Diffusion of dialkylnitrosamines into the rat esophagus as a factor in esophageal carcinogenesis

James Haorah1, Donald W. Miller2, Rhonda Brand5, Thomas C. Smyrk3, Xiaojie Wang1, Sheng Chong Chen1 and Sidney S. Mirvish1,2,4,6

1 Eppley Institute for Research in Cancer,
2 Department of Pharmaceutical Sciences, College of Pharmacy,
3 Department of Pathology and
4 Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA and
5 Department of Biological Systems Engineering, School of Engineering, University of Nebraska-Lincoln, Lincoln, NE 68583-0726, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To indicate how readily nitrosamines (NAms) diffuse into the esophagus, we measured diffusion rate (flux) through rat esophagus of dialkyl-NAms using side-by-side diffusion apparatuses. Mucosal and serosal flux at 37°C of two NAms, each at 50 µM, was followed for 90 min by gas chromatography–thermal energy analysis of NAms in the receiver chamber. Mucosal flux of one or two NAms at a time gave identical results. Mucosal flux was highest for the strong esophageal carcinogens methyl-n-amyl-NAm (MNAN) and methylbenzyl-NAm. Mucosal esophageal flux of 11 NAms was 18–280 times faster and flux of two NAms through skin was 13–28 times faster than that predicted for skin from the molecular weights and octanol:water partition coefficients, which were also measured. Mucosal: serosal flux ratio was correlated (P < 0.05) with esophageal carcinogenicity and molecular weight. For seven NAms tested for carcinogenicity by Druckrey et al. [(1967) Z. Krebsforsch., 69, 103–201], mucosal flux was correlated with esophageal carcinogenicity with borderline significance (P = 0.07). The MNAN:dipropyl-NAm ratio for mucosal esophageal flux was unaffected when rats were treated with phenethylisothiocyanate and was similar to that for forestomach, indicating no involvement by cytochromes P450. Mucosal esophageal flux of MNAN and dimethyl-NAm was reduced by >90% on enzymic removal of the stratum corneum, was unaffected by 0.1 mM verapamil and was inhibited 67–94% by 1.0 mM KCN and 82–93% by 0.23% ethanol. NAm flux through rat skin and jejunum was 5–17% of that through esophagus. Flux through skin increased 5–13 times after enzymic or mechanical removal of the epidermis; the histology probably explained this difference from esophagus. Hence, NAms could be quite rapidly absorbed by human esophagus when NAm-containing foods or beverages are swallowed, the esophageal carcinogenicity of NAms may be partly determined by their esophageal flux and NAm flux probably occurs by passive diffusion.

Abbreviations: DEN, diethylnitrosamine; DMN, dimethylnitrosamine; DPN, dipropylnitrosamine; EC, esophageal papillomas and carcinomas; ER, endoplasmic reticulum; GC, gas chromatography; MBZN, methylbenzylnitrosamine; MNAN, methyl-n-amylnitrosamine; MPN, methylpropylnitrosamine; NAm, nitrosamine; NNN, N'-nitrosonornicotine; PC, partition coefficient (octanol:water unless mentioned otherwise); PEITC, phenethylisothiocyanate; TEA, thermal energy analysis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We report here the diffusion rate (flux) through the rat esophagus of 11 unsymmetrical and symmetrical dialkyl-nitrosamines (-NAms) that vary in their ability to induce esophageal cancer in rats. Determination of flux through the esophagus may indicate the rate of diffusion into the esophagus and differs from the measurement of total uptake when tissue is placed in medium containing the test substance, as in studies on his-(2-oxopropyl)-NAm uptake by hepatocytes (1). Methyl-n-alkyl-NAms induce esophageal papillomas and carcinomas (EC) in rats when the alkyl group is propyl to heptyl, but not when it is methyl, ethyl or larger than heptyl (25). Methyl-n-amyl-NAm (MNAN), a well studied esophageal carcinogen in rats, is equally effective in both sexes and acts after oral or s.c. injection (2,6,7). The most potent known esophageal carcinogen, methylbenzyl-NAm (MBZN), induces EC in rats at ~10% of the molar dose required for MNAN (2,8). EC is also induced in rats by diethyl-, di-n-propyl- and di-n-butyl-NAm, though less efficiently than by the isomeric methylalkyl-NAms (8); by N'-nitrosonornicotine [(NNN) a NAm in cigarette smoke that probably initiates EC induction in smokers (9)], N-nitrosopiperidine and certain other heterocyclic NAms (8,10).

