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(CANCER RESEARCH (SUPPL.) 52. 2l08s-2113s. April I, 1992]

Metabolism of Food-derived Heterocyclic Amines in Human and Rabbit Tissues byP4503A Proteins in the Presence of Flavonoids1

Ross A. McKinnon, Wendy M. Burgess, Pauline de la M. Hall, Zaiminuddin Abdul-Aziz, and Michael E. McManus2

Departments of Clinical Pharmacology [R. A. McK.. W. M. B., Z. A-A., M. E. McM.] and Histopathology Â¡P.de la M. //./, School of Medicine, Flinders University ofSouth Australia, Bedford Park, SA 5042, Australia

Abstract

The ability of human and rabbit gastrointestinal-tract microsomes tometabolize the heterocyclic amine 2-amino-3,4-dimethylimidazo[4,5-/]-quinoline (MelQ) to a mutagen was determined with the Ames test.When human jejunal and ileal microsomes were used as the metabolicactivation source, MelQ produced 1675 and 388 revertants/mg of micro-soma! protein, respectively, and this increased to 29,230 and 17,963revertants/mg of microsoma! protein, respectively, in the presence of 100MMa-naphthoflavone. MelQ in the presence of control rabbit duodenal,jejunal, and ileal microsomes produced 2304 Â±1018, 988 Â±386, and444 Â±134 (mean Â±SD, four samples) revertants/mg of microsomalprotein, respectively. In the presence of a-naphthoflavone (100 n\\}.these activities increased >7-fold. P4S03A proteins were detectable onWestern blots of microsomes prepared from both human and rabbit smallintestine. Further, rifampicin-induced rabbit hepatic-microsomal activation of MelQ was completely inhibited at low concentrations of a-naphthoflavone, but at higher concentrations (/.

P450 ACTIVATION OF HETEROCYCLIC AMINES

Protein concentrations and P450 levels were determined as previouslydescribed (6-8).

Antibodies and Enzymes. Antibodies against rabbit P4503A6, rabbitP4501A1, and human P4503A3 were obtained or prepared as previously described (6-8). Rabbit P4501A1, P4501A2, P4503A6, andNADPH-cytochrome P450 reducÃ-asewere purified as described byMcManus et al. (l, 8). Reconstitution of rabbit P450 activity wasperformed as outlined previously (7, 8, 14). Immunoblotting of micro-somal and purified enzymes was carried out as described by McManuset al. (6-8).

Mutation Assay. The mutagenicity of MelQ was tested on Salmonellatyphimurium TA98; a detailed account of the methodology used inthese experiments was recently published (15).

Table 1 Effect of a-naphthoflavone (100 Â¡Â¡M)on the mutagenic activation ofMelQ by rabbit hepatic and gastrointestinalmicrosomesSource

ofmicrosomesLiver

StomachDuodenumJejunumIleumCecumColonMutagenicity

(no. ofrevertants/mgprotein)Control658.000

Â±374,000 (4)Â°ND* (2)

2,304 Â±1,018(4)988 Â±386 (4)444 Â±134 (4)

ND(3)ND(3)a-Naphthoflavone1

56,000 Â±12,000(4)ND(2)

16,826 Â±2,363 (4)10,256 Â±10,826(4)4,561 Â±1,632(4)4,248 Â±769 (3)1,678 Â±454 (3)

" Mean Â±SD. Numbers in parentheses, numbers of samples.* ND, activity not detected.

Results and Discussion

Heterocyclic amines were shown to be mutagenic in a rangeof short-term tests and are potent animal carcinogens (4, 5).These compounds are formed during the cooking of beef, lamb,chicken, pork, and fish (4, 5). Because the major portal of entryof heterocyclic amines into the body is oral, they may constitutea significant dietary risk to humans, particularly with regard tocancers of the gastrointestinal tract.

