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We examined the action of ricin and related protein abrin on hair follicle (HF) and re-growth of hair. Topical application of ricin in BALB/C and C57BL/6 mice after hair removal by waxing, resulted in sparse and delayed re growth of hair. Histopathological examination revealed hair follicle dystrophy and dose-dependent reduction in the number of hair follicles. Ricin-treated hair follicles showed immediate dystrophic catagen induction and were found to be dystrophic. The characteristic features of ricin-treated hair follicles resembled chemotherapy-induced hair follicle dystrophy such as presence of many ectopic melanin granules, follicular and hair shaft distortion, and irregular diameter of hair bulbs. During in vitro human hair follicle organ culture, ricin- and abrin-treated hair follicles showed inhibition of hair shaft elongation, premature HF regression, and hair follicle dystrophy. During preclinical toxicology studies, ricin was found to be safe for sub-acute dermal test for 90 days for a dose equivalent to the human dose of 160 μg/kg. Our novel observations propose ricin to be a promising candidate for inhibiting growth of hair follicle by inducing dystrophy without adversely affecting other skin structures.
Inhibition of unwanted hair growth is one of the major concerns in skincare, and a wide variety of approaches have been used toward this goal. We examined whether ribosome-inactivating protein (RIP) ricin and related abrin can be used to inhibit hair growth on topical application. Ricin, a well-known plant-derived protein, inactivates eukaryotic ribosomes by modifying two nucleoside residues, G4323 and A4324, in 28 S rRNA. Ricin and the other members of this toxin family are very efficient at cell killing. Ricin is known to activate ribotoxic stress-induced SAPK signaling pathways as well as p38. Moreover, ricin was shown to induce IGFBP-3 expression through the p38 signaling pathway leading to cytokine secretion. However, earlier reports indicated that amount of ricin absorbed through skin is so negligible that it is considered to be dermally inactive, as no dermal toxicity was observed at 50 μg/spot during skin tests on mice. Therefore, toxicity by this route is suggested to be unachievable by Schep et al.. Earlier studies using crude ricinus extracts and purified ricin had indicated that ricin inhibits hair growth when applied topically.
All mature hair follicles show a cyclic pattern consisting of phases of growth (anagen), regression (catagen), rest (telogen), and shedding (exogen). The anagen hair follicle consists of a so-called permanent portion at the bulge region above the muscle insertion and a cycling portion below. Though ricin and related cytotoxic lectin abrin were considered to be dermally inactive, the observations of the present study suggest that they have potential to induce hair follicle dystrophy resulting into reduced rate and altered character of hair regrowth, keeping all other skin structures intact. Present study corroborates earlier findings that cytotoxic lectins like ricin are not completely dermally inactive as perceived but instead inhibit the growth of hair follicle, keeping all other skin structures intact. The present findings provide a rationale for further investigations using hair follicle as a dynamic regenerative mini-organ with a cyclic growth pattern, to determine the dermal role of ricin and related proteins, as a model for programmed organ deletion. Phase I clinical trials of ricin and abrin have been reported earlier by intravenous route.
40 percent adult female population suffers from some degree of unwanted facial hair along its psychological and psychosocial impact. Therefore, non-invasive topical treatment for inhibition of unwanted hair growth is one of the major concerns in skincare. This is an effort to explore the therapeutic potential of ricin, following the footsteps of clinical success of botulinum toxin.
Purification of ricin
Ricin was purified by affinity chromatography (Fig. 1A), and purity of pooled ricin fractions was assessed by SDS-PAGE (Fig. 1B). LC-MS of aqueous extract of castor bean showed ricin peaks at 6.881 min (Fig. 1C). Deconvolution of LC-MS spectrum estimated a molecular weight of 64,831.42 Da for ricin (Fig. 1D).
