GASTROENTEROLOGY
1992;103:800-806
Inhibition of Rat Methylbenzylnitrosamine Metabolism by Dietary-Zinc and Zinc In Vitro DAVID H. BARCH, COLLEEN C. FOX, WILLIAM A. ROSCHE, LYNN M. RUNDHAUGEN, and STEVEN A. WRIGHTON Department of Medicine, Northwestern University Medical School and the Lake Side Veterans Administration Hospital, Chicago, Illinois; and Department of Drug Metabolism, Lilly Research Laboratories,
Eli Lilly & Company, Indianapolis, Indiana
Methylbenzylnitrosamine is an esophageal-specific carcinogen in the rat, and the incidence of methylbenzylnitrosamine-induced esophageal carcinoma is increased by dietary zinc deficiency. Methylbenzylnitrosamine requires activation by cytochrome P-450 to be mutagenic; the present study examined the role of dietary zinc deficiency and the in vitro addition of zinc on the cytochrome P-&&dependent microsomal metabolism of methylbenzylnitrosamine. Dietary zinc deficiency significantly increased the cytochrome P-&o-dependent esophageal and hepatic microsomal metabolism of methylbenzylnitrosamine. These changes occurred without alteration in the specific content of total microsomal cytochrome P-450 of the esophagus or liver. The addition of zinc in vitro, at concentrations found in normal tissues, irreversibly inhibited the esophageal and hepatic cytochrome P-450-dependent microsomal metabolism of methylbenzylnitrosamine. These results suggest that physiological levels of zinc may be an endogenous inhibitor of methylbenzylnitrosamine metabolism. Dietary zinc deficiency appears to reduce this inhibition of cytochrome P-450 activity, resulting in an increase in carcinogen activation.
E
xperimental studies have shown that methylbenzylnitrosamine (MBzN) is an esophageal-specific carcinogen in the raF3 and that rats raised on zinc-deficient diets have an increased incidence of MBzN-induced esophageal carcinoma compared with rats raised on zinc control diets.4-” MBzN is known to require cytochrome P-450-dependent activation to become mutagenic,7,8 and dietary zinc deficiency has been shown to increase the cytochrome P-450-dependent esophageal and hepatic microsomal metabolism of MBzN.‘This increase in the metabolism of MBzN is associated with an increase in MBzN-induced methylation of esophageal DNA in vivo.g Although the cytochrome P-450 enzyme responsible for the activation of MBzN has not been identified, MBzN is not activated by cytochrome
P-45011E1,‘0~” which activates the related hepatic carcinogen dimethylnitrosamine (DMN).12,13Esophageal epithelial cells alone are sufficient for the activation of MBzN, because MBzN can transform isolated esophageal epithelial cells in culture to a tumorigenic phenotype.14 In the present study we report that dietary zinc deficiency increases the microsomal metabolism of MBzN and DMN and that these changes occur without alterations in the total microsomal cytochrome P-450 of the esophagus or liver and without apparent alteration in the hepatic concentration of cytochrome P-450IIEl. The addition of zinc in vitro irreversibly inhibited the esophageal and hepatic cytochrome P-450-dependent microsomal metabolism of MBzN and the hepatic microsomal metabolism of DMN. This inhibition was concentration dependent, and significant inhibition was observed with concentrations of zinc found in normal tissues. Materials and Methods Animals Weanling, 21-day-old, male Sprague-Dawley rats purchased from Charles River (Portage, MI) were randomly assigned to a zinc-deficient diet [2.3 parts per million (ppm) zinc] or a zinc control diet (50 ppm zinc). These diets, prepared by Teklad (Madison, WI), contain the following components in grams per kilogram of diet; egg white solids, 200, DL-methionine, 1.5; dextrose monohydrate, 632.15; corn oil, 100; cellulose fiber, 30.0; sodium fluoride, 0.0022; chromium chloride, 0.002; sodium molybdate, 0.0001; Teklad Vitamin Mix TD 40060, 10.0; choline dihydrogen citrate, 0.655; biotin, 0.0046; and Teklad Mineral Mix TD81264-zinc deficient, 25.69. Zinc carbonate was added to yield a final concentration of 2.3 ppm or 50 ppm. The Teklad Vitamin Mix contains the following in grams per kilogram of mix: p-aminobenzoic acid, 11.0132; ascorbic acid, 101.66; biotin, 0.0441; vitamin B,, (0.1% trituration in mannitol), 2.974; calcium pantothenate, 6.608; This is a US. government work. There are no restrictions its use. 0016-5085/92/$0.00
on
September 1992
INHIBITION OF NITROSAMINE METABOLISM BY ZINC 801
choline dihydrogen citrate, 349.69; folate, 0.1982; inositol, 11.013; menadione, 4.956; niacin, 9.912; pyridoxine HCl, 2.203; riboflavin, 2.203; thiamine HCl, 2.203; dry vitamin A palmitate (5000,000 U/g), 3.965; dry vitamin D, (500,000 U/g), 0.4405; dry vitamin E acetate (500 U/g), 24.229; and corn starch, 466.688. The Teklad Mineral Mix contains the following in grams per kilogram of mix: CaHPO,, 769.44; KCl, 89.07; NaCl, 30.29; MgSO,, 96.35; MnSO, * H,O, 6.469; FeSO,. 7H,O, 7.785; KIO,, 0.0156; and &SO,, 0.5878. Rats raised on this zinc-deficient diet have a significant increase in MBzN-induced methylation of esophageal DNA’ and in the incidence of MBzN-induced esophageal carcinoma6 compared with zinc control rats. Animals were housed in individual suspended stainless steel cages with deionized water provided in zinc-free stainless-steel and polycarbonate water bottles (Nalgene; Nalge, Rochester, NY), with the diets and deionized water provided ad libitum.6 After 3 weeks on the diets, the animals were killed with carbon dioxide, and the esophagi and livers were rapidly removed and microsomes prepared as previously described! Homogenates of the esophageal mucosa from rats raised on the zinc control diet were pooled into three separate preparations of 30 animals each for the cytochrome P-450 difference spectra; three separate preparations of 30 animals each for the microsomal assays with the addition of zinc in vitro; three separate preparations of 15 animals each for the studies of the effects of preincubation with zinc; and three separate preparations of 10 animals each for the quantitation of microsomal zinc content. Similar numbers of esophagi were pooled to prepare the microsomes from zinc-deficient rats. For all esophageal studies, three independent preparations of esophageal microsomes were used. Homogenates from two to four livers were pooled to prepare the microsomes used in the hepatic studies. For all hepatic studies, at least four independent preparations of hepatic microsomes were used. Microsomal protein was determined by the method of Lowry et a1.15 Zinc contents of the microsomes prepared above as well as of the esophageal mucosa and livers of an additional six animals raised on each of the two diets were determined by atomic absorption spectroscopy.6
or varying concentrations of DMN from 0.33 to 10 mmol/ L; an NADPH-generating system (2 mmol/L NADP+, 10 mmol glucose-6-phosphate, and 1.2 U glucose-6-phosphate dehydrogenase); and 10 mmol/L MgCl,, 50 mmol/L Tris HCl (pH 7.4), and 0.1-3.0 mg microsomal protein in microsomal storage buffer (50 mmol/L Tris HCl and 20% glycerol, pH 7.4) in a final volume of 0.6 mL. Semicarbazide (20 mmol/L) was added to the MBzN assays to block nonenzymatic degradation of benzaldehyde to benzoic acid.” This concentration of semicarbazide does not inhibit the metabolism of MBzN to form benzaldehyde. To determine if zinc in vitro directly inhibited the microsomal metabolism of MBzN or DMN, zinc chloride was added to some of the assays to yield final zinc concentrations of 5 or 10 ppm. DMN, NADP+ (sodium), glucose-6-phosphate (monopotassium), and glucose-6-phosphate dehydrogenase (type XV) were purchased from Sigma (St. Louis, MO). MBzN was obtained from Ash Stevens (Detroit, MI). Samples were incubated for 30 minutes at 37°C under air with shaking. Controls were reaction mixtures without microsomal protein or without the NADPH-generating system. Reactions were terminated by placing the mixtures on ice, and protein was precipitated by the addition of 0.3 mL of 20% ZnSO,. 7H,O and 0.3 mL of saturated Ba(OH), solution. The reaction mixtures were then centrifuged (2000 X g) at 4’C for 10 minutes. Supernatants from the MBzN assays were stored in the dark at -20°C until analyzed by high-performance liquid chromatography (HPLC),’ and supernatants from the DMN assays were analyzed for formaldehyde by the method of Nash as modified by Chung et al.” To determine if the zinc effects were reversible or irreversible, preparations of esophageal and hepatic microsomes were pretreated with the addition of zinc chloride to a final concentration of 0, 5, or 10 ppm before their use in the nitrosamine metabolism assays. To reproduce the concentrations of the microsomes used in the metabolism assays, the esophageal microsomes to be pretreated with zinc were diluted with 50 mmol/L Tris HCl (pH 7.4) to a final concentration of approximately 0.5 mg protein/ml and the hepatic microsomes diluted to a concentration of approximately 5.0 mg protein/ml. Zinc chloride was then added to yield a final concentration of 0, 5, or 10 ppm, and the microsomes were incubated for 30 minutes at 37°C. The samples were then diluted with an additional two volumes of the Tris buffer and centrifuged at 100,000 X g for 60 minutes at 4”C, and the microsomal pellet was resuspended in microsomal storage buffer. Pretreatment of the microsomes with 5 and 10 ppm zinc increased the zinc content of the hepatic microsomes 4-5-fold and 7-lo-fold, respectively. The activity of these recentrifuged microsomes was then determined using the MBzN and DMN metabolism assay systems with a carcinogen substrate concentration of 5 mmol/L. Because of the very low to unmeasurable activity in esophageal control microsomes assayed in the presence of 10 ppm zinc, the control esophageal microsomes were not pretreated with 10 ppm zinc.
