Effects Of Phenethyl Isothiocyanate Research Articles

Transcriptomic alterations in human prostate cancer cell LNCaP tumor xenograft modulated by dietary phenethyl isothiocyanate

Temporal growth of tumor xenografts in mice on a control diet was compared to mice supplemented daily with 3 µmol/g of the cancer preventive compound phenethyl isothiocyanate. Phenethyl isothiocyanate decreased the rate of tumor growth. The effects of phenethyl isothiocyanate on tumor growth were examined by RNAseq to elucidate molecular changes that may contribute to tumor growth suppression. Bio-informatic analysis of differentially expressed genes identified changes in inflammation and extracellular matrix pathways that were modulated by treatment with phenethyl isothiocyanate. Specific gene expression changes in these pathways included up-regulation of insulin-like growth factor binding protein 3, fibronectin, thyroxine degradation enzyme, and down regulation of integrin beta 6. In addition, feeding phenethyl isothiocyanate induced alternative splicing of gene variants. This study represents the first use of RNAseq to analyze tumors from animals consuming dietary phenethyl isothiocyanate and to identify potential molecular signatures that may explain the cancer protective effect of this compound.

Phenethyl isothiocyanate induces Ca 2+ movement and cytotoxicity in PC3 human prostate cancer cells

The effect of phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived compound, on cytoplasmic free Ca 2+ concentrations ([Ca 2+] i) in prostate cells has not been explored. This study examined whether PEITC altered [Ca 2+] i levels in suspended human prostate PC3 cancer cells loaded with the Ca 2+-sensitive dye fura-2. PEITC caused [Ca 2+] i rises in a concentration-dependent manner with an EC 50 of 192 μM. Removing extracellular Ca 2+ reduced the Ca 2+ signal by 42%. PEITC-induced [Ca 2+] i rise in Ca 2+-containing medium was not affected by modulation of protein kinase C activity, but was inhibited by 90% by the phospholipase A2 inhibitor aristolochic acid (20 μM). In Ca 2+-free medium, the PEITC-induced [Ca 2+] i rise was changed by depleting store Ca 2+ with 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor). Conversely, PEITC pretreatment abolished thapsigargin-induced [Ca 2+] i rise. Chelation of cytosolic Ca 2+ with BAPTA did not reverse the decreased cell viability. Collectively, the data suggest that in PC3 cells, PEITC induced a [Ca 2+] i increase by causing Ca 2+ release from endoplasmic reticulum in a phospholipase A2-dependent fashion and by inducing Ca 2+ influx. PEITC decreased cell viability in a concentration-dependent, Ca 2+-independent manner. Moreover, the effect of allyl isothiocyanate (AITC), benzyl isothiocyanate (BITC) and PEITC on Ca 2+ signaling has also been compared.

Abstract 4624: Cleavage of HER2 by phenethyl isothiocyanate mediates apoptosis in breast cancer cells

Abstract Her2 is a well established marker of breast cancer and found to be overexpressed in around 20-30% of cases. It plays role in cell proliferation, angiogenesis, metastasis and drug resistance leading to poor prognosis and survival. Therefore development of novel agents targeting Her2 is highly imminent. Based on several epidemiological and experimental evidences, isothiocyanates have been shown to have anticancer effects. We evaluated the effect of phenethyl isothiocyanate (PEITC) in breast cancer cells. Treatment with PEITC for 24h significantly suppressed the survival of both MCF-7 (estrogen dependent) and MDA-MB-231 (estrogen independent) cells in a concentration dependent manner with an IC50 of about 12µM and 8µM respectively. Substantial apoptosis by PEITC treatment was observed in both the cell lines. Interestingly, PEITC treatment after 24h drastically reduced Her2 expression. The decrease in Her2 expression by PEITC treatment correlated with the cleavage of Her2. The low molecular weight fragments of Her2 increased with increasing PEITC concentration. Caspase-3 specific inhibitor blocked PEITC-mediated cleavage of Her2 and apoptosis indicating the involvement of caspase-3 in the cleavage of Her2. Her2 fragments have been shown to induce mitochondria-mediated apoptosis. In agreement, our results show the cleavage of Bid and increase in Bax level, whereas the expression of Bcl-XL was down regulated. Further, PEITC treatment resulted in the release of cytochrome c from the mitochondria into the cytosol, activating caspase 9/3 and PARP cleavage. Taken togther, our results suggests that cleavage of Her2 by PEITC activates mitochondrial death pathway in breast cancer cells. Further mechanistic studies are in progress. [Supported in part by R01 grants CA106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4624. doi:10.1158/1538-7445.AM2011-4624

