Cancer Letters. 61 (199’2)

129

129 - 134

Elsevier Scientific Publishers Ireland Ltd.

The effect of ellagic acid on xenobiotic metabolism by cytochrome P-450IIEl and nitrosodimethylamine mutagenicity T. Wilsona, ‘Eppley

68102

for Research

Institute

of Nebraska

Lewisb, K.L. Chab and B. Gold”

M.J.

Medical

in Cancer

Center,

Omaha,

and Allied

Diseases,

NE 68198-6805,

Department

High School,

of Pharmaceutical

124

North

20th

Sciences, Street,

Unioersity Omaha,

NE

(U.S.A.)

(Received

1 September

(Revision

received

(Accepted

1991)

18 September

19 September

1991)

1991)

Summary Ellagic acid (EA) is an inhibitor of the in uitro mutagenicity of N-nitrosodimethylamine (NDMA) in Salmonella typhimurium strain TAlOO using pyrazole-induced rat liver 9000 x g supernatant (S-9/. In order to understand this activity, the effect of EA on the metabolic hydroxylation of 4nitrophenol, a substrate, as is NDMA, for cytochrome P-45011E1 was studied using pyrazole induced rat S-9 and microsomal inhibitory

protein. effect

It is shown

on 4-nitrophenol

that EA has an hydroxylase

This effect on cytochrome P-45011E1 may be responsible, at least in part, for the inhibition of NDMA mutagenicity by EA. with

and

bCentral

both

enzyme

preparations.

Keywords: N-nitrosodimethylamine; ellagic acid; cytochrome P-4501IEl; metabolism; chemoprevention; mutagenicity. Introduction Ellagic acid (EA) inhibits the in vitro and in vivo genotoxicity of a wide variety of chemical Correspondence Nebraska 68195-6805.

to: B. Gold,

Medical Center,

600

Eppley

U.S.A.

0304.3835/92/$05.00

0

1992

Printed and Published in Ireland

Institute,

South 42 Street,

University Omaha,

of NE

carcinogens, including polycyclic aromatic hydrocarbons, N-nitroso compounds, aromatic amines and mycotoxins [ 1,4,5,10,12, 17,20,21,26,27,30]. The mechanism of action has been attributed to interference with metabolic activation and/or DNA adduction [2,3,7,9,21,28]. An inhibitory effect of EA on the mutagenicity of NDMA in the Ames assay (Salmonella typhimurium, strain TAlOO) has been reported [lo]. In that study EA was able to prevent the mutagenic activity of NDMA using pyrazoleinduced rat liver S-9 fraction for metabolic activation. In order to understand the mechanism of action of EA, the effect of EA on metabolism mediated by cytochrome P-450IIE1, the low K, P-450 isozyme responsible for the activation of NDMA [24,29,31], has been investigated using 4-nitrophenol hydroxylation as a monitor of cytochrome P-450IIEl catalytic activity. Because the primary metaboiites generated from NDMA activation, formaldehyde and methanol, are not stable in the presence of S-9 fractions, 4-nitrophenol, a specific marker for the P-450IIEl isozyme t&14,15,25], is used as a surrogate substrate. Due to its solubility characteristics, EA must be dissolved in DMSO or a similar solvent. These solvents invariably are inhibitors, competitive

Elsevier Scientific Publishers Ireland Ltd

130

or otherwise, of cytochrome P-450IIEl and this somewhat complicates, but does not prevent, the analysis of EA’s effect on metabolism. Material