Quantitation
of Cytochrome
P-450
Total cytochrome P-450. Total esophageal and hepatic microsomal cytochrome P-450 was quantitated by the method of Omura and Sate” using a Shimadzu UV-160 Spectrophotometer (Shimadzu, Kyoto, Japan) equipped with opal glass filters. Quantitation of specific cytochrome P-450 forms. The effect of dietary zinc deficiency on individual hepatic cytochrome P-450 forms was determined by immunoblot analysis of microsomal cytochrome P-450. Western blots were performed by the method of Wrighton et a1.l7 using form specific antibodies against cytochromes P-450IA1, P-450IA2, P-45OIIB1, P-450IIB2, P-450IIE1, and P-450111A.‘7-20 Microsomal
Assay
System
The microsomal incubation mixtures contained varying concentrations of MBzN from 0.025 to 20 mmol/L
Metabolite
Analysis
MBzN assay. The supernatant solutions were thawed and immediately chromatographed on a Beckman
802
BARCH ET AL.
GASTROENTEROLOGYVol. 103, No. 3
HPLC system
(Beckman, Fullerton, CA) in an Alltech Cl8 column (Alltech Associates, Deerfield, IL) eluted isocratitally with 17% (vol/vol) acetonitrile-3% (vol/vol) acetic acid in water, pH 2.6, at a flow rate of 1.5 mL/min; then the eluate was monitored at 254 nm as previously described.6 The control reactions yielded only the low background levels of benzaldehyde, which were present in the MBzN substrate. DMN assay. For quantitation of formaldehyde, 0.7 mL of the sample supernatant was added to 0.3 mL of the Nash reagent (15 gammonium acetate and 0.200 mL acetylacetone in 18 mL of 3% acetic acid), and the mixture was incubated at 50“C for 30 minutes in a shaking water bath under air.” Absorbance was measured at 412 nm, and the difference in absorbance between the sample and control tubes was compared with formaldehyde standards prepared in the assay buffer.
Table
I.
Characteristics of the Esophageal and Hepatic Tissues and Microsomes From Zinc-Deficient and Zinc Control Rats Zinc-deficient rats
Tissue zinc kg zinc/g tissue (ppm)] Esophagus Liver
21.4+ 0.43" 24.9f 2.4b
Esophageal microsomes” Hepatic microsomesd
158 *lo 72 + 8
193 t-lob 83 +6b
Total cytochrome P-450 (nmol/mg microsomal protein)
All data are expressed as mean + SEM. Statistical significance was determined by Student’s t test, paired or unpaired. In the metabolic analysis, n is equal to the number of independent preparations of microsomes.
Results Tissue Zinc Content Weanling rats raised on the zinc-deficient diet showed a significant reduction in esophageal and hepatic zinc content compared with zinc control rats (Table 1). The zinc content of the esophageal and hepatic microsomes prepared from zinc-deficient rats was also significantly lower than the zinc content of the microsomes prepared from the zinc control rats (Table 1). of Cytochrome
15.0Ik1.8 19.6 f 1.9
Microsomal zinc (,ug zinc/g microsomal protein)
Statistical Analysis
Quantification
Zinc control rats
P-450
The levels of total CO-binding cytochrome P-450 in esophageal and hepatic microsomes isolated from the zinc-deficient rats were not significantly different from the levels found in microsomes isolated from the zinc control rats (Table 1). In addition, immunoblot analysis of hepatic microsomes showed no detectable differences in the hepatic concentrations of cytochromes P-450IA1, P-450IA2, P-450IIB1, P-450IIB2, P-450IIE1, and P-450111A between the zinc-deficient and zinc control animals (data not shown). MBzN Metabolism It has been previously reportedz3 that at least two enzymes participate in the esophageal and hepatic microsomal metabolism of MBzN at the substrate concentrations examined in this study. The metabolism of MBzN by the esophageal and hepatic microsomes from the zinc-deficient animals was significantly greater (P < 0.05) than that of the zinc con-
Esophageal microsomes Hepatic microsomes
0.026 i 0.009 0.63 f 0.07
0.024zk0.005" 0.55 f 0.07e
NOTE. Results are expressed as means + SEM; esophageal and hepatic tissues, n = 6. “Significant difference between zinc-deficient and zinc control (P < 0.01). bSignificant difference between zinc-deficient and zinc control (P < 0.05). ‘n = 3,dn t 5. “Difference not significant between the zinc-deficient and zinc control microsomes.
trol microsomes, at all MBzN concentrations examined (Figures 1A and 2A). The differences between the zinc-deficient and zinc control microsomes are compatible with dietary zinc reducing the concentration of the cytochrome P-450 forms responsible for the metabolism of MBzN or with dietary zinc inhibiting the activity of these cytochrome P-450 forms. Because there were no apparent differences in the amount of cytochrome P-450 in the zinc-deficient and control microsomes, we next examined whether zinc was acting as a direct inhibitor of the cytochrome P-450-dependent metabolism of MBzN. Addition of zinc in vitro significantly inhibited (P < 0.05) the metabolism of MBzN by the esophageal and hepatic microsomes from both the zinc-deficient and zinc control animals; this inhibition was concentration dependent (Figures 1 and 2). The zincdependent inhibition of the microsomal metabolism of MBzN in vitro appears to reproduce the differences observed between the metabolism of MBzN by microsomes from the zinc-deficient and zinc control rats. The kinetics of this in vitro inhibition suggested that zinc was acting either as a noncompetitive inhibitor or as an irreversible inhibitor of the cytochrome P-W&dependent microsomal metabolism of MBzN. Pretreatment of the microsomes with zinc in vitro
September
INHIBITION OF NITROSAMINE
1992
A 25
-t -S
10 . +.
ZD + 10 ppm Zinc in vitro -R’ZD+5p mzincinvitro -6 Zinc De P. uent (ZD) microsomes
I
20
BY ZINC
803
C
B Zinc Contrpl \ZCbmicmsomes Zmc Defiaen (Z ) microsomes
METABOLISM
E-
50-
g g
4. -O-
ZC + 5 ppm Zinc in vitro Zinc Control (ZC) microsomes
/’ /’ /I d 0 10
20 i/ImM
30 40 of MBN]
50
0
10
20 30 40 l/[mM of MBN]
.