Abstract 5583: Differential effect of phenethyl isothiocyanate on expression of antioxidant defense genes between normal and cancerous human prostate cells

Abstract Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive constituent of cruciferous vegetables (e.g., watercress) as evidenced by efficacy against chemically-induced cancer in experimental rodents. The PEITC is also empowered to inhibit growth of cultured and xenografted human prostate cancer cells, which correlates with apoptotic and autophagic cell death. We have shown previously that reactive oxygen species (ROS) play a critical role in proapoptotic signal transduction by PEITC in human prostate cancer cells. Interestingly, a representative normal human prostate epithelial cell line (PrEC) is significantly more resistant to PEITC-induced apoptosis compared with cancerous prostate cells including PC-3 and LNCaP. However, the mechanism behind this differential response remains elusive. The present study was undertaken to test a hypothesis that differential sensitivity of normal versus cancerous human prostate cells to PEITC-mediated proapoptotic response is determined by differences in ROS production and/or basal or inducible expression of antioxidant defense genes. The PEITC treatment resulted in ROS production in PC-3 human prostate cancer cells as judged by flow cytometry and fluorescence microscopy using a chemical probe (MItoSOX Red) and EPR using a cell permeable sin probe (1-hydroxy-3-methoxy-carbonyl-2,2,5,5-tetramethylpyrrolidine). These effects were not observed in PrEC cells similarly treated with the PEITC. Basal antioxidant defense gene expression signature was strikingly different between PC-3 and PrEC cells. Moreover, PEITC treatment (2.5 μM, 6 h) caused up-regulation of 29 genes and down-regulation of 2 genes in PC-3 cells. Conversely only 4 genes were up-regulated and 10 genes were down-regulated by a similar PEITC treatment in the PrEC cell line. The results of the present study clearly indicate that differential sensitivity of PC-3 versus PrEC cells to proapoptotic effect of PEITC is likely attributable to both differences in ROS production and basal as well as altered expression of antioxidant defense genes. This investigation was supported by the USPHS grant CA101753-07, awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5583. doi:10.1158/1538-7445.AM2011-5583

Phenethyl isothiocyanate exhibits antileukemic activity in vitro and in vivo by inactivation of Akt and activation of JNK pathways

Effects of phenethyl isothiocyanate (PEITC) have been investigated in human leukemia cells (U937, Jurkat, and HL-60) as well as in primary human acute myeloid leukemia (AML) cells in relation to apoptosis and cell signaling events. Exposure of cells to PEITC resulted in pronounced increase in the activation of caspase-3, -8, -9, cleavage/degradation of PARP, and apoptosis in dose- and time-dependent manners. These events were accompanied by the caspase-independent downregulation of Mcl-1, inactivation of Akt, as well as activation of Jun N-terminal kinase (JNK). Inhibition of PI3K/Akt by LY294002 significantly enhanced PEITC-induced apoptosis. Conversely, enforced activation of Akt by a constitutively active Akt construct markedly abrogated PEITC-mediated JNK activation, Mcl-1 downregulation, caspase activation, and apoptosis, and also interruption of the JNK pathway by pharmacological or genetically (e.g., siRNA) attenuated PEITC-induced apoptosis. Finally, administration of PEITC markedly inhibited tumor growth and induced apoptosis in U937 xenograft model in association with inactivation of Akt, activation of JNK, as well as downregulation of Mcl-1. Taken together, these findings represent a novel mechanism by which agents targeting Akt/JNK/Mcl-1 pathway potentiate PEITC lethality in transformed and primary human leukemia cells and inhibitory activity of tumor growth of U937 xenograft model.

