The authors established a cell line from Incilius alvarius parotoid gland tissue and confirmed in vitro biosynthesis of 5‑MeO‑DMT by LC‑MS/MS. This provides a cruelty‑free, sustainable source of naturally derived toad secretions (and potentially accompanying entourage molecules) for research and drug development, potentially reducing pressure on wild populations.
There is growing interest in the therapeutic potential of 5-MeO-DMT (5-methoxy-N,N-dimethyltryptamine) for psychiatric disorders. While 5-MeO-DMT can be chemically synthesized, the parotoid gland secretions of Incilius alvarius (also known as the Colorado River or Sonoran Desert toad) contain 5-MeO-DMT and other molecules including bufotenine, bufagenins, bufotoxins, and indole alkylamines that may have individual clinical utility or act as entourage molecules to enhance the activity of 5-MeO-DMT. Incilius alvarius is currently under severe ecological pressure due to demand for natural 5-MeO-DMT and habitat loss. We established a cell line from tissue obtained by wedge biopsy of the Incilius alvarius parotoid gland and confirmed the cell-based biosynthesis of 5-MeO-DMT by LC-MS/MS. Cell-based biosynthesis of Incilius alvarius parotoid gland secretions is a potentially cruelty-free and sustainable source of naturally derived 5-MeO-DMT for research and drug development. Graphical Abstract
5-MeO-DMT (5-methoxy-N,N-dimethyltryptamine) is the principal psychoactive component of the parotoid gland secretion of Incilius alvarius, where it comprises an estimated 20-30% of the dry weight of the secretion. The compound acts primarily at serotonin receptors, with higher affinity for 5-HT1A than 5-HT2A, and is metabolised by CYP2D6 to bufotenine. Interest in serotonergic psychedelics has risen because preclinical and clinical work suggests they promote neuroplasticity and may have therapeutic potential for mood and anxiety disorders; 5-MeO-DMT is described as pharmacodynamically distinct from other psychedelics due to its rapid onset, short duration and particular transcriptomic effects. This study was undertaken against a backdrop of ecological concern: demand for natural toad secretion for recreational, self-medication and spiritual use places Incilius alvarius under pressure. Lerer and colleagues set out to establish a cell line derived from Incilius alvarius parotoid gland tissue and to determine whether those cells could synthesise 5-MeO-DMT in vitro, providing a potential cruelty-free and sustainable source of naturally derived 5-MeO-DMT and associated parotoid compounds (so-called entourage molecules).
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Parotoid gland tissue was obtained by partial gland biopsy from two anaesthetised Incilius alvarius toads following established anatomical descriptions and a modified version of a published full-thickness biopsy procedure. Glandular explants (~1 cm3) were rinsed with 70% isopropanol and phosphate-buffered saline and stored at ultra-low temperature until culture initiation; the procedure was reported to conform with best practices for handling laboratory animals. Cell isolation used enzymatic digestion with collagenase followed by an enzyme-free cell dissociation solution (ECDS). Dissociated material was strained through 0.40 μm and 0.22 μm filters and seeded into 24-well plates. Two surface treatments were compared: gelatin-coated wells and uncoated wells. Basal medium was adapted from a published recipe and comprised 2X L-15 medium with 10% fetal bovine serum and additional supplements (antibiotics, sugars, hormones, amino acids and ions). Cultures were incubated at 25°C with 5% CO2, with media changes every 3–4 days, and passaged on confluence. Cell immortalisation was performed using an SV40 T Antigen kit. Cultures were maintained for 45 days. For chemical analysis, culture media were filtered using an Amicon Ultra-0.5 centrifugal filter unit (10 kDa MWCO). 5-MeO-DMT quantification employed UPLC coupled to a Xevo TQ-S micro mass spectrometer. A calibration curve was prepared from a 5-MeO-DMT analytical standard in methanol over a concentration range of 1–2000 ng/mL. Monitored product/precursor ions included 130.25 m/z and 159.07 m/z. Media samples were analysed at two time points, 12 and 36 days after culture initiation, and compared to spontaneously secreted material collected from an Incilius alvarius toad.