Oxidation of aromatic amines to ./V-hydroxy derivatives is anobligatory step in the activation of these compounds to geno-toxins (16). These oxidative procarcinogens may be formeddirectly in the gastrointestinal tract or, alternatively, may beformed in the liver and then transported to the gastrointestinaltract, where they may undergo further metabolism by enzymessuch as sulfotransferase and A'-acetyltransferase (16-18) to the

ultimate carcinogen. If the former is correct, it appears likelythat those cytochromes P450 responsible for the activation ofheterocyclic amines must be localized in the gastrointestinaltract. Techniques used with liver cytochromes P450 includestudies of metabolic activities of purified P450s, antibody-inhibition studies; studies of correlations between immunore-active protein and metabolic activities of microsomal preparations, inhibitor studies, and studies of metabolic activities ofexpressed P450 complementary DNAs. By such techniques, theP4501A family of proteins was shown to be primarily responsible for the activation of heterocyclic amines in animals and

humans (6-9). However, when human gastrointestinal-tractmicrosomes were assayed by the Western blot technique andprobed with anti-rabbit P4501A1 antiserum, which recognizesboth human P4501A1 and P4501A2 proteins (6), no P4501Aprotein was detectable (data not shown). When the capacity ofhuman gastrointestinal-tract microsomes to activate MelQ toa mutagen in the Ames test was determined, the number ofrevenants formed by jejunal, Â¡leal,and colonie microsomes was1675, 388 (average of two subjects), and 2200 revertants/mg ofmicrosomal protein, respectively. Only one of four humancolons tested exhibited activity towards MelQ, whereas microsomes prepared from four stomach samples demonstrated noactivity. The human colonie microsomes that exhibited activitywere prepared from a patient undergoing surgical resection ofan adenocarcinoma. Human liver microsomes at the sameconcentration of MelQ formed 2,579,100 Â±2,368,725 (meanÂ±SD, five samples, range = 776,500 to 6,040,000) revenants/mg of microsomal protein. These immunoblot and metabolicdata suggest negligible expression of P4501A proteins in thehuman gastrointestinal tract.

When microsomes from control rabbit, duodenum, jejunum,and ileum were assayed by Western blots and probed with anti-

rabbit P4501A1 antiserum, a single band corresponding toP4501A1 was detectable (Fig. \A). The band recognized byP4501A1 antiserum in the ileum was barely detectable, however, despite the addition of 75 ng of microsomal protein to the

-â€¢â€¢â€¢â€¢Ii B

1A1 I I D TCDDLvCTCCCC CI JS E Lv

3A6 I I I S S Lv 3A6 3A6 Lv Lv Lv C C C Ce Ce 3A6 Lv

Fig. I. Typical Western blots of human and rabbit gastrointestinal and hepatic microsomes. Electrophoresis and immunoblotting were carried out as describedunder "Materials and Methods." The migration of protein was from the top to the bottom of the gel. The following symbols refer to microsomes from: Lv, liver; E,

esophagus; 5, stomach; D, duodenum; J, jejunum; /, ileum; Ce, cecum; C, colon; CT, colonie tumor; and TCDD, rabbit liver induced with TCDD. The following referto purified rabbit hepatic P450s: 3A6, P4503A6; and 1AI, P4501A1. A, rabbit P4501A1 (4 pmol) and hepatic (2 jig TCDD induced) and gastrointestinal (75 >

Fig. 2. Effect of n-naphthoflavone on theactivation of MelQ by human hepatic andgastrointestinal microsomes. a, effect of a-naphthoflavone on the activation of MelQ byhepatic microsomes from subjects H5, H6,H11, H17, and H23. Control activity was 2579Â±2369 revertants/Vg protein (mean Â±SD, fivesamples); b, c, and

P450 ACTIVATION OF HETEROCVCL1C AMINES

Fig. 3. Effect of flavonoids on the activation of MelQ by control (A), rifampicin-in-duced (O and â€¢¿�),and TCDD-induced (G) rabbit hepatic microsomes. a, a-naphthoflavone;b, flavone. Control activities for the two rifam-picin-induced microsomes were 989 (O) and631 (â€¢)revertants/0.5 /ig of microsomal protein, respectively. Activities for the control andTCDD-induced microsomes were 2955 revertants/0.5 ng and 2865 revertants/0.25 ng ofmicrosomal protein, respectively.

20-

0 .0001.001 .01

60-

ALPHA-NAPHTHOFLAVONE ( i:M )

1 1 0

FLAVONE ( nM )

1 000

isozyme is inhibited and a P4503A isozyme is activated. A concentrations it appears that MelQ becomes a substrate forsimilar pattern of a-naphthoflavone modulation of MelQ mu-

tagenicity was also observed in human jejuna! and ileal microsomes, except that stimulation was apparent at concentrationsas low as 0.1 MMa-naphthoflavone (Fig. 2, b and c), whereas inhuman hepatic microsomes stimulation was not observed untila concentration of 1 MMwas reached. This differential sensitivityto a-naphthoflavone stimulation may be because the pool ofP4503A proteins present in the gastrointestinal tract is differentfrom the pool in the liver. The degree of stimulation of MelQmutagenicity in the presence of human small intestinal microsomes was very pronounced. In human jejunal microsomes, thenumber of revenants in the absence of a-naphthoflavone was1675/mg of microsomal protein, increasing to 29,230 rever-tants in the presence of 100 MMa-naphthoflavone. Similarly,in ileal microsomes from two subjects, control activities were165 and 610 revertants/mg of microsomal protein, and in thepresence of 100 MMa-naphthoflavone these increased to 19,245and 16,680 revenants, respectively.

