John D. Clarke

Dietary Sulforaphane, a Histone Deacetylase Inhibitor for Cancer Prevention

The reversible acetylation of histones is an important mechanism of gene regulation. During prostate cancer progression, specific modifications in acetylation patterns on histones are apparent. Targeting the epigenome, including the use of histone deacetylase (HDAC) inhibitors, is a novel strategy for cancer chemoprevention. Recently, drugs classified as HDAC inhibitors have shown promise in cancer clinical trials. We have previously found that sulforaphane (SFN), a compound found in cruciferous vegetables, inhibits HDAC activity in human colorectal and prostate cancer cells. Based on the similarity of SFN metabolites and other phytochemicals to known HDAC inhibitors, we previously demonstrated that sulforaphane acted as an HDAC inhibitor in the prostate, causing enhanced histone acetylation, derepression of P21 and Bax, and induction of cell cycle arrest/apoptosis, leading to cancer prevention. The ability of SFN to target aberrant acetylation patterns, in addition to effects on phase 2 enzymes, may make it an effective chemoprevention agent. These studies are important because of the potential to qualify or change recommendations for high-risk prostate cancer patients and thereby increase their survival through simple dietary choices incorporating easily accessible foods into their diets. These studies also will provide a strong scientific foundation for future large-scale human clinical intervention studies.

Bioavailability and inter-conversion of sulforaphane and erucin in human subjects consuming broccoli sprouts or broccoli supplement in a cross-over study design

Broccoli consumption may reduce the risk of various cancers and many broccoli supplements are now available. The bioavailability and excretion of the mercapturic acid pathway metabolites isothiocyanates after human consumption of broccoli supplements has not been tested. Two important isothiocyanates from broccoli are sulforaphane and erucin. We employed a cross-over study design in which 12 subjects consumed 40 g of fresh broccoli sprouts followed by a 1 month washout period and then the same 12 subjects consumed 6 pills of a broccoli supplement. As negative controls for isothiocyanate consumption four additional subjects consumed alfalfa sprouts during the first phase and placebo pills during the second. Blood and urine samples were collected for 48h during each phase and analyzed for sulforaphane and erucin metabolites using LC-MS/MS. The bioavailability of sulforaphane and erucin is dramatically lower when subjects consume broccoli supplements compared to fresh broccoli sprouts. The peaks in plasma concentrations and urinary excretion were also delayed when subjects consumed the broccoli supplement. GSTP1 polymorphisms did not affect the metabolism or excretion of sulforaphane or erucin. Sulforaphane and erucin are able to interconvert in vivo and this interconversion is consistent within each subject but variable between subjects. This study confirms that consumption of broccoli supplements devoid of myrosinase activity does not produce equivalent plasma concentrations of the bioactive isothiocyanate metabolites compared to broccoli sprouts. This has implications for people who consume the recommended serving size (1 pill) of a broccoli supplement and believe they are getting equivalent doses of isothiocyanates.

Differential effects of sulforaphane on histone deacetylases, cell cycle arrest and apoptosis in normal prostate cells versus hyperplastic and cancerous prostate cells

SCOPE Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables such as broccoli. The ability of SFN to inhibit histone deacetylase (HDAC) enzymes may be one mechanism by which it acts as a chemoprevention agent. The ability of a chemopreventive agent to specifically cause cytotoxicity in cancer and not normal cells is an important factor in determining its safety and clinical relevance. METHODS AND RESULTS We characterized the effects of SFN in normal (PrEC), benign hyperplasia (BPH1) and cancerous (LnCap and PC3) prostate epithelial cells. We observed that 15 μM SFN selectively induced cell cycle arrest and apoptosis in BPH1, LnCap and PC3 cells but not PrEC cells. SFN treatment also selectively decreased HDAC activity, and Class I and II HDAC proteins, increased acetylated histone H3 at the promoter for P21, induced p21 expression and increased tubulin acetylation in prostate cancer cells. HDAC6 over-expression was able to reverse SFN-induced cyotoxicity. In PrEC cells, SFN caused only a transient reduction in HDAC activity with no change in any other endpoints tested. The differences in sensitivity to SFN in PrEC and PC3 are likely not due to differences in SFN metabolism or differences in phase 2 enzyme induction. CONCLUSION SFN exerts differential effects on cell proliferation, HDAC activity and downstream targets in normal and cancer cells.

Comparison of isothiocyanate metabolite levels and histone deacetylase activity in human subjects consuming broccoli sprouts or broccoli supplement.

Increased consumption of cruciferous vegetables such as broccoli may reduce the risk of various cancers. Myrosinase is required to convert dietary glucosinolates from broccoli into bioactive isothiocyanates. We evaluated isothiocyanate excretion profiles in healthy subjects who consumed broccoli sprouts or broccoli supplement (no myrosinase) with equivalent glucosinolate content. Urinary metabolites of two major isothiocyanates, sulforaphane and erucin, were measured by liquid chromatography coupled with tandem mass spectrometry. Peak excretion of sulforaphane and erucin was higher and occurred sooner in subjects who consumed broccoli sprouts as compared to subjects who consumed the supplement. A subject-dependent shift in the ratio of urinary sulforaphane to erucin metabolites was observed in both groups, indicating conversion of sulforaphane to erucin. Lower histone deacetylase activity was observed in the peripheral blood mononuclear cells only in subjects consuming sprouts. Fresh broccoli sprouts differ from broccoli supplements in regards to excretion of isothiocyanates and bioactivity in human subjects.