Parotoid-derived cells were successfully isolated and immortalised, and cultures were sustained for 45 days. Dried material from the culture media had a light tan appearance similar to dried natural parotoid secretion. Media samples taken at 12 and 36 days were analysed and compared with spontaneously secreted toad material. LC-MS/MS analysis showed that the media samples produced by the cell cultures exhibited mass spectral fragmentation consistent with 5-MeO-DMT, matching the fragmentation pattern of the natural secreted material (the authors refer to a figure demonstrating this conformity). The chromatograms also contained additional peaks that the investigators did not identify; they report that the small amount of material limited further characterisation. The extracted text does not clearly report quantified concentrations or yields of 5-MeO-DMT from the cultures.
Lerer and colleagues interpret their findings as the first successful establishment of an Incilius alvarius parotoid cell line and as preliminary evidence that such cells can biosynthesise 5-MeO-DMT in vitro. They suggest that cell-based production could provide a scalable, cruelty-free source of naturally derived 5-MeO-DMT for research and potential clinical use while reducing ecological pressure on wild toad populations. The investigators emphasise the potential importance of additional indole alkylamines and other constituents present in parotoid secretions (for example bufothionine, serotonin, cinobufotenine, bufotenine and dehydrobufotenine), noting that these entourage molecules might synergistically modulate the effects of 5-MeO-DMT; however, they acknowledge that the interactions and mechanistic roles of such compounds remain unknown. The authors position their work as enabling further study of both 5-MeO-DMT and co-occurring secreted molecules. Key limitations are acknowledged: only small quantities were produced in this initial work, and with the detection methods used the team could not determine concentration or yield. Additional chromatographic peaks were observed but not identified because of limited material. The authors state that further research is required to optimise and scale the cell lines for upscaled production and to better characterise the full complement of secreted compounds and their biological effects.
This report presents preliminary evidence that an immortalised cell line derived from Incilius alvarius parotoid tissue can produce 5-MeO-DMT in vitro. Lerer and colleagues propose that cellular agriculture could offer a sustainable and cruelty-free alternative to harvesting toad secretions, and that the cell line may also permit investigation of other parotoid constituents (entourage molecules). The authors stress the need for optimisation to increase yield, more comprehensive chemical characterisation of additional peaks observed, and further work to clarify the biological roles and therapeutic potential of the secretion's components.
5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is the primary psychoactive component of the parotoid gland secretion ("venom") of Incilius alvarius, the Sonoran Desert or Colorado River toad, and accounts for 20-30% of the dry weight of the secretion 1 . 5-MeO-DMT primarily acts as an agonist at the 5-HT 1A and 5-HT 2A receptors, with a higher affinity for the 5-HT 1A subtype 2,3 . 5-MeO-DMT is O-demethylated by polymorphic cytochrome P450 2D6 (CYP2D6) to an active metabolite, bufotenine. Neuropsychiatric disorders, including mood and anxiety disorders, are some of the leading causes of disability worldwide and place an enormous economic burden on society. Serotonergic psychedelics are receiving increasing attention as novel therapeutics for depression and other psychiatric and neurological disorders. Preclinical and clinical evidence support neuroplasticity as the convergent, downstream mechanism of action of psychedelics. Through their primary glutamate or serotonin receptor targets, psychedelics including psilocybin, lysergic acid diethylamide (LSD), 5-MeO-DMT, and N, N-dimethyltryptamine (DMT) induce synaptic, structural, and functional changes, particularly in pyramidal neurons in the prefrontal cortex 11 . 5-MeO-DMT appears to be pharmacodynamically unique as compared to other psychedelics in terms of the intensity and rapid onset of action, short duration of effect, transcriptomic and other parameters 12 . An optimized chemical method for the synthesis of 5-MeO-DMT has been described 13 . However, volunteers consuming toad secretion containing 5-MeO-DMT experienced a 20-30% increase in the magnitude for subjective effects like "ego dissolution" and "altered states of consciousness" compared to the effects reported by volunteers who used synthetic 5-MeO-DMT
It is possible that the non-psychedelic molecules in the toad secretion, also known as the entourage molecules, may be responsible for the enhanced effect of toad-derived 5-MeO-DMT, but whether or how the entourage molecules interact with the psychedelic molecules, thereby modulating the psychoactive experience and the neuroplastic effect, is currently unknown. While 5-MeO-DMT is present in several entheogenic plants, it should be noted that due to the high concentrations in the parotoid secretions of Incilius alvarius, the Sonoran Desert toad is currently under severe ecological pressure due to the demand for recreational, self-medication, and spiritual use 16 .