and Methods

Materials EA, DMSO, NDMA, 4-nitrophenol, 4-nitrocatechol, nicotinamide adenine dinucleotide phosphate (NADP), nicotinamide adenine dinucleotide phosphate, reduced (NADPH) and glucose 6-phosphate were purchased from Aldrich Chemicals (Milwaukee, WI), Pierce (Rockford, IL) or Sigma Chemicals (St. Louis, MO). The EA was crystallized from pyridine and dried in vacua. Animal treatment and enzyme isolation S-9 fractions were isolated from pyrazoleinduced (3 x i.p. injection of 150 mg pyrazole/kg per day) male Sprague - Dawley rats (- 200 g) (Sasco Inc., Omaha, NE) [29]. The rats were sacrificed 24 h after the last pyrazole treatment and S-9 fractions prepared [16] and stored in small aliquots at - 8OOC. Protein levels were determined as previously described [19]. Microsomes and the remaining supernatant were prepared from S-9 by low temperature centrifugation at 105 000 X g for 60 min. Metabolism studies The 4-nitrophenol hydroxylase assay followed the methods described by Koop [14] with incubation conditions similar to those used in the Ames assay [lo], in a final volume of 1 ml, water (control), DMSO or EA dissolved in DMSO was added to 25 mM sodium phosphate buffer (pH 7.4) containing 4 mM MgClz, 16.5 mM KCl, 5 mM glucose 6-phosphate, rat liver S-9 and 200 PM 4-nitrophenol (all concentrations are final concentrations). The reactions were initiated by the addition of 4 mM NADP or NADPH and incubation at 37OC. After 10 min the reactions were terminated by cooling in ice, adding 500 ~1 of trichloroacetic acid and vortexing. The

mixtures were centrifuged for 10 min and then 900 ~1 of supernatant removed and added to 100 ~1 of 10 N NaOH. If this solution appeared turbid an additional centrifugation step was included. The full visible spectrum was obtained and the concentration of 4-nitrocatechol calculated based on the AsI nm- AT0s nm and an extinction coefficient of 9650 M-l cm -I. For control purposes all experimental runs included control incubations with no S-9 or no NADP or NADPH. All results (mean f S.D.) are derived from at least three independent determinations and are corrected for background (no S-9 added). In the limited number of experiments with microsomes or 105 000 X g supernatants, 4 mg of protein/ml were used in place of 8 mg of S-9 protein and NADPH was used rather than NADP. Mutagenicity studies Mutagenicity studies were performed as previously described [10,22] with S. typhimurium strain TAlOO and 8.7 mg of pyrazoleinduced male Sprague - Dawley rat liver S-9 per plate. The order of addition was buffer, DMSO or EA in DMSO, bacteria, S-9, NDMA and NADP with a total final volume of 1 ml. After a 20 min incubation at 37OC the top agar was added and the mixture plated. Plates were kept at 37OC for 48 h and colonies counted. Results The metabolic oxidation of 200 FM 4-nitrophenol to 4-nitrocatechol is linear with time up to 20 min (data not shown) and in all experiments detailed below, a 10 min incubation time was used. The appearance of catechol is also dependent on addition of NADP (data not shown) and protein concentration (Fig. 1). The data shown are representative of three pyrazole-induced S-9 preparations obtained from different groups of animals (5 rats/group). As stated above, the conversion of 4-nitrophenol to the corresponding catechol with S-9 was NADP-dependent. However, in the ab-

90

a0 70

0

1

2

3

4 protein

5

6

7

8

0

(mg)

Fig. 1. Protein-dependency and effect of DMSO and EA on p-nitrophenol hydroxylase activity (pmol/min) with pyrazole-induced S-9. 0 , water control; v , 700 mM DMSO; v , 1 mM EA + 700 mM DMSO.

sence of added NADP the yield of catechol was approximately 20% of that formed in the presence of added NADP. There is an inhibition of hydroxylase activity by DMSO with a maximum effect seen between 150 and 250 mM (Fig. 2). When different concentrations of EA are co-administered with 700 mM DMSO there is a clear inhibition of hydroxylase activity (Fig. 3) above that observed with 700 mM DMSO alone (defined as 100% activity). This inhibition is observed at EA concentrations as low as 10 PM EA and appears to reach a maximum inhibition near 50 PM. The effect of S-9 concentration on catechol formation in the presence of 700 mM DMSO or 1 mM EA dissolved in 700 mM DMSO was studied (Fig. 1). The results show that EA has its own inhibitory activity with respect to P-450IIEl catalytic activity at all protein concentrations studied. Since the 4-nitrophenol hydroxylase assay measures steady-state levels of catechol me-

100

200

300

400

[DUSO]

500

600

(mu)

Fig. 2.

Dose-dependent inhibition of p-nitrophenol hydroxylase activity by DMSO with 8 mg/ml of pyrazoleinduced S-9 (100% activity is defined as hydroxylase activity in the absence of DMSO.