Pilot studies to assess topical effect of ricin and abrin
When compared to control patches (Fig. 2A), hair regrowth on all the test patches of BALB/C mice was delayed and sparse (Fig. 2B). The untreated hair follicles show normal ladder-like structure (Fig. 2C). The difference in terms of inhibition of hair regrowth and hair follicle dystrophy was visible in histological picture as early as 10 days after the topical application of ricin (Fig. 2D). Histopathology sections studied from all the biopsies showed no adverse reaction to other skin structures. All the ricin-treated sections after 30 days of treatment showed dose-dependent reduction in number of hair follicles as compared to the control (Fig. 2E-H). Some of the hair follicles from the test samples appeared empty, whereas some of them showed hair follicle dystrophy wherein the normal ladder-like structure of the hair shaft was disturbed. Similarly, as compared to control sections (Fig. 2I) abrin-treated sections show less number of HFs, and HF dystrophy (Fig. 2J).
Assessment of hair follicle dystrophy caused by ricin in mice
Effect of topical treatment of ricin was assessed in C57BL/6 female mouse model, which is a standard mouse model for hair growth studies (Fig. 3A). Difference between the dystrophy scores of normal healthy anagen hair follicles in control areas and the dystrophic anagen or dystrophic catagen hair follicles in the test areas in each animal persisted throughout the study period of 20 days (p <0.0032**) (n=60 C57BL/6 mice) (Fig. 3B). The HF growth cycle appeared to be shortened with early recovery followed by dystrophic hair follicles. In response to topical treatment of ricin for 10 days after inducing anagen by depilation, instead of entering a new growth cycle with a healthy anagen as compared to control section (Fig. 3C(a)), the ricin-treated HFs immediately responded by showing characteristic features of dystrophy on day 5 (Fig. 3C(k)). All the HFs in test groups at each time point showed various stages of dystrophic HFs with half of the HFs being in early dystrophic anagen VI. At each time point, the HFs of the control area of the same animal showed healthy stage of HF growth cycle (Fig. 3CA(a-j)). The ricin-treated HFs showed classical features of chemotherapy induced HF dystrophysuch as number of ectopic melanin granules, follicular and hair shaft distortion; and irregular diameter of hair bulbs (Suppl. Fig. 2). The point should be noted that these variations in HF growth stage and presence of HF dystrophy are observed in untreated and ricin-treated areas of the same animal and localized effect of HF dystrophy was achieved.
In vitro organ-cultured human HF studies
During the examination of cultured human HFs under a light microscope, HFs were assessed for changes in hair shaft elongation and classified according to classical parameters for hair cycle stage as described by Müller-Röver and Klopper. Parameters for this classification of hair follicle cycle stage were shape and size of hair matrix (HM) and dermal papilla (DP). The human HFs in vitro responded to treatment of ricin in line with our findings in animal studies. The hair shaft elongation was significantly less in both groups of ricin- and abrin-treated HFs with both the dose levels when compared to control group. (Mann-Whitney test for unpaired samples: ricin (R40- p <0.0001***, R100- p <0.0027**) and abrin (AB40- p <0.0003***, AB100- p <0.0001***)) (Fig. 4A). Ricin-treated HFs showed classic parameters of dystrophic catagen induction whereas the hair follicles in control group were healthy. Similarly, HFs treated with abrin too showed dystrophic catagen induction. Eflornithine-treated HFs are assessed for morphological parameters and compared with ricin and abrin-treated HFs. Eflornithine-treated HFs showed catagen induction. This observation was confirmed with DAPI staining for presence of less number of DAPI+ cells below Auber’s line (an imaginary line that separates the HF bulb at widest part of dermal papilla, the region below this line contains pluripotent stem cells with high mitotic activity and is the germination region) in ricin and abrin-treated group as compared to control and eflornithine group (Fig. 4B).Effect of topical ricin and abrin treatment in the form of dystrophy and reduced number of DAPI+ cells in this germination region indicates a possible mechanism of action on pluripotent stem cells.