Watercress and Water Quality: The Effect of Phenethyl Isothiocyanate on the Mating Behaviour ofGammarus pulex

Watercress releases phenethyl isothiocyanate (PEITC) upon wounding as a defence against herbivores. PEITC levels released from watercress farms are elevated due to cropping, washing, and processing and are thought to lead to adverse effects onGammarus pulexin chalk streams. This study elucidates the sublethal effect of PEITC on reproductive behaviour ofG. pulex, employingex situtests to investigate the disruption of precopular pairing under conditions simulatingin situexposure. Mean time to separation of precopular pairs was 89 ± 6 minutes for watercress wash water (1 g watercress per litre water) and 81 ± 15 minutes for pure PEITC (1 μL/L). Re-exposure to watercress wash water to simulate the pulsed operation at a watercress farm did not alter behavioural response. The repeated interruption of reproductive behaviour underin situconditions would impair long-term reproductive success and could explain in part low abundance ofG. pulexdownstream of watercress farms.

Phenethyl isocyanate is not the metabolite of phenethyl isothiocyanate responsible for mechanism-based inhibition of cytochrome P450

Phenethyl isothiocyanate is a chemopreventive phytochemical present in cruciferous vegetables where it exists as the glucosinolate gluconasturtiin. It is a mechanism-based inhibitor of both rat and human cytochrome P450 enzymes. The principal objective of the present study was to ascertain whether phenethyl isocyanate, formed by the cytochrome P450-mediated oxidative desulphuration of phenethyl isothiocyanate, is the metabolite responsible for the mechanism-based inhibition. Phenethyl isothiocyanate, following incubation with Aroclor 1254-induced rat liver microsomes in the presence of NADPH, markedly suppressed the CYP1A-mediated O-deethylation of ethoxyresorufin; extent of inhibition was directly related to the pre-incubation time and was antagonised by reduced glutathione. When human liver microsomes were used, the inhibitory effect of phenethyl isothiocyanate, which was once again related to the pre-incubation time, was even more pronounced. When the ability of phenethyl isothiocyanate and phenethyl isocyanate to directly inhibit the O-deethylation of ethoxyresorufin in rat microsomes was compared, the latter compound was only moderately more effective. In human microsomes, both compounds were equipotent. In phenobarbital-induced lung microsomes, phenethyl isothiocyanate was a direct and potent inhibitor of the O-depentylation of pentoxyresorufin; pre-incubation of the isothiocyanate had no impact. Human precision-cut liver slices were more effective than rat slices in metabolising phenethyl isothiocyanate. Pre-treatment of rats, however, with phenobarbitone significantly enhanced the metabolism of isothiocyanate. It may be inferred from the present studies that: (a) phenethyl isocyanate is not the metabolite of phenethyl isothiocyanate responsible for its mechanism-based inhibition, and (b) CYP2B is an important catalyst of the metabolism of phenethyl isothiocyanate.

Effect of phenethyl isothiocyanate (PEITC) on the urinary excretion of mutagens in rats treated with IQ (2-amino-3-methyl-3H-imidazo[4,5-f] quinoline)

Effect of phenethyl isothiocyanate (PEITC) on the urinary excretion of mutagens in rats treated with IQ (2-amino-3-methyl-3H-imidazo[4,5-f] quinoline)

Tissue differences in the modulation of rat cytochromes P450 and phase II conjugation systems by dietary doses of phenethyl isothiocyanate

Rats were fed diets supplemented with phenethyl isothiocyanate (PEITC) at 0.06 (low dose, dietary intake level), 0.6 (medium dose) and 6.0 μmole/g (high dose), and xenobiotic-metabolising enzymes were monitored in liver, lung and kidney. At the low dose, inhibition of the hepatic O-dealkylation of ethoxy- and methoxyresorufin was noted, whereas at the high dose increases in the O-depentylation of pentoxyresorufin and O-debenzylation of benzyloxyquinoline were observed, whereas p-nitrophenol hydroxylase was inhibited. Hepatic bioactivation of 2-amino-3-methylimidazo-[4,5- f]quinoline to mutagens was not influenced by the PEITC-treatment. In the lung, at the high dose, ethoxyresorufin dealkylation was elevated and that of pentoxyresorufin suppressed; no significant changes were seen in the kidney. Quinone reductase was markedly elevated at all doses in liver, but the lung enzyme was refractive whereas in the kidney a modest rise was observed at the high dose. Hepatic glutathione S-transferase activity was stimulated by PEITC-treatment, but no effect was evident in the lung or kidney. It is concluded that the effects of PEITC on xenobiotic-metabolising systems are dose- and tissue-dependent, with the liver being the most sensitive and the lung generally resistant. Increased detoxication rather than cytochrome P450 inhibition is the likely mechanism of the chemopreventive activity of PEITC.

