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.
In vitro organ-cultured human HF studies
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.