Preclinical toxicological studies of ricin and abrin
Acute oral toxicity test of ricin (200 μg/mL) solution were found to be safe at 2 mg/kg body weight. No clinical signs of intoxication were observed in any animal till the end of the study and no mortality observed. Acute dermal toxicity test (14 days) with ricin (200 μg/mL) was found to be safe at 2 mg/kg body weight. All the animals appeared normal and showed no clinical signs of intoxication. No change in food consumption was observed. Subacute dermal toxicity test (90 days) with ricin (200 μg/mL) and abrin (200 μg/mL) was found to be safe (Fig. 5). All the animals appeared normal and showed no clinical signs of intoxication. No change in food consumption was observed. No significant difference in hematology and blood chemistry parameters was observed in the test group as compared to the control group (Suppl. Fig. 1). The histopathology of liver, kidney, and heart did not show any toxicity (Suppl. Fig. 1). Ricin was found to be safe for sub-acute dermal test for 90 days for a dose equivalent to human dose of 160 μg/kg each.
It has been reported earlier that no dermal toxicity was observed during skin tests on mice at 50 μg/spot. Though ricin and related proteins were widely considered to be dermally inactive earlier, our observations indicate the dermal role of ricin and abrin in HF dystrophy at comparatively very low dose levels, resulting in hair growth inhibition. Our observations during in vivo animal studies and in vitro human HF organ culture studies indicate HF dystrophy induction by ricin. Observations during preclinical toxicology studies indicate that ricin by topical route of administration at given dose level was found to be safe for use without adversely affecting any other skin structures.
Repeated induction of dystrophy may result in reduction of the regrowth of unwanted HFs. Hence, we propose ricin to be a promising candidate for inhibiting growth of unwanted hair follicles.
We propose that common molecular players involved in ricin-induced cell death and those involved in growth modulation of hair follicle, such as IGF-1, IGFBP-3, Lhx2, LIM/CLIM,and Wnt may be involved in ricin-induced hair follicle dystrophy.
Current hair removal methods such as depilatory creams, plucking, shaving and waxing of facial and body hair have numerous adverse reactions and neither of these hair removal methods have any effect on hair growth. Our findings suggest topical ricin formulations have potential to reduce the rate and character of hair growth, offering a long-lasting relief to the user.
The scientific findings of this study present a possibility of creating a new treatment for unwanted hair, an unmet need in cosmetic industry.
So far, ricin was considered to be a toxic waste product of castor oil industry with no therapeutic value. Moreover, in spite of numerous reports and articles denying it having any such potential to be weaponized, it is perceived to be a weapon of mass destruction, labeled as an ugly duckling and classified as Category B select agent. With this new-found therapeutic potential by dermal route, this ugly duckling has matured and begun to look a little more like a swan.
These are preliminary observations showing the therapeutic potential of ricin and related proteins. The exact role of ricin on the molecular mechanisms of hair follicle is not known; however, the present findings provide a preliminary basis to investigate further the molecular mechanisms involved in HF dystrophy induced by ricin and related protein abrin. To propose a systematic molecular model of damage response of the hair follicle to topical treatment, extensive studies with a sufficiently large sample size is needed.
Assessment of apoptotic vs. cytotoxic markers in the hair follicle culture experiments would have been more insightful.
Dose-response analysis over a wide dose range is required to arrive at an optimum dose range of ricin.
Further studies are planned to explore the clinical applicability of ricin and related proteins in hirsutism treatment.
All chemicals used in these studies were of analytical grade.