’
0.I-:‘: 012345678
0 0
/’
50
i/[mM of MEN]
Figure 1. Double reciprocal plots of the cytochrome P-&B-dependent microsomal metabolism of MBzN as measured by benzaldehyde formation of esophageal microsomes from zinc-deficient and zinc control animals. Each data point represents the average of at least three independent assays with an SEM ~15% of the mean. (A) Comparison of the activity of esophageal microsomes from zinc-deficient and zinc control animals. Microsomal metabolism of MBzN by the zinc-deficient microsomes was significantly greater (P < 0.05) than that of the zinc control microsomes at all substrate concentrations examined. (B) Activity of esophageal microsomes from zinc-deficient rats with the addition of zinc in vitro. Activity was significantly reduced (P < 0.05) by zinc in vitro at all substrate concentrations examined. (C) Activity of esophageal microsomes from zinc control rats with the addition of zinc in vitro. Activity was significantly reduced (P< 0.05) by zinc in vitro at all substrate concentrations examined. Activity of the zinc control microsomes was below the level of detection at MBzN substrate concentrations GKZ~ mmol/L in the presence of 5 ppm zinc in vitro.
showed that zinc was acting as a concentration-dependent irreversible inhibitor of the cytochrome P-450-dependent metabolism of MBzN (Table 2). DMN Metabolism To determine if this effect was specific to the metabolism of MBzN or was more generalized to the microsomal metabolism of other nitrosamine carcinogens, we examined the effects of zinc on the hepatic microsomal metabolism of the related hepatic carcinogen DMN. At least three enzymes participate in the hepatic microsomal metabolism of DMN to form formaldehyde. 24*25At the substrate concentrations examined in this study, cytochrome P-450IIEl is responsible for approximately 90% of the hepatic mi-
A 8
-O-0-
crosomal metabolism of DMN.‘0,26 The microsomal metabolism of DMN by hepatic microsomes from the zinc-deficient animals was significantly greater (P < 0.05) than that of the zinc control microsomes at all DMN concentrations examined (Figure 3A). These differences are compatible with dietary zinc reducing the concentration of the cytochrome P-450 forms responsible for the metabolism of DMN or with dietary zinc inhibiting the activity of these cytochrome P-450 forms. Because there were no apparent differences in cytochrome P-450IIEl between the zinc-deficient and control microsomes, we next examined whether zinc was acting as a direct inhibitor of the cytochrome P-450 dependent metabolism of DMN. The addition of zinc to the in vitro assay signifi-
Zinc Control (ZC) microsomes Zinc Deficient (ZD) microsomes
*+ .C
B 8
-b ZD + IO ppm Zinc in vitro -Et- ZD + 5 ppm Zinc in vitro 6 + Zinc Deftaent (ZD) microsomes 1
2
6C
E g
+ +
ZC + 5 ppm Zinc in vitro Zinc Control (ZC) microsomes
40
.-
,/‘. 0’
20 4
Obp
012345678910 l/[mM of MBN]
.
I
0 1
4 l/[mM Ef Mi3N]3
Figure 2. Double reciprocal plots of the cytochrome P-&u-dependent microsomal metabolism of MBzN as measured by benzaldehyde formation of hepatic microsomes from zinc-deficient and zinc control animals. Each data point represents the average of at least four independent assays with an SEM 45% of the mean. (A)Comparison of the activity of hepatic microsomes from zinc-deficient and zinc control animals. Microsomal metabolism of MBzN by the zinc-deficient microsomes was significantly greater (P < 01~5) than the zinc control microsomes at all substrate concentrations examined. (B) Activity of hepatic microsomes from zinc-deficient rats with the addition of zinc in vitro. Activity was significantly reduced (P < 0.05) by zinc in vitro at all substrate concentrations examined. (C) Activity of hepatic microsomes from zinc control rats with the addition of zinc in vitro. Activity was significantly reduced (P < 0.05) by zinc in vitro at all substrate concentrations examined.
804 BARCH ET AL.
GASTROENTEROLOGY Vol. 103,No. 3
Table 2. Inhibition of the Microsomal Metabolism of MBzN by Pretreatment of Esophageal and Hepatic Microsomes With Zinc In Vitro Benzaldehyde formed @mol. min-’ . mg microsomal protein-‘)”
Pretreated with 0 ppm zinc Pretreated with 5 ppm zinc Pretreated with 10 ppm zinc
Esophageal microsomes Zinc-deficient diet
Esophageal microsomes Zinc control diet
0.154+ 0.006b
0.052f 0.008
0.106+ 0.013'
0.030It0.007d
0.099f o.014c
Not assayed
Benzaldehyde formed (nmol . min-’ . mg microsomal protein-‘)” Hepatic microsomes Zinc-deficient diet Pretreated with 0 ppm zinc Pretreated with 5 ppm zinc Pretreated with 10 ppm zinc
Hepatic microsomes Zinc control diet
0.202f 0.029b
0.085+ 0.002
0.097 f o.012c
0.054+ o.oo2c
0.038f 0.005d
0.020+ 0.002d
‘Microsomal incubation assays contained 5 mmol/L MBzN. bMean f SEM; esophageal microsomes, n = 3; hepatic microsomes, n = 4. “Significantly different from microsomes treated with 0 ppm zinc (P < 0.05). dSignificantly different from microsomes treated with 0 ppm zinc (P < 0.005).
cantly inhibited (P -C0.01 for the addition of 10 ppm zinc to the zinc-deficient microsomes, P < 0.01 for the additional of 5 ppm zinc to the control microsomes) the hepatic microsomal metabolism of DMN by the microsomes from both the zinc-deficient and zinc control animals; this inhibition was also concentration dependent (Figure 3). The zinc-dependent inhibition of the microsomal metabolism of DMN in vitro appears to reproduce the differences observed between the metabolism of DMN by microsomes from the zinc-deficient and zinc control rats. The kinetics of this in vitro inhibition once again suggested that zinc was acting either as a noncompetitive inhibitor or as an irreversible inhibitor of the cytochrome P-450-dependent microsomal metabolism of DMN. Pretreatment of the microsomes with zinc in vitro showed that zinc was also acting as a concentrationdependent irreversible inhibitor of the cytochrome P-450-dependent metabolism of DMN in microsomes from both zinc-deficient and zinc control animals (Table 3).
Discussion The current experiments show that zinc is a concentration-dependent irreversible inhibitor of the esophageal and hepatic microsomal metabolism of MBzN. These studies further show that the microsomal metabolism of MBzN is enhanced by dietary zinc deficiency without detectable alterations in the content of total esophageal or hepatic microsomal cytochrome P-450 and without detectable alterations in the profile of the forms of cytochromes P-450 expressed in the liver. To determine if this effect was specific to the metabolism of MBzN or was more generalized to the metabolism of other nitrosamine carcinogens, we examined the effects of zinc on the metabolism of the related hepatic carcinogen DMN. Of the various cytochrome P-4511s involved in the hepatic metabolism of DMN,24,25the ethanol-inducible cytochrome P-450IIEl appears to be the enzyme responsible for the hepatic activation of DMN to a mutagenic agent.12s13 This cytochrome P-450 form is different from the form of cytochrome P-450 responsible for the activation of MBzN.‘~*” At the substrate concentrations examined in this study, cytochrome P-450IIEl is responsible for approximately 90% of the hepatic microsomal metabolism of DMN.‘0,2” Similar to our results with MBzN, dietary zinc deficiency increased the hepatic microsomal metabolism of DMN without alterations in total hepatic cytochrome P-450 and without an apparent alteration in the hepatic concentration of cytochrome P-450IIEl. Zinc in vitro is also a concentration-dependent irreversible inhibitor of the hepatic microsomal metabolism of DMN. Previous studies have shown that zinc in vitro inhibits a variety of other cytochrome P-450-dependent reactions, including cytochrome P-450-dependent aniline hydroxylase, ethylmorphine demethylase, benzphetamine demethylase, benzo[a]pyrene hydroxylase, cytochrome P-450-dependent oxidation of NADPH, and NADPH-dependent reduction of cytochrome c.27-2g The concentrations of zinc required for the inhibition of these cytochrome P-450dependent reactions are also similar to the concentrations of zinc found in normal tissues. Jeffery2’ has reported that this inhibition appears to be caused by zinc effecting the electron transfer between the NADPH cytochrome P-450 reductase and the cytochrome P-450 complex or through alteration of the oxidation-reduction potential of the NADPH cytochrome P-450 reductase. A possible explanation for the increased metabolism of MBzN and DMN associated with dietary zinc deficiency is that physiological levels of zinc may be an endogenous inhibitor of the cytochrome P-450-
INHIBITION OF NITROSAMINE METABOLISM BY ZINC
September 1992
805
Figure 3. Double reciprocal plots of the cytochrome P-&O-dependent microsomal metabolism of DMN as measured by formaldehyde formation of hepatic microsomes from zinc-deficient and zinc control animals. Each data point represents the average of at least four independent assays with an SEM 40% of the mean. (A) Comparison of the activity of hepatic microsomes from zinc-deficient and zinc control animals. Microsomal metabolism of DMN by the zinc-deficient microsomes was significantly greater (P < 0.05) than the zinc control microsomes at all substrate concentrations examined. (B) Activity of hepatic microsomes from zinc-deficient rats with the by the addition of 10 ppm zinc in vitro at all substrate addition of zinc in vitro. Activity was significantly reduced (P < 0.01) concentrations examined. (C) Activity of hepatic microsomes from zinc control rats with the addition of zinc in vitro. Activity was significantly reduced (P< 0.01) by the addition of 5 ppm zinc in vitro at all substrate concentrations examined.
dependent metabolism of MBzN and DMN. The cytochrome P-450 enzymes in the tissues of the zinc control rats would be exposed to significantly higher concentrations of zinc than the cytochrome P-450 enzymes in the zinc-deficient tissues of zinc-deficient rats. Enzymes that irreversibly accumulated higher levels of zinc in vivo would then show lower enzymatic activity in vitro. Such an inhibition of cytochrome P-450 activity would explain the lower levels of MBzN and DMN metabolism observed with the microsomes from zinc control animals compared with microsomes from zinc-deficient animals. In summary, the current study shows that dietary zinc deficiency significantly increases the cy-
tochrome P-450-dependent microsomal metabolism of MBzN and DMN without alterations in the microsomal content of cytochrome P-450 of the esophagus or liver. The addition of zinc in vitro, at concentrations found in normal tissues, irreversibly inhibited the microsomal metabolism of MBzN and DMN in a concentration-dependent manner. These results suggest that physiological levels of zinc may be an endogenous inhibitor of MBzN metabolism. Dietary zinc deficiency appears to reduce this inhibition, resulting in an increase in carcinogen activation. This effect could explain the increased incidence of MBzN-induced esophageal carcinoma observed with dietary zinc deficiency. References
Table 3. Inhibition
of the Hepatic Microsomal Metabolism by Pretreatment ofHepatic Microsomes With Zinc In Vitro
ofDMN
1.
