Pyrrolizidine Alkaloids

 

Crotalaria spectabilis

Plants are not sitting ducks to humans and herbivores, and many of them produce toxins such as alkaloids to protect themselves. We have previously seen alkaloids that target the animal nervous system, and now we are exploring alkaloids that poison the DNA. In the last article, we have examined the alkaloid aristolochic acid, which is extremely cancer-causing, but thankfully only restricted to plants in the family Aristolochiaceae. This time, we are going to explore a huge group (>400 types) of cancer-causing, liver damaging plant toxins known as the pyrrolizidine alkaloids. The pyrrolizidine alkaloids are diverse, and they have independently evolved in unrelated plants families, including the daisy (Asteraceae), borage (Boraginaceae), dogbane (Apocynaceae), legume (Fabaceae), and even orchid (Orchidaceae). It is estimated that 3% of all plant species may contain pyrrolizidine alkaloids. While not all pyrrolizidine alkaloids are equally toxic, scientists believe that they function as anti-herbivorous toxins. To date, pyrrolizidine alkaloids are some of the most damaging plant toxins known to humans and the animal farming industry. Numerous outbreaks and loss of grazing livestock are attributed to pyrrolizidine alkaloid containing plants such as the ragwort (Senecio jacobaea), rattlepod (Crotalaria spp.) and Heliotrope (Heliotropium spp.). Pyrrolizidine alkaloid poisoning can be acute, chronic and cumulative. In acute poisoning, which occurs when a huge dose is consumed, victims rapidly develop massive liver damage, which is fatal within a few days. In chronic poisoning, victims exhibit enlarged liver, obstruction of hepatic vein leading to ascites, permanent scarring of the liver, development of liver cancer, lung hypertension (monocrotaline specific), often resulting in death. In parts of the world, pyrrolizidine alkaloid poisoning of cattle is known by many names, such as the Pictou disease and Winton disease. Humans are not safe from pyrrolizidine alkaloids either. Massive outbreaks of poisoning still occur recently in Western Afghanistan. The casualty is estimated to be as high as 8000, with 1600 deaths attributed to the seeds of Heliotropium popovii alone. 

 

Figure 1: Chemical structure of pyrrolizidine alkaloid.

Chemically speaking, pyrrolizidine alkaloids all contain this distinctive pyrrolizidine backbone, which is comprised of two pentagonal carbon-nitrogen rings (called pyrrolidine) merged back-to-back with a nitrogen atom at the center. Look carefully at the numbering of a pyrrolizidine ring in Figure 1, the carbon number 1,2,7 and 9, and nitrogen number 4 will be very important. Pyrrolizidine alkaloids can exist naturally in its free-base form, or as an N-oxide. An alkaloid N-oxide has a permanent positive charge on its nitrogen atom, and a negative charge its oxygen atom. There is no electron pair on the nitrogen atom of an alkaloid N-oxide, hence rendering it non-alkaline, and non-toxic. A simple pyrrolizidine backbone with a hydroxyl group at carbon-9 is called a necine base, and it is the precursor to all pyrrolizidine alkaloids. Take note of carbon-1 and carbon-2 of a necine base. If there is a double bond between carbon-1 and carbon-2, it’s called an unsaturated necine base, and it is this double bond that makes a pyrrolizidine alkaloid very toxic. In contrast, saturated necine base (without double bond at C-1 and C-2) are less poisonous, if not completely harmless. A full pyrrolizidine alkaloid is actually an ester between a necine base and an organic acid, which is usually a dicarboxylic acid. In other words, the necine base is esterified at carbon-9 and/or carbon-7 by a single organic acid containing one or two carboxylic acid functional groups. The full chemical structures of two of the most toxic pyrrolizidine alkaloids, monocrotaline and senecionine are shown in Figure 1, along with the 3D structure of monocrotaline.

 

Figure 2: Biosynthesis of pyrrolizidine alkaloids.

Plants make pyrrolizidine alkaloids from the simple amino acid L-ornithine. Examine Figure 2 carefully. L-ornithine can be converted by a decarboxylation step (removal of carboxylic acid group by forming carbon dioxide) into a diamine (diamine means a compound with two amine groups) called putrescine. Putrescine is so named because it is often generated when proteins and amino acids like ornithine putrefy, or decompose. Then, a special enzyme called homospermidine synthetase (HSS) condenses two molecules of putrescine into homospermidine, which is a symmetrical triamine. I’ve bolded the structure of homospermidine in Figure 2 and numbered it in a way that you can see the blueprint of a pyrrolizidine skeleton. In fact, all we need to do is to form two covalent bonds in homospermidine to obtain a pyrrolizidine ring. It is worth mentioning that scientists have sequenced the HSS gene from different pyrrolizidine alkaloid producing plants, and different plant families convergently evolved HSS from a common precursor gene. It is remarkable that unrelated plants have converged onto a same toxin producing strategy over aeons of evolution. Next, aminocarbon-8 of homopermidine is oxidised by enzyme into an aldehyde group. The electron pair of nitrogen-4 then attacks the aldehyde, forming an intermediate compound called a Schiff base, which bears the crucial chemical bond between carbon-8 and nitrogen-4. Note that the Schiff base carries a positive charge on its nitrogen atom, and that makes carbon-8 electrophilic. In other words, carbon-8 tends to accept electrons (from nucleophiles) to neutralise the positive charge at the adjacent nitrogen-4. We have thus completed one pyrrolidine ring, one more to go. Similar to carbon-8, aminocarbon 9 is also converted by enzyme into an aldehyde group. This allows the adjacent carbon-1 to become an enolate anion, which is a nucleophile. The nucleophile C-1 then attacks the electrophilic carbon 8, neutralising the positively charged nitrogen-4. Most importantly, a pyrrolizidine necine skeleton is generated. The necine molecule then undergoes oxidation at carbon 1, 2, and 7 to furnish its 1,2-unsaturated double bond, as well as an alcohol group at carbon-7. The aldehyde carbon-9 is concomitantly reduced into an alcohol. That gives retronecine, which is the parent of very toxic pyrrolizidine alkaloids. The final biosynthesis step is to esterify the alcohol groups at carbon 7 and 9 with a carboxylic acid. Different carboxylate esters define different pyrrolizidine alkaloids. 

