Effects of alkyl chain length on the inhibition of NNK-induced lung neoplasia in A/J mice by arylalkyl isothiocyanates

Six homologous arylalkyl isothiocyanates were evaluated fortheir abilities to inhibit pulmonary adenomas induced by thetobacco-specific nitrosamine 4-(methylnitrosamino)-l-(3-pyridyl)-1-butanone(NNK) in A/J mice. Four consecutive daily doses (5 µmol/mouse)of phenyl isothiocyanate (PITC), benzyl isothiocyanate (BITC),phenethyl isothiocyanate (PETTC), 3-phenylpropyl isothiocyanate(PPITC), 4-phenylbutyl isothiocyanate (PBITC), 4-oxo-4-(3-pyridyl)-butylisothiocyanate (OPBITC) and corn ofl were administered to miceby gavage. Two hours following the final dosing, mice were administeredsaline or 10 µmol of NNK in saline i.p. Pulmonary adenomaswere counted at 16 weeks after NNK administration. The miceadministered only corn oil prior to NNK developed an averagemultiplicity of 9.2 tumors/ mouse. Pretreatment with PITC, BITCand OPBITC had no significant effects on NNK-induced lung neoplasia.However, PEITC pretreatment resulted in a 64% reduction of lungtumor multiplicity, but did not affect the percentage of micethat developed tumors. Both PPITC and PBITC decreased tumormultiplicity by 96% and the percentage of tumor-bearing animalsby >60%. These results, in conjunction with our previouswork, demonstrate a general trend of increasing inhibition ofNNK-induced lung neoplasia by arylalkyl isothiocyanates withincreasing alkyl chain length. This study also demonstratesthe remarkable inhibitory activities of PPITC and PBITC, twoisothiocyanates that had not previously been tested as chemopreventiveagents.     

Urinary total isothiocyanates and colorectal cancer: a prospective study of men in Shanghai, China.

Laboratory and epidemiologic evidence suggests that dietary isothiocyanates (ITCs) may have a chemopreventive effect on cancer. Humans are exposed to ITCs primarily through ingestion of cruciferous vegetables that contain glucosinolates, the precursors to ITCs. The association between urinary total ITC level and colorectal cancer risk was examined in a cohort of 18,244 men in Shanghai, China, with 16 years of follow-up. Urinary total ITCs were quantified on 225 incident cases of colorectal cancer and 1,119 matched controls. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) were calculated using logistic regression models. High levels of urinary total ITCs were associated with a reduced risk of colorectal cancer 5 years after baseline measurements of ITCs, whereas a statistically nonsignificant increase in the risk of colorectal cancer was observed for cases within 5 years of post-enrollment (OR, 1.93; 95% CI, 0.85-4.39 for the upper three quartiles of urinary ITCs versus the lowest quartile). The inverse ITC-colorectal cancer association became stronger with a longer duration of follow-up. Compared with the first quartile, ORs (95% CIs) for the second, third, and fourth quartiles of total ITCs in urine collected 10 or more years before cancer diagnosis were 0.61 (0.35-1.05), 0.51 (0.29-0.92), and 0.46 (0.25-0.83), respectively, for risk of colorectal cancer (P for trend = 0.006). The present study suggests that dietary ITCs may exert tumor inhibitory effects, especially during earlier stages of the multistage process of carcinogenesis.     

Inhibition of rat liver cytochrome P450 isozymes by isothiocyanates and their conjugates: a structure-activity relationship study