In tandem with the human studies, the ability of controlrabbit gastrointestinal-tract and hepatic microsomes to activateMelQ in the presence of a-naphthoflavone was studied. Table1 demonstrates that 100 MMa-naphthoflavone stimulated themutagenicity of MelQ in rabbit small intestinal microsomes>7-fold, an increase similar to that observed in their humanequivalents. Rabbit colonie and cecal microsomes were unableto activate MelQ; however, in the presence of 100 MM Â«-naphthoflavone, 4248 and 1678 revertants/mg of microsomalprotein, respectively, were observed (Table 1). From our Western blot data, P4503A protein was detectable in rabbit coloniemicrosomes (Fig. ID) but not in human colonie microsomes(Fig. \B). Thus, the ability of a-naphthoflavone to stimulatethe activation of MelQ by rabbit colonie microsomes mostlikely reflects the presence of P4503A6. Similarly, P4503Aprotein was not detectable on Western blots of either human(Fig. \B) or rabbit stomach microsomes (Fig. 1C), and stomachmicrosomes from both species were not capable of activatingMelQ to a mutagen in the absence or presence of 100 MMÂ«-naphthoflavone. The observation, in a single sample, that a-naphthoflavone inhibits completely the ability of human coloniemicrosomes to activate MelQ suggests the presence of P4501Aproteins in this sample at levels below the limit of detection ofthe Western blot procedure (Fig. 2d). The pattern of a-naphthoflavone modulation of MelQ activation by control rabbithepatic microsomes was similar to that seen in human hepaticmicrosomes. At 1 MMa-naphthoflavone MelQ activation wascompletely inhibited, whereas at 100 MM a-naphthoflavoneactivity returned to 24% of control levels (Table 1). Again, atlow concentrations of a-naphthoflavone it is apparent thatinhibition of P4501A proteins is occurring, whereas at higher

members of the P4503A subfamily, in this case P4503A6.Because the aforementioned data point to P4503A proteins

being involved in MelQ activation, the ability of purified rabbitP4503A6 to carry out this process was determined. Whenpurita-d rabbit P4503A6 (100 pmol) was used as the activating

source in the Ames test, it was found to be a poor activator ofMelQ to a mutagen, resulting in only 304 revenants/100 pmolof P4503A6. This activation is negligible, compared with thatwith purified rabbit P4501A1 and P4501A2, which produce5,940 and 147,784 revenants/10 pmol of purified protein,respectively. The addition of 100 MMa-naphthoflavone to theincubation had no effect (313 revenants/100 pmol P4503A6)on the ability of P4503A6 to activate MelQ. Although thisresult may simply reflect low activity of P4503A6 toward MelQ,considerable difficulty has been encountered in reconstitutingP4503A6 activity in vitro (11, 23). For a comparable P4503Aprotein in humans, the addition of spermine to the incubationfacilitates reconstitution (24). We, therefore, studied the effectof spermine on the reconstitution of P4503A6 activity in vitro.Spermine (1 mivi) in the absence (252 revenants/100 pmolP4503A6) or presence (268 revenants/100 pmol P4503A6) of100 MMa-naphthoflavone had no effect on the ability of rabbitP4503A6 to activate MelQ to a mutagen. To overcome thisdifficulty, we compared the ability of control, rifampicin-in-duced, and TCDD-induced rabbit hepatic microsomes to activate MelQ (Fig. 3a) in the presence of a-naphthoflavone.Rifampicin is a known inducer of P4503A6, whereas TCDDinduces both P4501A1 and P4501A2 (17). The data from theseexperiments demonstrate that a-naphthoflavone at low concentrations almost completely inhibited the ability of rifampicin-induced microsomes to activate MelQ; however, at high concentrations (i.e., 100 MM)this returned to control levels. Incontrol and TCDD-induced hepatic microsomes, total inhibition was observed at low concentrations of a-naphthoflavone,whereas at higher concentrations (i.e., 100 MM), activity returned to only 8% (control) and 5% (TCDD) of the activitypresent in the absence of the modulator. These data indicatethat rabbit P4503A6 in the presence of a flavonoid is capableof metabolizing the food-derived heterocyclic amine MelQ to amutagen in the Ames test. It also enables us to discount thepossibility that, at higher concentrations of a-naphthoflavone,the ability of P4501A2 protein to carry out this process isstimulated. This finding appears significant because Huang etal. (21) reported that flavonoids stimulate the metabolism ofbenzo(a)pyrene by this isozyme. Indeed, in the present study100 MMa-naphthoflavone completely inhibited the ability ofpurified rabbit P4501A1 and P4501A2 to activate MelQ.