Increased Susceptibility to Methotrexate-Induced Toxicity in Nonalcoholic Steatohepatitis

Hepatic drug metabolizing enzymes and transporters play a crucial role in determining the fate of drugs, and alterations in liver function can place individuals at greater risk for adverse drug reactions (ADRs). We have shown that nonalcoholic steatohepatitis (NASH) leads to changes in the expression and localization of enzymes and transporters responsible for the disposition of numerous drugs. The purpose of this study was to determine the effect of NASH on methotrexate (MTX) disposition and the resulting toxicity profile. Sprague Dawley rats were fed either a control or methionine-choline-deficient diet for 8 weeks to induce NASH, then administered a single ip vehicle, 10, 40, or 100 mg/kg MTX injection followed by blood, urine, and feces collection over 96 h with terminal tissue collection. At the onset of dosing, Abcc1-4, Abcb1, and Abcg2 were elevated in NASH livers, whereas Abcc2 and Abcb1 were not properly localized to the membrane, similar to that previously observed in human NASH. NASH rodents receiving 40-100 mg/kg MTX exhibited hepatocellular damage followed by initiation of repair, whereas damage was absent in controls. NASH rodents receiving 100 mg/kg MTX exhibited slightly greater renal toxicity, indicating multiple organ toxicity, despite the majority of the dose being excreted by 6 h. Intestinal toxicity in NASH however, was strikingly less severe than controls, and coincided with reduced fecal MTX excretion. Because MTX-induced gastrointestinal toxicity limits the dose escalation necessary for cancer remission, these data suggest a greater risk for life-threatening MTX-induced hepatic and renal toxicity in NASH in the absence of overt gastrointestinal toxicity.

Synergistic interaction between genetics and disease on pravastatin disposition.

BACKGROUND & AIMS A genome wide association study and multiple pharmacogenetic studies have implicated the hepatic uptake transporter organic anion transporting polypeptide-1B1 (OATP1B1) in the pharmacokinetics and musculoskeletal toxicity of statin drugs. Other OATP uptake transporters can participate in the transport of pravastatin, partially compensating for the loss of OATP1B1 in patients carrying the polymorphism. Non-alcoholic steatohepatitis (NASH) in humans and in a diet-induced rodent model alter the expression of multiple OATP transporters. METHODS To determine how genetic alteration in one Oatp transporter can interact with NASH-associated changes in Oatp expression we measured the disposition of intravenously administered pravastatin in Slco1b2 knockout (Slco1b2(-/-)) and wild-type (WT) mice fed either a control or a methionine and choline deficient (MCD) diet to induce NASH. RESULTS Genetic loss of Oatp1b2, the rodent ortholog of human OATP1B transporters, caused a modest increase in pravastatin plasma concentrations in mice with healthy livers. Although a diet-induced model of NASH decreased the expression of multiple hepatic Oatp transporters, it did not alter the disposition of pravastatin compared to WT control mice. In contrast, the combination of NASH-associated decrease in compensatory Oatp transporters and Oatp1b2 genetic loss caused a synergistic increase in plasma area under the curve (AUC) and tissue concentrations in kidney and muscle. CONCLUSIONS Our data show that NASH alters the expression of multiple hepatic uptake transporters which, due to overlapping substrate specificity among the OATP transporters, may combine with the pharmacogenetic loss of OATP1B1 to increase the risk of statin-induced adverse drug reactions.

Modeling Human Nonalcoholic Steatohepatitis-Associated Changes in Drug Transporter Expression Using Experimental Rodent Models

Nonalcoholic fatty liver disease is a prevalent form of chronic liver disease that can progress to the more advanced stage of nonalcoholic steatohepatitis (NASH). NASH has been shown to alter drug transporter regulation and may have implications in the development of adverse drug reactions. Several experimental rodent models have been proposed for the study of NASH, but no single model fully recapitulates all aspects of the human disease. The purpose of the current study was to determine which experimental NASH model best reflects the known alterations in human drug transporter expression to enable more accurate drug disposition predictions in NASH. Both rat and mouse NASH models were used in this investigation and include the methionine and choline deficient (MCD) diet model, atherogenic diet model, ob/ob and db/db mice, and fa/fa rats. Pathologic scoring evaluations demonstrated that MCD and atherogenic rats, as well as ob/ob and db/db mice, developed NASH. Liver mRNA and protein expression analyses of drug transporters showed that in general, efflux transporters were induced and uptake transporters were repressed in the rat MCD and the mouse ob/ob and db/db models. Lastly, concordance analyses suggest that both the mouse and rat MCD models as well as mouse ob/ob and db/db NASH models show the most similarity to human transporter mRNA and protein expression. These results suggest that the MCD rat and mouse model, as well as the ob/ob and db/db mouse models, may be useful for predicting altered disposition of drugs with similar kinetics across humans and rodents.

