Characterization of xenobiotic-metabolizing enzymes and nitrosamine ...

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nitrosamine metabolism in the human esophagus. Theresa J.Smith1,4, Anita Liao1, Li-Dong Wang1,2,. Guang-yu Yang1, Sandra Starcic3, Martin A.Philbert3.
Carcinogenesis vol.19 no.4 pp.667–672, 1998

Characterization of xenobiotic-metabolizing enzymes and nitrosamine metabolism in the human esophagus

Theresa J.Smith1,4, Anita Liao1, Li-Dong Wang1,2, Guang-yu Yang1, Sandra Starcic3, Martin A.Philbert3 and Chung S.Yang1 1Laboratory

for Cancer Research, College of Pharmacy, 164 Frelinghuysen Road, Rutgers University, Piscataway, NJ 08855, 2Henan Medical University, Zhengzhou, Henan, China and 3Department of Pharmacology and Toxicology, College of Pharmacy, Rutgers University, Piscataway, NJ 08855, USA

4To

whom correspondence should be addressed Email: [email protected]

Esophageal cancer has been associated with tobacco smoking, and nitrosamines are possible causative agents for this cancer. The present study investigated the metabolism of the tobacco carcinogens N9-nitrosonornicotine (NNN), 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosodimethylamine (NDMA), as well as the presence of xenobiotic-metabolizing enzymes in human esophageal tissues from individuals in the United States and Huixian, Henan Province, China (a high-risk area for esophageal cancer). All esophageal microsomal samples activated NNN and the metabolic rate was 2-fold higher in the esophageal samples from China than the USA. All microsomal samples activated NDMA. However, most of the microsomal samples did not activate NNK. Troleandomycin (an inhibitor of cytochrome P450 3A) decreased the formation of NNNderived keto acid by 20–26% in the esophageal microsomes. The activities for NADPH: cytochrome c reductase, ethoxycoumarin O-deethylase, NAD(P)H: quinone oxidoreductase and glutathione S-transferase were present in the esophageal samples. Coumarin 7-hydroxylase (a representative activity for P450 2A6) activity was not detected in the esophageal microsomal samples. The activities for nitrosamine metabolism and xenobiotic-metabolizing enzymes were decreased (by 30–50%) in the squamous cell carcinomas compared with their corresponding non-cancerous mucosa. The presence of activation and detoxification enzymes in the esophagus may play an important role in determining the susceptibility of the esophagus to the carcinogenic effect of nitrosamines. Our results suggest that P450s 3A4 and 2E1 are involved in the activation of NNN and NDMA, respectively, in the human esophagus. Introduction Squamous cell carcinoma (SCC*) of the esophagus is one of the most common cancers worldwide (1–4). In the Western countries, cigarette smoking and alcohol intake are the major risk factors, whereas in some noted high-risk geographical *Abbreviations: SCC, squamous cell carcinoma(s); NNN, N9-nitrosonornicotine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NDMA, Nnitrosodimethylamine; P450, cytochrome P450; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; TAO, troleandomycin; CDNB, 1-chloro-2,4-dinitrobenzene; NQOR, NAD(P)H: quinone oxidoreductase. © Oxford University Press