Formaldehyde formed @mol. min-’ - mg mici-osomal protein-‘)O Hepatic microsomes Zinc-deficient diet Pretreated with 0 ppm zinc Pretreated with 5 ppm zinc Pretreated with 10 ppm zinc
2.
Hepatic microsomes Zinc control diet 3.
0.55 + 0.04b
0.36 + 0.04
0.38 + 0.04d
0.28 + 0.03’
0.16 + O.Ogd
0.11 ? 0.02d
“Microsomal incubation assays contained 5 mmol/L DMN. bMean + SEM; n = 4. “Significantly different from microsomes treated with 0 ppm zinc (P < 0.05). dSignificantly different from microsomes treated with 0 ppm zinc (P < 0.005).
4.
5.
6.
Druckrey H, Preussmann R, Ivankovic S, Schmahl IJ, Afkham J, Blum G, Mennel HD, Muller M, Petropoulos P, Schneider H. Organotrope Carcinogene Wirkungen bei 65 Verschiedenen N-Nitroso-Verbindungen an BD-Ratten. Krebsforschung 1967;69:103-201. Stinson SF, Squire RA, Sporn MD. Pathology of esophageal neoplasms and associated proliferative lesions induced in rats by N-methyl-N-benzylnitrosamine. 1 Nat1 Cancer Inst 1978;61:1471-1472. Druckrey H. Organospecific carcinogenesis in the digestive tract. In: Nakahara W, Takayama S, Sugimura T, Odashima S, eds. Topics in chemical carcinogenesis. Proceedings of the 2nd International Symposium of the Princess Takamatsu Cancer Research Fund. Baltimore, MD: University Park, 1972:73-101. Fong LYY, Sivak A, Newberne PM. Zinc deficiency and methylbenzylnitrosamine-induced esophageal cancer in rats. J Nat1 Cancer Inst 1978;61:145-150. Gabrial GN, Schrager TF, Newberne PM. Zinc deficiency, alcohol, and a retinoid: association with esophageal cancer in rats. J Nat1 Cancer Inst 1982;68:785-789. Barth DH, Kuemmerle SC, Hollenberg PF, Iannaccone PI.
806
7.
8.
9.
10.
11.
12.
13.
14.
15.
16. 17.
18.
19.
BARCH ET AL.
Esophageal microsomal metabolism of N-nitrosomethylbenzylamine in the zinc-deficient rat. Cancer Res 1984;44:56295633. Yahagi T, Nagao M, Seino Y, Matsushima T, Sugimura T, Okada M. Mutagenicities of N-nitrosamines on Salmonella. Mutation Res 1977;48:121-130. Pegg AE. Formation and removal of methylated nucleosides in nucleic acids of mammalian cells. Ret Results Cancer Res 1983;84:49-62. Barth DH, Fox CC. Dietary zinc deficiency increases the methylbenzylnitrosamine-induced formation of 06-methylguanine in the esophageal DNA of the rat. Carcinogenesis 1987;8:1461-1464. Barth DH, FOX CC, Koop DR. Different cytochrome P-450 enzymes are responsible for the activation of hepatic and esophageal specific nitrosamines (abstr). Hepatology 1988;8:1278. Ludeke B, Meier T, Kleihues P. Bioactivation of asymmetric N-dialkylnitrosamines in rat tissues derived from the ventral entoderm. IARC Sci Pub1 1991;105:286-293. Yang CS, Tu YY, Koop DR, Coon MJ. Metabolism of nitrosamines by purified rabbit liver cytochrome P-450 isozymes. Cancer Res 1985;45:1140-1145. Yoo J-SH, Yang CS. Enzyme specificity in the metabolic activation of N-nitrosodimethylamine to a mutagen for Chinese hamster V79 cells. Cancer Res 1985;45:5569-5574. Stoner GD, Babcock MS, Cothern GA, Klaunig JE, Gunning WT, Knipe SM. In vitro transformation of rat esophageal epithelial cells with N-nitrosobenzylmethylamine. Carcinogenesis 1982;3:629-634. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:205-225. Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomes. J Biol Chem 1964;239:2379-2385. Wrighton SA, Campanile C, Thomas PE, Maines SL, Watkins PB, Parker G, Mendez-Picon G, Haniu M, Shively J, Levin W, Guzelian PS. Identification of a human liver cytochrome P-450 homologous to the major isosafrole-inducible cytochrome P-450 in the rat. Mol Pharmacol 1986;29:405-410. Schuetz EG, Wrighton SA, Safe SH, Guzelian PS. Regulation of cytochrome P-450~ by phenobarbital and phenobarbitallike inducers in adult rat hepatocytes in primary monolayer culture and in vivo. Biochemistry 1986;25:1124-1133. Watkins PB, Wrighton SA, Maurel P, Schuetz EG, MendezPicon G, Parker GA, Guzelian PS. Identification of an induc-
GASTROENTEROLOGYVol.103,No.3
20.
21.
22.
23.
ible form of cytochrome P-450 in human liver. Proc Nat1 Acad Sci 1985;82:6310-6314. Wrighton SA, Thomas PE, Molowa DT, Haniu M, Shively JE, Maines SL, Watkins PB, Parker G, Mendez-Picon G, Levin W, Guzelian PS. Characterization of ethanol-inducible human liver N-nitrosodimethylamine demethylase. Biochemistry 1986;25:6731-6735. Labuc GE, Archer MC. Esophageal and hepatic microsomal metabolism of N-nitrosomethylbenzylamine and N-nitrosodimethylamine in the rat. Cancer Res 1982;42:3181-3186. Chung FL, Wang M, Hecht SS. Effects of dietary indoles and isothiocyanates on N-nitrosodimethylamine and 4-(methylnitrosamino)-I-(3-pyridyl)-I-butanone a-hydroxylation and DNA methylation in rat liver. Carcinogenesis 1985;6:539-543. Barth DH, Iannaccone PM. Role of zinc deficiency in carcinogenesis. In: Poirier L, Newberne P, Pariza M, eds. Essential nutrients in carcinogenesis. Advances in experimental medicine and biology. Volume 206. New York: Plenum, 1986:517-
527. 24. Lake BG, Heading
25. 26.
27.
28.
29.
CE, Phillips JC, Gangolli SD, Lloyd AG. Some studies on the metabolism in vitro of dimethylnitrosamine by rat liver. Biochem Sot Transac 1974;2:610-612. Farrelly J. A new assay for the microsomal metabolism of nitrosamines. Cancer Res 1980;40:3241-3244. Peng R, Tu YY, Yang CS. The induction and competitive inhibition of a high affinity microsomal nitrosodimethylamine demethylase by ethanol. Carcinogenesis 1982;3:1457-1461, Chvapil M, Ludwig JC, Sipes IG, Misiorowski RL. Inhibition of NADPH oxidation and related drug oxidation in liver microsomes by zinc. Biochem Pharmacol 1976;25:1787-1791. Colby HD, Johnson PB, Zulkoski JS, Pope MR, Miles PR. Comparative effects of cadmium, zinc, and lead in vitro on pulmonary, adrenal and hepatic microsomal metabolism in the guinea pig. J Toxic01 Environ Health 1981;8:907-915. Jeffery EH. The effect of zinc on NADPH oxidation and monooxygenase activity in rat hepatic microsomes. Mol Pharmacol 1983;23:467-473.
Received December 2, 1991. Accepted February 25, 1992. Address requests for reprints to: David H. Barth, Northwestern University Cancer Center, Room 8370 Olson, 303 East Chicago Avenue, Chicago, Illinois 60611. Supported in part by National Institutes of Health grant CA40487 and a Merit Review Grant from the Research Service of the Department of Veterans Affairs.