A series of arylalkyl and alkyl isothiocyanates, and their glutathione,cysteine, and N-acetylcysteine conjugates were used to studytheir inhibitory activity toward the dealkylation of ethoxyresorufin(EROD), pentoxyresorufin (PROD), and methoxyresorufin (MROD)in liver microsomes obtained from the 3-methylcholanthrene orphenobarbital-treated rats. These reactions are predominantlymediated by cytochrome P450 (P450) isozymes 1A1 and 1A2, 2B1and 1A2, respectively. All isothiocyanates inhibited PROD morereadily than EROD. Increases in the alkyl chain length of arylalkylisothiocyanates to C₆ resulted in an increased inhibitory potencyin these assays; at longer alkyl chain lengths (C₈-C₁₀) theinhibitory potency declined. The IC₅₀s for phenethyl isothiocyanate(PEITC) were 47, 46 and 1.8 µM for EROD, MROD and PROD,respectively. Substitution of an additional phenyl group onPEITC also increased the inhibitory potency; the IC₅₀s for 1,2-diphenylethyl isothiocyanate (1, 2-DPEITC) and 2, 2-diphenylethylisothiocyanate (2,2-DPEITC) were 0.9 and 0.26 µM for EROD,and 0.045 and 0.13 µM for PROD, respectively. The relativeinhibitory potency of PEITC and its conjugates was N-acetylcysteine-PEITC(PEITC-NAC) < glutathione-PEITC (PEITC-GSH) < cysteine-PEITC(PEITC-CYS) < PEITC. The observations that the parent isothiocyanateswere more potent inhibitors than the conjugates suggest thatdissociation of the conjugate is required for activity. Naturallyoccurring alkyl isothiocyanates, sulforaphane (SFO) and allylisothiocyanate (AITC), were very weak inhibitors in the assays.These results suggest the potential of isothiocyanates as structuralprobes for studying P450 isozymes. In addition, the inhibitoryactivity of isothiocyanates for PROD correlated with the previouslydemonstrated tumor inhibitory potency in (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) induced A/J mouse lung tumor bioassays, which supportsearlier findings that P450 2B1 is one of the major isozymesinvolved in NNK activation and that inhibition of this isozymeis an important mechanism for the chemopreventive activity ofisothiocyanates.     

The essential role of the functional group in alkyl isothiocyanates for inhibition of tobacco nitrosamine-induced lung tumorigenesis

The importance of the isothiocyanate group in alkyl isothiocyanatefor inhibition of tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)-induced lung tumorigenesis was examined in A/J mice. Ourprevious structure-activity relationship study of isothiocyanatesshowed that 1-dodecyl isothiocyanate [CH₃(CH₂)₁₁NCS], a simplealkyl isothiocyanate, is a potent inhibitor of NNK-induced lungtumorigenesis. It was chosenfor this study due to its structuralfeatures and potency. A single dose of 1-dodecyl isothiocyanategiven by gavage at 1 µmol/mouse 2 h prior to NNK administrationcompletely inhibited lung tumorigenesis, while removal of theisothiocyanate group or replacing it with a hydroxyl group abolishedthe inhibitory activity. These results demonstrate that theisothiocyanate functional group is critical for the inhibitoryactivity of isothiocyanates in NNK-induced lung tumorigenesis.To gain more insights into the relationship of in vivo inhibitionof tumorigenesis with the cytochrome P-450 enzyme inhibitoryactivity, the effects of these compounds on metabolism of NNKin mouse lung microsomes were studied. 1-Dodecyl isothiocyanateinhibited all three known oxidative pathways of NNK metabolism,with a stronger inhibitory activity toward NNK N-oxidation (IC₅₀430 nM) and keto alcohol formation (IC₅₀ 500 nM) than keto aldehydeformation (IC₅₀ 13 000 nM). 1-Dodecanol had a similar selectivityin inhibition of these metabolic pathways, but was less potentthan 1-dodecyl isothiocyanate. Dodecane showed little or noinhibitory activity in the same concentration range. These resultsindicate that the isothiocyanate group of 1-dodecyl isothiocyanateis important for inhibition of NNK-induced lung tumorigenesisand also for effective inhibition of cytochrome P-450 enzymesinvolved in NNK oxidation.     