People are exposed to preformed NAms in foods, beverages and cigarette smoke and in certain industries and to NAms produced in vivo (11). Such NAms may be significant inducers of human EC (11). NAms are activated by cytochromes P450 to give {alpha}-hydroxy-NAms, which decompose (are dealkylated) to give aldehydes and agents that can alkylate critical sites in DNA (8,11). NAm specificity for the esophagus is attributed to an esophageal P450, perhaps related to the rat nasal P450s CYP2A3 and CYP2A5 (1214). MNAN, MBZN and NNN are known to be {alpha}-hydroxylated by rat and (in the case of MNAN) human esophageal microsomes (1417). NAm activation occurs in the basal cells of the esophageal mucosa and leads to cancer derived from these cells (18).

Specific uptake of carcinogens has seldom been considered as a factor in their tissue specificity. However, Squier (19) studied NNN flux across porcine oral mucosa. Flux was most rapid for the non-keratinized floor-of-mouth and cheek mucosa (preferred sites for oral cancer in tobacco chewers), slower for keratinized gingival (gum) mucosa and slowest for skin. The present study used rats because esophageal carcinogenesis by NAms has been most studied in rats (8); mice and hamsters are generally less susceptible than rats to EC induction by NAms, e.g. by MNAN (20) and the rat esophagus is large enough to work with conveniently. The rat esophagus includes a mucosa (composed of an epithelium, lamina propria and muscularis mucosae), a submucosa and an outer muscle layer (muscularis propria) (Figures 1 and 2GoGo). All these layers contain blood vessels, except the epithelium. NAm uptake by the esophageal mucosa could occur from the mucosal side while NAm-containing food and beverages are being swallowed, or from blood in mucosal and submucosal blood vessels after NAms are administered and absorbed from the gut.



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Fig. 1. Diagram of rat esophagus taken from (55). Reprinted with permission of the publishers.

 



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Fig. 2. (A) Normal rat esophagus. Note the cornified layer on the epithelial surface. The lamina propria (solid arrow) and superficial submucosa (open arrow) are composed of loose connective tissue (165x). (B) Rat esophagus after enzyme treatment. The cornified layer is lost. The lamina propria and submucosa appear dense (66x).

 
In the present study, we used side-by-side diffusion chambers to study NAm flux through the rat esophagus from the mucosal side (`mucosal flux') and from the ablumenal side (`serosal flux'). To help understand the results, we determined the octanol:water partition coefficients (PCs) of the test NAms and studied effects of certain chemicals and treatments on the flux.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
The purest grades of chemicals other than NAms were purchased from Aldrich (Milwaukee, WI) unless mentioned otherwise. We obtained dimethyl-NAm (DMN) from Aldrich, methylethyl-, diethyl- and dibutyl-NAm from Sigma (St Louis, MO) and dipropyl-NAm (DPN) from Eastman Organic Chemicals (Rochester, NY). Experiments with NAms, which are potent volatile carcinogens, were performed with due care in a chemical hood. Excess NAms were destroyed with aluminum–nickel alloy in alkali (21).

Synthesis of methylalkylamines
All of these were purchased (methylamylamine from Carl Industries, Aurora, OH) except for methylheptylamine, which was synthesized as follows: 1-bromoheptane (1.57 ml, 1.02 g, 10 mmol) was added dropwise to 25 ml (50 mmol) of 2 M methylamine in tetrahydrofuran cooled in ice–salt. The mixture was stirred for 6 h at 0°C and then 16 h at room temperature. The solid (probably CH3NH3Br) was filtered off. The filtrate was mixed with 25 ml water and HCl to bring the pH to 1, extracted with 2x25 ml CH2Cl2, made basic with NaOH and extracted with 2x25 ml CH2Cl2 to give methylheptylamine (0.84 g, 6.5 mmol) as a floating oil. 1H-NMR in CDCl3: 0.88 (t, CH3·CH2, 3), 1.30 (m, CH2, 2), 2.42 (s, NCH3, 3) and 2.54 (t, NCH2, 2) p.p.m.

