BaP is an important environmental carcinogen presented in cigarette smoke, industrial waste by-products and food (Chen et al., 2002; Hecht, 1999b). Average daily intake of BaP ranges from non-detectable to 22.5 pg per person per day depending on individual lifestyle (Massaro, 1997). The induction of enzymes by BaP is mainly to enhance its biotransformation, primarily by CYPl especially C Y PlA l (Drahushuk et al., 1998; Roberts- Thomson et al., 1993; Shimada et al., 1996). Such induction is believed to enhance the generation of the genotoxic BaP 7,8-diol-9,10-epoxide that binds covalently to cellular DNA and initiates carcinogenesis (Grover and Sims, 1968; Nebert et al., 2004; Whitlock, Jr., 1999).
BaP is an avid ligand of the Ah receptor and this binding leads to up-regulation o f CYP enzyme involved in its metabolism such as C Y PlA l, CYPIBI and phase II enzyme such as NQOl and GST (Nebert et al., 2004; Vondracek et al., 2009). Utilising precision-cut rat and human liver slices the aim of the study was to determine whether PEITC can antagonise BaP- mediated enzyme induction, possibly by interfering with its interaction with Ah receptor. BaP at 0.1 pM was employed for investigating a possible antagonistic effect o f PEITC, and 10 pM BaP was employed to achieve a maximal enzyme induction in rat liver slices, according to previous studies conducted in this laboratory under identical incubation conditions (Pushparajah et al., 2008a). EROD and NQOl activities were determined in rat liver slices whereas in human liver slices MROD and GST, monitored using CDNB as substrate, were additionally investigated.
Rat C Y PlA l as exemplified by EROD activity, was induced by BaP, and PEITC was capable of preventing the rise in activity especially at the low concentration o f BaP. Western blot analysis showed that this was a consequence of decrease of enzyme availability. Human EROD activity from only Donors 3 and 4 was susceptible to BaP treatment, and concurrent
Chapter 5: Interaction between PEITC and chemical carcinogens
treatment with PEITC antagonised BaP-mediated enzyme induction. The inhibition at all PEITC concentrations in Donor 2 where no BaP induction was achieved, indicating that PEITC can also suppress basal activity as observed previously in Chapter 3 (Fig. 3.7).
MROD activity in human liver slices was moderately induced by BaP only in Donors 1 and 4 and this induction was inhibited by PEITC at all concentrations employed. CYP1A2 apoprotein levels, on the other hand, were markedly induced by BaP in all donors. The co
treatment with PEITC led to a decrease in BaP-induced CYP1A2 apoprotein levels at all concentrations in Donor 4 whereas that in Donor 1 was resistant to this treatment, the latter case contradictory the observations at the activity level. A decrease in CYP1A2 apoprotein levels was observed at only the highest PEITC concentration in Donors 2 and 3, presumably due to cytotoxicity effect of PEITC as exemplified by increasing of LDH leakage (Chapter 3).
It is relevant to note that exposure of slices to BaP at the concentrations used in the current study, did not influence slice viability (C. loannides, unpublished data). The discrepancy in induction of CYP1A2 apoprotein levels by BaP in Donors 2 and 3 but without concomitant increase in MROD activity may be related to a CYP1A2 variant expressing low activity (Murayama et al., 2004). M oreover, the decrease in MROD activity while CYP1A2 apoprotein levels remained unchanged in Donor 1 implies mechanism-based inactivation of PEITC towards BaP-induced MROD activity and this resistant CYP1A2 expression may imply that PEITC failed to interfere the binding o f BaP to Ah receptor.
Induction o f CYPl is known to be regulated through the ligand-activated aryl hydrocarbon receptor (AhR) (Whitlock, Jr., 1999). Prior to dissociation from its ligand-binding partners, AhR translocates into the nucleus, subsequently binds to a specific nucleotide sequence of general transcription factors and initiates gene transcription o f enzymes including CYPl (Whitlock, 1999). As a result of suppression of apoprotein levels, the antagonistic effects of
Chapter 5: Interaction between PEITC and chemical carcinogens
PEITC in the current study may involve modulation of the binding of BaP by PEITC, although the underlying mechanism remains to be established.
The inter-individual variability in human liver from the four donors by BaP treatment was remarkable. Polymorphism in AhR where the different alleles have differing affinities for carcinogen binding and resulting in differences in induction effect have been reported (Kouri et al., 1974; Nebert et al., 2004) and are believed to be responsible for the different response of the enzymes to BaP in each individual. The higher inducibility type o f C Y PlA l enhanced susceptibility to cancer risk such as lung cancer (Kawajiri et al., 1990a; 1990b).
