Comparative effects of phenylenebis(methylene)selenocyanate isomers on xenobiotic metabolizing enzymes in organs of female CD rats
Ock Soon Sohn1,
Emerich S. Fiala,
Pramod Upadhyaya,
Young-Heum Chae and
Karam El-Bayoumy
American Health Foundation, 1 Dana Road, Valhalla, NY 10595, USA
 |
Abstract
|
---|
The cancer chemopreventive agent 1,4-phenylenebis-(methylene)selenocyanate (p-XSC) inhibits various chemically induced tumors in laboratory animals. We examined the effects of p-XSC and its o- and m-isomers on xenobiotic metabolizing enzymes in vivo. Six-week-old female CD rats were given diets containing o-, m- or p-XSC (5 or 15 p.p.m. as Se), or equimolar amounts (30 or 90 µmol/kg) of 1,4-phenylenebis(methylene)thiocyanate (p-XTC, the sulfur analog of p-XSC) for 1 week. At termination, substrate-specific assays for enzymes of xenobiotic metabolism in various organs were performed. Overall, o-XSC was a more potent enzyme inducer than m- or p-XSC. In hepatic microsomes, o-XSC significantly induced CYP2E1 as detected by increased N-nitrosodimethylamine N-demethylase activity and also by western blot. The activities of CYP1A1 (ethoxyresorufin-O-dealkylase) and CYP1A2 (methoxyresorufin-O-dealkylase) were not affected, but a significant decrease in the activity of CYP2B1 (pentoxyresorufin-O-dealkylase) was observed at the 15 p.p.m. Se level of o-XSC. With the m- and p-XSC isomers or with p-XTC, no significant effect on phase I enzymes was noted. Hepatic UDP-glucuronosyltransferase activities were increased 1.5- to 2-fold by all three XSC isomers at the higher dose level (15 p.p.m. Se), but not by p-XTC; o-XSC again was the most effective. All three XSC isomers were found to increase the
, µ and
isozymes of glutathione S-transferases in the liver, kidney, lung, colon and mammary gland to varying degrees. The XSC isomers also significantly increased glutathione peroxidase in the colon and mammary gland. Although o-XSC was the most powerful in stimulating the enzyme activities, especially in the liver, atomic absorption spectrometry showed that the selenium levels were highest in organs of rats given p-XSC. Thus, the level of tissue distribution of the XSC isomers and/or their metabolite(s) does not correlate with their effects on enzyme activities. The present study demonstrates that individual XSC isomers are capable of modulating specific phase I and/or phase II enzymes involved in the activation and/or detoxification of chemical carcinogens, and provides some mechanistic basis for the cancer chemopreventive efficacy of these organoselenium compounds at the stage of tumor initiation.
Abbreviations: AAS, atomic absorption spectrometry; AOM, azoxymethane; B[a]P, benzo[a]pyrene; BSC, benzyl selenocyanate; CDNB, 1-chloro-2,4-dinitrobenzene; CYP1A1, ethoxyresorufin-O-dealkylation; CYP1A2, methoxyresorufin-O-dealkylation; CYP2E1, N-nitrosodimethylamine-N-demethylase; DCNB, 1,2-dichloro-4-nitrobenzene; DMBA, 7,12-dimethylbenz[a]anthracene; GSH peroxidase, glutathione peroxidase; GST, glutathione S-transferase; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; o-XSC, m-XSC and p-XSC, 1,2- 1,3- and 1,4-phenylenebis(methylene)selenocyanate; p-NP, p-nitrophenol; p-XTC, 1,4-phenylenebis(methylene)thiocyanate; UDPGT, UDP-glucuronosyltransferase.
