Mechanism of enhancement of esophageal tumorigenesis by 6-phenylhexyl isothiocyanate

CANCER LETTERS Cancer Letters 112 (1997) 119-125

Mechanism of enhancement of esophageal tumorigenesis by 6-phenylhexyl isothiocyanate Mark A. MorseaT*, Jerry Lua, Rajaram Gopalakrishnana, Gary D. Stonera

Lisa A. Petersonc, Gulzar Wanib,

aDivision of Environmental Health Sciences, The Ohio State University School of Public Health, and The Ohio State University Comprehensive Cancer Center, The Ohio State University, CHRI Suite 1148. 300 West Tenth Avenue, Columbus, OH 43210, USA bDivision of Radiobiology, Department of Radiology, The Ohio State University, 103 Wiseman Hall, 400 West Twelfth Avenue, Columbus, OH 43210, USA ‘Division of Chemical Carcinogenesis, American Health Foundation, 1 Dana Road, Valhalla, NY 10595. USA

Received 23 July 1996; revision received 7 November 1996; accepted 8 November 1996

Abstract 6-Phenylhexyl isothiocyanate (PHITC) enhances esophageal tumorigenesis induced by the carcinogen N-nitrosomethylbenzylamine (NMBA) in rats while its shorter chain analog, phenethyl isothiocyanate (PEITC), inhibits NMBA-induced esophageal tumorigenesis. A significant increase in 06-methylguanine levels in esophageal DNA at 72 h after NMBA administration to rats pretreated with PHITC suggested that PHITC might enhance NMBA metabolic activation or inhibit DNA repair. To test this hypothesis, groups of 20 rats were administered PEITC or PHITC at concentrations of 0, 1.0, or 2.5 mmol/kg in modified AIN-76A diet for 2 weeks. The esophagi were removed from rats, stripped, split, and maintained in HEPES buffered saline (HBS) for assays of NMBA metabolism (n = 5 per group) or were snap frozen for DNA repair assays (n = 15 per group). The principal metabolites of NMBA produced by esophageal explants were: two unidentified peaks, benzyl alcohol (at 4 h only), and benzoic acid. Esophageal explants from PEITC-treated animals showed a significantly decreased ability to metabolize NMBA as expected. PHITC-treated animals showed a slight inhibition in the formation of most NMBA-related metabolites, rather than an overall increase in NMBA activation. This inhibition was less than that observed with PEITC. No inhibitory effects were observed on 06-alkylguanine transferase (AGT) activity in the esophagi of rats treated with 1.0 pmol/g or 2.5 pmol/g PHITC. Thus, effects of PHITC on esophageal metabolism and DNA repair do not account for the enhancement of NMBA tumorigenicity by PHITC. 0 1997 Elsevier Science Ireland Ltd. All rights reserved Keywords:

Esophagus; N-Nitrosomethylbenzylamine,

Phenethyl isothiocyanate;

1. Introduction Worldwide, esophageal cancer ranks seventh in incidence in men and women [l]. Major risk factors *Corresponding

author. Tel.: +I 614 2933713; fax: +l 614

2933333.

0304-3835/97/$17.00 PII

isothiocyanate

for esophageal cancer are: smoking [2], alcohol consumption [3,4], and consumption of salt-pickled and moldy foods [5]. Investigations conducted in the Transkei region of South Africa and northern China, areas of high esophageal cancer incidence, indicate that N-nitroso compounds and their precursors may be etiological factors [6,7]. In rats, several asymmetric