J 04

0.05 ellogic

ocld

05

(mM)

Fig. 3. Dose-dependent inhibition of p-nitrophenol hydroxylase activity by EA in the presence of 700 mM DMSO with 8.0 mg/ml of pyrazole-induced S-9 (100% activity is defined as hydroxylase activity in the presence of 700 mM DMSO without any EA).

132

tabolite, it was determined whether DMSO, or EA in DMSO, had any effect on the disappearance of product via secondary metabolism; conjugation of the catechol to the glucuronide has been reported in perfused liver of EtOH treated rats [25]. The recovery of 21 PM 4-nitrocatechol added to the incubation system was 78.0 f 5.2% and was not affected by the addition of 700 mM DMSO or 1 mM EA in DMSO (700 mM). Recovery of catechol in the absence of S-9 enzyme system was 89.7 * 2.0%. Similar incubations were performed using 4 mg/ml of microsomes obtained from the same pyrazole-induced S-9. With 700 mM DMSO the activity was reduced to 173 =t 34 pmol/min per mg protein ( - 20% of control incubations with no DMSO) .The addition of 1 mM EA dropped the rate by an additional - 50% to 82 =t 11 pmol/min per mg protein (- 10% of control). The effect of EA on the mutagenicity of NDMA (50-400 PM) was tested in the Ames assay using strain TAlOO in the presence of 8.7 mg/plate of pyrazole-induced S-9 protein (Fig. 4). The results of three independent ex-

0

50

100

150

200

[NDMA]

Fig. 4. The effect of ty of IV-nitrosodimethyl in the presence of 8.7 water control; v , 700 mM DMSO.

250

300

350

400

(~4)

DMSO and EA on the mutageniciamine in S. typhimurium TAlOO mg of pyrazole-induced S-9. 0 , mM DMSO; v , 1 mM EA + 700

periments, each run in triplicate, consistently show a significant difference between control (water), DMSO (700 mM) and EA (1 mM) + DMSO (700 mM). The potentiality that DMSO or EA could alter the growth of revertant colonies by a mechanism not directly related to metabolism or genotoxicity was addressed by adding them with the top agar after the 20 min incubation period. The results (not shown) indicate that neither agent has an effect on NDMA-induced revertant colony growth. Discussion Because of its activity against a variety of chemical carcinogens and because it is a naturally occurring compound found in relatively high amounts in certain foods [5], EA has attracted a fair amount of scientific interest as a chemopreventive agent. It is still not clear how EA inhibits genotoxicity although one suggested mechanism is its ability to block the metabolic activation of carcinogens, and thereby, decrease DNA damage. The metabolism of NDMA by microsomes yields CHzO as a primary activation metabolite and it is routine to simply measure CHzO formation via hydrazone or semicarbazone formation as an indicator of NDMA metabolism. However, this primary product is not stable in the presence of S-9 and it is further metabolized, at least in part, to CO*. Since the Ames assay used to evaluate the in vitro genotoxicity of nitrosamines and other potential carcinogens usually employs S-9 for the purposes of metabolic activation, it seemed necessary to use a substrate that has the same isozyme NDMA for cytochrome selectivity as P-450IIE1, but which affords a stable metabolic product that would allow studies to assess EA’s effect on metabolism using S-9 fractions. It is well documented that the hydroxylation of 4-nitrophenol to the corresponding catechol is a selective marker for cytochrome P-450IIEl activity [14,15,25] and that the catechol is relatively resistant to secondary metabolism [25]. Pyrazole was used to induce the rats because it enhances the level of

