Almudena García

Myricetin, quercetin, (+)-catechin and (-)-epicatechin protect against N-nitrosamines-induced DNA damage in human hepatoma cells.

The aim of this study was to investigate the protective effect of myricetin, quercetin, (+)-catechin and (-)-epicatechin, against N-nitrosodibutylamine (NDBA) and N-nitrosopiperidine (NPIP)-induced DNA damage in human hepatoma cells (HepG2). DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or Comet assay. (+)-Catechin at the lowest concentration (10 microM) showed the maximum reduction of DNA strand breaks (23%), the formation of endonuclease III (Endo III, 19-21%) and formamidopyrimidine-DNA glycosylase (Fpg, 28-40%) sensitive sites induced by NDBA or NPIP. (-)-Epicatechin also decreased DNA strand breaks (10 microM, 20%) and the oxidized pyrimidines/purines (33-39%) induced by NDBA or NPIP, respectively. DNA strand breaks induced by NDBA or NPIP were weakly reduced by myricetin at the lowest concentration (0.1 microM, 10-19%, respectively). Myricetin also reduced the oxidized purines (0.1 microM, 17%) and pyrimidines (0.1 microM, 15%) induced by NDBA, but not the oxidized pyrimidines induced by NPIP. Quercetin did not protect against NDBA-induced DNA damage, but it reduced the formation of Endo III and Fpg sensitive sites induced by NPIP (0.1 microM, 17-20%, respectively). In conclusion, our results indicate that (+)-catechin and (-)-epicatechin at the concentrations tested protect human derived cells against oxidative DNA damage effects of NDBA and NPIP. However, myricetin at the concentrations tested only protects human cells against oxidative DNA damage induced by NDBA and quercetin against oxidative DNA damage induced by NPIP.

Protective effects of isothiocyanates towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay.

The aim of this study was to investigate the protective effect of isothiocyanates towards N‐nitrosamine‐induced DNA damage in the single‐cell gel electrophoresis (SCGE)/HepG2 assay. None of the isothiocyanates (ITCs) concentrations tested in the presence or absence of formamidopyrimidine‐DNA glycosylase (Fpg) caused DNA damage per se. Combined treatments of HepG2 cells with phenethyl isothiocyanate (PEITC), allyl isothiocyanate (AITC) or indol‐3‐carbinol (I3C) and N‐nitrosopyrrolidine (NPYR) or N‐nitrosodimethylamine (NDMA) reduced the genotoxic effects of the N‐nitrosamines in a dose‐dependent manner. The protective effect of the three ITCs tested was higher towards NPYR‐induced oxidative DNA damage than against NDMA. The greatest protective effect towards NPYR‐induced oxidative DNA damage was shown by I3C (1 µM, 79%) and by PEITC (1 µM, 67%) and I3C (1 µM, 61%) towards NDMA (in presence of Fpg enzyme). However, in absence of Fpg enzyme, AITC (1 µM, 72%) exerted the most drastic reduction towards NPYR‐induced oxidative DNA damage, and PEITC (1 µM, 55%) towards NDMA. Our results indicate that ITCs protect human‐derived cells against the DNA damaging effect of NPYR and NDMA, two carcinogenic compounds that occur in the environment. Copyright

Organosulfur compounds alone or in combination with vitamin C protect towards N-nitrosopiperidine- and N-nitrosodibutylamine-induced oxidative DNA damage in HepG2 cells.

The aim of this study was to investigate the protective effects of organosulfur compounds (OSCs) alone or in combination with vitamin C towards N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA)-induced oxidative DNA damage in the single cell gel electrophoresis (SCGE)/HepG2 assay. Diallyl sulfide (DAS) did not protect against NDBA-induced oxidized purines, but it reduced the oxidized purines induced by NPIP (1 microM, 29%). The formation of formamidopyridine-DNA glycosylase (Fpg) sensitive sites induced by NPIP or NDBA was prevented by dipropyl sulfide (DPS) at concentrations of 1-10 microM (55-24% and 66-15%, respectively). The maximum reduction of the formation of Fpg sensitive sites induced by NPIP was observed at the highest concentration of diallyl disulfide (DADS) (2.5 microM, 38%). However, the oxidative DNA damage induced by NDBA was strongly reduced by DADS at the lowest concentration tested (0.1 microM, 92%). The oxidative DNA damage induced by NPIP or NDBA was prevented by all the concentrations of dipropyl disulfide (DPDS) (0.1-2.5 microM, 59-80% and 51-64%, respectively). DADS and DPDS, in combination with vitamin C showed an overall protective effect towards the formation of Fpg sensitive sites induced by NPIP and NDBA. However, the contribution of OSCs to the protective effect found in combined experiments might not be relevant, because it could be caused by vitamin C alone. One feasible mechanism by which OSCs exert their protective effects towards N-nitrosamine-induced oxidative DNA damage could be by modulation of phase I and II enzyme activities. DADS and DPDS (0.1-2.5 microM) exerted greater inhibition on CYP2E1 and CYP2A6 activity than DAS and DPS (1-50 microM). However, DAS and DADS (1 microM) exerted greater inhibition on CYP1A1 activity than DPS and DPDS (1 microM). DAS/DPS (50 microM) and DADS (2.5 microM) exerted a moderate increase of UDP-glucuronyltransferase (UGT1A4) activity, whereas DPDS (2.5 microM) had the most pronounced effect.

