Handbook of Vitamin C Research

Vitamin C Protects from Oxidative DNA Damage… 93vitamin C towards N-Nitrosamines-induced apoptosis. Recent data indicate that exposure toN-Nitrosamines is related with excess occurrence of leukemia [55, 56]. Therefore, in additionto HepG2 cells, the leukemia cell line HL-60 has been also tested in our apoptosis studies[44, 46]. The human leukemia HL-60 cell line has proven to be a good model for studying theapoptotic process induced by chemicals in lymphoid organs [57] and to express relativelyhigh levels of enzymatic isoforms of cytochrome P450 [58].ChemicalsMaterial and MethodsN-Nitrosamines (Figure 3): N-Nitrosodimethylamine (NDMA), N-nitrosopyrrolidine(NPYR), N-Nitrosopiperidine (NPIP), N-Nitrosodibuthylamine (NDBA), vitamin C (Lascorbicacid), phenethyl isothiocyanate (PEITC), allyl isothiocyanate (AITC), indole-3-carbinol (I3C), diallyl disulfide (DADS), dipropyl sulfide (DPS), dipropyl disulfide (DPDS),dimethyl sulfoxide (DMSO), low melting point agarose (LMP), -nicotinamide adeninedinucleotide phosphate, glucose-6-phosphate dehydrogenase, D-glucose-6-phosphate, UDPglucuronicacid, p-nitrophenol, 4-nitrocatechol, coumarin, 7-hydroxycoumarin, 7-ethoxyresorufin, resorufin, 4-methylumbellipherone, 4-methylumbellipheryl- , D-glucuronide, diethyldithiocarbamate (DEDTC), tranylcypromine and -naftoflavone werepurchased from Sigma-Aldrich (Spain). Formamidopyrimidine-DNA glycosylase (Fpg) andEndonuclease III (Endo III) were obtained from Trevigen Inc. (Gaithersburg, MD).Magnesium chloride 6-hydrate, di-potassium hydrogen phosphate anhydrous, trichloroaceticacid solution 20% (w/v) and hydrogen peroxide (H 2 O 2 ) were obtained from Panreac. Tris(hydroxymethyl) amino methane was purchased from Qbiogene (Montreal, Quebec). 2´,7´dichlorodihydrofluoresceindiacetate (H 2 DCFDA) was obtained from Molecular Probes(Eugene, Oregon, USA). All other chemicals and solvents were of the highest gradecommercially available.N-Nitrosamines and vitamin C, PEITC, AITC, I3C, DPS, DADS and DPDS weredissolved in sterile DMSO (0.1%). The stock solutions were stored deep-frozen (−80 ◦C).Cell CultureHuman hepatocellular carcinoma cells (HepG2) and human leukemia cells (HL-60) wereobtained from the Biology Investigation Center Collection (BIC, Madrid, Spain). HepG2cells were cultured as a monolayer in Dulbecco‘s modified Eagle‘s medium and HL-60 cellswere maintained in RPMI 1640 medium. The media were supplemented with 10% v/v heatinactivatedfetal calf serum, 50 g/ml streptomycin, 50 UI/ml penicillin and 1% v/v L-glutamine. Culture medium and supplements required for the growth of the cell lines werepurchased from GIBCO Laboratories (Life Technologies Inc., Gaithersburg, MD, USA). Cellcultures were incubated at 37°C and 100% humidity in a 5% CO 2 atmosphere.