A novel mechanism of chemoprotection by sulforaphane: inhibition of histone deacetylase

Sulforaphane (SFN), a compound found at high levels in broccoli and broccoli sprouts, is a potent inducer of phase 2 detoxification enzymes and inhibits tumorigenesis in animal models. SFN also has a marked effect on cell cycle checkpoint controls and cell survival and/or apoptosis in various cancer cells, through mechanisms that are poorly understood. We tested the hypothesis that SFN acts as an inhibitor of histone deacetylase (HDAC). In human embryonic kidney 293 cells, SFN dose-dependently increased the activity of a beta-catenin-responsive reporter (TOPflash), without altering beta-catenin or HDAC protein levels. Cytoplasmic and nuclear extracts from these cells had diminished HDAC activity, and both global and localized histone acetylation was increased, compared with untreated controls. Studies with SFN and with media from SFN-treated cells indicated that the parent compound was not responsible for the inhibition of HDAC, and this was confirmed using an inhibitor of glutathione S-transferase, which blocked the first step in the metabolism of SFN, via the mercapturic acid pathway. Whereas SFN and its glutathione conjugate (SFN-GSH) had little or no effect, the two major metabolites SFN-cysteine and SFN-N-acetylcysteine were effective HDAC inhibitors in vitro. Finally, several of these findings were recapitulated in HCT116 human colorectal cancer cells: SFN dose-dependently increased TOPflash reporter activity and inhibited HDAC activity, there was an increase in acetylated histones and in p21(Cip1/Waf1), and chromatin immunoprecipitation assays revealed an increase in acetylated histones bound to the P21 promoter. Collectively, these findings suggest that SFN may be effective as a tumor-suppressing agent and as a chemotherapeutic agent, alone or in combination with other HDAC inhibitors currently undergoing clinical trials.     

Oral cytology assessment by flow cytometry of DNA adducts, aneuploidy, proliferation and apoptosis shows differences between smokers and non-smokers

Oral cytology and morphometric staining is used to identify malignant keratinocytes in oral premalignant or malignant lesions. To detect and to begin to assess changes in oral keratinocytes exposed to tobacco-derived carcinogens, which are at risk for malignant transformation, a novel method is required. The approach uses oral cytology harvested oral keratinocytes analyzed using flow cytometry (FC) for changes in DNA content, damage, cell cycle and apoptosis. Six smoker and six non-smoker oral keratinocytes were evaluated using flow cytometry in the form of laser scanning cytometry (LSC) and laser microdissection (LMD). Among smokers compared to non-smokers, the method detected and assessed DNA damage from tobacco smoke exposure quantifying an enhanced formation of DNA adducts, such as, 8-hydroxy-2′-deoxyguanine (8-OHdG) which creates oxidation lesions and benzo[a]pyrene(B[a]P), which produces a B[a]P)-N2-dG bulky adduct. Increased DNA content, aneuploidy, percentage of cells in synthesis (S) and G₂+Mitosis (M), and apoptosis were recorded. Tissue and cell controls were used to verify these relationships. Data suggested healthy smokers were at increased risk for malignant transformation of oral keratinocytes because of the changes stated above. Using identical methods, keratinocytes exposed to the tobacco derived carcinogen, B[a]P parallel results obtained from smoke exposure indicating a direct link. Flow cytometric evaluation of oral cytology harvested keratinocytes can be used to measure exposure to tobacco carcinogens, and possibly establish a link to premalignant and malignant transformation before a lesion is noted.     

Glutathione depletion enhances the formation of endogenous cyclic DNA adducts derived from t-4-hydroxy-2-nonenal in rat liver

Earlier, we detected the cyclic adducts of deoxyguanosine (dG) derived from t-4-hydroxy-2-nonenal (HNE), a long chain alpha,beta-unsaturated aldehyde (enal) product from oxidation of omega-6 polyunsaturated fatty acids, in tissue DNA of rats and humans as endogenous DNA damage. Recent evidence implicates the cyclic HNE adducts in human liver carcinogenesis. Because glutathione (GSH) protects against oxidative stress, we undertook a study to examine the effect of GSH depletion on the HNE-derived cyclic adducts in vivo. Four F344 rats were administered L-buthionine-(S,R)-sulfoximine (BSO), a potent inhibitor of GSH biosynthesis, at 10 mM in drinking water for 2 weeks. Rats in the control group were given water only. Livers were harvested, and each liver was divided into portions for GSH and DNA adduct analyses. The BSO treatment depleted hepatic GSH by 77%; the GSH levels were reduced from 6.3 +/- 0.3 in the control rats to 1.5 +/- 0.1 micromol/g tissues in the treated group. The formation of HNE-dG adducts, analyzed by an HPLC-based 32P-postlabeling assay, was increased by 4-fold, from 6.2 +/- 2.2 nmol/mol dG in liver DNA of control rats to 28.5 +/- 16.1 nmol/mol dG in the rats treated with BSO (p <0.05). The formation of 8-oxodG in liver DNA was also increased as a result of BSO treatment, although the increase was not statistically significant. These results further support the endogenous origin of HNE-dG adducts and, more importantly, indicate a critical role that GSH plays in protecting against in vivo formation of the promutagenic cyclic DNA adducts derived from HNE.     

