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Introduction
Retrorsine (12,18-DIHYDROXYSENECIONAN-11,16-DIONE) is a naturally pyrrolizidine alkaloid (PA), present in Senecio with origins in South African. PAs contain a branched C10 necic acid derived from the C5 carbon skeletons of two molecules of isoleucine or more uncommonly from leucine. Pyrrolizidine alkaloids are an important group of secondary metabolites characteristic of some angiosperm plant families. They are synthesised by many plants as a strategy of defense against herbivores and pathogens. It specifically inhibits the proliferation of hepatocytes coupled with induction of polyploidy and megalocytosis, and subsequently induces liver injury. It has been reported that the mediated bioactivation is necessary for the toxicity of PAs and that CYP34A4 is the major isoform involved in its metabolism. Together with CYP3A4, Organic cation transporter 1 (OCT 1) mediates the liver-specific uptake of retrorsine and plays an important role in retrotsine induced hepatotoxicity.

History
It has been known for a long time alkaloids compounds can be toxic. The first reported poisoning with Pyrrolizidine alkaloids in humans was in 1920, Western Cape of South Africa. Humans were diagnosed with liver cirrhosis after eating bread. The wheat was likely contaminated with Senecio burchelli, witch is family of Senecio retrorsum, the plant which produce retrorsine. Pyrrolizidine alkaloids can enter the human food chains by contamination of grain because they grow between the cultivated crops. Especially in countries with poor rainfall, plants which produce PA can develop very fast. The best known PA contamination was reported in Afghanistan in 1974. Approximately 35.000 were exposed to the toxic compounds resulting in many deads. One of the first times retrorsine was scientifically reported was by Davidson in 1935. . He injected rats with the toxic compound to research the severity. Despite it was clearly toxic, no exact values were determined.

Structure and Reactivity
Retrorsine belongs to the class of pyrrolizidine alkaloid (PAs) toxins. These naturally occurring secondary metabolites (phanerogams) represent a large collection of toxic alkaloids. Their chemical structure is based on a bicyclic tertiary amine the so called pyrrolizidine from which the name of this alkaloid toxin class is also derived. The heterocyclic ring structure of pyrrolizidine shows the typical characteristic of PAs due to the fact that it serves as precursor for retronecine during the biosynthesis of several different pyrrolizidine alkaloids. Retronecine is composed of necine and two hydroxyl (OH) moieties. Based on the esterification possibilities of both hydroxyl moieties, most of the PAs are derived from retronecine.

The toxicity of these toxic alkaloids (including retrorsine) originates from its incorporated 1,2 double bond (unsaturated) within the nicine moiety, which can make those compounds highly reactive. Due to the 1,2 double bond in the nicine residue, PAs are classified as hepatotoxic compounds that can crosslink cellular DNA, can disrupt hepatocyte division or can lead to liver cancer. In general the toxic effect of PAs depend on several biological and non-biological factors like: species, health status, general condition of organism, hormonal status, exposure time, volume of dose or enzyme (oxidases) induction. During biotransformation pyrrolizidine alkaloids can be converted to reactive pyrroles by oxidases. Those reactive pyrroles as well as their further catalyzed metabolites can react with nucleic acids (DNA) and proteins. On the other hand detoxification of reactive PAs will be catalyzed by liver esterases that break down ester bonds of PAs and generate a compound with necine and a necine acid moieties. Lack of detoxification can lead to liver damage, which can be indicated by abdominal pain, nausea, vomiting diarrhea and edema in animals and human.

Synthesis
The synthesis of the secondary metabolite retrorsine that is generated by Senecio retrorsum, is a two-step metabolic process (primary and secondary metabolic pathway). The toxicity of this pyrrolizidine alkaloid as well as their biological activity depends on the structural feature of the naturally occurring necine base, which is formed during those subsequent metabolic processes. The formation of retrorsine as well as intermediate metabolic products are described in step 1 to 9.