areas, including certain areas in northern China, Iran and South Africa, dietary carcinogen exposure and nutritional deficiencies are major etiological factors (2,4–8). Because nitrosamines have been shown to induce esophageal tumors readily in animals, this class of carcinogens have been suspected to be causative agents for this cancer (9–11). The formation of DNA adducts in the esophagus of nitrosamine-treated rats and in esophageal cancer tissues of humans have been observed (12–14). The tobacco-specific nitrosamines, N9-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) have been suggested to play a role in human tobaccorelated cancers (15,16). N-Nitrosodimethylamine (NDMA) is present in cigarette smoke, as well as many other sources (17,18), and induces liver and kidney tumors in animals (11,19). NNN has been shown to induce esophageal and nasal cavity tumors in rats (15,16). On the other hand, NNK has been demonstrated to induce lung tumors but not esophageal tumors in all laboratory animals tested (15,16). In order to exert their carcinogenic effect, nitrosamines need to be metabolically activated by way of α-hydroxylation, leading to the formation of diazohydroxides, which can result in DNA alkylation. NNN and NNK share the common pathway leading to the formation of pyridyloxobutylated DNA adducts (15) (Figure 1). Pyridyloxobutylation of DNA is believed to be important in NNN and NNK carcinogenesis (15,16,20). With the pyridyloxobutylating agent 4-(acetoxymethylnitrosamino)1-(3-pyridyl)-1-butanone, it was demonstrated that pyridyloxobutylated DNA increased the persistence of O6-methylguanine adducts by inhibiting O6-alkylguanine transferase (21). Although the 59-hydroxylation pathway of NNN leads to the formation of pyridylformylpropyldiazohydroxide, DNA adducts have not been detected (15,22). On the other hand, activation of NNK and NDMA, but not NNN, can lead to the methylation of DNA. Studies have suggested that DNA methylation is more important for NNK lung carcinogenesis (23,24). The molecular events leading to human esophageal carcinogenesis are not known. Cytochrome P450 (P450) enzymes are involved in the activation of NNN, NNK and NDMA (25–30). Recently, heterologously expressed human P450 2A6 has been shown to metabolize NNN to lactol (a 59-hydroxylation product) and P450 3A4 catalyzed the formation of keto alcohol (a 29hydroxylation product) (27). Moreover, a low Km enzyme is present in the esophagus of rats that activates NNN (25). However, the specific P450s that are responsible for the activation of NNN in human esophagus are not known. The organ specificity of NNK and NNN may be due to the presence of specific enzymes in the lung and esophagus, respectively, which selectively bioactivates these tobacco-specific nitrosamines. Glutathione S-transferases (GST) exist in multiple forms and are important in the detoxification of xenobiotics. GSTs are grouped into four classes (alpha, pi, mu and theta) based 667

T.J.Smith et al.

Fig. 1. Metabolic pathways for NNN and NNK (15).

upon their substrate specificities, amino acid composition and sequence, and immunological cross-reactivities (31–33). Of these enzymes, GSTs mu (GSTM1) and theta are polymorphic (34–36). GSTs are expressed in several tissues, including the esophagus (37–39). Although GSTs are important for the detoxification of xenobiotics, conjugation may also be a means of transporting activated metabolites from the liver to extrahepatic tissues where it could be reactivated into reactive metabolites (40). Furthermore, conjugation to glutathione is known to be involved in the activation of some halogenated compounds (31,41,42). Since the esophagus is a major tissue for carcinogen exposure, xenobiotic-metabolizing enzymes could play an important role in determining the susceptibility of the esophagus to carcinogens. The present study investigated the metabolism of NNK, NNN and NDMA, and the presence of xenobioticmetabolizing enzyme activities in esophageal mucosa microsomes from esophageal cancer patients in Huixian, Henan Province, China (a high-risk area for esophageal cancer) and individuals in the United States. These activities in microsomes from esophageal carcinomas and non-cancerous epithelia were compared. Materials and methods Chemicals [5-3H]NNK (2.4 Ci/mmol; purity .97%) and [5-3H]NNN (3.42 Ci/mmol; purity .95%) were purchased from Chemsyn Science Laboratories (Lenexa,

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KS). The radiolabeled NNK and NNN were further purified by reverse phase high performance liquid chromatography (HPLC) before use. NNK and NNN metabolite standards were kindly provided by Dr Stephen Hecht (University of Minnesota Cancer Center, Minneapolis, MN). [14C]NDMA (49 mCi/ mmol) was custom synthesized by SRI International Co. (Menlo Park, CA). [14C]Formaldehyde (10 mCi/mmol) was purchased from New England Nuclear (Boston, MA). Coumarin, troleandomycin (TAO), cytochrome c, menadione, 1-chloro-2,4-dinitrobenzene (CDNB), reduced glutathione, hematoxylin and eosin were purchased from Sigma Chemical Co. (St Louis, MO). Unlabeled NDMA, 7-ethoxycoumarin and 7-hydroxycoumarin were from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals were of reagent grade. Human esophagus Esophageal samples from the USA were from individuals who died from accidents and were obtained from The National Disease Research Interchange (Philadelphia, PA). The US esophageal samples were snap-frozen 1.5 to 4 h post-mortem, from subjects ages 21–64 years, eight were from males and six were from females, and all the samples were morphologically normal. Esophageal mucosa microsomes were prepared by homogenizing the tissues in 0.05 M Tris–HCl, 1.15% KCl (pH 7.4) buffer using a motor-driven polytron (Brinkmann, Switzerland) and centrifuging at 9000 g for 30 min at 4°C. The supernatants were removed and further centrifuged at 105 000 g for 90 min at 4°C to separate the cytosolic fractions and microsomes. The microsomes were washed [resuspended in 1.15% KCl–10 mM EDTA (pH 7.4) and centrifuged] once and then suspended in 0.25 M sucrose. Both microsomal and cytosolic samples were stored in aliquots at –80°C. Surgically resected human esophagi from esophageal cancer patients were obtained from Huixian, Henan Province, China. The resected samples were frozen in liquid nitrogen 1 to 4 h after surgery. Esophageal mucosa microsomes were prepared from non-cancerous (normal) and cancerous sections of the samples from the same individuals. The ages varied from 40–62 years and seven were from males and one was from a female. For histological analysis, esophageal sections were prepared from the USA and China samples.