1992;103:800-806
Inhibition of Rat Methylbenzylnitrosamine Metabolism by Dietary-Zinc and Zinc In Vitro DAVID H. BARCH, COLLEEN C. FOX, WILLIAM A. ROSCHE, LYNN M. RUNDHAUGEN, and STEVEN A. WRIGHTON Department of Medicine, Northwestern University Medical School and the Lake Side Veterans Administration Hospital, Chicago, Illinois; and Department of Drug Metabolism, Lilly Research Laboratories,
Eli Lilly & Company, Indianapolis, Indiana
Methylbenzylnitrosamine is an esophageal-specific carcinogen in the rat, and the incidence of methylbenzylnitrosamine-induced esophageal carcinoma is increased by dietary zinc deficiency. Methylbenzylnitrosamine requires activation by cytochrome P-450 to be mutagenic; the present study examined the role of dietary zinc deficiency and the in vitro addition of zinc on the cytochrome P-&&dependent microsomal metabolism of methylbenzylnitrosamine. Dietary zinc deficiency significantly increased the cytochrome P-&o-dependent esophageal and hepatic microsomal metabolism of methylbenzylnitrosamine. These changes occurred without alteration in the specific content of total microsomal cytochrome P-450 of the esophagus or liver. The addition of zinc in vitro, at concentrations found in normal tissues, irreversibly inhibited the esophageal and hepatic cytochrome P-450-dependent microsomal metabolism of methylbenzylnitrosamine. These results suggest that physiological levels of zinc may be an endogenous inhibitor of methylbenzylnitrosamine metabolism. Dietary zinc deficiency appears to reduce this inhibition of cytochrome P-450 activity, resulting in an increase in carcinogen activation.
E
xperimental studies have shown that methylbenzylnitrosamine (MBzN) is an esophageal-specific carcinogen in the raF3 and that rats raised on zinc-deficient diets have an increased incidence of MBzN-induced esophageal carcinoma compared with rats raised on zinc control diets.4-” MBzN is known to require cytochrome P-450-dependent activation to become mutagenic,7,8 and dietary zinc deficiency has been shown to increase the cytochrome P-450-dependent esophageal and hepatic microsomal metabolism of MBzN.‘This increase in the metabolism of MBzN is associated with an increase in MBzN-induced methylation of esophageal DNA in vivo.g Although the cytochrome P-450 enzyme responsible for the activation of MBzN has not been identified, MBzN is not activated by cytochrome
P-45011E1,‘0~” which activates the related hepatic carcinogen dimethylnitrosamine (DMN).12,13Esophageal epithelial cells alone are sufficient for the activation of MBzN, because MBzN can transform isolated esophageal epithelial cells in culture to a tumorigenic phenotype.14 In the present study we report that dietary zinc deficiency increases the microsomal metabolism of MBzN and DMN and that these changes occur without alterations in the total microsomal cytochrome P-450 of the esophagus or liver and without apparent alteration in the hepatic concentration of cytochrome P-450IIEl. The addition of zinc in vitro irreversibly inhibited the esophageal and hepatic cytochrome P-450-dependent microsomal metabolism of MBzN and the hepatic microsomal metabolism of DMN. This inhibition was concentration dependent, and significant inhibition was observed with concentrations of zinc found in normal tissues. Materials and Methods Animals Weanling, 21-day-old, male Sprague-Dawley rats purchased from Charles River (Portage, MI) were randomly assigned to a zinc-deficient diet [2.3 parts per million (ppm) zinc] or a zinc control diet (50 ppm zinc). These diets, prepared by Teklad (Madison, WI), contain the following components in grams per kilogram of diet; egg white solids, 200, DL-methionine, 1.5; dextrose monohydrate, 632.15; corn oil, 100; cellulose fiber, 30.0; sodium fluoride, 0.0022; chromium chloride, 0.002; sodium molybdate, 0.0001; Teklad Vitamin Mix TD 40060, 10.0; choline dihydrogen citrate, 0.655; biotin, 0.0046; and Teklad Mineral Mix TD81264-zinc deficient, 25.69. Zinc carbonate was added to yield a final concentration of 2.3 ppm or 50 ppm. The Teklad Vitamin Mix contains the following in grams per kilogram of mix: p-aminobenzoic acid, 11.0132; ascorbic acid, 101.66; biotin, 0.0441; vitamin B,, (0.1% trituration in mannitol), 2.974; calcium pantothenate, 6.608; This is a US. government work. There are no restrictions its use. 0016-5085/92/$0.00
on
September 1992
INHIBITION OF NITROSAMINE METABOLISM BY ZINC 801
choline dihydrogen citrate, 349.69; folate, 0.1982; inositol, 11.013; menadione, 4.956; niacin, 9.912; pyridoxine HCl, 2.203; riboflavin, 2.203; thiamine HCl, 2.203; dry vitamin A palmitate (5000,000 U/g), 3.965; dry vitamin D, (500,000 U/g), 0.4405; dry vitamin E acetate (500 U/g), 24.229; and corn starch, 466.688. The Teklad Mineral Mix contains the following in grams per kilogram of mix: CaHPO,, 769.44; KCl, 89.07; NaCl, 30.29; MgSO,, 96.35; MnSO, * H,O, 6.469; FeSO,. 7H,O, 7.785; KIO,, 0.0156; and &SO,, 0.5878. Rats raised on this zinc-deficient diet have a significant increase in MBzN-induced methylation of esophageal DNA’ and in the incidence of MBzN-induced esophageal carcinoma6 compared with zinc control rats. Animals were housed in individual suspended stainless steel cages with deionized water provided in zinc-free stainless-steel and polycarbonate water bottles (Nalgene; Nalge, Rochester, NY), with the diets and deionized water provided ad libitum.6 After 3 weeks on the diets, the animals were killed with carbon dioxide, and the esophagi and livers were rapidly removed and microsomes prepared as previously described! Homogenates of the esophageal mucosa from rats raised on the zinc control diet were pooled into three separate preparations of 30 animals each for the cytochrome P-450 difference spectra; three separate preparations of 30 animals each for the microsomal assays with the addition of zinc in vitro; three separate preparations of 15 animals each for the studies of the effects of preincubation with zinc; and three separate preparations of 10 animals each for the quantitation of microsomal zinc content. Similar numbers of esophagi were pooled to prepare the microsomes from zinc-deficient rats. For all esophageal studies, three independent preparations of esophageal microsomes were used. Homogenates from two to four livers were pooled to prepare the microsomes used in the hepatic studies. For all hepatic studies, at least four independent preparations of hepatic microsomes were used. Microsomal protein was determined by the method of Lowry et a1.15 Zinc contents of the microsomes prepared above as well as of the esophageal mucosa and livers of an additional six animals raised on each of the two diets were determined by atomic absorption spectroscopy.6
or varying concentrations of DMN from 0.33 to 10 mmol/ L; an NADPH-generating system (2 mmol/L NADP+, 10 mmol glucose-6-phosphate, and 1.2 U glucose-6-phosphate dehydrogenase); and 10 mmol/L MgCl,, 50 mmol/L Tris HCl (pH 7.4), and 0.1-3.0 mg microsomal protein in microsomal storage buffer (50 mmol/L Tris HCl and 20% glycerol, pH 7.4) in a final volume of 0.6 mL. Semicarbazide (20 mmol/L) was added to the MBzN assays to block nonenzymatic degradation of benzaldehyde to benzoic acid.” This concentration of semicarbazide does not inhibit the metabolism of MBzN to form benzaldehyde. To determine if zinc in vitro directly inhibited the microsomal metabolism of MBzN or DMN, zinc chloride was added to some of the assays to yield final zinc concentrations of 5 or 10 ppm. DMN, NADP+ (sodium), glucose-6-phosphate (monopotassium), and glucose-6-phosphate dehydrogenase (type XV) were purchased from Sigma (St. Louis, MO). MBzN was obtained from Ash Stevens (Detroit, MI). Samples were incubated for 30 minutes at 37°C under air with shaking. Controls were reaction mixtures without microsomal protein or without the NADPH-generating system. Reactions were terminated by placing the mixtures on ice, and protein was precipitated by the addition of 0.3 mL of 20% ZnSO,. 7H,O and 0.3 mL of saturated Ba(OH), solution. The reaction mixtures were then centrifuged (2000 X g) at 4’C for 10 minutes. Supernatants from the MBzN assays were stored in the dark at -20°C until analyzed by high-performance liquid chromatography (HPLC),’ and supernatants from the DMN assays were analyzed for formaldehyde by the method of Nash as modified by Chung et al.” To determine if the zinc effects were reversible or irreversible, preparations of esophageal and hepatic microsomes were pretreated with the addition of zinc chloride to a final concentration of 0, 5, or 10 ppm before their use in the nitrosamine metabolism assays. To reproduce the concentrations of the microsomes used in the metabolism assays, the esophageal microsomes to be pretreated with zinc were diluted with 50 mmol/L Tris HCl (pH 7.4) to a final concentration of approximately 0.5 mg protein/ml and the hepatic microsomes diluted to a concentration of approximately 5.0 mg protein/ml. Zinc chloride was then added to yield a final concentration of 0, 5, or 10 ppm, and the microsomes were incubated for 30 minutes at 37°C. The samples were then diluted with an additional two volumes of the Tris buffer and centrifuged at 100,000 X g for 60 minutes at 4”C, and the microsomal pellet was resuspended in microsomal storage buffer. Pretreatment of the microsomes with 5 and 10 ppm zinc increased the zinc content of the hepatic microsomes 4-5-fold and 7-lo-fold, respectively. The activity of these recentrifuged microsomes was then determined using the MBzN and DMN metabolism assay systems with a carcinogen substrate concentration of 5 mmol/L. Because of the very low to unmeasurable activity in esophageal control microsomes assayed in the presence of 10 ppm zinc, the control esophageal microsomes were not pretreated with 10 ppm zinc.