In the case o f phase II detoxifying NQOl activity, an apparent modest synergistic effect of PEITC was observed in rat liver slices, albeit at the low BaP concentration only. However, immunoblot analysis revealed marked synergistic effect o f PEITC on NQOl expression at both BaP concentrations. Diminishing of protein level and enzyme activity at the highest concentration of PEITC was observed, presumably due to toxicity o f isothiocyanate as already discussed. In human liver slices, induction of NQOl activity by BaP was observed only in Donors 3 and 4. The lowest activity in the presence o f BaP was seen in Donor 2, similar to the lowest basal activity among the other donors. A possible reason has already been discussed, i.e. the presence of an allele type having low activity. Treatment o f slices with PEITC and BaP in combination modestly inhibited human NQOl activity in Donor 3, whereas that in other donors was unchanged. At protein level, concurrent treatment with PEITC suppressed the BaP-modulated NQOl expression in Donors 2 and 3 whereas that in Donors 1 and 4, a synergistic effect was observed, however, at only 10 pM PEITC.
Clearly, PEITC synergistically increased NQOl activity and expression in rat, but only minor modulation in activity was observed. In contrast, inconsistent effects were observed in human
Chapter 5: Interaction between PEITC and chemical carcinogens
PEITC to enhance phase II is thought to be through nuclear factor-E2-related factor 2 (Nrf2), a key regulator of ARE-mediated gene expression of phase II detoxifying enzymes (Xu et ah, 2006). Induction mechanism by PEITC is believed to involve covalent binding of the isothiocyanate with the thiol groups of Keapl hence liberation of Nrf2 from Keapl leading to gene transcription initiation (Xu et ah, 2006).
GST is involved in the detoxification of many polycyclic aromatic hydrocarbons including BaP (Lamy et ah, 2008; Steinkellner et ah, 2001b), and polymorphism of this enzyme has been reported (Jourenkova-Mironova et ah, 1999). Activity of human GST assessed using CDNB as a broad substrate was elevated by BaP in only Donors 3 and 4. It has been demonstrated that BaP failed to modulate hepatic GST (CDNB) in vitro assessed, however, in only one donor (Pushparajah et ah, 2008b). In the current study, treatment with PEITC did not modulate BaP-mediated GST activity.
Collectively, PEITC suppressed BaP-induced CYPIA activity and apoprotein levels in rat liver slices and, at certain concentration, it also increased NQOl induction mediated by BaP.
Even though both CYPIA and NQOl are known to be regulated through the same AhR- driven gene expression, the differential response of these enzymes may be attributed to the fact that induction of phase II enzyme can occur via two different pathways; in addition to AhR, induction of NQOl expression by BaP may be also Nrf2 dependent as demonstrated in mice (Hu et ah, 2006). Moreover, PEITC is more likely to induce phase II enzyme through Nrf2 transcription factor as already discussed (Bonnesen et ah, 2001), implying that synergistic increase o f NQOl by BaP and PEITC is mediated through NrfZ rather than AhR.
On the other hand, antagonistic mechanism of PEITC on CYPIA may be the result of interactions at the site o f the AhR, preventing the binding o f BaP and/or mechanism-based inhibition leading to loss of activity. It was demonstrated in Chapter 4 that PEITC was a potent mechanism-based inhibitor towards CYPlAl-mediated EROD activity, even at low
Chapter 5: Interaction between PEITC and chemical carcinogens
concentrations employed in the current study. However, bearing in mind that uptake of PEITC by the slices may lead to lower levels, direct inhibition is unlikely. Moreover, more pronounced inhibition effect in mechanism-based inhibition study than the current study was anticipated since it was carried out in Aroclor 1254-induced microsomes.
Even though PEITC antagonised the BaP-mediated rise in hepatic CYPIA, it has been reported that PEITC failed to prevent BaP-induced lung tumorigenesis (Adam-Rodwell et al., 1993; Hecht et al., 1995; 1996b; Lin et al., 1993). Although there is experimental evidence that the liver bioactivates BaP and the reactive intermediates are transported to the lung (Wall et al., 1991), it may be that in situ activation is also important. It would be worthwhile to determine whether interactions between BaP and PEITC are also manifested in lung slices.
Furthermore, tissue-specific effect of PEITC on carcinogen-metabolising enzymes was demonstrated (Chapter 4) where the High dose of PEITC inhibited hepatic C Y PlA l activity in a mechanism-based fashion but the same dose induced both C Y PlA l apoprotein levels and activity in rat lung.
5.5 Conclusions
• Long-tem exposure of rats to PEITC even at dietary doses, decreased IQ-induced urinary mutagenicity. This was not related to changes in CYPIA and GST activities.
• In rat liver slices, PEITC antagonised the BaP-mediated rise in C Y PlA l whereas at
certain concentration of PEITC an apparent synergism on BaP-mediated NQOl was noted.
The effects of PEITC on BaP-mediated enzymes in human liver slices from four donors were inconsistent.