0 1997 Elsevier Science Ireland Ltd. All rights reserved

SO304-38j5(96)04556-9

Phenylhexyl

120

M.A. Morse el ~1. I Cancer- Letter-s 112 (1997) 119-125

dialkylnitrosamines are potent inducers of esophageal tumors [8]. Among the most potent of these nitrosamines is N-nitrosomethylbenzylamine (NMBA) [9] (Fig. I). The induction of esophageal tumors by NMBA has proven to be a valuable model for screening the chemopreventive efficacy of several naturallyoccurring compounds, including diallyl sulfide [9], ellagic acid [lo,1 I], green and black tea [12], and phenethyl isothiocyanate [ 13,141. Isothiocyanates are potent inhibitors of nitrosamine-induced tumorigenesis in a variety of animal models. Phenethyl isothiocyanate (PEITC) is an effective inhibitor of esophageal tumorigenesis induced by NMBA when administered in the diet at concentrations of 1.0 pmol/g diet or more [ 13-1.51. When isothiocyanates are tested against the tobaccospecific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)- 1-butanone (NNK) in mouse lung, increasing the

length of the alkyl chain separating the isothiocyanate moiety from the phenyl group results in greater inhibitory efficacy [16,17]. In the rat esophageal tumor model, the structure-activity relationships for inhibition of NMBA-induced tumors by isothiocyanates are different. While PPITC is more potent than PEITC, PBITC has very little effect on esophageal tumorigenesis [ 151. 6-Phenylhexyl isothiocyanate (PHITC) actually enhances NMBA-induced esophageal tumor multiplicity in rats at dietary concentrations of 1.O and 2.5 pmol/g [18]. Associated with this increase in tumor multiplicity is a significant increase in 06methylguanine levels in esophageal DNA at 72 h after NMBA dosing. These data suggested that the increased tumor formation caused by NMBA was due to induction of NMBA activation and/or inhibition of repair of 06-methylguanine. Accordingly, we have investigated the effects of PHITC on the metabolic activation of NMBA and on repair of 06-methylguanine by 06-alkylguanine transferase (AGT). PEITC was included for comparison with PHITC.

CH2-YcH3 N=O

2. Materials

N-nitrosomethylbenzylamine

(NMBA)

and methods

2.1. Chemicals PEITC and DMSO were purchased from Aldrich Chemical Company (Milwaukee, WI). PHITC was synthesized as described [17]. previously [3-3H]NMBA was prepared by reductive tritiation of 3-bromobenzylmethylamine followed by nitrosation as described previously [19]. Unlabeled NMBA was purchased from Ash Stevens (Detroit, MI). [3H]NMethyl-N-nitrosourea was obtained from Amersham (Arlington Heights, IL). All unlabeled chemicals were analyzed for purity by reversed-phase HPLC and were found to be 97-99% pure.

crN=c=s phenethyl

isothiocyanate

(PEITC)

2.2. Animals

N=C=S /

6-phenylhexyl

isothiocyanate

Fig. 1. Structures of NMBA,

(PHITC)

PEITC, and PHITC

All experimental protocols were in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee of the Ohio State University. Five-weekold male F344 rats were purchased from Harlan Sprague-Dawley, and were allowed to acclimate to the animal facility for 2 weeks. Animals were housed three per cage, and maintained under standard condi-

M.A. Morse et al. / Cancer Letters 112 (1997) 119-125

tions (20 f 2°C; 50 + 10% relative humidity; 12-h light/dark cycle). Modified AIN-76A diet (Dyets, Bethlehem, PA) and water were provided ad libitum. Modified AIN-76A diet consists of 20% casein, 0.3% D,L-methionine, 52% corn starch, 13% dextrose, 5% cellulose, 5% corn oil, 3.5% AIN salt mix, 1.O% AIN vitamin mix, and 0.2% choline bitartrate. 2.3. Experimental