Tea and cancer prevention: studies in animals and humans

The role of tea in protection against cancer has been supported by ample evidence from studies in cell culture and animal models. However, epidemiological studies have generated inconsistent results, some of which associated tea with reduced risk of cancer, whereas others found that tea lacks protective activity against certain human cancers. These results raise questions about the actual role of tea in human cancer that needs to be addressed. This article is intended to provide a better perspective on this controversy by summarizing the laboratory studies in animals and humans with emphasis on animal tumor bioassays on skin, lung, mammary glands and colon, and the molecular and cellular mechanisms affected by tea. Finally, a recent small pilot intervention study with green tea in smokers is presented.     

Formation of the cyclic 1,N2-glyoxal-deoxyguanosine adduct upon reaction of N-nitroso-2-hydroxymorpholine with deoxyguanosine

N-nitroso-2-hydroxymorpholine, a mutagenic metabolite of N-nitrosomorpholineand N-nitrosodiethanolamine, reacted with deoxyguanosine atpH 7, 37°C, to give 3-(2-deoxy-ß-D-erythropentofuranosyI)-6,7-dihydro-6, 7-dihydroxyimidazo-[1, 2-a]purine-9(3H)one, whichis also formed by reaction of deoxyguanosine with glyoxal. Theresults suggest that this adduct might be involved in DNA bindingby N-nitrosomorpholine and N-nitrosodiethanolamine.     

Inhibition by phenylethyl and phenylhexyl isothiocyanate of metabolism of and DNA methylation by N-nitrosomethylamylamine in rats

We investigated the effect of 2-phenylethyl and 6-phenylhexylisothiocyanate (PEITC and PHITC) on the metabolism of the ratesophageal carcinogen, N-nitrosomethylamylamine (NMAA). PEITCwas administered orally to MRC-Wistar rats as single doses of0.1 or 1.0 mmol/kg, or by other regimens. When esophagi andliver slices from the treated rats were incubated with 23 µMNMAA, the formation of 2-to 5-hydroxy-NMAA was inhibited by45–90% for esophagus and by 14–19% for liver slices.In contrast, when esophagi and liver slices from untreated MRC-Wistarrats were incubated in vitro with NMAA and 10 µM PEITC,the PEITC inhibited hydroxy-NMAA formation similarly (by 79–89%)in the two tissues. Also, PEITC inhibited the formation fromNMAA of the hydroxy-NMAAs, formaldehyde and pentaldehyde byesophageal and liver microsomes to similar extents. In studieson DNA methylation by NMAA, 7- and O⁶-methylguanine (O⁶-MeG)were determined by HPLC with fluorimetric detection. Guaninemethylation in esophageal and liver DNA was generally closeto linear for doses of 5–50 mg NMAA/kg. With 50 mg NMAA/kg,guanine methylation in esophageal and liver DNA peaked after5 h, and 8–11% of the peak O⁶-MeG persisted after 72 h.A single dose of 0.1 or 1.0 mmol PEITC/kg reduced the O⁶-MeGlevels by 44–51% in the esophagus but by only 7–22%in the liver. Administration of the PEITC homolog, PHITC, inhibitedNMAA metabolism by liver slices from the treated rats and themethylation of guanine in liver DNA, but had little effect inthe esophagus, i.e. PHITC tended to have the opposite tissuespecificity to PEITC. The finding that administration of PEITCspecifically inhibited NMAA metabolism in the rat esophagussupports the view that PEITC may be a useful chemopreventiveagent against esophageal carcinogenesis in humans.