Modulating testosterone stimulated prostate growth by phenethyl isothiocyanate via Sp1 and androgen receptor down‐regulation

The effects of phenethyl isothiocyanate (PEITC), present naturally in cruciferous vegetables, on androgen-influenced growth of the prostate such as benign hyperplasia, was investigated. Rats dosed with cyproterone acetate and testosterone, were fed at the same time with either PEITC or vehicle control. The growth of the prostates was compared to untreated rats. While testosterone increased the prostate mass (30%) and hyperplastic seminiferous tubules as compared to the untreated rats, PEITC feeding decreased the prostate mass and hyperplasia to roughly the levels of untreated rats (P < 0.05). PEITC negated the testosterone-mediated enhancement of the androgen receptor (AR), via down-regulating transcription factor Sp1 expression and Sp1 binding complex formation. Cell cycle progression was attenuated with decreases of cyclins, Rb, and up-regulates p27. PEITC modulates the testosterone-influenced growth by repressing Sp1, thus down-regulating AR and proliferation. PEITC from cruciferous vegetables may represent a regulator for hormone-dependent growth of the prostate.

Repression of androgen receptor in prostate cancer cells by phenethyl isothiocyanate

Prostate cancer usually progresses to androgen refractory after an initial anti-androgen treatment. The androgen receptor (AR) is a pivotal factor for the androgen-mediated growth and maintenance of the prostate. Abnormality of the AR, such as overexpression has been postulated to be related to the hormone independent growth of the cancer. Although we previously demonstrated that the AR expression could be modulated by isothiocyanates, which are natural constituents of cruciferous vegetables, the mechanism, however, remained to be clarified. We have since investigated the mechanism of phenethyl isothiocyanate (PEITC) in AR regulation. A human androgen dependent prostate cancer cell line LNCaP (AD) and its sub-line LNCaP (AI), i.e. androgen independent but overexpressing AR, were exposed to PEITC. The effects of PEITC on cell growth and AR expression/transcription were analyzed with MTT assay, real-time PCR and western blotting. The AR promoter activity was analyzed with the reporter activity after transfection with pAR-luc. The effects on Sp1, the major transcription factor of the AR, were tested with Sp1-luc activity, western blotting and electrophoretic mobility shift assay. PEITC induced a significant growth inhibition, with equal IC(50), in both AD and AI cells. The AR present in both cells was repressed as demonstrated with real-time PCR and western blot. PEITC mediates dual effects at transcriptional and post-translational levels to regulate the AR. At transcriptional level the AR level was reduced via inhibition of the transcription factor Sp1, and at post-translational level by accelerating protein degradation. PEITC represses AR transcription and expression, and mediates growth arrest in androgen dependent and independent prostate cancer cells. With the AR modulation and growth attenuation, PEITC and possibly other isothiocyanates, may prevent and inhibit hormone sensitive and refractory prostate cancer.

Identification of Nrf2-regulated genes induced by chemopreventive isothiocyanate PEITC by oligonucleotide microarray

