Acrylamide is an industrially produced chemical with known neurotoxic, reproductive toxin and carcinogenic effects. The carcinogenicity associated with acrylamide is mostly attributed to its metabolism by liver CYP2E1. However, studies investigating the effects of acrylamide on CYP2E1 enzyme are limited. In this study, it was aimed to investigate in vivo interaction of carcinogenic acrylamide on microsomal cytochrome P450 enzyme activities, and protein levels, and on cytosolic NQO1 and GST enzyme activities of rabbit liver, kidney and lung of acrylamide-treated rabbits. The in vivo protective effect of resveratrol, a phenolic compound, was also investigated on acrylamide toxicity. New Zealand male rabbits were treated with acrylamide and resveratrol, separately in different doses and conditions. Their combined effects were also investigated. CYP2E1-dependent p-Nitrophenol hydroxylase, NDMA N-demethylase and aniline 4-hydroxylase activities were found to be significantly increased in acrylamide-treated rabbit liver (1.80-3.0 fold) and kidney (1.6-fold). Rabbit liver and kidney CYP2E1 protein levels (determined by western blot analyisis) also increased approximately 2-fold due to acrylamide treatment. In rabbit liver, resveratrol was found significantly effective in decreasing both acrylamide-induced CYP2E1-dependent enzyme activities (approximately 1.5-1.80 fold) and CYP2E1 protein levels (approximately 1.5-1.70 fold). Additionally, resveratrol significantly decreased acrylamide-induced CYP2E1 protein level (2-2.5 fold) in rabbit kidney. However, no significant change was observed in rabbit lung CYP2E1-dependent enzyme activities and CYP2E1 protein levels due to acrylamide, resveratrol or their combined treatments. Furthermore, it was found that acrylamide treatment significantly increased CYP3A6-dependent erythromycin N-demethylase enzyme activity (1.85-fold) and CYP3A6 protein levels in rabbit liver (1.69-fold). No change was observed in CYP2B4-dependent benzphetamine N-demethylase enzyme activities of rabbit liver, kidney and lung by in vivo acrylamide, resveratrol or their combined treatments. Moreover, total GST and GST-Mu activities of rabbit kidney (1.5-fold, respectively) and total GST activity of rabbit lung (1.6-fold) were increased significantly only in resveratrol treated group. NQO1 enzyme activity of rabbit kidney was significantly increased by acrylamide treatment (1.6-fold). The results of the present study have demonstrated for the first time that acrylamide induces rabbit liver and kidney CYP2E1-dependent enzyme activities and CYP2E1 protein levels. The induction of CYP2E1 enzyme activity and protein level by acrylamide treatment can stimulate formation of other toxic compounds and procarcinogens metabolized by CYP2E1 which in turn further potentiates the risk of hepatotoxicity, mutagenicity and carcinogenicity. In the present study, it was also demonstrated for the first time that acrylamide treatment also increases CYP3A6 enzyme activity in rabbit liver which may lead to alterations in drug metabolism. The results of this study have also suggested that resveratrol may have protective effects on acrylamide induced toxicity / however, further in vivo studies are required to clarify the effect of resveratrol on both acrylamide-induced toxicity and anti-oxidant enzymes.