Parotoid cells obtained from biopsies of anesthetized Incilius alvarius toads were successfully immortalized. Cultures were maintained for 45 days. Dried media from the cell culture had a light tan appearance that was similar to the known appearance of dried Incilius alvarius parotoid secretion. Well plate media samples were collected at 12 and 36 days from initiation and were analyzed for the presence of 5-MeO-DMT using LC-MS/MS and compared to spontaneously secreted material from an Incilius alvarius toad. MS fragmentation of the media samples and secreted material showed conformity in structure with 5-MeO-DMT (Figure). Additional chromatographic peaks were present but were not identified due to the paucity of the material. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
To our knowledge, this is the first report of the successful establishment of an Incilius alvarius parotoid cell line. These findings constitute preliminary evidence of the feasibility of cell-based 5-MeO-DMT production as a scalable source of research and clinical material. In addition to 5-MeO-DMT, toad secretions contain other indole alkylamines, such as bufothionine, serotonin, cinobufotenine, bufotenine, and dehydrobufotenine that could synergistically modulate the effects of 5-MeO-DMT with possible therapeutic applications. The fact that the immortalized cells, may in addition to 5-MeO-DMT, produce other compounds (entourage molecules) contained in the toad parotoid secretion, makes research into these additional components possible. While psychedelics may have therapeutic potential in mental health disorders, further research is needed to better understand their biological effects on the various receptors they bind to and their mechanisms of action, including the role of the entourage molecules as modulators of receptors and downstream signaling pathways. In this initial study, only small quantities were produced and it was not possible, with the detection methods used, to determine concentration or yield. Further research is required to optimize the cell lines for upscaled production. The potential availability of "natural" 5-MeO-DMT produced through cellular agriculture, as opposed to the cruel and was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which this version posted May 22, 2022. ;doi: bioRxiv preprint 8 destructive practice of "milking" Incilius alvarius, also supports efforts to ensure the protection of our planet's entheogen heritage.
Incilius alvarius parotoid anatomy and morphology were established according to O ́Donohoe et al.. A partial parotoid gland biopsy was undertaken under aseptic conditions in two anesthetized Incilius Alvarius toads in conformity with the current best practice for the handling of laboratory animals. A slightly modified procedure as described by Semoncelli et al. (2015) 19 was performed; full-thickness ventral skin was removed, and glandular tissue was cut with a scalpel into approximately 1 cm 3 sections. Explants were then rinsed with 70% isopropanol, and phosphatebuffered saline and stored in an ultra-cold freezer until culture initiation.
Basal media preparation described by Ellinger et al.was employed with modification and composed of 2X L-15 (GIBCO) with an addition of 10% fetal bovine serum. Media was then supplemented with antibiotics, sugars, hormones, amino acids, and ionic compounds. Two treatment groups were established; gelatin (Sigma-Aldrich, St Louis, MO, USA) was applied to two 24-well plates while no adherent was applied to two 24-well plates. Explants were subsequently thawed in a 65°C water bath, rinsed with amphibian Ringers solution prepared as described by Handler et al., resuspended in fresh media, and prepared for connective tissue was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which this version posted May 22, 2022. ;doi: bioRxiv preprint 9 digestion. Two treatments of cell isolators were used, collagenase was employed similarly to Hitoshi et al.followed by ECDS (enzyme-free cell dissociation solution). Upon completion, dissociated cells were strained via both 0.40 and 0.22 μm cell strainers. Isolated cells were aseptically seeded into 24-well plates and incubated at 25°C and 5% CO 2 . Upon confluence, cells were disassociated with the initial reagent used and passaged to additional well plates. Media exchange occurred approximately every 3-4 days and cell immortalization was achieved using the SV40 T Antigen Cell Immortalization Kit (Alstem, Richmond, CA, USA). Cell culture was undertaken in an incubator at 25℃ and 5% CO 2 . Upon confluence, the cells were disassociated and passaged with media exchange every 3-4 days. The culture was maintained for 45 days.
Before analysis, cell media was filtered with an Amicon Ultra-0.5 Centrifugal Filter Unit (MWCO 10 kDa, Millipore Sigma, Burlington, MA, USA). 5-MeO-DMT was quantified using a UPLC with Xevo TQ-S micro MS (Waters Corporation, Milford, MA, USA). The calibration curve was prepared using the 5-MeO-DMT analytical standard, in methanol, from Cerilliant Corporation (Round Rock, Texas, USA) using a concentration range of 1-2000 ng/mL. For quantification, monitored ions at 130.25 m/z and 159.07 m/z were used. The column used was a Phenomenex
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