Synthesis and analysis of methylalkyl-NAms
Five ml of 12 N (60 mmol) HCl was added to 30 mmol methylalkylamine in 5 ml water cooled in ice. NaNO2 (4.14 g, 60 mmol) in 5 ml water was added over 30 min. The mixture was reacted for 1 h in ice, 1 h at room temperature and 1 h at 50°C and cooled. The upper layer was collected to give >50% yields of the NAm, which was dried over Na2SO4. Methylpropyl-NAm (MPN) did not separate well and was extracted with ether. The extract was dried over Na2SO4 and freed of ether with a N2 stream. All NAms were >90% pure as determined by 1H-NMR spectrometry of CDCl3 solutions and by gas chromatography (GC) with detection by thermal energy analysis (TEA). The 1H-NMR spectra in CDCl3 of DMN, methylethyl-NAm, MPN, diethyl-NAm (DEN), methylbutyl-NAm and MBZN were similar to the published spectra measured in CCl4 or as neat compounds (22). The remaining NAms gave the following 1H-NMR peaks in CDCl3, shown with multiplicity and assignment for the major syn-anti isomer: MPN: 0.93 (t, CH3·CH2), 1.77 (m, CH2·CH3), 3.07 (s, CH3N) and 4.10 (t, CH2N) p.p.m.; methylbutyl-NAm: 0.99 (t, CH3·CH2), 1.38 and 1.73 (m, CH2), 3.04 (s, CH3N) and 4.15 (t, CH2N) p.p.m.; MNAN: 0.92 (t, CH3·CH2), 1.34 and 1.75 (m, CH2), 3.06 (s, CH3N) and 4.14 (t, CH2N) p.p.m.; methylhexyl-NAm: 0.89 (t, CH3·CH2), 1.32 (broad, CH2), 3.06 (s, CH3N) and 4.14 (s, CH3N) p.p.m.; methylheptyl-NAm: 0.88 (t, CH3·CH2), 1.29 (m, CH2), 3.05 (s, CH3N) and 4.13 (t, CH2N) p.p.m.

For GC–TEA, 5 µl samples were analyzed on a GC apparatus (model 5890) connected to an automatic injector (model 7673A) and an integrator (model 3392A), all from Hewlett-Packard (Avondale, PA), and a thermal energy analyzer (model 502) from Thermedics (Chelmsford, MA). GC was performed on a packed column (6 ftx4 mm o.d.) of 10% Carbowax 20M-TPA on 80–100 Chromosorb, heated from 130 to 150°C over 15 min with helium at a flow rate of 20 ml/min. Table IGo (column 2) gives the GC retention times. The 2-, 3- and 4-hydroxy-MNANs were determined similarly but with the GC run at 185°C (23).


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Table I. Flux through rat esophagus and other parameters for 11 dialkyl-NAms, arranged in order of increasing molecular weight
 
Determination of PC
Solutions of 10 mg NAm/10 ml n-octanol were shaken with 10 ml water in a separating funnel for 4 min and left to stand for 30 min. The two phases were allowed to separate (centrifugation did not affect the results). The octanol phase was dried over Na2SO4, diluted with CH2Cl2 and analyzed by GC–TEA. The water phase (5 ml) was extracted with 3x10 ml CH2Cl2. The extract was dried over Na2SO4 and analyzed by GC–TEA. NAm solutions in CH2Cl2 served as standards. Apparent PCs were calculated. Extraction efficiency of each NAm from 5 ml water containing a known NAm level was determined by extraction with 3x10 ml CH2Cl2 and analysis of the extracts. PCs were calculated from the equation: true PC = apparent PCxpercent extraction efficiency/100.

Flux studies
Male MRC-Wistar rats 9–12 weeks old from the Eppley Institute breeding colony were used. Body weights were 300–350 g. The rats were killed with CO2. Each esophagus (weight, 220–250 mg; length, 8.5–9.0 cm) was dissected out, stripped of outer connective and muscle tissue with two forceps, blotted, weighed (weight of stripped esophagus, 70–80 mg), placed on an inverted Petri dish, slit open longitudinally, cut into distal and proximal halves and kept moist with high-glucose Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, NY) (`incubation medium'). Each half of the esophagus was placed between the two parts of a side-by-side diffusion chamber manufactured with 3x9 mm slits (Crown Bio-Scientific, Somerville, NJ) (Figure 3Go). Each part contained a 9x4 mm magnet for stirring the contents and was surrounded by circulating water at 37°C. Four chambers were used in each experiment. The esophagi were placed with the mucosa or submucosa facing the left (`donor') chambers (mucosal or serosal flux, respectively). The mucosal side could be distinguished because it glistened more than the serosal side. Incubation medium (total volume, 3 ml) was added to each chamber. In most tests, two NAms, each 50 µM, were added to the donor chamber by adding 300 µl stock solution of each NAm in medium to 2.4 ml medium. In many tests, one of the NAms was MNAN. In others, the NAms were paired randomly. The chambers were left unstoppered to allow access to air and were incubated for 90 min.



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Fig. 3. Diagram of side-by-side diffusion apparatus, each half of which was 40 mm longx27 mm in diameter.

 
In a few tests, 300 µl samples from the donor chamber were analyzed at 0 and 90 min; little change in NAm concentration was seen. In all tests, 300 µl samples were taken from the right (`receiver') chamber after 30, 60 and 90 min; or (in later tests) after 45 and 90 min, with two samples taken at 90 min and results expressed as the mean values. The samples were extracted in 15 ml tubes with 3x3 ml CH2Cl2. The combined extracts were concentrated to 3 ml in fresh 15 ml tubes with a gentle stream of N2 directed over the surface with Pasteur pipettes, dried with Na2SO4, transferred to graduated 4 ml conical tubes, concentrated to 300 µl with a stream of N2 and analyzed by GC–TEA (see above). NAm solutions in CH2Cl2 served as standards for the GC–TEA.