Inhibition by vitamin C of apoptosis induced by N-nitrosamines in HepG2 and HL-60 cells

The aim of this study was to evaluate the effect of vitamin C towards N‐nitrosopyrrolidine (NPYR)‐ and N‐nitrosodimethylamine (NDMA)‐induced apoptosis in human hepatoma (HepG2) and leukemia (HL‐60) cell lines using flow cytometry analysis and the terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling assay (TUNEL). None of the vitamin C concentrations tested (1–100 µm) caused cytotoxicity in HepG2 cells. However, there were significant losses of HL‐60 cells viability, measured by MTT assay, 72 h after treatment with 50 and 100 µm vitamin C (29 and 46%, respectively). Moreover, an increase of lactate dehydrogenase release was significant with 50 µm at 72 h (28%) and with 100 µm of vitamin C at 48 and 72 h (27 and 36%, respectively). Also, the percentage of apoptotic HL‐60 cells found in TUNEL assay increased to 21% when they were treated with 100 µm vitamin C for 72 h. Thus, in subsequent simultaneous treatments with NPYR (30 and 50 mm) or NDMA (27 and 68 mm) and vitamin C, concentrations of 5–50 µm vitamin C were used. Our results revealed that vitamin C, at all concentrations and times tested, reduced the apoptosis induced by NPYR and NDMA in both cell lines, showing a similar effect in HepG2 and HL‐60 cells towards NPYR (50 mm) — 65 and 63% of reduction, respectively — whereas towards NDMA (27 mm) the inhibition was higher in HL‐60 than in HepG2 cells — 75 and 57%, respectively. Therefore, our findings suggest that inhibition of apoptosis may be one of the mechanisms by which vitamin C exerts its protective effect. Copyright

N-Nitrosopiperidine and N-Nitrosodibutylamine induce apoptosis in HepG2 cells via the caspase dependent pathway

The human hepatoma cell line (HepG2) exhibited a dose and time‐dependent apoptotic response following treatment with N‐Nitrosopiperidine (NPIP) and N‐Nitrosodibutylamine (NDBA), two recognized human carcinogens. Our results showed a significant apoptotic cell death (95%) after 24 h treatment with NDBA (3.5 mM), whereas it was necessary to use high doses of NPIP (45 mM) to obtain a similar percentage of apoptotic cells (86%). In addition, both extrinsic (caspase‐8) and intrinsic pathway (caspase‐9) could be implicated in the N‐Nitrosamines‐induced apoptosis. This study also addresses the role of reactive oxygen species (ROS) as intermediates for apoptosis signaling. A significant increase in ROS levels was observed after NPIP treatment, whereas NDBA did not induce ROS. However, N‐acetylcysteine (NAC) did not block NPIP‐induced apoptosis. All these findings suggest that NPIP and NDBA induce apoptosis in HepG2 cells via a pathway that involves caspases but not ROS.

N-nitrosopiperidina y N-nitrosodibutilamina (II): relevancia en la carcinógenesis química y genotoxicidad

La N-nitrosopiperidina (NPIP) y la N-nitrosodibutilamina (NDBA) han sido clasificadas como posibles carcinogenos en humanos. La NPIP causa tumores en esofago, y tambien en cavidad nasal, higado y estomago, mientras que la NDBA es carcinogeno de vejiga urinaria. Ambas N-nitrosaminas son consideradas carcinogenos genotoxicos indirectos puesto que necesitan una bioactivacion para generar metabolitos que reaccionen con el DNA. La principal lesion al DNA inducida por las N-nitrosaminas es el dano alquilativo. En el caso de la NDBA, la posicion de alquilacion es en el O6 de la guanina, formando la O6 butilguanina y la O6-4-hidroxibutilguanina. Sin embargo, esta N-nitrosamina alquila preferentemente proteinas. Por otra parte, la bioactivacion de la NPIP genera metabolitos que reaccionan con el N2 de la guanina in vitro, aunque se desconocen sus efectos in vivo. Ademas, durante la activacion metabolica pueden tambien producirse especies reactivas del oxigeno (EROs) y del nitrogeno (ERNs). Las lesiones oxidativas y nitrativas mas comunes son la 8- hidroxideoxiguanosina (8 OHdG) y la 8-nitroguanina, respectivamente, que producen mutaciones y conducen a la carcinogenesis.

N-nitrosopiperidina y N-nitrosodibutilamina (I): formación, exposición humana y metabolismo

Los vegetales, el agua de bebida y los productos carnicos son las principales fuentes de exposicion de nitratos en humanos. La toxicidad de estos compuestos es resultado de su conversion en nitritos que actuan como agentes nitrosantes en la formacion de las Nnitrosaminas. Las N-nitrosaminas son uno de los grupos de agentes carcinogenicos mas estudiados y existe una gran preocupacion debido a su presencia en nuestra vida diaria. Sin embargo, son muy escasos los estudios realizados con dos N-nitrosaminas, la Nnitrosopiperidina (NPIP) y la N-nitrosodibutilamina (NDBA). La NPIP se encuentra en productos carnicos que incluyan especias en la formulacion de sus mezclas y la NDBA en productos carnicos envasados en goma y en chupetes y tetinas para biberones. Ademas, la NPIP y la NDBA son compuestos genotoxicos indirectos que necesitan una activacion metabolica para danar el DNA y ejercer su efecto carcinogenico. Este proceso es llevado a cabo por el citocromo P450, en concreto, por las isoformas enzimaticas CYP 2A6 y el CYP 1A1, respectivamente.