Quantitation
of Cytochrome
P-450
Total cytochrome P-450. Total esophageal and hepatic microsomal cytochrome P-450 was quantitated by the method of Omura and Sate” using a Shimadzu UV-160 Spectrophotometer (Shimadzu, Kyoto, Japan) equipped with opal glass filters. Quantitation of specific cytochrome P-450 forms. The effect of dietary zinc deficiency on individual hepatic cytochrome P-450 forms was determined by immunoblot analysis of microsomal cytochrome P-450. Western blots were performed by the method of Wrighton et a1.l7 using form specific antibodies against cytochromes P-450IA1, P-450IA2, P-45OIIB1, P-450IIB2, P-450IIE1, and P-450111A.‘7-20 Microsomal
Assay
System
The microsomal incubation mixtures contained varying concentrations of MBzN from 0.025 to 20 mmol/L
Metabolite
Analysis
MBzN assay. The supernatant solutions were thawed and immediately chromatographed on a Beckman
802
BARCH ET AL.
GASTROENTEROLOGYVol. 103, No. 3
HPLC system
(Beckman, Fullerton, CA) in an Alltech Cl8 column (Alltech Associates, Deerfield, IL) eluted isocratitally with 17% (vol/vol) acetonitrile-3% (vol/vol) acetic acid in water, pH 2.6, at a flow rate of 1.5 mL/min; then the eluate was monitored at 254 nm as previously described.6 The control reactions yielded only the low background levels of benzaldehyde, which were present in the MBzN substrate. DMN assay. For quantitation of formaldehyde, 0.7 mL of the sample supernatant was added to 0.3 mL of the Nash reagent (15 gammonium acetate and 0.200 mL acetylacetone in 18 mL of 3% acetic acid), and the mixture was incubated at 50“C for 30 minutes in a shaking water bath under air.” Absorbance was measured at 412 nm, and the difference in absorbance between the sample and control tubes was compared with formaldehyde standards prepared in the assay buffer.
Table
I.
Characteristics of the Esophageal and Hepatic Tissues and Microsomes From Zinc-Deficient and Zinc Control Rats Zinc-deficient rats
Tissue zinc kg zinc/g tissue (ppm)] Esophagus Liver
21.4+ 0.43" 24.9f 2.4b
Esophageal microsomes” Hepatic microsomesd
158 *lo 72 + 8
193 t-lob 83 +6b
Total cytochrome P-450 (nmol/mg microsomal protein)
All data are expressed as mean + SEM. Statistical significance was determined by Student’s t test, paired or unpaired. In the metabolic analysis, n is equal to the number of independent preparations of microsomes.
Results Tissue Zinc Content Weanling rats raised on the zinc-deficient diet showed a significant reduction in esophageal and hepatic zinc content compared with zinc control rats (Table 1). The zinc content of the esophageal and hepatic microsomes prepared from zinc-deficient rats was also significantly lower than the zinc content of the microsomes prepared from the zinc control rats (Table 1). of Cytochrome
15.0Ik1.8 19.6 f 1.9
Microsomal zinc (,ug zinc/g microsomal protein)
Statistical Analysis
Quantification
Zinc control rats
P-450
The levels of total CO-binding cytochrome P-450 in esophageal and hepatic microsomes isolated from the zinc-deficient rats were not significantly different from the levels found in microsomes isolated from the zinc control rats (Table 1). In addition, immunoblot analysis of hepatic microsomes showed no detectable differences in the hepatic concentrations of cytochromes P-450IA1, P-450IA2, P-450IIB1, P-450IIB2, P-450IIE1, and P-450111A between the zinc-deficient and zinc control animals (data not shown). MBzN Metabolism It has been previously reportedz3 that at least two enzymes participate in the esophageal and hepatic microsomal metabolism of MBzN at the substrate concentrations examined in this study. The metabolism of MBzN by the esophageal and hepatic microsomes from the zinc-deficient animals was significantly greater (P < 0.05) than that of the zinc con-
Esophageal microsomes Hepatic microsomes
0.026 i 0.009 0.63 f 0.07
0.024zk0.005" 0.55 f 0.07e
NOTE. Results are expressed as means + SEM; esophageal and hepatic tissues, n = 6. “Significant difference between zinc-deficient and zinc control (P < 0.01). bSignificant difference between zinc-deficient and zinc control (P < 0.05). ‘n = 3,dn t 5. “Difference not significant between the zinc-deficient and zinc control microsomes.
trol microsomes, at all MBzN concentrations examined (Figures 1A and 2A). The differences between the zinc-deficient and zinc control microsomes are compatible with dietary zinc reducing the concentration of the cytochrome P-450 forms responsible for the metabolism of MBzN or with dietary zinc inhibiting the activity of these cytochrome P-450 forms. Because there were no apparent differences in the amount of cytochrome P-450 in the zinc-deficient and control microsomes, we next examined whether zinc was acting as a direct inhibitor of the cytochrome P-450-dependent metabolism of MBzN. Addition of zinc in vitro significantly inhibited (P < 0.05) the metabolism of MBzN by the esophageal and hepatic microsomes from both the zinc-deficient and zinc control animals; this inhibition was concentration dependent (Figures 1 and 2). The zincdependent inhibition of the microsomal metabolism of MBzN in vitro appears to reproduce the differences observed between the metabolism of MBzN by microsomes from the zinc-deficient and zinc control rats. The kinetics of this in vitro inhibition suggested that zinc was acting either as a noncompetitive inhibitor or as an irreversible inhibitor of the cytochrome P-W&dependent microsomal metabolism of MBzN. Pretreatment of the microsomes with zinc in vitro
September
INHIBITION OF NITROSAMINE
1992
A 25
-t -S
10 . +.
ZD + 10 ppm Zinc in vitro -R’ZD+5p mzincinvitro -6 Zinc De P. uent (ZD) microsomes
I
20
BY ZINC
803
C
B Zinc Contrpl \ZCbmicmsomes Zmc Defiaen (Z ) microsomes
METABOLISM
E-
50-
g g
4. -O-
ZC + 5 ppm Zinc in vitro Zinc Control (ZC) microsomes
/’ /’ /I d 0 10
20 i/ImM
30 40 of MBN]
50
0
10
20 30 40 l/[mM of MBN]
.
