissue destruction in their wake. If you want to learn more about Toxoplasma parasite, you can do so here.

In the center of this heart tissue is a tissue cyst filled with latent Toxoplasma parasites. These parasites can start growing again if immunity is compromised, causing rapid damage to the heart. The parasite cysts also reside in the brain, creating serious neurological problems if the disease is reactivated.
Toxoplasmosis can be life-threatening if not treated promptly. So how does Daraprim help save lives?

Daraprim is the trade name for pyrimethamine, the chemical structure shown here. $750 will buy you a single pill. A standard round of treatment for toxoplasmosis is usually a month, putting the cost at ~$22,500. Unfortunately, this drug only puts the disease into remission, so the patient typically needs additional courses throughout his or her lifetime.
Daraprim is an antifolate drug; as you may surmise, taking it leads to a depletion of the B-vitamin called folate. Folates are absolutely required by all living cells to make genetic material such as RNA and DNA. Everyone knows we need folate in our diet or else we may suffer from anemia, digestive issues, cognitive defects, or growth problems. But what you may not know is that all living things need folate – bacteria, fungi, and parasites like Toxoplasma.

Pyrimethamine targets an important enzyme in the folate metabolic pathway called DHFR (dihydrofolate reductase). By inhibiting DHFR, pyrimethamine stops the conversion of dihydrofolic acid to tetrahydrofolic acid, the latter of which is needed to make purines and thymidylate, molecules that are required for DNA and RNA synthesis. If germs can’t make DNA or RNA, they simply cannot grow anymore.

Like trimethoprim, pyrimethamine is an inhibitor of the enzyme DHFR. Sulfa drugs (sulfonamides) are also added to the treatment regimen as they serve to block the upstream step of folate metabolism, delivering a one-two punch to the pathogen.
Hopefully you can now see why Daraprim kills parasites like Toxoplasma, but you may be wondering why the drug doesn’t also kill the patient. Truth is, at high enough concentrations for a long enough time, Daraprim can kill a person. But the risk is offset for two main reasons. One, pyrimethamine binds to the parasite form of DHFR much better than to the human form of DHFR. Both Toxoplasma and human DHFR function the same way, but there are subtle differences in the enzyme’s structure between the pathogen and the patient that make it bind to the drug with different affinity. Said another way, if you consider Daraprim to be like a piece of metal, then the parasite DHFR enzyme is like a stronger magnet than the human DHFR enzyme. Two, a patient taking Daraprim is usually given folinic acid (leucovorin), which converts to tetrahydrofolic acid without the need for DHFR.

Pyrimethamine was developed by Nobel-Prize winning scientist Dr. Gertrude Elion in the early 1950s. Dr. Elion was also instrumental in the development of acyclovir for herpes viruses. I wonder what she would say to Mr. Shkreli.
The discovery of pyrimethamine as a potent anti-parasitic agent many years ago marked a great advance in the medical sciences. It is a shame we let the clay from the best minds get molded by the worst hands.

Contributed by:  Bill Sullivan, Ph.D.

Roos DS (1993). Primary structure of the dihydrofolate reductase-thymidylate synthase gene from Toxoplasma gondii. The Journal of biological chemistry, 268 (9), 6269-80 PMID: 8454599

Sullivan WJ Jr, & Jeffers V (2012). Mechanisms of Toxoplasma gondii persistence and latency. FEMS microbiology reviews, 36 (3), 717-33 PMID: 22091606