design

Groups of twenty 7-S-week-old male rats were fed control diet (AIN-76A from Dyets, Bethlehem, PA) or diets containing 1.0 or 2.5 I.cmol/g PEITC or PHITC for 14 days. After this 2 week feeding period, the esophagi and livers of each animal were removed. Esophagi were stripped, split, and transferred to the laboratory in HEPES buffered saline (HBS) for metabolism assays (n = 5 per group) or were snap frozen for DNA repair assays (n = 15 per group). 2.4. Analysis of NMBA metabolism by cultured esophagi Esophagi were immersed in ethanol for 5- 10 s followed by HBS (without penicillin/streptomycin/fungizone, PSF) for 2-3 min. Tissues were transferred to another dish containing HBS-PSF and then finally, to PFMR4 medium (without fetal bovine serum, FBS). Each dish of PFMR4 medium (without FBS) contained 0 or 10 PM of [3H]NMBA. Samples were incubated for 24 h at 37°C. Aliquots were removed at 4 and at 24 h, filtered through Acrodiscs, and analyzed by reversed-phase HPLC. The HPLC system consisted of an Alcott 738 autosampler, a Waters gradient controller, two Waters 510 pumps, a Burdick and Jackson 0.46 x 15 cm octadecyl column, an Eppendorf column heater, a Waters 484 UV detector, and an IN/US P-Ram radioflow detector. Filtered aliquots were injected on a Burdick and Jackson 5 p Cl8 (4.6 mm x 25 cm) column and eluted with 2.4% acetic acid (pH 3.7) in 20% acetonitrile. The column temperature was maintained at 30°C. The eluent flow rate was 1.5 ml/min and the scintillation cocktail flow rate was 3.0 ml/min. Detection was by UV (254 nm) and flow-through radioactivity. The duration of each chromatographic run was 55 min. Authentic standards of NMBA, benzyl alcohol, benzaldehyde, and benzoic acid were co-injected with aliquots of each sample.

121

2.5. Analysis of esophageal 06-alkylguanine transferase activity Preparation of the alkylated DNA substrate and analyses of AGT activity were performed by modifications of a previously published method [20]. Alkylated substrate for AGT assays was prepared by reaction of DNA with [3H]N-Methyl-N-nitrosourea (MNU) in 100 mM Tris-HCl (pH 7.0). Dialyzed DNA was heated to 70°C overnight to remove 7methyl purines and 3-methyl purines. The protein content of esophageal homogenates was determined by the Bio-Rad protein assay. Each 0.2-ml AGT assay mixture contained 0, 250, or 500 pg of esophageal protein, and 20 ~1 (5200 dpm) of methylated DNA in HEPES buffer; samples were run in triplicate. Following incubation for I h at 37”C, 500 pg of proteinase K was added to two of the three replicates, and vehicle only to the third replicate. The samples were incubated for an additional 2 h. The reaction was terminated by adding carrier DNA, BSA, and ethanol. Following precipitation of DNA and protein, samples were centrifuged at 12 000 x ,g for 30 min. The replicates treated with proteinase K yielded a supernatant that contained small peptide fragments of tritiumlabeled AGT, while the third replicate for each sample served as an indicator of spontaneous release of tritium. The radioactivity in the supematants was quantitated by scintillation counting. 2.6. Statistical analysis Initial tests of homogeneity of variances were performed by Bartlett and Kendall log s’ analysis of variance. Comparisons among groups of the production of individual metabolites or for AGT-specific activities were performed by analysis of variance followed by Newman-Keuls’ ranges test where appropriate. Statistically significant differences among different groups were declared when P-values were less than

0.05. 3. Results 3.1. Effects oj’ PEITC and PHITC on metabolism of NMBA by esophageal explants The major radioactive peaks found by HPLC were

122

M.A. Morse et al. I Cancer Letters I12 (1997) 119-125

Fig. 2. HPLC analysis of NMBA metabolism by rat esophageal explants. Depicted are the results for an untreated esophagus incubated for 4 h with NMBA. Reversed-phase HPLC was performed as indicated in Section 2. Peak 1 and Peak 2 refer to uncharacterized radioactive peaks. Identified peaks co-elute with authentic UV standards as indicated.

two unidentified peaks (1 and 2) as well as peaks that coeluted with benzyl alcohol, benzoic acid, and NMBA (Fig. 2). No significant quantities of benzaldehyde were detected under the present conditions. Aliquots removed 4 h after incubation of PEITC-treated esophagi with NMBA showed inhibition of peak 1 and benzoic acid by 43-47% and 52-.56%, respectively, for the 1.0 and 2.5 pmol/g concentrations of

