Tiny lines on the tooth enamel reveal a previously unknown biological rhythm. If the data are confirmed, this finding will help researchers understand why larger animals grow slower and live longer than smaller ones.
One summer last year, Timothy Bromage, a paleontologist at New York University, was chewing on a lamb chop while vacationing in Cyprus. Suddenly he heard a crunch. When the sound was followed by a sharp pain, he realized that he had broken a tooth.
When he returned to New York, his dentist told him that he would have to endure three months of agony if he wanted to have a tooth restored. "Or give me just five minutes," the doctor said, "and I'll pull it out right now."
Bromage preferred deletion. Thus, he was able to make a thin cut of the tooth, which is what he had wanted to do for several years, in order to measure a new kind of biorhythm, which he studied in the permanent teeth of mammals. This is not a well-studied circadian biorhythm, but a longer one, differing from species to species, lasting from two days to two weeks. Bromage believes that this rhythm can set the growth rate of animals and their lifespan.
In rats, the biorhythm lasts one day; in macaques - four, in sheep - five, in humans - from six to 12 days. Bromage confirmed this relationship in dozens of other living and extinct mammals, including Asian elephants, which have a biorhythm that lasts 14 days. (There are exceptions: for example, dogs do not show this relationship.)
In general, the slower rhythm in larger mammalian species is justified: large animals grow more slowly than smaller animals, spending longer periods. Bromage believes that the rhythm of teeth and bones reflects a growth signal that stimulates the rate of cell division, which the cells of the body receive this signal at regular intervals. The more often such signals are received, the faster the animal grows.
Rhythmic interval increases not only with body weight, Bromage found that it increases with other characteristics that increase along with body weight, for example, with life expectancy, lactation duration, metabolic rate, estrous cycle duration and even kidney size. This suggests that by measuring the growth rate of just one tooth, even if it is an extinct animal, it will be possible to determine not only the size of its body, but also many of its other features.
“Give me any tooth, any permanent primate tooth - just throw it to me, don't tell me what primate it is - and I will reconstruct what size it had kidneys, how long it lived, all those features,” says Bromage. “It’s incredible what a window of opportunity this material opens up for finding the key to life.”
Promotional video:
After receiving the prestigious Max Planck Science Prize with a colleague in 2010, Bromage spent the € 750,000 on research to determine whether animal blood samples reflect the same rhythms as teeth. The research was expensive and time-consuming, partly because mice and rats (the cheap workhorses of biology) do not have a multi-day rhythm and therefore cannot be used as experimental subjects.
The results of his research, published in 2016, are not yet solid enough to become a discovery. Many chronobiologists are skeptical about them.
But “What if he's right after all?” Asks Robin Bernstein, an anthropologist biologist at the University of Colorado at Boulder who has studied the evolution of body size and is now studying the growth of humans and non-human primates. “In my opinion, he is one of those people who are ahead of his time,” she says. “Maybe there’s nothing special here, but it’s original, really interesting, and I think a lot could be done about it.”
Dental connections
Bromage became interested in teeth when he was a graduate student in the mid-1980s. At the time, scientists knew that just as trees form annual rings, daily growth streaks form on the tooth enamel. In the 1930s and 1940s, Japanese scientists discovered them on the teeth of dogs, rats, pigs and macaques.
Mammals also have prominent stripes called Retzius stripes. In the early hominids studied at the time by Bromage, seven daily bands separated each Retzius lineage. No one knew how or why they formed, but Bromage was able to use them as a marker to show that the first permanent molars appeared in early hominids around the age of three, like chimpanzees, much earlier than modern humans. This meant that the early hominids were not just miniature versions of modern humans, as was then believed, but were closer to apes.
In 1991, Bromage confirmed that Retzius' lines in macaques were separated by only four daily growth lines, as opposed to seven in early hominids. Then in 2000, he realized that bones also have a pattern of periodic growth. He found that stripes, called lamellae, formed on the bones of rats in just one day. How could this be possible if human bones grow much more slowly than rat bones?
“It hasn't gone out of my head for years,” says Bromage. And then one day in 2008, he read in a dissertation of one of his students that lamellae in the bones of macaques are formed in four days, that is, in the same way as the Retzius lines, which he found in the teeth of macaques in 1991. “This 1991 memory came into my mind the very second I saw the number four,” he recalls. Could it be possible, he wondered, that mammals have the same growth periods in teeth and bones? If this is the case, then lamellae in humans should also form in seven days, which is much longer than in rats, which only takes one day to do so.
Bromage called this idea "a whole new paradigm." Until that time, it was believed that there was no connection between how teeth and bones grow; bones were never thought of as tissue that develops in gradual, measurable stages, like teeth and trees. Any possible connection between the rate of development of teeth and bones was so fundamental that I could not tell anyone for a whole week,”says Bromage, even to his wife. He checked the histological structure of bones and teeth in his laboratory and found that the rhythms of growth of teeth and bones did coincide in macaques, sheep and humans.
The rhythm of the brain
If the rhythms Bromage saw in the growth bands of mammalian teeth and bones were a response to a growth signal, where could that signal come from? Bromage believes that its source is the same part of the brain that, as is already known, sets the circadian biorhythm, that is, the hypothalamus. After all, the length of the biorhythms he studied is always a multiple of a whole day, and the biological clock, as has already been established, affects the rate of cell division. The hypothalamus is capable of performing this function, so “why invent another, completely new instrument?” - a question arose in him. Something, perhaps a substance accumulating in the hypothalamus, can vary the biological clock in a multi-day cycle. Whichever part of the brain is responsible for this, “it’s just meant to count,” says Bromage.