Human esophageal enzymes and nitrosamine metabolism NNK and NNN metabolism analyses Incubation mixtures consisted of 100 mM sodium phosphate, pH 7.4, 1 mM EDTA, 3 mM MgCl2, an NADPH-generating system (5 mM glucose 6phosphate, 1 mM NADP1, and 1.5 units glucose 6-phosphate dehydrogenase), 10 µM [5-3H]NNK or [5-3H]NNN and 0.1 mg esophageal mucosa microsomal protein in a total volume of 0.2 ml. Reactions were carried out for 30 min at 37°C and terminated with 50 µl each of 25% zinc sulfate and saturated barium hydroxide. Each sample was centrifuged and filtered, and 0.1 ml was coinjected with 5 µl of metabolite standards onto a reverse-phase HPLC system equipped with a radioflow detector (Radiomatic Instruments and Chemical Co., Tampa, FL) (43). For NNK analysis, it was eluted with a linear gradient of 92% A (0.02 M Tris–HCl, pH 6) and 8% B (methanol) to 75% A and 25% B over a 45-min period followed by a 10-min period of 100% B at a flow rate of 1 ml/min. For NNN analysis, it was eluted with a linear gradient of 100% A (0.02 M Tris–HCl, pH 7) to 70% A and 30% B (methanol) over a 60-min period at a flow rate of 1 ml/min. For the inhibition studies, coumarin was dissolved in methanol and used at 0.5% of the total incubation volume. At this concentration, methanol had no effect on NNN metabolism. When TAO was used, the microsomes were preincubated with 1 mM NADPH and 50 µM TAO for 30 min at 37°C. The reaction was then initiated with NNN and 1 mM NADPH. Other enzyme assays The radiometric assay for NDMA demethylase activity was determined as described (44) with 30 µM [14C]NDMA and 0.1 mg microsomal protein. Microsomal NADPH: cytochrome c reductase was determined by measuring the change in absorbance for the reduction of cytochrome c by NADPH at 550 nm (45). Coumarin and ethoxycoumarin metabolism were determined as described (46). NAD(P)H: quinone oxidoreductase (NQOR; EC 1.6.99.2) activity was assayed based on the reduction of cytochrome c by NADH at 550 nm with menadione (10 µM) as the substrate (47). Total GST activity was determined spectrophotometrically with CDNB as the substrate according to the method of Benson et al. (48). Histopathological analysis Human esophageal samples were fixed in 80% ethanol, embedded in paraffin, sectioned (6 µm) onto slides and stained with hematoxylin and eosin. Histopathological diagnosis was made according to cellular morphologic changes and tissue architecture using previously established criteria (49). Statistical analyses The level of significance was tested by the Student’s paired t-test between non-cancerous and cancerous esophageal mucosa samples. The Student’s t-test was used between the USA and China normal esophageal mucosa samples.

Results Nitrosamine metabolism The metabolism of NNN in the esophageal microsomes resulted in the formation of keto acid (29-hydroxylation product) and hydroxy acid (59-hydroxylation product). The formation of hydroxy acid was observed in all samples analyzed and keto acid formation was observed in 16 (73%) of the microsomal esophageal samples. The lack of detection of keto alcohol and lactol suggests that these metabolites were completely oxidized to keto acid and hydroxy acid, respectively. The rate for total α-hydroxylation of NNN was 2.3-fold higher in the noncancerous esophageal mucosa microsomes from China than the USA (Table I). When the samples that had no keto acid formation were excluded from the analysis, the rate for total α-hydroxylation of NNN was 1.8-fold higher in the esophageal mucosa microsomes from China than the USA (Table I). Hydroxy acid and keto acid formation in the microsomes from esophageal carcinomas was 33–43% lower than in microsomes from non-cancerous esophageal mucosa. In esophageal mucosa microsomes, NNK was metabolized to NNAL (carbonyl reduction product) in all samples analyzed. The formation of hydroxy acid (from α-hydroxylation of NNAL) was only observed in seven (32%) of the microsomal esophageal samples. The formation of keto aldehyde, keto alcohol, and keto acid was not observed in any of the samples examined, suggesting that different enzymes are responsible