In a test of the method, 300 µl medium containing DMN and MNAN (each 50 µM) was extracted with 3x3 ml CH2Cl2 and the extract was dried over Na2SO4 and analyzed by GC–TEA. NAm recovery was 106 ± 1% for DMN and 93 ± 1% for MNAN (n = 6, A ± B refers to mean ± SE throughout the paper). In another test, NAm recovery was 92 ± 1% for DMN and 85 ± 3% for MNAN (n = 9) when samples of 50 µM DMN and 50 µM MNAN in 9 ml CH2Cl2 were evaporated to 300 µl with a stream of N2 and the concentrate was analyzed by GC–TEA. Both tests used the same procedures as in the flux experiments. Results were not corrected for these losses. Differences between full duplicate analyses of diffusates were expressed by calculating differences between the individual results and the mean of the two results and expressing them as percentages of the mean results; these values were 4 ± 1% (n = 22) for MNAN, 4 ± 1% (n = 10) for MBZN and 11 ± 5% (n = 8) for DMN. All fluxes were expressed as µM/h.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
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Initial flux experiments
The stripped rat esophagi that were used in these studies retained about one third of the submucosa and all the mucosa (Figure 2Go). When fluorescein was added to the donor chamber of the diffusion apparatus to test for mechanical breaks in the esophagus (24), none was ever detected in the receiver chamber. NAm concentration in the receiver chamber increased linearly with time for 90 min (Figure 4Go). For flux from both sides of the esophagus and under all conditions, the observed 90 min values were very close to those calculated from the best-fitting straight line for NAm concentration at 30, 60 and 90 min. Therefore, only the 90 min values are presented from here on. The following pairs of results refer to 21–22 rats/group that were, respectively, 7–8 and 9–12 weeks old: 2.65 ± 0.20 and 2.57 ± 0.25 µM/h for mucosal flux of MNAN; 1.63 ± 0.15 and 2.15 ± 0.13 µM/h for serosal flux of MNAN (significantly different, P < 0.01); and 0.35 ± 0.10 and 0.70 ± 0.17 µM/h for mucosal flux of DMN. Hence, at least for serosal flux of MNAN, flux rates were higher in 9–12-week-old than in 7–8-week-old rats. All subsequent tests used rats that were 9–12 weeks old.



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Fig. 4. Effect of diffusion time on mucosal esophageal flux for five NAms. MBN, methylbutyl-NAm; MPN, methylpropyl-NAm; MHPN, methylheptyl-NAm.

 
Mucosal flux for MNAN was 0.51 ± 0.08 (n = 8) at 20°C and 0.39 ± 0.1 (n = 2) µM/h at 0°C, compared with 2.80 ± 0.09 µM/h at the standard 37°C (Table IGo). Mucosal flux for DMN was 0.48 ± 0.17 µM/h (n = 8) at 20°C, zero at 0°C (n = 2) and 0.65 ± 0.05 µM/h at 37°C (Table IGo). MNAN concentrations of 0.2, 0.4, 0.8 and 1.6 mM showed mucosal fluxes of 1.8, 2.1, 2.9 and 3.3 µM/h, respectively, i.e. flux did not reach a maximum even at 0.8 mM. When 23 µM MNAN in 5 ml medium is incubated for 3 h with an adult rat esophagus, ~3% of the MNAN is converted into 2-, 3- and 4-hydroxy-MNAN, which can be determined by GC–TEA (23). However, hydroxy-MNANs were not detected when the diffusate, collected after flux of 50 µM MNAN through three rat esophagi for 90 min, was analyzed by GC–TEA. Probably, the area of esophagus covering the slit was too small and contact time too short to yield detectable metabolites.

In the flux studies, the level of 50 µM NAm was chosen to minimize toxic effects, e.g. the LD50 for MNAN is 620 µmol/kg (20), though that for MBZN is only 120 µmol/kg (2). [NAm-treated rats can die from acute esophageal damage, but only 3–6 days after treatment (20).] In most studies, two NAms were added at the same time to the donor chamber because flux was thought to occur by passive diffusion. This reduced the work involved and the number of rats used. Passive flux of one NAm should not affect that of a second NAm, provided that toxic effects do not alter the structures through which diffusion occurs. If active transport was involved, one NAm could saturate the system and inhibit transport of a second NAm. Accordingly, we examined mucosal esophageal flux of MNAN and MBZN and of MNAN and DMN, when these NAms were tested individually and together in the same experiment. The two conditions gave almost identical results (Table IIGo), justifying the testing of two NAms at a time.


