P LANIFICAR LA GESTIÓN DEL ALCANCE DEL PROYECTO

ITCs may cause toxicity, both in vivo and in vitro. Short chain length isothiocyanates such as allyl isothiocyanate (AITC) have been demonstrated to cause bladder cancer in male rats (Dunnick et al., 1982). In rats treated with AITC (40 mg/kg) for 4 weeks, the isothiocyanate gave rise to renal dysfunction as exemplified by an increase in urinaiy excretion o f aspartate

Chapter 1: Introduction

urine animo-transferase, reduction in urine volume and a change in the specific gravity of uri (Lewerenz et al., 1988). The same researchers also reported toxicity for BITC in rats treated with high dose (50-200 mg/kg) for 4 weeks, causing reduction o f weight gain in a dose- dependent manner, renal dysfunction and increase in the weight of many organs (Lewerenz et al., 1992). In the case of PEITC, no adverse effects were observed following administration of a diet supplemented with PEITC (0.75-6 pmole/g diet) for 13 weeks to F344 rats (Morse et al., 1989). Nevertheless, genotoxicity at high concentrations o f PEITC has been reported in the Ames test employing Salmonella typhimurium, DNA damage in Escherichia Coli and micronucleus assay in HepG2 cells (Kassie and Knasmuller, 2000).

L l l Pharmacokinetics and bioavailability of phenethyl isothiocyanate

In order to understand the mechanism of the chemopreventive action o f isothiocyanates, bioavailability and pharmacokinetic studies were undertaken by several laboratories. The fate of glucosinolates very much depends on the adopted cooking process (Johnson and Williamson, 2003). Raw cruciferous vegetable intake leads to further degradation of glucosinolates inside the oropharyngeal or small intestine by plant myrosinase or may eventually undergo hydrolysis by bacterial myrosinase in the colon. The degradation products will then be excreted into faeces or may be partly absorbed into the bloodstream (Krul et al., 2002). In the case of cooked vegetables, only bacterial myrosinase will bring about the degradation process, so that the body is exposed mostly to glucosinolates. Accordingly, urinary excretion of biomarker metabolites of ITCs was higher in humans that consumed raw vegetables compared with cooked vegetables (Getahun and Chung, 1999). In the case of isothiocyanates, they are absorbed from the small bowel and colon and the rate o f absorption from gastro-intestinal tract (GIT) to bloodstream and organs depends largely on their lipophilicity.

Chapter 1: Introduction

The mercapturic acid pathway is the major route of ITC metabolism (Brusewitz et al., 1977;

Chung et al., 1992; Mennicke et al., 1987). Although all types o f ITC metabolites, i.e. ITC- cysteinylglycine, ITC-cysteine and mercapturate have been found in urine, such as following administration of sulforaphane (Al Janobi et al., 2006), metabolism is subject to species differences; a cyclic mercaptopyruvic acid conjugate was demonstrated as the major urinary metabolite o f PEITC in mouse (Eklind et al., 1990), whereas following gluconasturtiin administration to humans the V-acetylcysteine conjugate was the most important metabolite (Chung et al., 1992). After metabolism, distribution of ITCs through the plasma membrane cannot occur in the glutathione conjugate form. Thus dissociation o f ITC conjugates to the parent compounds is the rate limiting step of both their import or export from cells. About 9- 15% o f ITC conjugates was converted to free ITCs within 15 min when incubated with rat hepatocytes in the buffer (pH 7.4), depending on the individual structure o f ITCs (Bruggeman et al., 1986).

The chemoprevention of ITCs is species- and tissue-dependent (Guo et al., 1992).

Correspondingly, bioavailability of ITCs in the target organs may be an important key in determining their protection potency. For example, a synthetic homologue 6-phenylhexyl isothiocyanate (PHITC) was a more potent inhibitor o f tumour development in lung than other tissues in A/J mice and F344 rats compared with PEITC due to its higher effective concentration in these organ (Conaway et al., 1999); this is possibly related to the higher lipophilicity of PHITC associated with the slower rate o f elimination. Species differences in ITC bioavailability and, therefore, in their effectiveness has been described, presumably due to distinct pattern o f XMEs, and tissue disposition and metabolism of ITCs in the different species (Chung et al., 1996).

In our laboratory, determination of plasma concentrations of SFN in Wistar albino rats revealed a rapid absorption and 82% absolute bioavailability after an oral dose o f 2.8

Chapter 1: Introduction

|imole/kg which, however, decreased with increasing dose, similar to the half-life and volume of distribution (Hanlon et al., 2008a). PEITC (19 nM) was detected in the plasma o f human volunteers 3 hr after consumption of 200 g raw broccoli (Song et al., 2005) whereas plasma concentration o f PEITC (928 nM) in human volunteer was detected following consumption of 100 g watercress (Ji and Morris, 2003). In studies conducted in rats, PEITC was rapidly absorbed with peak plasma occurring at 0.4 and 2 hr following oral doses of 10 and 100 pmole/kg, and oral bioavailability o f PEITC was 115% and 93%, respectively (Ji and Morris, 2005). However, these studies employed doses higher than human daily intake. In order to evaluate the pharmacokinetic characteristics of PEITC from a therapeutic point of view, pharmacokinetic studies following repeated administration are pivotal and remain to be investigated.

1 . 1 2 Precision-cut tissue slices in the study of the regulation of xenobiotic-metabolising enzymes

The tissue slicing technique was first established in 1923, and in 1980 the Krumdieck sheer was developed, enabling widespread use of this technique for various purposes such as investigations into xenobiotic-metabolism, mechanism of toxicity, organ specificity (Catania et al., 2001; Harrigan et al., 2004; Olinga et al., 2001; Price et al., 1996), enzyme regulation (Pushparajah et al., 2008b) and enzyme-mediated toxicity (Lake et al., 1999; van de et al., 2005). Slices have been prepared from a number of tissues including liver, kidney (Parrish et al., 1998) and lung (Pushparajah et al., 2007), and from a number o f species, e.g. rat, (Lake et al., 1996), human, dog (Connors et al., 1996), guinea pig, monkey (Price et al., 1996), deer and cattle (Sivapathasundaram et al., 2004). Cryopreservation o f tissue slices has been demonstrated as a feasible method to alleviate the scarcity o f tissue (De Graaf et al., 2007; De Kanter et al., 1998; Fisher et al., 1993). Optimum slicing method in terms of suitable incubation medium, and slice thickness and diameter has been studied (Fisher et al., 1995;

Parrish et al., 1995). Certainly, precision-cut tissue slice is a well-developed in vitro system. It

Chapter 1: Introduction

mimics features o f the whole tissue and has a number of advantages over other in vitro systems, specifically isolated cell culture, i.e. maintaining the functional heterogeneity and cellular communication and interactions of the parent tissue (Toutain et al., 1998). Moreover, this system has been reported as a suitable model for investigation of xenobiotic-metabolising enzymes as exemplified by the stability of enzyme activities during culture period such as phase II enzymes (epoxide hydrolase, GST and glutathione reductase) which apparently were more stable than cytochrome P450 enzymes (C Y PlA l, 1A2 and 2B1), in rat liver slices (Hashemi et al., 1999b; Hashemi et al., 2000).

Precision-cut slices have been successfully employed to establish modulation of cytochrome P450 and phase II enzymes by aliphatic isothiocyanates, such as sulforaphane and erucin, in rat liver and lung (Hanlon et al., 2008c; 2009a) as well as in human liver (Hanlon et al., 2008b).