Table I. NNN metabolism in human esophageal mucosa microsomesa Hydroxy acid pmol/min per mg protein

Keto acid pmol/min per mg protein

USA (9)b (5) Total USA (14)

0.19 6 0.12 0.10 6 0.04 0.16 6 0.10c

0.33 6 0.25 0 0.21 6 0.25c

China Normal (7) (1) Total normal (8) Tumor (7) (1) Total tumor (8)

0.34 0.43 0.35 0.21 0.15 0.20

6 0.07

0.58 0 0.51 0.39 0 0.34

6 0.07 6 0.16 6 0.15d

6 0.18 6 0.26 6 0.20 6 0.23d

aIncubation

conditions are described under Materials and methods. Values are the mean 6 SD, including zeros where applicable. bNumber in parentheses refers to the number of esophageal samples analyzed. cSignificantly (P , 0.05) different from normal esophageal mucosa microsomes from China. dSignificantly (P , 0.05) different from the corresponding normal esophageal mucosa microsomes.

Table II. NNK and NDMA metabolism in human esophageal mucosa microsomes (pmol/min per mg protein) a Sample

NNK

NDMA

Hydroxy acid

NNAL

Formaldehyde

USA (3)b (11) Total USA (14)

0.34 6 0.25 0 0.07 6 0.18c

2.31 6 1.21 4.71 6 2.74 4.20 6 2.66c

14.7 6 3.4c (10)

China Normal (4) (4) Total normal (8) Tumor (4) (4) Total tumor (8)

1.31 0 0.66 0.73 0 0.36

6 0.43 6 0.77 6 0.20 6 0.41d

9.84 5.27 7.55 4.15 3.59 3.87

6 6 6 6 6 6

4.44 0.78 3.83 2.85 0.83 1.96d

10.7 6 6.4 (8) 7.2 6 5.0d (8)

contained 10 µM NNK or 30 µM NDMA and 0.1 mg microsomal protein and were carried out as described under Materials and methods. Values are the mean 6 SD, including zeros where applicable. bNumber in parentheses refers to the number of samples analyzed. cSignificantly (P , 0.05) different from normal esophageal mucosa microsomes from China. dSignificantly (P , 0.05) different from the corresponding normal esophageal mucosa microsomes. aIncubations

for the activation of NNK and NNN in the esophagus. The rate of formation of NNAL and hydroxy acid was significantly (P , 0.05) greater in the microsomal samples from China than the USA (Table II). In the microsomes from esophageal carcinomas, the rate of formation of NNAL and hydroxy acid was ~50% lower than their counterparts from non-cancerous epithelium. The demethylation of NDMA was observed in all esophageal microsomal samples analyzed. The rate of formation of formaldehyde was 1.4-fold higher in the microsomal samples from the USA than China (Table II). Formaldehyde formation in the microsomes from esophageal carcinomas was 33% lower than in microsomes from non-cancerous esophageal mucosa. Effect of inhibitors on NNN metabolism In order to determine the enzymes that are responsible for the oxidation of NNN in the human esophagus, chemical inhibitors 669

T.J.Smith et al.

Table III. Effect of coumarin and troleandomycin (TAO) on NNN metabolism in human esophageal microsomes (pmol/min per mg protein)a

Table V. Glutathione S-transferase and NADPH-quinone oxidoreductase activities in human esophageal mucosa cytosol (nmol/min per mg protein)a

Inhibitor

Hydroxy acid

Keto acid

Sample

Glutathione S-transferase

NADPH-quinone oxidoreductase

China (7) Control Coumarin TAO

0.42 6 0.10 0.44 6 0.09 0.45 6 0.12

0.60 6 0.11 0.61 6 0.08 0.48 6 0.09b (2–38%)c

USA

180 6 114b (14)

53 6 28b (13) 0 (1)

China Normal

433 6 290 (8)

