In the present study, we conducted experiments using Nrf2−/−
and Nrf2 wild-type mice to determine the metabolism and tissue distribution of SFN following oral administration. Using LC-MS/MS analysis we sought to quantify SFN and four major metabolites (SFN-GSH, SFN-CG, SFN-Cys and SFN-NAC) in plasma, SI, colon, liver, kidney, lung, brain and prostate. Most studies in rodents and humans have reported peak plasma concentrations of SFN and its metabolites occurring between 1 and 3 h after SFN administration (11
). Following SFN gavage, SFN metabolites were detected in all tissues at 2 and 6 h, and a dose dependent increase in tissue concentrations was observed in all tissues except for the prostate. In liver, kidney, lung, brain and plasma the highest concentrations were at 2 h, but in SI, colon and prostate the highest concentrations were at 6 h. This indicated that SFN metabolites may be accumulating in certain tissues, and that peak plasma concentrations do not always align precisely with target tissues of major cancers, such as prostate and colon. Although no gender difference was observed in SFN metabolism in the wild-type mice, in Nrf2−/−
mice at the higher dose and 6 h time point there was a marked increase in tissue concentrations in the female mice compared to the male mice, and a similar trend also was seen at the lower SFN dose. Interestingly, the relative abundance of each metabolite was not strikingly different between genders and genotypes, despite some variability on a case-by-case basis. This study is the first to show detailed LC-MS/MS analysis of SFN metabolites in mouse tissues, and to compare SFN metabolite profiles between Nrf2−/−
and wild-type mice.
Although there have been several studies showing PK of SFN in rodents and humans, tissue distribution is still largely unknown. Herein we report that the tissue concentrations of SFN metabolites vary as much as 100 fold between different tissues. For example, SI had the highest concentration at 0.355 nmole SFN metabolites/mg of tissue whereas brain had 0.003 nmole SFN metabolites/mg of tissue (). It has been reported that 74% of a SFN dose was absorbed in human jejunum (21
), so high concentrations in the SI are likely not a result of poor bioavailability, although we did not directly test that here. Currently little is known regarding the ability of SFN metabolites to cross the blood-brain barrier, but here we report low concentration in the brain which likely indicates that SFN metabolites do not readily cross the blood-brain barrier, Tissues such as liver, kidney, lung and prostate had comparable concentrations at 2h, ranging from 0.075 nmole SFN metabolites/mg of tissue in lung to 0.041 nmole SFN metabolites/mg of tissue in liver. The abundance of individual metabolites also varied between tissues. SFN-GSH, SFN-Cys and SFN-NAC represented the highest proportion of SFN metabolites in most tissues. For some tissues, such as prostate and lung, SFN-NAC and SFN-GSH were the most abundant metabolites, respectively. The in vivo
bioactivity of each metabolite is still unclear, although it has been reported that the SFN-Cys and SFN-NAC metabolites are the bioactive intermediates targeting histone deacetylases (22
Interestingly, because ITC thiol conjugates can dissociate into free ITCs under physiological conditions (23
), it has been postulated that these thiol conjugates can be considered prodrugs of the parent compound (24
). Studies have shown similar efficacy from either free ITC or the N
-acetylcysteine (NAC) conjugated ITC in vitro
, in cancer cells, or in rodent cancer models in vivo
). The metabolism of the ITC and ITC-NAC compounds in the latter studies is unknown, but from the current investigation it can be expected that tissue concentrations in the mice that received the free ITCs were predominately in the form of thiol conjugates. The metabolism of orally administered ITC-NAC conjugates is unknown and would be an interesting area of future research. These differences in total and individual SFN metabolite tissue concentrations could impact the bioactivity of SFN in different tissues, and merits further investigation. From the current data it is clear that thorough accounting of SFN in vivo
requires analysis of at least four of the five main metabolites. These data also support the hypothesis that repeated consumption of cruciferous vegetables is required to maintain SFN metabolite concentrations in tissues.