Purification of ricin and abrin
Ricinus communis and Abrus precatorious beans were purchased from local market in Pune, India. To ascertain that the crude castor bean extract has sufficiently higher concentration of ricin for purification, the aqueous extract (1 mg of bean powder/mL) was subjected to LC-MS analysis and deconvolution data analysis (Agilant 6450 Accurate mass QTOF MS, Agilent Bioconfirm software). Ricin and abrin were purified by affinity chromatography essentially following a combination of the protocol for the guar gum gel matrix and that of eluting the column using galactose gradient. Castor beans were delipidated by grinding in equal volumes of petroleum ether and centrifuged at 3000 × g for 10 min. The supernatant was discarded and the pellet was resuspended and again grounded in equal volume of ether. This procedure was repeated 4 to 5 times. The final pellet was air-dried. Delipidated castor bean pellet was dissolved in saline (0.9 N NaCl, pH 7.2). The crude ricin preparation was further diluted with saline 0.9 N NaCl as 1:10 and filtered and stored at -20°C till use. Under these conditions (galactose residues available on the partially acid-hydrolyzed matrix), ricin binds to the gel matrix. The gel matrix-bound proteins were eluted with beta-D galactose gradient. The ricin fractions eluted here were pooled (fractions 16–62, Fig. 1A) and extensively dialyzed against 0.9 N NaCl, and used for experiments as ricin. Abrin was purified in the same manner. Abrus precatorious seeds were soaked and homogenized with ten times volume of 5% acetic acid, pH was neutralized and the centrifuged extract was loaded onto cross- linked guar gum. Under these conditions, abrin binds to the gel matrix. The gel matrix bound proteins were eluted with beta-D galactose gradient. The abrin fractions eluted here were pooled, extensively dialyzed against 0.9 N NaCl and used for experiments as abrin. SDS-PAGE (Fig. 1B), using standard molecular weight marker and BSA as a standard, was performed to assess the purity of both the proteins. Ricin and abrin were loaded (2 μg protein/20 mL loading buffer each) on to gel. Ricin and abrin concentrations were estimated by Folin Lowry method and UV spectrometry at 280 nm.
Pilot studies of hair growth inhibition by topical ricin and abrin
Ricin of varying concentrations (2, 20 and 200 μg/mL) was applied (0.2 mL) (effective dose being 0.4, 4 and 40 μg, respectively), and rubbed on skin patches prepared by removing hair by waxing of BALB/C mice. Skin biopsies and histopathology (hematoxylin-eosin staining) were done after 10 days of treatment for the first group and after 30 days for the second group (6 animals in each group).
Assessment of hair follicle dystrophy by topical ricin in mice
This study was designed to assess the response of hair follicles to the damage induced by topical treatment of ricin according to the animal model of Hendrix et al. with some variations. This protocol was modified for topical ricin application (Fig. 2) in which control and test patches were made by waxing on the same animal to reduce error, due to animal-to-animal variation in hair growth cycle. Assessment of hair follicles in distinct hair cycle stages was done utilizing essential basic criteria according to Müller-Röver and Hendrix et al.. Hair follicle dystrophy score (Fig. 3) was calculated. For calculating “dystrophy score,” every stage of dystrophic anagen or catagen is assigned a factor in ascending numerical order: healthy anagen = factor 0, early dystrophic anagen = factor 1, mid-dystrophic anagen = factor 2, late dystrophic anagen = factor 3, early dystrophic catagen = factor 4, mid-dystrophic catagen = factor 5, late dystrophic catagen = factor 6, dystrophic telogen = factor 7. The number of hair follicles in each specific stage is multiplied by the corresponding factor. The results of each sum are totaled and divided by the overall number of hair follicles counted. This gives a final value between 0 and 7, thus defining the average stage of all hair follicles within the entire group according to Hendrix.
Thirty C57BL/6 female mice (6–8 weeks old) were procured from National Toxicology Centre, Pune. For each mouse, two patches were made on the dorsal skin by waxing on day 0. Ricin (200 μg/mL) 0.1 mL was applied and rubbed in from day 1 for 10 days to one patch marked as Test, whereas the other untreated patch served as control. 3 animals were euthanized and skin harvested for serial biopsies (10×3 = 30 animals) at each time point (day 5, 9, 11, 12, 13, 14, 15, 16, 18, 20) and processed for histopathology using standardized longitudinal sections of hair follicles based on harvesting and embedding technique described by van der Veen et al.. The slides were stained with hematoxylin-eosin (Fig. 3C). Various criteria such as morphological assessment of hair follicle matrix and the dermal papilla, i.e. number of DAPI+ cells, DP stalk fibroblasts, and calculation of “dystrophy score” were used to distinguish normal healthy anagen VI, dystrophic anagen, early catagen and dystrophic catagen hair follicles.