82 0 61 0

USA (11) Control Coumarin TAO

0.23 6 0.07 0.25 6 0.08 0.24 6 0.07

0.26 6 0.20 0.28 6 0.19 0.19 6 0.16b (13–44%)c

contained 10 µM NNN, 0.1 mg microsomal protein, 1 mM NADPH and 50 µM coumarin or TAO. For incubations containing TAO, microsomes were pre-incubated with 1 mM NADPH and 0 µM or 50 µM TAO for 30 min at 37°C. NNN and additional NADPH (1 mM) were then added to start the reaction. Incubations were carried out for 30 min at 37°C. Values are the mean 6 SD of seven (China) or 11 (USA) samples. bSignificantly (P , 0.05) different from the control group as determined by Student’s t-test. cThe numbers are the range of percentage inhibition by TAO. aIncubations

Table IV. P450 enzyme activities in human esophageal mucosa microsomesa Sample

NADPH-cyt. c reductase nmol/min per mg

Ethoxycoumarin dealkylase pmol/min per mg

Coumarin hydroxylase pmol/min per mg

USA

2.2 6 0.9b (10)

45.59 6 12.58b (11)

NDc (11)

China Normal Tumor

271 6 169c (8)

aValues are the mean 6 SD; number of samples analyzed are in parentheses. bSignificantly (P , 0.05) different from normal esophageal mucosa cytosolic fractions from China. cSignificantly (P , 0.05) different from the corresponding normal esophageal mucosa cytosolic fractions.

coumarin hydroxylase, were present in the human esophageal samples. Two of the esophageal cytosolic samples had no NQOR activity. The activity for ethoxycoumarin dealkylase was significantly greater (by 62%) in the microsomal samples from the USA than China (Table IV). Whereas, the activities for NADPH-cytochrome c reductase, GST and NQOR were 1.5- to 3-fold higher in the esophageal samples from China than the USA (Tables IV and V). All enzyme activities were significantly lower (by 26–42%) in the samples from esophageal carcinomas than non-cancerous esophageal mucosa samples, suggesting an alteration in the regulation of the genes in the cancer. Discussion

6.7 6 2.5 (8) 3.9 6 0.9d (8)

28.08 6 11.49 (7) 16.71 6 13.67d (7)

ND (7) ND (7)

are the mean 6 SD; number of samples analyzed are in parentheses. bSignificantly (P , 0.05) different from normal esophageal mucosa microsomes from China. cND, activity was not detectable. dSignificantly (P , 0.05) different from the corresponding normal esophageal mucosa microsomes. aValues

were used. Expressed human P450s 2A6 and 3A4 have been shown to catalyze the formation of lactol and keto alcohol, respectively, from NNN (27) and correlation studies suggested that P450 2A6 was involved in the 59-hydroxylation of NNN in human liver microsomes (26,27). Therefore, coumarin and TAO were used as inhibitors for P450 2A6 and P450 3A, respectively. Coumarin had no effect on the metabolism of NNN in all of the microsomal esophageal samples analyzed. TAO significantly decreased the rate of formation of keto acid by an average of 20–26% (Table III). α-Napthoflavone (an inhibitor of P450 1A) had no effect on NNN oxidation in the five human esophageal microsomal samples analyzed (data not shown). These results suggest that P450 3A plays a partial role in the activation of NNN in the human esophagus. Xenobiotic-metabolizing enzyme activities To investigate the presence of various xenobiotic-metabolizing enzymes in the human esophagus, the activities for NADPHcytochrome c reductase, ethoxycoumarin dealkylase, coumarin hydroxylase, GST and NQOR were determined (Tables IV and V). The activities for all of the enzymes, except for 670

Tumor

6 35 (7) (1) 6 34c (7) (1)

The present study demonstrates that human esophageal microsomes are capable of metabolizing NNN via the 29- and 59hydroxylation pathways. Similar results have been observed with human and rat esophageal culture explants (50–52), and rat esophageal microsomes (25). In comparison with rat esophageal microsomes, the rate of NNN oxidation by the human esophageal microsomes were 5- to 10-fold lower. The rate for total α-hydroxylation of NNN was 2.3-fold greater in the human esophageal microsomes from China than the USA (Table I). In addition, the NADPH cytochrome c reductase activity was 3-fold greater in the esophageal microsomes from China (Table IV). The higher level of P450 reductase, which is an essential component for P450 catalysis, could contribute to the increased oxidation of NNN. Although the esophageal samples were obtained within 4 h of surgery or death, and precautions were taken in the handling and processing of the esophageal samples, one cannot disregard the possibility that the freshness of the esophageal samples may affect the activity of nitrosamine metabolism. The esophageal microsomes metabolizes NNK primarily through the carbonyl reduction pathway (Table II). Similar results were observed with human esophageal explants (50). In human lung microsomes, NNAL formation is also the major metabolite formed, but lung microsomes also exhibits αhydroxylation of NNK (28,29). Furthermore, the carbonyl reduction of NNK is higher (by 3- to 8-fold) with human lung microsomes (28,29) than with human esophageal microsomes. In comparing the metabolism of NNK and NNN in the esophageal microsomes, the major difference is the lack of α-hydroxylation products (keto alcohol and keto acid) with