PHITC (Table 1). Peak 2 was reduced by 38% and 42% by the low and high concentrations of PEITC, while both concentrations of PEITC resulted in a 20% inhibition of the formation of benzyl alcohol, a minor metabolite in this system. Concentrations of unmetabolized NMBA were significantly higher in esophageal explants from PEITC-treated rats than in those of untreated controls. With the exception of enhanced production of benzyl alcohol in PHITC-treated tissues incubated with NMBA for 4 h, there were no indications that PHITC enhanced NMBA metabolism. Instead, PHITC pretreatment tended to inhibit overall NMBA metabolism, albeit to a lesser degree than PEITC . By 24 h, no detectable benzyl alcohol was present in the medium of esophageal explant cultures and almost all of the NMBA had been metabolized (Table 2). As a consequence of the longer incubation time, fewer differences were apparent between the control and isothiocyanate-treated groups. However, in the 2.5 pmol/g PEITC group, concentrations of peak 1 and benzoic acid were significantly lower than those of controls and unmetabolized NMBA levels were significantly higher. There were no significant differences between PHITC-treated groups and controls in the levels of any of the metabolites or of unmetabolized NMBA at 24 h. 3.2. Effects of PEITC and PHITC on esophageal AGT activity The ability of untreated and treated esophageal tissue to repair &-methylguanine is illustrated in Fig. 3. All esophageal tissues displayed repair capacity above

Table 1 Effects of PEITC and PHITC pretreatment Addition

to diet

Metabolite

(nmol/ml

Peak 1 None PElTC (1 .O pmol/g) PEITC (2.5 pmollg) PHITC (1.0 nmol/g) PHITC (2.5 pmollg)

f f f f

metabolism by rat esophageal explants incubated for 4 h”

medium)

NMBA remaining (nmobml)

Peak 2

1.20 f 0.08d 0.69 0.58 0.92 0.98

on NMBA

0.02b 0.08b 0.03' 0.06'

0.80 0.50 0.46 0.58 0.69

+ f k f +

0.06d 0.02b 0.05b 0.03b.C 0.07C.d

Benzyl alcohol

Benzoic acid

0.15 f 0.01b.c 0.12 f O.Olb

2.49 k 0.14d

0.12 f O.Olb 0.17 f 0.01L.d 0.20 k O.Old

1.09 * 0.14b

1.33 * 0.07b 1.89 + 0.08” 2.06 + 0.10”

“Values are mean f SE for five esophagi. b.c.dValues in the same column that have no numerical superscripts in common are statistically determined by ANOVA and Newman-Keuls’ ranges test.

different

4.78 6.70 7.08 5.74 5.50

k 0.26b 5~ 0.09d k 0.29d * o.12c k o.17c

from each other (P < 0.05) as

123

MA. Morse et al. I Cancer Letters I12 (1997) 119-125 Table 2 Effects of PElTC and PHITC pretreatment Addition

to diet

Metabolite

on NMBA

(nmol/ml

Peak 1 None PEITC (1.0 pmol/g) PEITC (2.5 pmol/g) PHITC (1.0 pmol/g) PHITC (2.5 pmollg)

1.96 1.80 1.69 2.05 2.05

+ f + f f

metabolism

NMBA remaining (nmol/ml)

medium) Peak 2

0.08’ 0.03b.c 0.09b 0.07’ 0.07’

by rat esophageal explants incubated for 24 ha

2.49 2.54 2.35 2.59 2.91

f f f f +

0.32b 0.13 0.4Sb 0.23b 0.34b

Benzyl alcohol

Benzoic acid

N.D. N.D. N.D. N.D. N.D.