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Table II. Mucosal flux through the esophagus for mixtures of two NAms compared with that for one of these NAms run alone in the same experimenta
 
Flux of 11 NAms through esophagus
Table IGo shows flux through the esophagus, log PC and other parameters for 11 dialkyl-NAms. Mucosal flux was 1.84–2.80 µM/h for the strong esophageal carcinogens MNAN and MBZN, 0.48–1.00 µM/h for eight NAms and 0.13 µM/h for one NAm. Serosal flux was 2.38 µM/h for MNAN, 1.17–1.37 for two NAms and <=0.71 µM/h for eight NAms. The mucosal flux of 2.80 µM/h for MNAN corresponds to a loss of 5.6%/h of the 50 µM MNAN in the donor chamber.

Flux through forestomach and effect of phenethylisothiocyanate (PEITC)
We hypothesized that P450-catalyzed metabolism of NAms was involved in the more rapid flux of MNAN, a strong esophageal carcinogen, compared with that of its isomer DPN, a weak esophageal carcinogen. To test this view, we examined the mucosal flux of MNAN–DPN mixtures in two situations.

(a) Flux through forestomach.
Most NAms, including MNAN, do not induce tumors of the rat forestomach (7,8). Adult rat forestomach is far less able than rat esophagus to convert MNAN to 2-, 3- and 4-hydroxy-MNAN (23,25). Formation of these metabolites in a 1:3:2 ratio is probably due to the same P450 that activates MNAN by 1-hydroxylation (11). Therefore, the adult rat forestomach probably has low levels of the relevant P450 and should show a lower MNAN:DPN flux ratio than the esophagus if this P450 was involved in the flux. However, this ratio was ~2.0 in both the esophagus and forestomach (Table IIIGo). For both MNAN and DPN, flux through the esophagus was 1.9–2.0 times faster than that through the forestomach, although their thicknesses were similar (weight of 1x2.5 cm pieces stripped of outer tissue was 20 mg for esophagus and 23 mg for forestomach, corresponding to thicknesses of ~0.1 mm).


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Table III. Mucosal flux (for esophagus, forestomach and jejunum) or flux from epidermal side (for skin) of MNAN plus DPN or MNAN plus DMN in tissues from untreated rats (section a), or for the esophagus of rats treated with PEITC (section b)
 
(b) Esophageal flux after PEITC treatment.
PEITC probably inhibits NAm metabolism by the esophageal P450 because treatment of rats with PEITC strongly inhibited esophageal carcinogenesis by MBZN (26), esophageal DNA methylation by MNAN and methylbutyl-NAm (26,27) and MNAN conversion to hydroxy-MNANs by the excised esophagus (27). If the esophageal P450 were involved in NAm flux, PEITC treatment should reduce the MNAN/DPN flux ratio. Table IIIGo shows that the mucosal esophageal flux of MNAN and DPN, and the MNAN:DPN ratio, were not affected by gavage of 1.0 mmol PEITC/kg 2 h before the rats were killed [the same treatment that inhibited hydroxy-MNAN formation (27)]. Hence P450s are probably not involved in the esophageal flux of MAns.

Effect of enzyme treatment on esophageal flux
Treatment with hyaluronidase and elastase facilitates removal of mucosal tissue from the skin (28) and esophagus (29). Although this treatment was expected to increase the esophageal flux of NAms because they would have to diffuse through less tissue, it reduced mucosal flux by 91% for MNAN and 97% for DMN and reduced serosal flux of these NAms by 53–54% (Table IVGo). Enzyme-treated esophagus showed loss of the stratum corneum above an otherwise intact mucosa and made the lamina propria and submucosa appear denser, as though the normally loose network of elastin and collagen fibers had condensed (Figure 2Go). Three tests for flux of fluorescein after removing the mucosa showed diffusion after 90 min of 0, 0.6 and 0.6% of the fluorescein, indicating a few breaks in the tissue, but apparently not sufficient to cause rapid flux of NAms.


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Table IV. Various effects on NAm diffusion through rat esophagus
 
Flux through jejunum and skin
We measured MNAN and DMN flux from the mucosal side of rat jejunum and from the epidermal side of rat skin (Table IIIGo). For the jejunum, DMN flux was similar to that for the esophagus, but MNAN flux was 20% of that for the esophagus. Flux through skin increased 4.4 times for MNAN and 8.8 times for DMN after enzyme treatment, opposite to the findings for esophagus. Enzyme treatment of the skin changed the appearance of the dermis, similar to our findings for esophageal submucosa, and (in contrast to esophagus) removed the entire mucosa (Figure 5Go). Mechanical scraping of the skin removed the mucosa, similar to the effect of enzyme treatment, except that more of the basal layer was preserved. The results with this method were similar to those obtained by the enzyme method (Table IIIGo).