Mechanistic Basis of Altered Morphine Disposition in Nonalcoholic Steatohepatitis

Morphine is metabolized in humans to morphine-3-glucuronide (M3G) and the pharmacologically active morphine-6-glucuronide (M6G). The hepatobiliary disposition of both metabolites relies upon multidrug resistance-associated proteins Mrp3 and Mrp2, located on the sinusoidal and canalicular membrane, respectively. Nonalcoholic steatohepatitis (NASH), the severe stage of nonalcoholic fatty liver disease, alters xenobiotic metabolizing enzyme and transporter function. The purpose of this study was to determine whether NASH contributes to the large interindividual variability and postoperative adverse events associated with morphine therapy. Male Sprague-Dawley rats were fed a control diet or a methionine- and choline-deficient diet to induce NASH. Radiolabeled morphine (2.5 mg/kg, 30 µCi/kg) was administered intravenously, and plasma and bile (0–150 or 0–240 minutes), liver and kidney, and cumulative urine were analyzed for morphine and M3G. The antinociceptive response to M6G (5 mg/kg) was assessed (0–12 hours) after direct intraperitoneal administration since rats do not produce M6G. NASH caused a net decrease in morphine concentrations in the bile and plasma and a net increase in the M3G/morphine plasma area under the concentration-time curve ratio, consistent with upregulation of UDP-glucuronosyltransferase Ugt2b1. Despite increased systemic exposure to M3G, NASH resulted in decreased biliary excretion and hepatic accumulation of M3G. This shift toward systemic retention is consistent with the mislocalization of canalicular Mrp2 and increased expression of sinusoidal Mrp3 in NASH and may correlate to increased antinociception by M6G. Increased metabolism and altered transporter regulation in NASH provide a mechanistic basis for interindividual variability in morphine disposition that may lead to opioid-related toxicity.

Experimental Nonalcoholic Steatohepatitis Increases Exposure to Simvastatin Hydroxy Acid by Decreasing Hepatic Organic Anion Transporting Polypeptide Expression

Simvastatin (SIM)-induced myopathy is a dose-dependent adverse drug reaction (ADR) that has been reported to occur in 18.2% of patients receiving a 40- to 80-mg dose. The pharmacokinetics of SIM hydroxy acid (SIMA), the bioactive form of SIM, and the occurrence of SIM-induced myopathy are linked to the function of the organic anion transporting polypeptide (Oatp) hepatic uptake transporters. Genetic polymorphisms in SLCO1B1, the gene for human hepatic OATP1B1, cause decreased elimination of SIMA and increased risk of developing myopathy. Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease, and is known to alter drug transporter expression and drug disposition. The purpose of this study was to assess the metabolism and disposition of SIM in a diet-induced rodent model of NASH. Rats were fed a methionine- and choline-deficient diet for 8 weeks to induce NASH and SIM was administered intravenously. Diet-induced NASH caused increased plasma retention and decreased biliary excretion of SIMA due to decreased protein expression of multiple hepatic Oatps. SIM exhibited increased volume of distribution in NASH as evidenced by increased muscle, decreased plasma, and no change in biliary concentrations. Although Cyp3a and Cyp2c11 proteins were decreased in NASH, no alterations in SIM metabolism were observed. These data, in conjunction with our previous data showing that human NASH causes a coordinated downregulation of hepatic uptake transporters, suggest that NASH-mediated transporter regulation may play a role in altered SIMA disposition and the occurrence of myopathy.

Circulating microRNA 122 in the methionine and choline-deficient mouse model of non-alcoholic steatohepatitis

Non‐alcoholic steatohepatitis (NASH) is a progressive form of non‐alcoholic fatty liver disease (NAFLD) and is a major cause of liver cirrhosis and hepatic failure. The methionine choline‐deficient diet (MCD) is a frequently used hepatotoxicity animal model of NASH that induces hepatic transaminase (ALT, AST) elevations and hepatobiliary histological changes similar to those observed in human NASH. Liver‐specific microRNA‐122 (miR‐122) has been shown as a key regulator of cholesterol and fatty acid metabolism in adult liver, and has recently been proposed as a sensitive and specific circulating biomarker of hepatic injury. The purpose of this study was to assess miR‐122 serum levels in mice receiving an MCD diet for 0, 3, 7, 14, 28 and 56 days and compare the performance vs. routine clinical chemistry when benchmarked against the histopathological liver findings. MiR‐122 levels were quantified in serum using RT‐qPCR. Both miR‐122 and ALT/AST levels were significantly elevated in serum at all timepoints. MiR‐122 levels increased on average by 40‐fold after 3 days of initiating the MCD diet, whereas ALT and AST changes were 4.8‐ and 3.3‐fold, respectively. In general, miR‐122 levels remained elevated across all time points, whereas the ALT/AST increases were less robust but correlated with the progressive severity of NASH as assessed by histopathology. In conclusion, serum levels of miR‐122 can potentially be used as a sensitive biomarker for the early detection of hepatotoxicity and can aid in monitoring the extent of NAFLD‐associated liver injury in mouse efficacy models. Copyright