Primary metabolism
1. The synthesis of arginine, which is generated from its metabolic precursor ornithine is the first step in PAs synthesis. 2. Decarboxylation of arginine leads to agmatine that is further transformed into N-carbamoylputrescine. 3. Through elimination of the amido group from N-carbamoylputrescine, putrescine is formed. 4. Putrescine is further converted into two metabolites, spermine and spermidine. 5. Homospermidine, which formation is based on the metabolites spermidine and putrescine can be synthesized in two ways. Either the dimerization of putrescine or the combination of spermidine and putrescine. The combination of both metabolites is catalyzed by the NAD+ dependent enzyme homospermidine synthase (HSS). Homospermidine can be seen as the first biosynthetic precursor of the necine base residue of PAs including retrosine. Due to this SHH represents the link between primary and secondary metabolism.

Secondary metabolism
6. The reaction step leading from homospermidine to the necine base moiety has not yet been characterized at the enzymatic level. Most likely, the intermediate dialdehyde is formed by an enzyme with diamine oxidase activity. 7. During the cyclization of the carbon skeleton of the intermediate iminium ion, four stereoisomers of 1-hydroxymethylpyrrolizidine are metabolized, which are specific PAs biosynthetic precursors. It could be shown that the 1-hydroxymethylpyrrolizidine stereoisomer [5-3H]a is the direct retrorsine precursor and that it is incorporated in its formation. 8. For the biosynthesis of the di-acid residue from retrorsine it could be shown that this necic acid is derived from the α-amino acid L-isoleucine. Further it could be shown that L-isoleucine is the specific precursor of senecic acid and isatinecic acid. 9. Finally it could be proven by isolation and crystallization experiments that the pyrrolizidine alkaloid product retrorsine is composed of isatinecic acid and the necine base retronecine.

Use and mechanism of action
Retrorsine has been shown to induce a variety of acute chronic pathological lesions in the livers of animals like acute hepatocellular necrosis, inhibition of hepatocyte division and bile ductular proliferation In the past recent years it has been used to block proliferation of mature hepatocytes in order to stimulate liver repopulation by transplanted hepatocytes in various animal models. After a hepatectomy, small hepatocyte-like progenitor cells (SHPCs) seem to proliferate in retrorsine-exposed rats and give rise to expanding clusters of cells that eventually will repopulate the liver. Liver regeneration proceeds through a cellular response mediated by the expansion of the less differentiated SHPCs. Progenitor cells that proliferate have decreased expression of one or all CYP enzymes, so these cells can’t catalyze the bioactivation of retrorsine and therefore can escape the toxic effects of retrorsine. The mechanism by which hepatocytes are blocked in their proliferative state after retrorsine treatment is still under debate. However, the existing data suggests that the block is located very late in the mitotic cycle in late S or early G2 phase and that is due to various forms of DNA damage related to alkylation and/or crosslinking.