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Fig. 5. (A) Normal rat skin (165x). (B) Rat skin after enzyme treatment. Some areas of the epithelium are completely stripped away (66x).

 
Effect of verapamil
The multidrug resistance P-glycoprotein system (30), which transports drugs out of resistant cancer cells, could have pumped NAms that are potent esophageal carcinogens into the esophagus or pumped weakly carcinogenic NAms out of the esophagus. Verapamil inhibits P-glycoprotein action (30). However, mucosal and serosal fluxes were not affected when 0.1 mM verapamil [a level used by others (30)] was added to the donor chamber containing MNAN and DMN or MNAN and methylheptyl-NAm (Table IVGo).

Effect of potassium cyanide (KCN)
To help decide if NAm flux was an active process, 1.0 mM KCN [a level used by others (31,32)] was added to the donor chamber containing MNAN and DMN. KCN inhibition of esophageal flux was 89–93% for DMN and 58–67% for MNAN and was similar from both directions (Table IVGo). The difference between the strong inhibition of DMN and the moderate inhibition of MNAN is intriguing.

Effect of ethanol
Ethanol consumption and cigarette smoking synergistically induce EC in humans (33). Ethanol enhanced EC induction by NAms in rats, partly because it inhibits first-pass clearance of NAms by the liver (34). In pigs, 5% ethanol increased NNN flux across the floor-of-mouth mucosa 4.6-fold, though it did not much affect flux across the buccal mucosa (35). Here we examined the effect on esophageal flux of adding 20 or 50 mM ethanol to the donor chamber. These ethanol concentrations are 0.09 and 0.23%, close to the blood level of 0.1% commonly defined as intoxicated. Twenty millimolar ethanol had no effect, but 50 mM ethanol significantly inhibited the mucosal flux of MNAN and DMN by 82–93% and their serosal flux by 68–72% (Table IVGo).


    Discussion
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 Abstract
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 Materials and methods
 Results
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 References
 
Facility of NAm flux through the esophagus
Mucosal flux of NAms through the esophagus was surprisingly rapid, 11 (for DMN) and 21 (for MNAN) times as fast as flux through skin (Table IIIGo), up to 3.5 times as fast as serosal esophageal flux (Table IGo) and nearly twice as fast as diffusion through the forestomach (Table IIIGo). Using an in vitro system, others have demonstrated diffusion from cosmetic vehicles through human skin of N-nitrosodiethanolamine, a constituent of industrial cutting oils (36); DMN (37); and methyldodecyl-NAm, a potential contaminant of cosmetics (38).

Despite the short time for which swallowed materials are in contact with the esophagus, our findings suggest that significant amounts of NAms could be absorbed directly by the human esophagus when NAm-containing foods and beverages are swallowed. This may partly explain how NAms induce EC in humans, even though human esophagus differs from rat esophagus in that it is non-keratinizing and lacks a stratum corneum. Pig esophagus could provide a better model for flux than rat esophagus because it is non-keratinized and its structure and ultrastructure are similar to that of human esophagus (C.Squier, personal communication). Direct absorption of NAms also could occur in other internal squamous epithelia of humans, such as the cervix and urinary bladder, both of which may be exposed to NAms (11,39) and may be far more rapid for these tissues than for skin, which has evolved to be an efficient barrier to xenobiotic absorption.

Our results indicate that NAms and, perhaps, other compounds can be absorbed by the rat esophagus from food or drinking water during swallowing. Direct absorption by the rodent esophagus was suggested by four reports: (i) MBZN induced EC in mice when given in drinking water but not when injected i.p., though it induced forestomach tumors under both conditions (40); (ii) methylheptyl-NAm induced EC in 100% of rats fed this NAm in drinking water, but did not induce EC when it was repeatedly gavaged in corn oil, leading the authors to suggest direct esophageal absorption of the NAm (3); (iii) carcinogens were absorbed and induced tumors when their solutions in ethanol were applied to the esophagus (41); and (iv) catechol given in the drinking water or diet enhanced EC induction by MNAN, suggesting that it was directly absorbed by the esophagus (42). Von Hofe et al. (43) gavaged rats with aqueous solutions or suspensions of methylalkyl-NAms (alkyl = methyl to dodecyl). They found 7-methylguanine in the esophageal DNA after gavage with methylpropyl-NAm, methylbutyl-NAm and MNAN, but not after gavage with the esophageal carcinogens methylhexyl-NAm and methylheptyl-NAm (Table IGo). Our findings (Table IGo) and the carcinogenesis test of methylheptyl-NAm (3) referred to in point (ii) suggest that methylhexyl- and methylheptyl-NAm would have methylated esophageal DNA if they had been given in drinking water.