’
0.I-:‘: 012345678
0 0
/’
50
i/[mM of MEN]
Figure 1. Double reciprocal plots of the cytochrome P-&B-dependent microsomal metabolism of MBzN as measured by benzaldehyde formation of esophageal microsomes from zinc-deficient and zinc control animals. Each data point represents the average of at least three independent assays with an SEM ~15% of the mean. (A) Comparison of the activity of esophageal microsomes from zinc-deficient and zinc control animals. Microsomal metabolism of MBzN by the zinc-deficient microsomes was significantly greater (P < 0.05) than that of the zinc control microsomes at all substrate concentrations examined. (B) Activity of esophageal microsomes from zinc-deficient rats with the addition of zinc in vitro. Activity was significantly reduced (P < 0.05) by zinc in vitro at all substrate concentrations examined. (C) Activity of esophageal microsomes from zinc control rats with the addition of zinc in vitro. Activity was significantly reduced (P< 0.05) by zinc in vitro at all substrate concentrations examined. Activity of the zinc control microsomes was below the level of detection at MBzN substrate concentrations GKZ~ mmol/L in the presence of 5 ppm zinc in vitro.
showed that zinc was acting as a concentration-dependent irreversible inhibitor of the cytochrome P-450-dependent metabolism of MBzN (Table 2). DMN Metabolism To determine if this effect was specific to the metabolism of MBzN or was more generalized to the microsomal metabolism of other nitrosamine carcinogens, we examined the effects of zinc on the hepatic microsomal metabolism of the related hepatic carcinogen DMN. At least three enzymes participate in the hepatic microsomal metabolism of DMN to form formaldehyde. 24*25At the substrate concentrations examined in this study, cytochrome P-450IIEl is responsible for approximately 90% of the hepatic mi-
A 8
-O-0-
crosomal metabolism of DMN.‘0,26 The microsomal metabolism of DMN by hepatic microsomes from the zinc-deficient animals was significantly greater (P < 0.05) than that of the zinc control microsomes at all DMN concentrations examined (Figure 3A). These differences are compatible with dietary zinc reducing the concentration of the cytochrome P-450 forms responsible for the metabolism of DMN or with dietary zinc inhibiting the activity of these cytochrome P-450 forms. Because there were no apparent differences in cytochrome P-450IIEl between the zinc-deficient and control microsomes, we next examined whether zinc was acting as a direct inhibitor of the cytochrome P-450 dependent metabolism of DMN. The addition of zinc to the in vitro assay signifi-
Zinc Control (ZC) microsomes Zinc Deficient (ZD) microsomes
*+ .C
B 8
-b ZD + IO ppm Zinc in vitro -Et- ZD + 5 ppm Zinc in vitro 6 + Zinc Deftaent (ZD) microsomes 1
2
6C
E g
+ +
ZC + 5 ppm Zinc in vitro Zinc Control (ZC) microsomes
40
.-
,/‘. 0’
20 4
Obp
012345678910 l/[mM of MBN]
.
I
0 1
4 l/[mM Ef Mi3N]3
Figure 2. Double reciprocal plots of the cytochrome P-&u-dependent microsomal metabolism of MBzN as measured by benzaldehyde formation of hepatic microsomes from zinc-deficient and zinc control animals. Each data point represents the average of at least four independent assays with an SEM 45% of the mean. (A)Comparison of the activity of hepatic microsomes from zinc-deficient and zinc control animals. Microsomal metabolism of MBzN by the zinc-deficient microsomes was significantly greater (P < 01~5) than the zinc control microsomes at all substrate concentrations examined. (B) Activity of hepatic microsomes from zinc-deficient rats with the addition of zinc in vitro. Activity was significantly reduced (P < 0.05) by zinc in vitro at all substrate concentrations examined. (C) Activity of hepatic microsomes from zinc control rats with the addition of zinc in vitro. Activity was significantly reduced (P < 0.05) by zinc in vitro at all substrate concentrations examined.
804 BARCH ET AL.
GASTROENTEROLOGY Vol. 103,No. 3
Table 2. Inhibition of the Microsomal Metabolism of MBzN by Pretreatment of Esophageal and Hepatic Microsomes With Zinc In Vitro Benzaldehyde formed @mol. min-’ . mg microsomal protein-‘)”
Pretreated with 0 ppm zinc Pretreated with 5 ppm zinc Pretreated with 10 ppm zinc
Esophageal microsomes Zinc-deficient diet
Esophageal microsomes Zinc control diet
0.154+ 0.006b
0.052f 0.008
0.106+ 0.013'
0.030It0.007d
0.099f o.014c
Not assayed
Benzaldehyde formed (nmol . min-’ . mg microsomal protein-‘)” Hepatic microsomes Zinc-deficient diet Pretreated with 0 ppm zinc Pretreated with 5 ppm zinc Pretreated with 10 ppm zinc
Hepatic microsomes Zinc control diet
0.202f 0.029b
0.085+ 0.002
0.097 f o.012c
0.054+ o.oo2c
0.038f 0.005d
0.020+ 0.002d
‘Microsomal incubation assays contained 5 mmol/L MBzN. bMean f SEM; esophageal microsomes, n = 3; hepatic microsomes, n = 4. “Significantly different from microsomes treated with 0 ppm zinc (P < 0.05). dSignificantly different from microsomes treated with 0 ppm zinc (P < 0.005).
cantly inhibited (P -C0.01 for the addition of 10 ppm zinc to the zinc-deficient microsomes, P < 0.01 for the additional of 5 ppm zinc to the control microsomes) the hepatic microsomal metabolism of DMN by the microsomes from both the zinc-deficient and zinc control animals; this inhibition was also concentration dependent (Figure 3). The zinc-dependent inhibition of the microsomal metabolism of DMN in vitro appears to reproduce the differences observed between the metabolism of DMN by microsomes from the zinc-deficient and zinc control rats. The kinetics of this in vitro inhibition once again suggested that zinc was acting either as a noncompetitive inhibitor or as an irreversible inhibitor of the cytochrome P-450-dependent microsomal metabolism of DMN. Pretreatment of the microsomes with zinc in vitro showed that zinc was also acting as a concentrationdependent irreversible inhibitor of the cytochrome P-450-dependent metabolism of DMN in microsomes from both zinc-deficient and zinc control animals (Table 3).
Discussion The current experiments show that zinc is a concentration-dependent irreversible inhibitor of the esophageal and hepatic microsomal metabolism of MBzN. These studies further show that the microsomal metabolism of MBzN is enhanced by dietary zinc deficiency without detectable alterations in the content of total esophageal or hepatic microsomal cytochrome P-450 and without detectable alterations in the profile of the forms of cytochromes P-450 expressed in the liver. To determine if this effect was specific to the metabolism of MBzN or was more generalized to the metabolism of other nitrosamine carcinogens, we examined the effects of zinc on the metabolism of the related hepatic carcinogen DMN. Of the various cytochrome P-4511s involved in the hepatic metabolism of DMN,24,25the ethanol-inducible cytochrome P-450IIEl appears to be the enzyme responsible for the hepatic activation of DMN to a mutagenic agent.12s13 This cytochrome P-450 form is different from the form of cytochrome P-450 responsible for the activation of MBzN.‘~*” At the substrate concentrations examined in this study, cytochrome P-450IIEl is responsible for approximately 90% of the hepatic microsomal metabolism of DMN.‘0,2” Similar to our results with MBzN, dietary zinc deficiency increased the hepatic microsomal metabolism of DMN without alterations in total hepatic cytochrome P-450 and without an apparent alteration in the hepatic concentration of cytochrome P-450IIEl. Zinc in vitro is also a concentration-dependent irreversible inhibitor of the hepatic microsomal metabolism of DMN. Previous studies have shown that zinc in vitro inhibits a variety of other cytochrome P-450-dependent reactions, including cytochrome P-450-dependent aniline hydroxylase, ethylmorphine demethylase, benzphetamine demethylase, benzo[a]pyrene hydroxylase, cytochrome P-450-dependent oxidation of NADPH, and NADPH-dependent reduction of cytochrome c.27-2g The concentrations of zinc required for the inhibition of these cytochrome P-450dependent reactions are also similar to the concentrations of zinc found in normal tissues. Jeffery2’ has reported that this inhibition appears to be caused by zinc effecting the electron transfer between the NADPH cytochrome P-450 reductase and the cytochrome P-450 complex or through alteration of the oxidation-reduction potential of the NADPH cytochrome P-450 reductase. A possible explanation for the increased metabolism of MBzN and DMN associated with dietary zinc deficiency is that physiological levels of zinc may be an endogenous inhibitor of the cytochrome P-450-
INHIBITION OF NITROSAMINE METABOLISM BY ZINC
September 1992
805
Figure 3. Double reciprocal plots of the cytochrome P-&O-dependent microsomal metabolism of DMN as measured by formaldehyde formation of hepatic microsomes from zinc-deficient and zinc control animals. Each data point represents the average of at least four independent assays with an SEM 40% of the mean. (A) Comparison of the activity of hepatic microsomes from zinc-deficient and zinc control animals. Microsomal metabolism of DMN by the zinc-deficient microsomes was significantly greater (P < 0.05) than the zinc control microsomes at all substrate concentrations examined. (B) Activity of hepatic microsomes from zinc-deficient rats with the by the addition of 10 ppm zinc in vitro at all substrate addition of zinc in vitro. Activity was significantly reduced (P < 0.01) concentrations examined. (C) Activity of hepatic microsomes from zinc control rats with the addition of zinc in vitro. Activity was significantly reduced (P< 0.01) by the addition of 5 ppm zinc in vitro at all substrate concentrations examined.
dependent metabolism of MBzN and DMN. The cytochrome P-450 enzymes in the tissues of the zinc control rats would be exposed to significantly higher concentrations of zinc than the cytochrome P-450 enzymes in the zinc-deficient tissues of zinc-deficient rats. Enzymes that irreversibly accumulated higher levels of zinc in vivo would then show lower enzymatic activity in vitro. Such an inhibition of cytochrome P-450 activity would explain the lower levels of MBzN and DMN metabolism observed with the microsomes from zinc control animals compared with microsomes from zinc-deficient animals. In summary, the current study shows that dietary zinc deficiency significantly increases the cy-
tochrome P-450-dependent microsomal metabolism of MBzN and DMN without alterations in the microsomal content of cytochrome P-450 of the esophagus or liver. The addition of zinc in vitro, at concentrations found in normal tissues, irreversibly inhibited the microsomal metabolism of MBzN and DMN in a concentration-dependent manner. These results suggest that physiological levels of zinc may be an endogenous inhibitor of MBzN metabolism. Dietary zinc deficiency appears to reduce this inhibition, resulting in an increase in carcinogen activation. This effect could explain the increased incidence of MBzN-induced esophageal carcinoma observed with dietary zinc deficiency. References
Table 3. Inhibition
of the Hepatic Microsomal Metabolism by Pretreatment ofHepatic Microsomes With Zinc In Vitro
ofDMN
1.