Occipital non-inflamed human scalp skin was obtained from a female volunteer after informed consent form. Anagen VI hair follicles were isolated according to the Philpot method and Helsinki Guidelines. Isolated human hair follicles were maintained with culture medium in 24 well plates according to Kloepper et al.. Isolated HFs were maintained in 500 ll serum-free Williams E medium (Sigma-Aldrich) in a 24 well plate supplemented with 10 lg⁄mL insulin (Sigma-Aldrich), 2 mmol⁄l l-glutamine (Sigma-Aldrich), 10 ng⁄mL hydrocortison (Sigma-Aldrich), and 1% antibiotic⁄antimycotic mixture. All the HFs were checked daily. Anagen hair follicles and hair follicles that have already entered catagen were fixed with 10% formaldehyde and processed for hematoxylin-eosin and DAPI staining. Test groups received ricin, abrin, and eflornithine as per the concentrations given, and the control HFs were treated daily with the same amount of water instead. (Hair follicles were divided into 7 groups with 1 control group (distilled water); eflornithine (Calbiochem) (two doses: 400 and 1,000 μg/mL); ricin (two doses: 40 and 100 μg/mL); and Abrin (two doses: 40 and 100 μg/mL)). Each group contains around 12–24 hair follicles. Untreated hair follicles were maintained as control group and eflornithine was used as positive control. Hair follicles were maintained for 6 days. Hair follicles were divided into 7 groups (according to Table 1) with 12–24 hair follicles per group. As all the hair follicles in the test group entered catagen on day 5, all the follicles were harvested. Eflornithine-treated HFs (n = 12) were assessed only for qualitative morphological parameters. Morphological and hair length parameters were analyzed by two independent researchers. Data was analyzed using Mann-Whitney test (two-tailed) for unpaired samples.
Preclinical toxicological studies of ricin
Acute oral, acute dermal, and sub-acute dermal toxicity testing of ricin was done. For acute oral toxicity test of ricin, 12 female Wistar rats were orally administered a single dose of 2 mg/kg body weight of suspension containing ricin (200 μg/mL). (For example, a rat with 225 gm body weight received 2.25 mL suspension containing 450 μg toxin.) The treated animals were observed for 14 days for mortality, clinical signs, and symptoms. For acute dermal toxicity test of ricin, 40 Wistar rats were used. Twenty female Wistar rats were topically administered a single dose of 2 mg/kg body weight of suspension containing ricin (200 μg/mL). (For example, a rat with 225 gm body weight received 2.25 mL suspension containing 450 μg toxin.) The treated animals were observed for 14 days for mortality, clinical signs, and symptoms. For sub-acute repeated dose dermal toxicity test of ricin, 5 male and 5 female rats were used for each group (total 40 animals), and ricin (200 μg/mL) suspension was applied over not less than 10% of the body surface area respectively. The treated animals were observed for 28 days for mortality, clinical, signs and symptoms. Weekly body weight and food consumption data were monitored. At the end of 28 days, blood was withdrawn to perform hematological and biochemical parameters. Histopathology of liver, kidney, and heart were performed with the standard procedure.
This study was self-funded by first author.
National Toxicology Center, Pune staff; Dr. Avinash Pradhan (Histopathologist-KEM Hospital, Pune); Maharashtra Medical Research Society, Pune and Dr. Dhanashree Bhide and Dr. Sharad Mutalik (Joshi Hospital, Pune); Dr. Ghaskadabi’s lab (Agharkar Research Institute, Pune), Dr. Ajeet Singh (CAMS, Venture Center, Pune).
All the animal experiments were approved by institutional animal ethics committee according to CPCSEA guidelines. The protocols to purify ricin and abrin were approved by Institutional Bio-Safety Committee according to (Department of Biotechnology, Government of India) guidelines. The in vitro human HF organ culture protocol was approved by Institutional Ethics Committee of Maharashtra Medical Research Society, Pune (India).