The hypothalamus also does another job: it regulates the pituitary gland, a hormone-producing pituitary gland, the front of which regulates body size and the back of which regulates the duration of the estrous cycle. Perhaps not coincidentally, these are the only two physiological features that Bromage discovered are directly correlated with the duration of the new biorhythm.
Bromage began to test his theory. If the signal generated in the brain regulates the rate of growth, Bromage speculated, then the blood must carry traces of this signal.
Bromage spent two weeks collecting six milliliters of blood samples from pigs. Then he handed over 1,700 samples he collected from 33 pigs to an independent laboratory to identify 995 different metabolites, biochemical substances produced by the body.
After spending 300 thousand dollars, he received the answer: of the 159 most concentrated metabolites with a specific biological function, 108 reflected the circadian rhythm. The next most frequent rhythm was the same five-day rhythm that Bromage identified in the teeth and bones of pigs. Only 55 out of 159 metabolites passed through this cycle, and only in 20 did the cycle coincide with other rhythms.
To his surprise, Bromage identified two five-day cycles three days apart. The first contained metabolites associated with growth, and the second - metabolites formed during the breakdown of biological molecules. This made sense: when growth is over, metabolites must undergo breakdown in order to become available for processing in the next growth cycle. What an exquisitely designed system, Bromage thought. I would never have believed it if I hadn't seen it with my own eyes!
He named the new biorhythm "Havers-Halberg Oscillations". The name is given in honor of Clopton Havers, who, in the late 17th century, first described the lamellae of bones and what would later become known as the Retzius stripes; and Franz Halberg, a chronobiologist who died in 2013 at the age of 93.
The pig problem
Looking back, we realize that naming the rhythm after Halberg was not the smartest decision.
Chronobiologists have become extremely skeptical about the discovery of multi-day biorhythms, says Roberto Refinetti, a physiologist at the University of Boise and author of a textbook on circadian physiology. And we owe a lot to Halberg for this. He introduced the very concept of "circadian". However, in the future, he announced the discovery of longer rhythms, without presenting substantial evidence. "He was truly, as he liked to say, a broad-minded man," Refinetti said. "Some thought he was even out of bounds."
Refinetti himself tried (and failed) to identify a weekly rhythm in blood pressure and lactic acid concentration in horses. He believes that Bromage's five-day rhythm in pigs may be the result of a human working week, a relatively new social invention. Moreover, he says, nothing in the environment could have been a prerequisite for the development of a weekly rhythm over millions of years. Contrast this with the circadian rhythm, which obviously arose as a reaction to the change of day and night.
Bromage replied that the rhythms he identified most likely could not be caused by the work week, because the pigs were kept in constant conditions all the time. Moreover, if Bromage's theory is correct, then these rhythms would not need a multi-day external signal to develop, since they are based on daily hours that can be counted. Refinetti, he added, probably didn’t measure weekly rhythm in horses because he didn’t measure the entire complex associated with growth.
In terms of criticism of Halberg's data, Bromage said he named the rhythm after him because he "championed long-term rhythms when no one else on earth thought about it." But that, says Bromage, doesn't mean "I agree with all of his statements."
It is more difficult to argue, perhaps, with the statistics according to Bromage's data. Due to the cost and complexity, the experiment had to be carried out on a shorter timeframe than Bromage had hoped. Since there were too few cycles, he could not statistically objectively check the rhythms. Instead, the situation prompted him to assume a five-day rhythm, and then check if that assumption was statistically relevant. If you're claiming a five-day cycle, you have to measure many cycles to have a statistical basis, says Andrew Liu, a chronobiologist at the University of Memphis.
Bromage agreed that the experiment had its own flaws. “We really sped it up,” he says. It would be difficult to measure the blood of pigs over a longer period: the animals became more stressed and by the end of the study they began to develop infections. “It was a totally new experience for everyone, so it wasn't perfect and we learned a lot,” says Bromage.
To get more accurate data, he plans to include more cycles in his next study, during which he will measure blood in rhesus monkeys (they have a rhythm of four days) for a month. Macaques are accustomed to blood sampling, he added, meaning scientists will take blood samples from animals that do not experience stress-related problems like pigs.
Bromage noted that regardless of this, he identified a five-day rhythm in another type of molecules circulating in pigs' blood: small RNAs, and most of those with a five-day cycle also have a biological function related to growth. He doesn't think this discovery is a coincidence. "The chance that this could happen is astronomically small," he says.
Two day old rat
Blood tests are not the only way scientists can track biorhythms. Liu, from the University of Memphis, says that if he had money, he would be interested in determining the multi-day rhythm in a large animal using the daily reporter gene. These genes are triggered by the circadian rhythm and produce a molecule that biologists can measure with high precision in real time. The association of such a gene with the animal's hypothalamus may reveal that the circadian rhythm somehow varies over the multi-day schedule,”says Liu. "It's doable," he says, "and very interesting."
However, even if the rhythm of the metabolites is confirmed, Liu and other scientists say, this does not mean that he is responsible for body size. Rather, it may simply reflect different growth rates in animals of different sizes. As Liu explained, “just because you mark something in the blood that has rhythms doesn’t necessarily mean,” that is the reason.
Bromage agreed. "This is just a hypothesis," he said. "It can be experimentally tested." To do this, he wants to subject the grown cells, dividing once a day, to biological factors that could turn the circadian rhythm into a multi-day rhythm. Once that works, he says, scientists will see if they can turn a "whole rat into a two-day-old animal."
Andreas von Bubnoff