Synthesis of sulforaphane and inhibition of cytochrome P450 enzymes as a basis for antigenotoxicity

Many dietary factors have been associated with a decreased risk of developing cancer. One potential mechanism by which these factors, chemopreventors, protect against cancer may be via alteration of carcinogen metabolism. The broccoli constituent sulforaphane (1-isothiocyanate-4-methylsulinylbutane) (CH3-S0-(CH2)4-NCS) has been isolated as a potential inducer of phase II detoxification enzymes and also protects rodents against 9,10-dimethyl-1,2-benz[aJanthracene-induced mammary tumours. The ability of sulforaphane to also modulate phase I activation enzymes (cytochrome P450) (CYP450) was studied here. Sulforaphane was synthesised with an overall yield of 15%, essentially via 1-methylsulfinylphthalimidobutane, which was oxidised to the sulfoxide moiety. Deprotective removal of phthalimide yielded the amine, which was converted into sulforaphane by reaction with N,N'-thionocarbonyldiimidazole. Purity (95 %) was checked by 1H-NMR,13C-NMR and infrared and mass spectrometry.Sulforaphane was a competitive inhibitor of CYP2E1 in acetone-induced Sprague-Dawley rat microsomes (Ki 37.9 ± 4.5μM), as measured by the p-nitrophenol hydroxylase assay. Ethoxyresorufin deethylase activity (EROD), a measurement of CYP1A activity, was also inhibited by sulforaphane (100μM) but was not competitive, and a preincubation time-dependence was observed. In view of these results, the capacity of sulforaphane to inhibit N-nitrosodimethylamine (NDMA)-induced genotoxicity (CYP2E1-mediated) was studied using mouse liver activation systems. Sulforaphane (>0.8μM) inhibited the mutagenicity of NDMA (4.4 mg/plate) in Salmonella typhimurium strain TA100 after pre-incubation for 45 min with acetone-induced liver 9000 g supernatants from Balb/c mice. Unscheduled DNA synthesis induced by NDMA (33μ5 M) in mouse hepatocytes was also reduced by sulforaphane in a concentration-dependent manner (0.064-20μM). Sulforaphane was not genotoxic itself in any of these systems and cytotoxic only at high concentrations (>0.5 mM and > 40μM respectively). The ability of sulforaphane to modulate the orthologous human enzymes was studied using a human epithelial liver cell line (THLE) expressing individual human CYP450 isoenzymes. Using the Comet assay (a measurement of DNA strand breakage under alkaline conditions), NDMA (0.01-1μg/ml) and IQ (0.1-10μg/ml) were used to produce strand breaks in T5-2E1 cells (expressing human CYP2E1) and T5-1A2 cells (expressing human CYP1A2) respectively, however no response was observed in T5-neo cells (without CYP450 cDNA transfection). Sulforaphane inhibited both NDMA and IQ-induced DNA strand breakage in a concentration-dependent manner (0.1-10μM).The inhibition of metabolic activation as a basis for the antigenotoxic action of sulforaphane in these systems (bacteria, rodent hepatocytes and human cells) is further supported by the lack of this chemopreventor to influence NaN3 mutagenicity in S. typhimurium and H202-induced DNA strand breakage in T5-neo cells. These findings suggest that inhibition of CYP2E1 and CYP1A by sulforaphane may contribute to its chemoprotective potential.

Molecular basis for reduced estrone sulfate transport and altered modulator sensitivity of transmembrane helix (TM) 6 and TM17 mutants of multidrug resistance protein 1 (ABCC1)

Multidrug resistance protein 1 (MRP1) confers drug resistance and also mediates cellular efflux of many organic anions. MRP1 also transports glutathione (GSH); furthermore, this tripeptide stimulates transport of several substrates, including estrone 3-sulfate. We have previously shown that mutations of Lys(332) in transmembrane helix (TM) 6 and Trp(1246) in TM17 cause different substrate-selective losses in MRP1 transport activity. Here we have extended our characterization of mutants K332L and W1246C to further define the different roles these two residues play in determining the substrate and inhibitor specificity of MRP1. Thus, we have shown that TM17-Trp(1246) is crucial for conferring drug resistance and for binding and transport of methotrexate, estradiol glucuronide, and estrone 3-sulfate, as well as for binding of the tricyclic isoxazole inhibitor N-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo-[4,3-c]quinolin-5-yl)-cyclohexylmethyl]-benzamide (LY465803). In contrast, TM6-Lys(332) is important for enabling GSH and GSH-containing compounds to serve as substrates (e.g., leukotriene C(4)) or modulators (e.g., S-decyl-GSH, GSH disulfide) of MRP1 and, further, for enabling GSH (or S-methyl-GSH) to enhance the transport of estrone 3-sulfate and increase the inhibitory potency of LY465803. On the other hand, both mutants are as sensitive as wild-type MRP1 to the non-GSH-containing inhibitors (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571), 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl]-ethanone (LY171883), and highly potent 6-[4'-carboxyphenylthio]-5[S]-hydroxy-7[E], 11[Z]14[Z]-eicosatetrenoic acid (BAY u9773). Finally, the differing abilities of the cysteinyl leukotriene derivatives leukotriene C(4), D(4), and F(4) to inhibit estradiol glucuronide transport by wild-type and K332L mutant MRP1 provide further evidence that TM6-Lys(332) is involved in the recognition of the gamma-Glu portion of substrates and modulators containing GSH or GSH-like moieties.