Electrophiles generated during metabolic activation of carcinogens and reactive oxygen species formed from endogenous and exogenous sources might play a significant role in carcinogenesis. Cancer chemoprevention by induction of phase II detoxifying enzymes to counteract the insults of these reactive intermediates is under intensive investigation. Nrf2, a bZIP transcription factor, plays a central role in the regulation of phase II genes by binding to the antioxidant response element (ARE) in their promoters. Identification of novel Nrf2-regulated genes is likely to provide insight into cellular defense systems against the toxicities of electrophiles and oxidants and may define effective targets for achieving cancer chemoprevention. Phenethyl isothiocyanate (PEITC) is a promising chemopreventive agent that exerts its effects by induction of phase II enzymes via activation of Nrf2. In the present study, a transcriptional profile of liver of the wild-type ( Nrf2+/+) and knock-out ( Nrf2−/−) mice after treatments with vehicle or PEITC at 3 h and at 12 h was generated using the Affymetrix Mouse Genome 430 2.0 Array. Comparative analysis of gene expression changes between different treatment groups of wild-type and Nrf2-deficient mice facilitated identification of numerous genes regulated by Nrf2. These Nrf2-dependent and PEITC-inducible genes include known detoxication enzymes, as well as novel xenobiotic-metabolizing genes regulated by Nrf2 such as CYP 2c55, CYP 2u1 and aldehyde oxidase. Unexpected clusters included genes for heat shock proteins, ubiquitin/26 S proteasome subunits, and lipid metabolism molecules. Collectively, the identification of these genes not only provides novel insight into the effect of PEITC on global gene expression and chemoprevention, but also reveals the role of Nrf2 in those processes, which would confer cancer chemopreventive future.

Genotoxic effects of allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC)

Two isothiocyanates (ITCs) commonly found in human diet, allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC), were tested for genotoxic effects in a battery of assays: Salmonella/microsome assay with TA 98 and TA 100, differential DNA repair assay with E. coli and micronucleus (MN) induction assay with human derived Hep G2 cells. Albeit to a different degree, both ITCs induced genotoxic effects in all test systems. AITC was more genotoxic in bacterial test systems than in Hep G2 cells; in contrast, the effect of PEITC was stronger in Hep G2 cells. In in vivo assays with E. coli indicators in which mice were exposed to relatively high doses of the compounds (90 and 270 mg/kg), AITC induced moderate but significant effects; PEITC failed to induce significant effects in any of the organs. To find out the reason for the weak genotoxicity of AITC and PEITC under in vivo test conditions, we exposed E. coli indicator cells to the test substances in the absence or presence of rat liver homogenate (with and without cofactors), bovine serum albumin (BSA) and human saliva. All of them markedly attenuated the genotoxicity of AITC and PEITC, implying that the test substances are detoxified by direct non-enzymatic binding to proteins. Additional experiments carried out on the mechanistic aspects of AITC and PEITC-induced genotoxicity showed that the compounds induce the formation of thiobarbituric acid reactive substances (TBARS) in Hep G2 cells. Furthermore, in in vitro assays with E. coli, radical scavengers reduced the differential DNA damage induced by AITC and PEITC. The latter two findings give a clue that reactive oxygen species might be involved in the genotoxic effect of the ITCs. Although ITCs have been repeatedly advocated as very promising anticancer agents, the data presented here indicate that the compounds are genotoxic, and probably carcinogenic, in their own right.

Effects of phenethyl isothiocyanate and benzyl isothiocyanate, individually and in combination, on lung tumorigenesis induced in A/J mice by benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Phenethyl isothiocyanate (PEITC) is an effective inhibitor of lung tumorigenesis induced in rats and mice by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) while benzyl isothiocyanate (BITC) inhibits lung tumorigenesis induced in mice by another tobacco smoke carcinogen, benzo[a]pyrene (BaP). However, little is known about the inhibitory effects of PEITC and BITC in combination, or about the effects of PEITC or BITC on tumorigenesis by a mixture of NNK and BaP. In this study, we carried out a series of experiments pertinent to these questions. In Experiment 1, treatment of A/J mice with PEITC (6 μmol), BITC (6 μmol), or a combination of the two (6 μmol each) by gavage, 2 h prior to each of eight weekly gavage treatments with a mixture of BaP and NNK (3 μmol of each), had no effect on lung tumor multiplicity. In Experiment 2, we evaluated the inhibitory potential of four different mixtures of PEITC and BITC, administered by gavage 2 h prior to each of eight weekly doses of BaP and NNK, as given in Experiment 1. Mixtures of PEITC and BITC (12 μmol of each, or 12 μmol PEITC and 9 μmol BITC) significantly reduced lung tumorigenesis induced by a mixture of BaP and NNK. In Experiment 3, we investigated the effects of dietary PEITC (3 μmol/g diet), BITC (1 μmol/g diet), or a mixture of PEITC (3 μmol/g diet) and BITC (1 μmol/g diet). These compounds were started 1 week before, and continued through to 1 week after the eight weekly treatments with BaP and NNK. PEITC, and PEITC plus BITC, both significantly inhibited lung tumor multiplicity; inhibition was due mainly to PEITC. In Experiment 4, we tested dietary PEITC (3, 1, or 0.3 μmol/g diet) as an inhibitor of lung tumorigenesis induced by BaP, NNK, or BaP plus NNK using a protocol identical to that in Experiment 3. PEITC was an effective inhibitor of lung tumor multiplicity induced by NNK and a mixture of BaP plus NNK, but not by BaP. Dietary PEITC, or PEITC plus BITC, was more effective in these experiments than the compounds given by gavage. The results of this study demonstrate that proper doses of dietary PEITC and dietary as well as gavaged PEITC plus BITC are effective inhibitors of lung tumorigenesis induced in A/J mice by a mixture of BaP and NNK.