In our study we observed striking differences between male and female Nrf2−/−
mice at the 20 µmole dose and 6 h time point. It has been reported that female Nrf2−/−
mice have significantly higher morbidity and mortality, even in the absence of apparent exogenous stress (30
). Interestingly, Ma et al.
reported that 88% and 47% of the death in Nrf2−/−
female and male mice, respectively, were caused by renal failure and severe glomerulonephritis. In the present study, the Nrf2−/−
female mice in the 20 µmole SFN/6 h group had apparent toxicity after the SFN treatment manifested as lethargy and non-responsiveness, and as much as 23-fold higher concentrations of SFN metabolites in all tissues (except for the SI and colon) compared to the corresponding wild-type females (). The observation that the SI and colon were the only tissues that had similar concentrations as observed in the wild-type mice indicates that the differences in tissues concentrations occur post-absorption. We speculate that the female Nrf2−/−
mice in our study had some degree of glomerulonephritis as a result of lacking Nrf2, and upon receipt of the higher dose (20 µmole SFN) underwent acute kidney failure and could not excrete the SFN metabolites in the urine, thus leading to an accumulation in tissues and ultimately toxicity. Furthermore, it is possible that this potential kidney damage is compounding the impact of reduced inducibility of phase III efflux transporters on clearance of SFN compounds from the tissues, thus contributing to the high tissue concentrations observed. Further study confirming degree of glomerulonephritis and/or altered phase III transporter expression is an important area for future research to understand the mechanisms accouonting for this response in the Nrf2−/− females. Also, although differences between wild-type female and male mice in basal and inducible GST activity as well as hepatotoxicity have previously been reported (31
), we did not observe a difference in SFN metabolism between wild-type males and wild-type females. Taken together, these data illuminate the need for caution when selecting either male or female Nrf2−/−
mice for xenobiotic studies.
The importance of Nrf2 in drug metabolism is well documented (34
). As expected, it has been reported that Nrf2−/−
mice have much lower expression and lack the inducibility of phase I, II, and III enzymes (30
). Several groups have shown that Nrf2−/−
mice are more susceptible to experimentally induced colon cancer (36
). In the context of SFN treatment, one study reported that the protective effects of SFN administration in a Parkinson’s disease model was lost in Nrf2−/−
). Similarly, another group reported that topical SFN administration in a UVB induced skin inflammation model was only able to restore sunburn cells back to basal levels in mice that were wild-type for Nrf2 (17
). The working hypothesis is that the loss in SFN-mediated protection in these models is partially attributed to altered metabolism of SFN in Nrf2−/−
mice. In the current report we show that SFN metabolism and tissue distribution is nearly identical between wild-type and Nrf2−/−
mice ( and ), with the exception of female Nrf2−/−
mice given 20 µmole for 6 h (see discussion above regarding these mice). Also, across genotypes there were no drastic differences in the relative abundance of each metabolite, even though the Nrf2−/−
females at the high dose and 6 h time point had dramatically higher tissue concentrations. Several aspects of drug metabolism could contribute to this apparent disconnect between SFN metabolism and Nrf2 status. For example, cross talk between the many different nuclear receptors involved in drug metabolism has been reported, and several members of the GST family are regulated independently of Nrf2 (39
). Indeed, it has been shown that dextran sulfate sodium treatment caused induction of GSTM1 protein in colonic tissues of Nrf2−/−
), indicating either a separate pathway for induction or retention of inducibility of GSTM1 in the colon of these mice. Also, it has been shown that SFN metabolites can undergo facile thiol exchange reactions (41
), indicating that interconversion between SFN metabolites may occur, independent of enzymatic activity in vivo
. Future research will be important to elucidate exactly what factors are contributing to this disconnect between SFN metabolism and Nrf2 driven phase II enzyme induction. From these data we conclude that Nrf2 status does not have a marked impact on SFN metabolism and tissue distribution in mice, and therefore the differences in SFN efficacy observed in other studies are likely not related to metabolism and biodistribution.