4.49 4.00 3.41 4.17 3.98

“Values are mean f SE for five esophagi. b.cValues in the same column that have no numerical superscripts in common are statistically determined by ANOVA and Newman-Keuls’ ranges test.

background levels. Pretreatment with PEITC or PHITC at 1.0 or 2.5 pmol/g diet did not affect AGT activity in rat esophageal tissue, although 2.5 pmol/g PEITC produced a 43% reduction of repair capacity in

f * * k +

0.18’ O.OSb.” 0.24b 0.17’ 0.21b.’

different

0.26 0.80 1.76 0.42 0.34

k k f f *

O.lOb 0.09b.C 0.72’ 0.09b 0.14b

from each other (P < 0.05) as

500 pg esophageal protein samples. Further experiments performed with in vitro addition of isothiocyanates indicate that isotbiocyanates had effect on AGT activity (data not shown). 4. Discussion

-0

control

+

1 .O ~md/g

0

2.5 pmol/g PEITC

+

1.0 pmol/g

PHITC

-&

2.5 pmdlg

PHITC

PEITC

200 vg Esophageal

400 Protein

Fig. 3. Effects of PEITC and PHITC on repair of U6-methylguanine in DNA by esophageal AGT. Assays were performed as described in Section 2. Values represent the mean f SE of four pooled esophageal samples (three esophagi per sample). There were no statistically significant differences among groups when compared by analysis of variance.

PEITC induces CYP 2B 1 in rat liver [21,22]. When present in rodent tissues, CYP 2Bl plays a major role in the metabolism of NNK [23,24]. In addition, PEITC is known to be a potent inhibitor of this enzyme [24]. Thus, we hypothesized that the observed increase in NMBA-induced U6-methylguanine formation caused by PHITC could be due to an induction by PHITC of the cytochrome P450 enzyme principally responsible for NMBA metabolism. As our results indicate, PHITC has only a slight inhibitory effect on esophageal metabolism of NMBA. There is no evidence of a substantial increase in any metabolite in esophageal explant cultures with the exception of benzyl alcohol after incubation for 4 h. These results are qualitatively similar to those of Huang et al., who found that PEITC was a more effective inhibitor than PHITC of the metabolism of the asymmetric nitrosamine, N-nitrosomethylamylamine (NMAA), in esophageal microsomes [25]. It is likely that the cytochrome P450 enzymes principally responsible for the activation of NMAA and NMBA are identical. Ultimately, knowledge of the specific cytochrome P450 enzymes (currently unidentified) responsible for NMBA metabolism would be extremely useful in the design of further experiments to probe the effects of isothiocyanates on esophageal metabolism of NMBA.

124

MA. Morse et al. / Cancer Letters I12 (1997) 119-125

Another logical possibility for the observed increase caused by PHITC in esophageal @-methylguanine levels at 72 h after administration of NMBA would have been an effect on esophageal AGT activity. Isothiocyanates can interfere with a variety of protein functions, especially the catalytic activities of various enzymes [26,27]. In many cases, this enzyme inhibition is directly related to covalent binding of the isothiocyanate to a protein [26,27]. The critical amino acid residue of the active site of AGT is a cysteine residue, and isothiocyanates react preferentially with sulfhydryl groups [27-301. To test this hypothesis, we examined the repair capacities of esophagi from rats treated with dietary PHITC or PEITC for 2 weeks. Again, our results indicated that isothiocyanates had little inhibitory effect. The protocol used in our original experiment involved consumption of PHITC-containing diets by rats for the duration of the experiment. Thus, a component of the enhancement of NMBA tumorigenicity by PHITC may reside in the promotion stage of esophageal tumorigenesis. Enhanced arachidonic acid metabolism generally plays a role in the promotional stage of tumorigenesis. Rao et al. [31] demonstrated that PHITC enhances azoxymethane-induced colon tumorigenesis. Furthermore, dietary PHITC increased levels of various cyclooxygenase products, including prostaglandin E,; PHITC also increased production of various lipoxygenase products [31]. Thus, it may be wise to consider the possible effects of PHITC on promotion of esophageal tumorigenesis. However, such effects cannot explain the enhancement of 06methylguanine formation caused by PHITC in NMBA-treated rats. Future experiments should also consider the possible interaction between NMBA and PHITC in the toxicity induced in the esophageal mucosa, a characteristic feature of NMBA tumorigenicity [32].

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[toI

[Ill WI

[I31

2068. Cl41 Morse, M.A., Zu, H.. Galati, A.J., Schmidt, C.J. and Stoner,

Acknowledgements Supported 59887.

by NIH

grants CA-46535

and CA-

[W

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