 

Figure 3: Pyrrolizidine alkaloid toxicosis, mechanism of action.

  

In analogy to aristolochic acid, the pyrrolizidine alkaloid itself is actually non-toxic. It is the animal liver that transforms pyrrolizidine alkaloids into highly reactive chemical species that damage the DNA, and in this case the DNA or proteins of the liver, where toxic metabolites are generated in the first place. Think about it, this is also a way plants prevent their own toxins from damaging themselves. These DNA damaging toxins are like a grenade, plant allow their enemies who consume them to pull the trigger, smart! Examine Figure 3 carefully. Enzymes in the liver first convert the pyrrolizidine alkaloids into a highly reactive metabolite called dihydropyrrolizine. Note that the double bond at carbon 1 and 2 facilitates this reaction by promoting the loss of leaving groups, which in turn generates two double bonds at carbon 2–3 and carbon 1–8, respectively. Without a double bond at carbon 1 and 2, the dihydropyrrolizine metabolite will not be formed, hence the lack of toxicity in saturated necine bases. Another group of liver enzymes called esterases then remove the ester component of the pyrrolizidine alkaloid, transforming carbon-7 and carbon-9 back into alcohol groups. Owing to the two double bonds in the dihydropyrrolizine moiety, the electron pair of nitrogen can resonate between carbon 1, 2, 3, 7, 8, and 9, and result in the loss of the alcohol groups at carbon 7 and carbon 9 as water. Whenever that happens, the nitrogen itself becomes positively charged, while carbon 7 and carbon 9 become strongly electrophilic. Any nucleophiles such as DNA and negatively protein will attack carbon 7 and 9 of the dihydropyrrolizine metabolite, forming a permanent adduct with it. One molecule of pyrrolizidine alkaloid can potentially ‘capture’ two molecules of DNA bases or proteins. Similar to aristolochic acid, human serum dihydropyrrolizine-protein adduct can be used as a reliable biomarker of pyrrolizidine alkaloid exposure.The resulting DNA damage can cause cells to undergo programmed cell death, or necrosis, if not becoming cancerous. The liver cells are most affected, but in some instances like monocrotaline toxicosis, even the blood vessels of the lungs are damaged. Monocrotaline is currently used in biomedical research to induce pulmonary hypertension (high blood pressure of the lung) in animal models. In the event of pyrrolizidine alkaloid toxicosis, the hepatic veins  that drain the liver of deoxygenated blood become inflamed and obstructed (Budd-Chiari syndrome and/or hepatic veno-occlusive disease). This obstruction (blockade) causes blood to back up in the liver, resulting in an increased blood pressure, a condition called portal hypertension. The liver itself becomes enlarged, while liver cells turn fibrous or die due to a lack of oxygenation (cirrhosis). Besides, portal hypertension forces liquid (water) into the peritoneum, causing the abdomen to swell (ascites), which is a sign of serious liver disease. Uncontrolled portal hypertension can even swell the veins of the esophagus to the point where they rupture, the prognosis of which is dire. Eventually, liver failure occurs and the victim can die of severe gastrointestinal bleeding, or brain damage (hepatic encephalopathy) due to toxins like ammonia in the blood. Without a liver transplant, chronic and severe pyrrolizidine alkaloid toxicosis is invariably fatal.  It is worth remembering that pyrrolizidine alkaloid poisoning is chronic and somewhat cumulative, as it can happen over months or even years of exposure. However, low levels of pyrrolizidine alkaloid (less than a few micrograms) can be detoxified by the human/ animal body, by converting it into the N-oxide or conjugate the electrophilic carbon 7 and 9 with groups other than important DNA or proteins. The availability of an electron pair in the pyrrolizidine nitrogen atom is crucial to its toxicity. Rendering the electron pair unavailable detoxifies pyrrolizidine alkaloids.

Nonetheless, pyrrolizidine alkaloids remain one of the most hazardous plant toxins known to humans because it is extremely widespread and insidious.  DNA damaging toxins are not just restricted to alkaloids of the flowering plants. In my next article, we will explore an ancient toxin that is just as reactive, and it hails from an ancient lineage of non-flowering plants.