Human esophageal enzymes and nitrosamine metabolism

NNK. The α-hydroxylation pathway leading to the formation of keto alcohol and keto acid is the common pathway shared by NNK and NNN (Figure 1). However, human esophagus has an enzyme that only catalyzes the α-hydroxylation of NNN, but not of NNK. The ability of esophageal microsomes to activate NNN to carcinogenic species may account for the susceptibility of the esophagus to tumor formation by NNN. Studies have shown that the esophagus contains P450s that activate nitrosamines (8,25). The inhibition of keto acid (product from the further oxidation of keto alcohol) formation by TAO (Table III) suggests that P450 3A plays a partial role in NNN oxidation in the human esophageal microsomes. The 29-hydroxylation of NNN has been demonstrated to be catalyzed by expressed human P450 3A4, displaying Km and Vmax values of 334 µM and 4.6 nmol/min per nmol P450, respectively, for keto alcohol formation (27). Although NNN can be oxidized to lactol by expressed human P450 2A6 and human liver microsomes containing high levels of P450 2A6 (26,27), P450 2A6 does not appear to play a role in NNN oxidation in the human esophageal microsomes. The lack of an inhibitory effect of coumarin on NNN metabolism (Table III), the absence of coumarin hydroxylase activity (Table IV), and the lack of P450 2A6 immunoreactivity in the esophageal samples (data not shown) suggests that P450 2A6 is not present in the human esophagus. Other enzyme(s) with a high affinity for NNN are responsible for a major portion of NNN oxidation in the human esophageal microsomes. These enzymes remain to be characterized. The activation and detoxification of a carcinogen will depend on the amount, activity, and presence of xenobioticmetabolizing enzymes in the tissue. In the present study, NDMA demethylation was observed in the esophageal microsomal samples (Table II). P450 2E1 is known to play an important role in the metabolism of NDMA. The esophageal samples were immunoreactive for P450 2E1 (data not shown). Thus, esophageal microsomes are capable of metabolizing nitrosamines that are catalyzed by P450 2E1, as was observed with NDMA. Immunoblotting studies have shown that P450 2E1 is present at low levels in human esophageal microsomes (38) and much lower than the P450 2E1 level in the human liver. Murray et al. (53) showed that there was no expression of P450 3A in non-cancerous esophageal samples, only in squamous carcinomas and adenocarcinomas. However, P450 3A mRNA has been demonstrated to be present in normal human esophageal samples (54). In the present study, variability in P450 3A4 activity was observed for NNN metabolism in the human esophageal microsomes. In the presence of TAO, a P450 3A inhibitor, the range of percent inhibition of NNNderived keto acid formation was 2% to 38% (Table III). Evidence for polymorphism in P450 3A4 at the phenotypic level has been shown (38,55); however, no genetic polymorphism has presently been demonstrated. Whether the results in the present study are due to a polymorphism is not known and requires further investigation. NQOR and GSTs are important in the detoxification of carcinogens and both enzymes are polymorphic (34–36,56,57). In the present study, two of the esophageal mucosa cytosolic samples had no NQOR activity (Table V). It remains to be determined whether the lack of NQOR activity in these samples is because of a reported polymorphism of NQOR, which is known to result in the loss of function (56,57). In the esophagus, NQOR and GST activities were 1.5- to 2.4-fold higher for the cytosolic samples from China than the USA (Table V). The

increased NQOR and GST activities in the samples from China could be caused by an ethnic difference or to an induction of these enzymes by different dietary factors, cancer therapy, or the trauma state of cancer. In conclusion, P450s 3A4 and 2E1 are involved in the activation of NNN and NDMA, respectively, in the human esophagus. Further studies are needed to characterize the specific enzymes that play an important role in the activation of esophageal carcinogens in humans to gain insight on the possible causes of esophageal carcinogenesis and to develop strategies for its prevention. In addition, polymorphisms that may be associated with an increased susceptibility to esophageal cancer and the functional significance of these polymorphisms need to be identified. Acknowledgements This study was supported by NIH grants CA46535 and CA37037, and NIEHS Center Grant ES05022.

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