Formaldehyde formed @mol. min-’ - mg mici-osomal protein-‘)O Hepatic microsomes Zinc-deficient diet Pretreated with 0 ppm zinc Pretreated with 5 ppm zinc Pretreated with 10 ppm zinc
2.
Hepatic microsomes Zinc control diet 3.
0.55 + 0.04b
0.36 + 0.04
0.38 + 0.04d
0.28 + 0.03’
0.16 + O.Ogd
0.11 ? 0.02d
“Microsomal incubation assays contained 5 mmol/L DMN. bMean + SEM; n = 4. “Significantly different from microsomes treated with 0 ppm zinc (P < 0.05). dSignificantly different from microsomes treated with 0 ppm zinc (P < 0.005).
4.
5.
6.
Druckrey H, Preussmann R, Ivankovic S, Schmahl IJ, Afkham J, Blum G, Mennel HD, Muller M, Petropoulos P, Schneider H. Organotrope Carcinogene Wirkungen bei 65 Verschiedenen N-Nitroso-Verbindungen an BD-Ratten. Krebsforschung 1967;69:103-201. Stinson SF, Squire RA, Sporn MD. Pathology of esophageal neoplasms and associated proliferative lesions induced in rats by N-methyl-N-benzylnitrosamine. 1 Nat1 Cancer Inst 1978;61:1471-1472. Druckrey H. Organospecific carcinogenesis in the digestive tract. In: Nakahara W, Takayama S, Sugimura T, Odashima S, eds. Topics in chemical carcinogenesis. Proceedings of the 2nd International Symposium of the Princess Takamatsu Cancer Research Fund. Baltimore, MD: University Park, 1972:73-101. Fong LYY, Sivak A, Newberne PM. Zinc deficiency and methylbenzylnitrosamine-induced esophageal cancer in rats. J Nat1 Cancer Inst 1978;61:145-150. Gabrial GN, Schrager TF, Newberne PM. Zinc deficiency, alcohol, and a retinoid: association with esophageal cancer in rats. J Nat1 Cancer Inst 1982;68:785-789. Barth DH, Kuemmerle SC, Hollenberg PF, Iannaccone PI.
806
7.
8.
9.
10.
11.
12.
13.
14.
15.
16. 17.
18.
19.
BARCH ET AL.
Esophageal microsomal metabolism of N-nitrosomethylbenzylamine in the zinc-deficient rat. Cancer Res 1984;44:56295633. Yahagi T, Nagao M, Seino Y, Matsushima T, Sugimura T, Okada M. Mutagenicities of N-nitrosamines on Salmonella. Mutation Res 1977;48:121-130. Pegg AE. Formation and removal of methylated nucleosides in nucleic acids of mammalian cells. Ret Results Cancer Res 1983;84:49-62. Barth DH, Fox CC. Dietary zinc deficiency increases the methylbenzylnitrosamine-induced formation of 06-methylguanine in the esophageal DNA of the rat. Carcinogenesis 1987;8:1461-1464. Barth DH, FOX CC, Koop DR. Different cytochrome P-450 enzymes are responsible for the activation of hepatic and esophageal specific nitrosamines (abstr). Hepatology 1988;8:1278. Ludeke B, Meier T, Kleihues P. Bioactivation of asymmetric N-dialkylnitrosamines in rat tissues derived from the ventral entoderm. IARC Sci Pub1 1991;105:286-293. Yang CS, Tu YY, Koop DR, Coon MJ. Metabolism of nitrosamines by purified rabbit liver cytochrome P-450 isozymes. Cancer Res 1985;45:1140-1145. Yoo J-SH, Yang CS. Enzyme specificity in the metabolic activation of N-nitrosodimethylamine to a mutagen for Chinese hamster V79 cells. Cancer Res 1985;45:5569-5574. Stoner GD, Babcock MS, Cothern GA, Klaunig JE, Gunning WT, Knipe SM. In vitro transformation of rat esophageal epithelial cells with N-nitrosobenzylmethylamine. Carcinogenesis 1982;3:629-634. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:205-225. Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomes. J Biol Chem 1964;239:2379-2385. Wrighton SA, Campanile C, Thomas PE, Maines SL, Watkins PB, Parker G, Mendez-Picon G, Haniu M, Shively J, Levin W, Guzelian PS. Identification of a human liver cytochrome P-450 homologous to the major isosafrole-inducible cytochrome P-450 in the rat. Mol Pharmacol 1986;29:405-410. Schuetz EG, Wrighton SA, Safe SH, Guzelian PS. Regulation of cytochrome P-450~ by phenobarbital and phenobarbitallike inducers in adult rat hepatocytes in primary monolayer culture and in vivo. Biochemistry 1986;25:1124-1133. Watkins PB, Wrighton SA, Maurel P, Schuetz EG, MendezPicon G, Parker GA, Guzelian PS. Identification of an induc-
GASTROENTEROLOGYVol.103,No.3
20.
21.
22.
23.
ible form of cytochrome P-450 in human liver. Proc Nat1 Acad Sci 1985;82:6310-6314. Wrighton SA, Thomas PE, Molowa DT, Haniu M, Shively JE, Maines SL, Watkins PB, Parker G, Mendez-Picon G, Levin W, Guzelian PS. Characterization of ethanol-inducible human liver N-nitrosodimethylamine demethylase. Biochemistry 1986;25:6731-6735. Labuc GE, Archer MC. Esophageal and hepatic microsomal metabolism of N-nitrosomethylbenzylamine and N-nitrosodimethylamine in the rat. Cancer Res 1982;42:3181-3186. Chung FL, Wang M, Hecht SS. Effects of dietary indoles and isothiocyanates on N-nitrosodimethylamine and 4-(methylnitrosamino)-I-(3-pyridyl)-I-butanone a-hydroxylation and DNA methylation in rat liver. Carcinogenesis 1985;6:539-543. Barth DH, Iannaccone PM. Role of zinc deficiency in carcinogenesis. In: Poirier L, Newberne P, Pariza M, eds. Essential nutrients in carcinogenesis. Advances in experimental medicine and biology. Volume 206. New York: Plenum, 1986:517-
527. 24. Lake BG, Heading
25. 26.
27.
28.
29.
CE, Phillips JC, Gangolli SD, Lloyd AG. Some studies on the metabolism in vitro of dimethylnitrosamine by rat liver. Biochem Sot Transac 1974;2:610-612. Farrelly J. A new assay for the microsomal metabolism of nitrosamines. Cancer Res 1980;40:3241-3244. Peng R, Tu YY, Yang CS. The induction and competitive inhibition of a high affinity microsomal nitrosodimethylamine demethylase by ethanol. Carcinogenesis 1982;3:1457-1461, Chvapil M, Ludwig JC, Sipes IG, Misiorowski RL. Inhibition of NADPH oxidation and related drug oxidation in liver microsomes by zinc. Biochem Pharmacol 1976;25:1787-1791. Colby HD, Johnson PB, Zulkoski JS, Pope MR, Miles PR. Comparative effects of cadmium, zinc, and lead in vitro on pulmonary, adrenal and hepatic microsomal metabolism in the guinea pig. J Toxic01 Environ Health 1981;8:907-915. Jeffery EH. The effect of zinc on NADPH oxidation and monooxygenase activity in rat hepatic microsomes. Mol Pharmacol 1983;23:467-473.
Received December 2, 1991. Accepted February 25, 1992. Address requests for reprints to: David H. Barth, Northwestern University Cancer Center, Room 8370 Olson, 303 East Chicago Avenue, Chicago, Illinois 60611. Supported in part by National Institutes of Health grant CA40487 and a Merit Review Grant from the Research Service of the Department of Veterans Affairs.