Effects of phenethylisothiocyanate on the expression of glutathione S-transferases and hepatotoxicity induced by acetaminophen

1. The effect of PEITC on the expression of hepatic glutathione S-transferases (GST) and the glutathione (GSH) conjugation has been investigated in the Sprague-Dawley rat, and it has been determined whether hepatotoxicity of acetaminophen (AA) could be inhibited through the induction of GST expression in mouse. 2. The hepatic GST activity and protein levels of alpha class (Ya, Yc) and mu class (Yb1, Yb2) of GST were elevated in a dose-dependent manner after treatment with PEITC (0, 3.16, 10, 31.6, 100 and 200 mg/kg, p.o., 3 days). The mRNA levels of GST Ya and GST Yb1 were also markedly increased 1 day after treatment with PEITC at dosages ranging from 31.6 to 200 mg/kg. The hepatic GSH content was significantly increased to 200% of control at dose of 200 mg/kg PEITC. 3. Pretreatment with 100 mg/kg PEITC significantly enhanced the biliary excretion of glutathione conjugate of AA 2-fold, whereas treatment with 200 mg/kg did not affect it. 4. In mouse, PEITC (100 and 200 mg/kg, 3 days) decreased the lethality and hepatotoxicity caused by AA. 5. These results indicate that (1) the induction of GST by PEITC is presumably under transcriptional regulation, and (2) PEITC may have a protective function against AAinduced hepatotoxicity by induction effect on GST, in combination of enhancement of hepatic GSH.

The effects of phenethyl isothiocyanate, N-acetylcysteine and green tea on tobacco smoke-induced lung tumors in strain A/J mice.

Male and female strain A/J mice were exposed to a mixture of cigarette sidestream and mainstream smoke at a chamber concentration of total suspended particulates of 82.5 mg/m3. Exposure time was 6 h/day, 5 days/week for 5 months. The animals were allowed to recover for another 4 months in filtered air before sacrifice and lung tumor count. Male animals were fed either 0.2% N-acetylcysteine (NAC) or 0.05% phenethyl isothiocyanate (PEITC) in diet AIN-76A with 5% corn oil added. Female animals received normal laboratory chow and were given a 1.25% extract of green tea in the drinking water. Corresponding control groups were fed diets without NAC or PEITC or given plain tap water. Exposure to tobacco smoke increased lung tumor multiplicity to 1.1-1.6 tumors/lung, significantly higher than control values (0.5-1.0 tumors/lung). None of the putative chemopreventive agents (NAC, PEITC or green tea extract) had a protective effect. In positive control experiments, PEITC significantly reduced both lung tumor multiplicity and incidence in mice treated with the tobacco smoke-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In mice treated with three different doses of urethan and fed NAC in the diet, a significant reduction in lung tumor multiplicity was found only at one dose level. Green tea extract did not reduce lung tumor multiplicity in animals treated with a single dose of NNK. It was concluded that successful chemoprevention of tobacco smoke-induced lung tumorigenesis might require administration of several chemopreventive agents rather than just a single one.