Correlations between esophageal flux and other parameters
Permeability, Kp, through skin is defined as flux/donor concentration (44). Potts and Guy (45) developed equation (1), which rather accurately predicted Kp for epidermal flux through hairless mouse skin in studies using aqueous solutions [e.g., 100 µM (46)] of chemicals ranging from water and simple alkyl alcohols to those with molecular weights exceeding 750 (4548). Kp rises as molecular weight falls and PC rises. Tissue thickness should only affect the time needed to reach a steady-state flux. Equation 2 converts our results for mucosal flux D in µM/h (Table IGo) into observed Kp in cm/s.





In equation (2), 2.7 ml (cm3) is the average volume of the receiver chamber, converting D to amount diffused in nmol/h; h are converted to s; 0.27 cm2 is the area of the 0.9x0.3 cm diffusion slit, converting flux to nmol/s/cm2; and 50 µmol/l is initial NAm concentration in the donor chamber.

The ratio of observed to predicted Kp for mucosal esophageal flux (Table IGo, antilog column 8/antilog column 5) varied from 18–22 for methylhexyl- and methylheptyl-NAm to 204 for MNAN and 280 for DMN, i.e. mucosal esophageal flux was 18–280 times faster than that predicted for skin by equation (1). Fluxes through skin (Table IIIGo) were 13 (for MNAN) and 26 (for DMN) times faster than those predicted from equation (1). Thus, the rapid flux of NAms through the esophagus is due partly to a particularly rapid flux of NAms and partly to a more rapid flux through esophagus than through skin. Another factor might be that we used rats, whereas the tests quoted by Potts and Guy used mice (4548).

Equation (3) defines the relative esophageal carcinogenicity of each NAm (Table IGo, column 11). This parameter was based on reports by Druckrey et al. (2) and Lijinsky et al. (3,4), each of whom tested a series of dialkyl-NAms by chronic administration in drinking water.



In equation (3), D50 is the dose producing a 50% incidence of all tumors. The factor of 4.65 (percent incidence of EC/D50 for MBZN) relates esophageal potency to that of MBZN taken as 100. See also footnote e of Table IGo.

Table VGo lists the correlation coefficient (r) and probability (P) for correlations between the various parameters, mostly for all 11 test NAms. As expected from equation 1, molecular weight, log PC and log predicted Kp were highly correlated with each other (r >= 0.84, P = 0). There were significant correlations (r >= 0.59, P <= 0.05) of the ratio of mucosal to serosal flux with molecular weight and with esophageal carcinogenicity and of esophageal carcinogenicity with log PC and log predicted Kp. The mucosal/serosal flux ratio might reflect the advantage given to mucosal over serosal flux by solution of the NAm in lipids of the stratum corneum. There was a trend toward significant correlations (r > 0.5, P = 0.05–0.10) of mucosal with serosal flux and of carcinogenicity with molecular weight. When only the seven NAms tested by Druckrey et al. (2) were considered (Table VGo, last column), there was a trend (r >= 0.70, P = 0.05–0.10) toward significant correlations of carcinogenicity with mucosal flux and with log predicted Kp.


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Table V. Spearman correlation coefficients (r) and probability (P) values for correlations between various parametersa
 
The significant correlation between esophageal carcinogenicity and log PC agrees with our 1976 observation (10) that, within groups of related heterocyclic and dialkyl NAms, a raised ether:water PC correlated with an increase in esophageal carcinogenicity. Presumably, the correlation of flux with esophageal carcinogenicity (Table IGo) was imperfect because flux is only one factor determining carcinogenicity, the other being ability of the esophageal P450 to activate NAms. The latter factor probably explains the 19-fold higher carcinogenicity (relative potency—see equation 3 and Table IGo) of MBZN compared with that of MNAN, despite their similar mucosal fluxes and the >25-fold differences in carcinogenicity between the strongly carcinogenic unsymmetrical NAms, MNAN and methylheptyl-NAm and the isomeric symmetrical NAms, DPN and dibutyl-NAm. PC and molecular weight (indicating molecular size) may help determine both esophageal flux and specificity of the esophageal P450, the active site of which may be lipophilic, favoring NAms with high PCs and of limited size, favoring NAms with low molecular weights [the two parameters in equation (1)].