Flavonoids are natural constituents of many food plants, andit is estimated that the average daily intake of these compounds

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P450 ACTIVATION OF HETEROCVCL1C AMINES

Fig. 4. Effect of flavone on the activationof MelQ by human and control rabbit smallintestinal microsomes. a and Â¿>.human jejunaland ileal microsomes. respectively; c and igof microsomal protein, respectively.

>â€¢t->

5ocO

10 100 1000 O

2000

10 100 1000 O 10 100 tooo

FLAVONE(uM )

is approximately 1 g. Flavones, flavonols, and flavanones account for approximately 17% of flavonoids consumed daily(25). Therefore, to determine whether heterocyclic amines interact with flavonoids in the diet, the effect of flavone on themutagenic activation of MelQ by gastrointestinal-tract microsomes was investigated. Fig. 4 shows that flavone stimulatesthe activation of MelQ by both human and rabbit small-intestinal microsomes at least 12-fold. Flavone had effects on theactivation of MelQ by control, rifampicin-induced, and TCDD-induced rabbit hepatic microsomes similar to those observedwith a-naphthoflavone (Fig. 3). However, the increase in MelQactivation observed at higher concentrations of modulator wasless pronounced with flavone than with a-naphthoflavone.These data suggest that in the presence of flavone, as with a-naphthoflavone, members of the P4503 A subfamily are capableof activating MelQ to a mutagen in both rabbit and humangastrointestinal-tract microsomes. Flavone has also been shownto stimulate the metabolism of benzo(a)pyrene and the activation of aflatoxin by human liver microsomes (26). Recently,human hepatic P4503A3 and P4503A4/P450NF were shownÃ®oactivate both benzo(a)pyrene-7,8-diol (27) and aflatoxin Bl(28,29) to mutagens. Lasker et al. (30) also showed that flavonestimulates oxidative drug metabolism in vivo in neonatal rats,which highlights the possibility of an interaction occurringbetween dietary flavonoids and heterocyclic amines in humans.

This present study demonstrates that, in control rabbits,P4501A1 and P4503A6 are present in the small intestine butonly P4503A6 is found in the large intestine. P4503A proteinis also present in the small intestine of humans. Other than theability of a-naphthoflavone to inhibit completely the activationof MelQ by the one human colon demonstrating activity, nodata were obtained suggesting that P4S01 Al is a major constitutive isozyme of the human gastrointestinal tract. The declinein immunoreactive P4503A levels from the small to the largeintestine is consistent with the distribution of NADPH-cyto-chrome P450 reductase in the human gastrointestinal tract (31).The presence of P4503A6 immunoreactive protein in all segments of the rabbit gastrointestinal tract tested except stomach

may be a characteristic of herbivores and may indicate that adietary component is inducing P4503A6 in these tissues. Collectively, the present data indicate that a potential interactionmay exist between the diet-derived heterocyclic amines andflavonoids. The significance of such an interaction in the etiology of human cancer is unclear, given that the vast majority ofhuman gastrointestinal cancers occur in the colorectal region(2) and no P4503A protein was detected in colonie microsomesprepared from four different subjects. However, a role for thisinteraction in tumors of the small intestine and other diseasesof the gastrointestinal tract, such as inflammatory bowel disorders, cannot be discounted. Elucidation of the specificP4503A proteins involved in this heterocyclic amine-flavonoidinteraction in the human gastrointestinal tract will necessitatethe use of P450 complementary DNA-expression systems todefinitively determine what isozymes are being stimulated byflavonoids.

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1992;52:2108s-2113s. Cancer Res Ross A. McKinnon, Wendy M. Burgess, Pauline de la M. Hall, et al. FlavonoidsRabbit Tissues by P4503A Proteins in the Presence of Metabolism of Food-derived Heterocyclic Amines in Human and

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