Effects of Phenethyl Isothiocyanate on Acetaminophen Metabolism and Hepatotoxicity in Mice

Phenethyl isothiocyanate (PEITC), a compound derived from cruciferous and other vegetables, is a potent inhibitor of cytochrome P450 2E1. This enzyme catalyzes the bioactivation of acetaminophen (APAP) and many other xenobiotics. The present study investigated the effects of PEITC on APAP metabolism and associated hepatotoxicity in Swiss–Webster mice. When PEITC (19–150 μmol/kg) was given to mice intragastrically 1 hr before or immediately prior to a toxic dose of APAP, the APAP-induced hepatotoxicity was significantly decreased or was completely prevented. The extent of toxicity was evaluated by mortality, serum levels of glutamic–pyruvic transaminase, lactate dehydrogenase, and liver histopathology. Pretreatment of mice with ethanol enhanced APAP hepatotoxicity; this enhanced toxicity could also be prevented by the administration of PEITC. PEITC treatment prevented the depletion of hepatic glutathione levels caused by oxidized APAP metabolites. PEITC treatment also significantly decreased the plasma levels of oxidized APAP metabolites (analyzed as APAP–glutathione, APAP–cysteine, and APAP–N-acetylcysteine) and reduced the urinary excretion of APAP–cysteine. In microsomal incubations, PEITC effectively inhibited the rate of APAP–glutathione formation from APAP as well as the P450 2E1-dependentN-nitrosodimethylamine demethylase and the P450 1A2-dependent ethoxyresorufinO-deethylase activities. The protective action of PEITC against APAP toxicity is attributed to the blocking of APAP activation through inhibition of P450 enzymes.

Effects of phenethyl isothiocyanate on metabolism and on genotoxicity of dimethylnitrosamine and 2-amino-1-methyl-6-phenylimidazo[4, 5− b]pyridine (PhIP)

Phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables, inhibited the genotoxic effects of N-nitrosodimethylamine (DMN) and of 2-amino-1-methyl-6-phenylimidazo[4,5− b]pyridine (PhIP) in differential DNA repair assays with E. coli K−12 strains in vitro and in animal mediated assays with mice. In Salmonella typhimurium, the mutagenic activities of DMN and PhIP measured after activation with S−9 homogenates from several organs of PEITC-treated mice were substantially lower than those obtained with homogenates of untreated animals as well. PEITC also reduced the formatio of micronuclei by DMN in metabolically competent Hep-G-2 cells of human origin but was ineffective in combination with PhIP. Biochemical investigations showed that the prevention of genotoxic effects of DMN by PEITC results from an inhibition of its α-hydroxylation. The effect of oral administration of PEITC on the formation of DNA adducts of PhIP was examined in the colon and liver of mice. No inhibition of adduct formation was observed in these experiments. Biochemical experiments showed that PEITC reduces not only the metabolic activation of PhIP via 2-hydroxyamino PhIP but also inhibits a detoxification pathway (formation of 4-hydroxy PhIP). The present results can be taken as an indication that the anticarcinogenic activities of isothiocyanates towards nitrosamines are paralleled by antimutagenic effects, and that probably no such protective effects occur in combination with heterocyclic amines. Furthermore, our findings show that the effects of chemopreventive agents demonstrated in bacteria in vitro cannot always be extrapolated to reactions occurring in intact mammalian cells.

Effects of phenethyl isothiocyanate, a carcinogenesis inhibitor, on xenobiotic-metabolizing enzymes and nitrosamine metabolism in rats.

Phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables, has been shown to inhibit chemical carcinogenesis, possibly due to its ability to block the activation or to enhance the detoxification of chemical carcinogens. The present study was conducted to elucidate the biochemical mechanisms involved by characterizing the effects of PEITC on phase I and phase II xenobiotic-metabolizing enzymes. A single dose of PEITC to F344 rats (1 mmol/kg) decreased the liver N-nitrosodimethylamine demethylase (NDMAd) activity (mainly due to P450 2E1) by 80% at 2 h and the activity of NDMAd remained decreased by 40% at 48 h after treatment. The liver pentoxyresorufin O-dealkylase (PROD) activity and P450 2B1 protein level were elevated 10- and 7-fold at 24 h after treatment respectively. The liver microsomal ethoxyresorufin O-dealkylase (EROD) (mainly due to P450 1A) and erythromycin N-demethylase (mainly due to P450 3A) activities were decreased at 2-12 h after treatment and recovered afterwards. The lung microsomal PROD and EROD activities were not significantly affected; whereas, the nasal microsomal PROD and EROD activities were decreased by 40-50%. After a treatment with PEITC, the rates of oxidative metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were decreased in liver microsomes by 40-60% at 2 h and recovered gradually; the rates in lung microsomes were markedly decreased by 60-70% at 2 h and remained at the decreased level at 24 h; and the rates in nasal mucosa microsomes were decreased gradually with the lowest activities observed at 18 h (50%) followed by a gradual recovery. Furthermore, the treatment with PEITC resulted in a maximal 5-fold increase of NAD(P)H:quinone oxidoreductase and 1.5-fold increase of glutathione S-transferase activities in the liver, but the activities of these two enzymes were not significantly affected in the lung and nasal mucosa. The sulfotransferase activity in the liver was decreased by 32-48% at 24-48 h after treatment; the nasal activity was increased by 1.8- to 2.5-fold, but the lung activity was not significantly changed. The hepatic UDP glucuronosyltransferase activity was slightly decreased at 2 h but slightly increased at 48 h after treatment, but no changes were observed for the lung and nasal activities. The study demonstrates that PEITC selectively affects xenobiotic-metabolizing enzymes in the liver, lung and nasal mucosa and it is especially effective in inhibiting the P450-dependent oxidation of NNK in the lung and of NDMA in the liver.

Inhibition of repairable DNA-damage in Escherichia coli K-12 cells recovered from various organs of nitrosamine-treated mice by vitamin A, phenethylisothiocyanate, oleic acid and triolein.

The influence of various dietary constituents--phenethylisothiocyanate (PEITC), oleic acid (OA), triolein (TO), and vitamin A (ROL)--on the genotoxic activity of nitrosamines (NDMA, NDELA, NPYR) was investigated. For this purpose differential DNA repair assays with Escherichia coli K-12 strains were performed in vitro and in vivo with mice. Under in vitro conditions (liquid holding), all compounds reduced nitrosamine induced DNA-damage in the indicator bacteria in the dose range 1-10 micrograms/ml, the ranking order of efficiency being PEITC greater than OA greater than ROL greater than or equal to TO. In animal-mediated assays, acute oral treatment with PEITC (17-150 mg/kg), 2 h before nitrosamine administration, resulted in a marked decrease of nitrosamine genotoxicity in liver, kidneys, lungs and in the blood. Also in other organs (spleen, testes) an increase in differential survival (which serves as a measure for repairable DNA damage) occurred. With ROL only a comparatively moderate antigenotoxic effect was obtained at a high dose level (250 mg/kg) under identical experimental conditions. OA (2000 mg/kg) and TO (16,000 mg/kg) were completely inactive. Upon repeated treatment (consecutive oral administration of the putative antigenotoxins over 4 days, a final treatment 24 h before nitrosamine administration) PEITC (150 mg/kg/day), ROL (80 mg/kg/day) and OA (2000 mg/kg/day) had no influence on the genotoxic effects of the nitrosamines. Repeated treatment with TO (4000-16,000 mg/kg/day) resulted in a moderate dose-dependent reduction of NDMA-induced DNA-damage in the indicator bacteria, whereas in combination with NPYR only a marginal effect was observed. Biochemical experiments indicated that the antigenotoxic effects of PEITC seen under in vivo conditions were due to inhibition of alpha-hydroxylation of the nitrosamines, whereas ROL and TO appeared not to interfere strongly with this metabolic activation step. Our results indicate that in vitro assays do only partly reflect the antigenotoxic properties of the different food constituents in vivo and that animal-mediated DNA repair assays with E. coli strains are an appropriate approach to study the effects of modifiers of nitrosamine genotoxicity in the living animal.