Vinblastine sulphate

Reference genotoxins (mitomycin C, M7949, vinblastine sulphate, V1377, and benzo(a)pyrene, B1760) were purchased from Sigma Aldrich (St Louis, MO). Phenformin HCl (PHR1573, Supelco), purchased from Sigma Aldrich (St Louis, MO) was used as non-genotoxic negative control. Test compound stock solutions were prepared in DMSO (D161802, Sigma Aldrich, St Louis, MO). For the 3D tissues, dilutions were made in complete culture media yielding ≤ 0.1% DMSO final concentration. Media containing 0.1% DMSO (i.e. without test article) were applied to tissues to serve as the vehicle control. All 3D tissues were treated apically every alternate day (from day 0–6) and daily (from day 7–9), while basolateral wells contain untreated culture media to support the tissue growth. TK6 cells were exposed for 24 h with the test materials prior to harvest. S9 liver fraction (11-402L, MolTox, Boone, NC) was added to TK6 cells 3 h prior to benzo(a)pyrene exposure to activate cellular metabolism (Cox et al. 2016 (link)).

Standards substances of vincristine sulfate and vinblastine sulphate used as an internal standard (IS) were obtained from Sigma Aldrich Corp. (St. Louis, MO, USA) and ChromaDex, Inc. (Irvine, CA, USA). The methanolic stock solutions were stored at 18 °C and brought to room temperature prior to use. LC-MS ultra-grade methanol was purchased from Honeywell LabReady™. Formic acid (98%, w/w) was purchased from VWR Inter-national LLC. Zinc sulfate heptahydrate (HPLC purity) from Sigma-Aldrich was applied. The water used in the entire analysis was prepared using a MilliQ water purification system from Millipore.

Paclitaxel, vinblastine sulphate, podophyllotoxin, colchicine, 5,5′‐dithiobis‐2‐nitrobenzoic acid (DTNB), sulforhodamine B (SRB), Hoechst 33342, guanosine 5′‐triphosphate (GTP), propidium iodide, EGTA, MgCl₂, piperazine‐N,N′‐bis (2‐ethanesulphonic acid) (PIPES), mouse monoclonal anti‐α‐tubulin IgG and FITC‐conjugated anti‐mouse IgG, fluorescein isothiocyanate isomer (FITC) and dimethylformamide were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Foetal bovine serum (FBS) and Alexa Fluor 568‐conjugated anti‐mouse IgG were purchased from Invitrogen (Thermo Scientific, Massachusetts, USA). Acridine orange (AO), hydroxysuccinimide, minimal essential medium (MEM), cell culture tested antibiotic solution, and phosphate‐buffered saline (PBS) were purchased from HiMedia (Mumbai, India). Dichloromethane, n‐hexane, hydroxylamine hydrochloride and triethylamine were purchased from Merck, India. 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride was purchased from SRL chemicals. All other reagents used in the study were of analytical grade.