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Sulforaphane is an isothiocyanate found in high concentrations in cruciferous vegetables like broccoli. Sulforaphane has received much attention due to the evidence that it inhibits phase I carcinogen-bioactivating enzymes and/or induces phase II antioxidant enzymes as well as metallothioneins (MTs) (Perocco et al., 2006; Clarke et al., 2008; Yeh & Yen, 2009). Since MTs and antioxidant enzymes are involved in the scavenging of reactive oxygen species (ROS), the question was raised whether sulforaphane can provide protection against increased oxidative stress and if sulforaphane exposure of a human hepatocellular carcinoma cell line, like HepaRG cells, will have a negative impact on phase I and II biotransformation in these cells. Oxidative stress was exogenously induced in HepaRG cells with tert- Butyl hydroperoxide (t-BHP). Phase I and phase II biotransformation pathways were assessed with caffeine, paracetamol, aspirin, sodium benzoate, and paraaminobenzoic acid, respectively, as probe substances. Through the use of a liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) assay, the biotransformation of caffeine in phase I and the formation of paracetamol, aspirin, sodium benzoate and para-aminobenzoic acid conjugates in phase II were investigated. This involved elucidating the time it took for the whole probe to be completely biotransformed during phase I biotransformation and the unique conjugates formed during phase II biotransformation in HepaRG cells. The optimal t-BHP concentration and exposure time in HepaRG cells were standardized with a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. LC-ESI-MS/MS assays to monitor phase I and phase II biotransformation were optimized and validated. The optimal sulforaphane concentration and exposure time in HepaRG cells were standardized with a MTT assay. To evaluate the possible protective effect of sulforaphane against oxidative stress, HepaRG cells were pre-incubated with sulforaphane followed by the induction of oxidative stress with t-BHP and the quantification of the amount of viable cells with a MTT assay. To investigate the effect of sulforaphane on phase I and phase II biotransformation pathways, HepaRG cells were first pre-incubated with sulforaphane followed by the addition of a specific probe substance and the assessment of the biotransformation of the probe with a LC-ESI-MS/MS assay. The results partially supported the hypothesis of the study that sulforaphane will protect HepaRG cells against oxidative stress without negatively influencing phase I and phase II biotransformation. The results indicated that sulforaphane provided partial protection against t-BHP induced oxidative stress and had no effect on phase II paracetamol biotransformation in HepaRG cells. / Thesis, MSc (Biochemistry), North-West University, Potchefstroom Campus, 2013.

Sulforaphane is an isothiocyanate found in high concentrations in cruciferous vegetables like broccoli. Sulforaphane has received much attention due to the evidence that it inhibits phase I carcinogen-bioactivating enzymes and/or induces phase II antioxidant enzymes as well as metallothioneins (MTs) (Perocco et al., 2006; Clarke et al., 2008; Yeh & Yen, 2009). Since MTs and antioxidant enzymes are involved in the scavenging of reactive oxygen species (ROS), the question was raised whether sulforaphane can provide protection against increased oxidative stress and if sulforaphane exposure of a human hepatocellular carcinoma cell line, like HepaRG cells, will have a negative impact on phase I and II biotransformation in these cells. Oxidative stress was exogenously induced in HepaRG cells with tert- Butyl hydroperoxide (t-BHP). Phase I and phase II biotransformation pathways were assessed with caffeine, paracetamol, aspirin, sodium benzoate, and paraaminobenzoic acid, respectively, as probe substances. Through the use of a liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) assay, the biotransformation of caffeine in phase I and the formation of paracetamol, aspirin, sodium benzoate and para-aminobenzoic acid conjugates in phase II were investigated. This involved elucidating the time it took for the whole probe to be completely biotransformed during phase I biotransformation and the unique conjugates formed during phase II biotransformation in HepaRG cells. The optimal t-BHP concentration and exposure time in HepaRG cells were standardized with a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. LC-ESI-MS/MS assays to monitor phase I and phase II biotransformation were optimized and validated. The optimal sulforaphane concentration and exposure time in HepaRG cells were standardized with a MTT assay. To evaluate the possible protective effect of sulforaphane against oxidative stress, HepaRG cells were pre-incubated with sulforaphane followed by the induction of oxidative stress with t-BHP and the quantification of the amount of viable cells with a MTT assay. To investigate the effect of sulforaphane on phase I and phase II biotransformation pathways, HepaRG cells were first pre-incubated with sulforaphane followed by the addition of a specific probe substance and the assessment of the biotransformation of the probe with a LC-ESI-MS/MS assay. The results partially supported the hypothesis of the study that sulforaphane will protect HepaRG cells against oxidative stress without negatively influencing phase I and phase II biotransformation. The results indicated that sulforaphane provided partial protection against t-BHP induced oxidative stress and had no effect on phase II paracetamol biotransformation in HepaRG cells. / Thesis, MSc (Biochemistry), North-West University, Potchefstroom Campus, 2013.