133

the low K, form of NDMA demethylase, without causing a substantial increase in other cytochrome P-450 isozymes [ 11,291. The data presented in this study show that EA has an inhibitory effect on 4-nitrophenol hydroxylase activity, indicating that EA can block cytochrome P-450IIEl-mediated metabolism (Figs. 1,3). This result is observed with either S-9 or microsomal fractions. Because the solvent used to dissolve EA in this study was DMSO and DMSO and related polar solvents are known to be inhibitors of cytochrome P-450 [13,23,32] it is possible that DMSO rather than EA suppressed NDMA metabolism. While the presence of DMSO solvent does tend to mask the activity of EA, based on a molar dose the inhibitory effect of EA on the metabolism of 200 PM 4-nitrophenol is - 3 orders of magnitude greater than that of DMSO (Fig. 3). The inhibition of 4nitrophenol hydroxylase plateaus at -50 - 60% of control in S-9 incubations with either DMSO or EA + DMSO (Fig. 3). In contrast, inhibition of 80 and 90% are seen with DMSO and EA + DMSO, respectively, with microsomal preparations. The origin of this difference is unclear, but 4-nitrophenol hydroxylase activity in the absence of added NADP or NADPH co-factor with S-9 is - 20% of control while it is barely detectable with microsomes in the absence of NADPH. Experiments using the 105 000 x g supernatant (4 mg/ml) indicate that soluble enzymes alone do not catalyze catechol formation (data not shown). It is possible that there is some synergistic effect between soluble and microsomal proteins or that the DMSO and EA bind with and are sequestered by components of the supernatant which results in a lowering of their interaction with cytochrome P-450. At this time it is not known how EA inhibits 4-nitrophenol metabolism, however its activity is related to phase I metabolism since EA does not affect the stability of the catechol product. Finally, using pyrazole-induced S-9 fractions from three separate groups of rats, there is a significant difference between DMSO (700 mM) and 1 mM EA + 700 mM DMSO (Fig. 4). This reconfirms the antimutagenic proper-

ties of EA on NDMA mutagenicity with rat enzyme activation. The previous report that EA had no effect on the mutagenicity of NDMA in the presence of ethanol-induced hamster liver S-9 may be due to the difference in animals, inducing agents and/or experimental methods

Dl. In conclusion, EA has been shown to inhibit cytochrome P-450IIEl mediated metabolism in S-9 enzyme preparations and this effect may account for, at least in part, the decrease in NDMA-mediated mutagenicity. Acknowledgements We gratefully acknowledge Ms. Jan Williamson for the Ames assay results. This work was supported by Tobacco Research Council Grant 2449R1, NIH Minority High School Student Research Apprentice Program Grant RR0 3408 (M.J.L. and K.L.C.), USPHS Laboratory Cancer Support Grant CA36727, and American Cancer Society Core Grant SIG-16. References Barth. D.H. and Fox. C.C. (1988) Selective inhibition of methylbenzylnitrosamine-induced formation of esophageal 06-methylguanine by dietary ellagic acid in rats. Cancer Res.. 48, 7088- 7092. Barth. D.H. and Fox, C.C. (1989) Dietary ellagic acid reduces the esophageal microsomal metabolism of methylbenzylnitrosamine. Cancer Lett.. 44, 39 - 44. Castonguay, A., Allaire. L., Charest. M., Rossignol. G. and Boutet. M. (1989) Metabolism of 4-(methylnitrosamino)-1-(3-pyridylj-l-butanone by hamster respiratory tissues cultured with ellagic acid. Cancer Lett., 46, 93 - 105. Chang, R.L., Huang, M.T.. Wood, A.W.. Wang, C.-Q.. Newmark, H.L., Yagi, H.. Sayer, J.M.. Jerina. D.M. and Conney, A.H. (1985) Effect of ellagic acid and hydroxylated flavonoids on the tumorigenicity of benzo[a]pyrene and (~)-7~,8wdihydroxy-9o,lOwepoxy-7.8.9,lO-tetrahydrobe nzo[a]pyrene on mouse skin in the newborn mouse. Carcinogenesis, 6. 1127 - 1133. Daniel, E.M.. Krupnick, A.S., Heur, Y.-H., Blinzler. J.A., Nims. R.W. and Stoner, G. D. (1990) Extraction, stability, and quantitation of ellagic acid in various fruits and nuts. J. Food Comp. Anal., 2, 338-349. Daniel, E.M. and Stoner, G.D. (1991) The effects of ellagic acid and 13.cis-retinoic acid on N-nitrosobenzylmethylamine-induced esophageal tumongenesis in rats. Cancer

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The effect of ellagic acid on xenobiotic metabolism by cytochrome P-450IIE1 and nitrosodimethylamine mutagenicity.

Ellagic acid (EA) is an inhibitor of the in vitro mutagenicity of N-nitrosodimethylamine (NDMA) in Salmonella typhimurium strain TA100 using pyrazole-...
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