Metabolism and toxicity
Pyrrolizidine alkaloids can cause severe damage such as liver hepatic sinusoidal obstruction syndrome, cirrhosis, and cancer in both humans and animals. Humans are exposed to them through food intake. Retrorsine is a 12-membered macrocyclic diester pyrrolizidine alkaloid with an α, β – unsaturated double bond linked to the ester group at C-7 position of the retronecine base. The structural features that produce toxicity are the unsaturation site at the C1 and C2 positions in the pyrrolizidine ring, a branched acid moiety and some degree of esterification in the hydroxyl groups. However, pyrrolizidine alkaloids, including retrorsine, are not toxic per se, they need metabolic activation to form a pyrrolic metabolite in order to exert their toxicity. Metabolism of retrorsine leads to the formation of isatinecic acid, pyrrolic metabolites, retrorsine N-oxide, and retronecine. The three main metabolic pathways in vivo that have been elucidated are N-oxidation, hydrolysis and dehydrogenation to pyrrolic derivatives. The third pathway is the responsible for the toxicity, the pyrrolic metabolites produced are thought to play an important role in the carcinogenic and mutagenic effects of pyrrolizidine alkaloids in animals, since they can bind to macromolecules such as DNA and proteins causing abnormal functions of tissues. The bioactivation of PAs is mediated by the cytochrome P450 (CYP450) located in the liver, and therefore the liver is the main target of PAs induced toxicity and experiences the most injury. CYP3A4 is the major isoform involved in the metabolism of retrorsine and it primarily catalyses the dehydrogenation of pyrrolizidine alkaloids and leads to the formation of pyrrolizidine intermediates. The transporters located in the liver also play important roles in the disposition of xenobiotics. Retrorsine is a hydrophilic weak base, therefore is a substrate of organic cation transporters, specifically of OCT 1. Through passive diffusion and OCT1 retrorsine is taken up into hepatocytes then, is metabolized mainly by CYP3A4 to its reactive metabolites, and eventually exerts toxicity. A small part of retrorsine not metabolised could be excreted by P-glycoprotein transporter (P-gp). The formation of necic acids can take place by the hydrolysation by carboxylesterase or the formation of dehydroalkaloids followed by a reaction with water or cellular nucleophiles. The necic acid is cleaved by hydrolysis and gives rise to 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP). The formation of dehydropyrrolizidine intermediate is followed by the formation of pyrrolizinium ions, afterwards the nucleophilic substitution of glutathione (GSH) leads to 7-glutathionyl or 9-glutathionyl dihydropyrrolizidines, however the C-9 position is the preferred site in DHP for the substation by GSH. These reactive alkylating species are captured by liver cells and bound to proteins and DNA. The resulting protein- and/or DNA adducts cause liver damage as VOD/cirrhosis and/or tumors, respectively In the liver of rats treated with retrorsine DHP-derived DNA adducts formed indicate that the tumorigenicity of retrorsine takes place also through a genotoxic mechanism. .

In female pregnant individuals, the metabolite transport through the placenta into the fetus is an important fact to take into account. As told before, retrorsine requires metabolic activation by CYP450 3A to exhibit hepatotoxicity. The metabolites formed during the bioactivation are pyrrolic esters, which can permeate the placenta barrier and therefore enter into the fetal circulation. It was seen in rats that, metabolic activity of the mother’s liver to the placenta gets lower and therefore causes fetal growth retardation and placental and liver injury for the pregnant rat. The drug used as an antidote to prevent and cure a retrorsine overdose is Astaxanthin, which is an antioxidative substance which has anti-inflammatory, antitumor and free radical scavenging activity.

Indications
In plant material, PAs can be analysed with High-performance liquid chromatographic analysis.

Humans
Retrorsine is classified in group 3 according to the IARC (“The agent is not classifiable based on its carcinogenicity to humans”). No clear data is present of the toxicity to humans. Nevertheless it is known that a grain contamination in South Africa has caused serious problems. Contaminated grain ended up in bread. This lead to symptoms such as; rapidly developing ascites, abdominal pain and hepatomegaly.

Animals
Exposure of retrorsine to rats with 40 mg/kg body weight caused rapid inhibition of liver and serum protein synthesis. Data of animals more closely related to humans has been reported by scientist, who have done research on vervet monkeys. The following data was reported for a vervet monkey, LD50 of 46mg/kg body weight. In both animals centrilobular hemorrhagic necrosis occurred in the liver and central and hepatic veins, due to the toxic effects of retrorsine. The microscopic findings of the surviving vervet monkeys exposed to 46mg/kg showed; hepatocellular midzonal, central necrosis, bile stasis, mild bile duct proliferation, congestion and megalocytosis. Portal tract oedema was noticeable as well as portal vein wall distention and disruption. Similar change were noticeable in the large central vein. Monkeys administered to a retrorsine dose of 100mg/kg died after exposure.

According to Lewis (1996) :
 * LD50 mouse iv 59mg/kg
 * LD50 rat ip 34mg/kg (male), 153mg/kg (female)
 * LD50 rat iv 38 mg/kg

CYP 2B1/2, CYP2E1, and CYP 1A1/2 proteins were increased in rat liver microsomes after exposure to retrorsine. One or all of these isoforms are involved in the bioactivation of retrorsine. Chronic exposure of toxic pyrrolizidine alkaloids to laboratory animals induces cancer. Within a few hours, only a small part of the given dose is left in the body. The most of the metabolites are tissue bound. PAs disappeared from animal serum with half-lives of 3-20 minutes.