Effect of enzyme treatment
Enzymic removal of the esophageal stratum corneum decreased mucosal flux of MNAN and DMN by 91–97% and their serosal flux by 53–54%, whereas removal of the epidermis increased MNAN and DMN flux through skin 11- to 14-fold (Tables III and IVGoGo). If flux is passive (as we think probable), the lipid-rich stratum corneum could have enhanced mucosal and, to some extent, serosal flux because lipid-soluble NAms would be concentrated in the stratum corneum. Hence flux would be diminished by removing this layer. In contrast, removal of the entire epidermis from the skin probably increased flux because much of the physical barrier to flux had been removed. This difference may have arisen because enzyme treatment was carried out for 30 min with esophagus but for 1 h with skin. Another factor retarding flux through the enzyme-treated esophagus may have been the apparently increased density of the lumina propria and submucosa. However, (i) the same change was noted in enzyme-treated skin and (ii) mechanically stripped skin, which did not exhibit changes in the submucosa, showed fluxes similar to those for enzyme-treated skin (Table IIIGo). These findings suggest that deeper levels of the mucosa, including the basal cells and basement membrane, are the major barrier to flux of lipophilic compounds. Clearly, further experiments will be needed to explain the effect of enzyme treatment.

Active versus passive flux
The following findings and arguments suggest that NAm flux through the esophagus was a passive process: (i) mucosal and serosal fluxes were similar except for MBZN (Table IGo); (ii) mucosal flux of MNAN did not reach a maximum as its concentration was raised (see Results); (iii) flux of one NAm was not affected by the presence of a second NAm (Table IIGo); (iv) the verapamil test indicated that a P-glycoprotein system was not involved (Table IVGo); and (v) the higher flux through esophagus than through skin could be due to differences in the structure and composition of lipid lamellae in the intercellular space of the stratum corneum [as found for porcine skin versus oral mucosa (49)], or to differences between the keratins of each tissue (50,51).

The following findings suggest that esophageal flux was an active process: (i) The observed Kp for esophageal flux (Table IGo) was up to 280 times higher than Kp predicted for skin by equation (1) and up to 21 times higher than the observed NAm flux through skin (Table IIIGo); (ii) esophageal flux was twice that through forestomach, despite their similar structures (Table IIIGo); (iii) the inhibition of esophageal mucosal flux by enzyme treatment (Table IVGo) could have been due to removal of an active transport system; (iv) the metabolic inhibitor KCN inhibited flux by up to 96% (Table IVGo); (v) flux dropped sharply at 20 and 0°C (see Results); and (vi) an active transport system was indicated for bis-(2-oxopropyl)-NAm uptake by hepatocytes (1).

Conclusions
We think the following are our most interesting findings: (i) mucosal flux of some NAms through the rat esophagus is quite rapid, especially that of the strong esophageal carcinogens MNAN and MBZN. This suggested that NAms can be absorbed by the human esophagus from NAm-containing foods and beverages. However, structural differences between the rat and human esophagus reduce the validity of this extension to humans; (ii) mucosal flux may help determine NAm carcinogenicity in the rat esophagus because MBZN and MNAN, which are most commonly used to induce high incidences of EC, showed the highest mucosal fluxes and because, for seven NAms tested by Druckrey et al. (2), carcinogenicity was almost significantly correlated with mucosal flux (Table VGo). For the NAms tested by Druckrey's group, carcinogenicity also tended to be correlated with log predicted Kp, but this parameter could directly affect esophageal P450 activation of NAms; (iii) enzymic removal of the esophageal stratum corneum reduced NAm flux, perhaps because of an affinity of NAms for mucosal lipids; (iv) NAm diffusion through the esophagus was up to 21 times faster than that through skin; (v) KCN (1 mM) and ethanol (2.3 g/l) inhibited NAm flux through the esophagus; and (vi) the evidence favors passive rather than active NAm diffusion through the esophagus.


    Acknowledgments
 
We thank T.A.Lawson (Eppley Institute) for suggesting that flux could help determine esophageal carcinogenicity, W.Lijinsky (Columbia, MD) for suggesting that we try to explain why methylhexyl- and methylheptyl-NAm induce EC but have not methylated esophageal DNA, and E.R.Lyden and J.Kollath (Department of Preventive and Societal Medicine, University of Nebraska Medical Center) for the statistical analyses. This project was supported by grant R01-CA-35628 and core grant P30-CA-36727 from the National Cancer Institute, grant 97B-125 from the American Institute for Cancer Research, a National Overseas Fellowship (to J.H.) from the Ministry of Social Justice and Empowerment, Government of India and core grant SIG-16 from the American Cancer Society. Some of these results were reported at three meetings (5254)


    Notes
 
6 To whom correspondence should be addressed Email: smirvish{at}unmc.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 19, 1998; revised November 1, 1998; accepted December 4, 1998.