Recombinant β glucuronidase

NNK, [¹³C₆]NNK, [CD₃]-O⁶-mG, and [4-CD₂,CD3]NNAL-O-Gluc were purchased from Toronto Research Chemicals (Toronto, ON). NNAL and [¹³C₆]NNAL were synthesized from NNK or [¹³C₆]NNK via sodium borohydride reduction.^{10 (link)} (±)-DHM and (±)-DHK were synthesized in house with a slight modification of a reported procedure.²⁵ AIN-G powdered diet was purchased from Harlan Teklad (Madison, WI). Recombinant β-glucuronidase was purchased from Sigma-Aldrich (St. Louis, MO). Ethoxyresorufin was purchased from Sigma-Aldrich. NADPH was purchased from RPI (Mount Prospect, IL). The qPCR primers were purchased from IDT (Coralville, IA), and the sequences are as follows: Gapdh, 5′-AACTTTGGCATTGTGGAAGG-3′ (sense) and 5′-ACA-CATTGGGGGTAGGAACA-3′ (antisense); Ahr, 5′-AGCCGGTGC-AGAAAACAGTAA-3′ (sense) and 5′-AGGCGGTCTAACTCTGT-GTTC-3′ (antisense); Cyplal, 5′-GGTTAACCATGACCGGGA-ACT-3′ (sense) and 5′-TGCCCAAACCAAAGAGAGTGA-3′ (antisense); Cyp1a2, 5′-TGGAGCTGGCTTTGACACAG-3′ (sense) and 5′-CGTTAGGCCATGTCACAAGTAGC-3′ (antisense). All other chemicals or solvents were purchased from either Fisher Scientific (Fairlawn, NJ) or Sigma-Aldrich, unless stated otherwise.

Cotinine, [¹³CD₃]Cotinine, NNAL, and [¹³C₆]NNAL (Figure 1), were procured from Toronto Research Chemicals (Ontario, Canada). Recombinant β-glucuronidase (catalog # G8295) was purchased from Sigma-Aldrich (Milwaukee, WI). True Taper® 96-well plates for sample processing and analysis were from Analytical Sales & Services (Pompton Plains, NJ) while silicone cap mats used to cover the 96-well plates were purchased from Phenomenex (Torrance, CA). Four hundred μL Isolute SLE+ diatomaceous earth solid-phase extraction 96-well plates were from Biotage (Charlotte, NC) while Oasis MCX 10 mg, 30 μm solid-phase extraction 96-well plates were from Waters (Milford, MA). A Cerex 96-well positive pressure processor (Chromtech, Apple Valley, MN) was utilized during sample processing. Dulbecco’s PBS was purchased from Invitrogen (Grand Island, NY) and the rest of the chemicals were obtained either from Fisher Scientific (Fairlawn, NJ), Sigma-Aldrich (Milwaukee, WI) or Alfa Aesar (WardHill, MA) and used without further purification unless otherwise noted. An Eppendorf multichannel pipettor was used during sample processing.