 |
Introduction
|
---|
Bioassays using various animal tumor models have demonstrated that benzyl selenocyanate (BSC), 1,4-phenylenebis-(methylene)selenocyanate (p-XSC) and related organo-selenium compounds are effective in protecting against chemically induced tumorigenesis, including azoxymethane (AOM)-induced colon tumors (13), 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors (47), benzo[a]pyrene (B[a]P)-induced forestomach tumors (8), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumors (9,10) and 4-nitroquinoline-1-oxide-induced oral cancer (11). At the stage of initiation, chemopreventive agents may act by inhibiting activation pathways or stimulating detoxification pathways (1215). Thus, for instance, BSC was found to inhibit DNA guanine methylation by AOM in the rat colon by significantly increasing the CYP2E1-mediated metabolism of the carcinogen in the liver (13). This organoselenium compound also inhibited 2-nitropropane-induced DNA modifications in rat liver (15). The inhibitory effect of p-XSC during the initiation phase of DMBA-induced mammary carcinogenesis was accompanied by decreased DMBADNA adduct formation in mammary tissue but not in the liver (7). In a subsequent study, using the positional isomers of p-XSC, Chae et al. (4) reported that o- and m-XSC were equally effective, and appeared to be stronger than p-XSC, in inhibiting DMBADNA binding in rat mammary tissues. Using human liver microsomes and recombinant human cytochrome P450 enzymes, Shimada et al. (16) recently examined the effects of several synthetic organoselenium compounds on the metabolism of xenobiotics, and demonstrated that all three XSC isomers were potent inhibitors of the metabolic activation of pro-carcinogenic substrates in vitro. However, the effects of these compounds on xenobiotic metabolism in vitro, where the organoselenium compounds are directly added to the incubation mixtures (16), could be totally different from the effects observed when the chemopreventive agents are ingested by animals in bioassays. In order to evaluate the effects of feeding of XSC isomers and the sulfur analog of p-XSC [1,4-phenylenebis(methylene)thiocyanate (p-XTC); see Figure 1
for structures] on select phase I and phase II enzymes, we carried out studies under conditions similar to those used in chemoprevention bioassays.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1. Structures of p-XSC and its positional isomers o-XSC and m-XSC. The sulfur analog of p-XSC (p-XTC) is also shown.
|
|
 |
Materials and methods
|
---|
Chemicals
p-XSC and its isomers, o- and m-XSC, were synthesized by reacting the corresponding
,
-dibromo-p-xylene,
,
-dibromo-o-xylene and
,
-dibromo-m-xylene with KSeCN (4,7); p-XTC was prepared by reacting
,
-dibromo-p-xylene with KSCN (4). The purity of all XSC isomers and p-XTC was >99.9%, as determined by HPLC analysis (4,7). NADPH, cytochrome c, UDPglucuronic acid, p-nitrophenol (p-NP), glutathione (GSH, reduced form), 1-chloro-2,4-dinitrobenzene (CDNB), N-nitrosodimethylamine, resorufin, ethoxyresorufin, pentoxyresorufin, glutathione reductase and ethacrynic acid were obtained from Sigma (St Louis, MO). 1,2-Dichloro-4-nitrobenzene (DCNB), hydrogen peroxide, t-butyl hydroperoxide, PdCl2, and hydroxylamine hydrochloride were from Aldrich (Milwaukee, WI). Methoxyresorufin was obtained from Molecular Probes (Eugene, OR) and 5-androsten-3,17-dione was from Steraloids (Wilton, NH).
Animals and treatments
Thirty-six 5-week-old female CD rats [Crl:CD® (SD)BR], purchased from Charles River Breeding Laboratories (Kingston, NY), were maintained in the animal-holding room under controlled environmental conditions (12:12 h lightdark cycle, 50% humidity and 21°C) and fed the AIN-76 high-fat (23.5% corn oil) diet ad libitum. The high fat diet was employed in this study to mimic the Western human diet and to duplicate the conditions utilized in our previous efficacy studies (2,4,7). Starting at 6 weeks of age, rats were divided into nine groups (four rats/group) and fed the high fat diets containing an XSC isomer or p-XTC for 1 week. Rats in group 1 were fed the control diet, and rats in groups 29 were given the experimental diet supplemented with two dose levels (5 or 15 p.p.m. Se) of o-, m- or p-XSC, or equimolar levels (30 or 90 µmol/kg) of p-XTC. Diets were replaced with a new batch after 3 days; the stability of XSC isomers and p-XTC in the diet under the conditions of feeding protocol was confirmed by HPLC analysis (8). At the end of 1 week of feeding, the rats were killed under CO2 euthanasia, blood was collected from cardiac puncture and separated for plasma. Liver, kidney, lung, mammary glands and colon were excised, quickly frozen in liquid nitrogen and stored at 75°C until analyses for enzymes and/or for selenium determination.
Preparation of microsomes and/or cytosols of various organs
Both microsomes and cytosol were prepared from livers, but only cytosols were prepared from other organs. For liver, kidney and mammary gland, 1.0 ml aliquots of tissue homogenates were reserved for the determination of selenium content; however, because of the limited amounts of material, the same could not be done with homogenates from lung and colon. The liver was homogenized in 3 weight-vol of ice-cold 0.25 M sucrose in 0.01 M potassium phosphate buffer, pH 7.5, and centrifuged. Microsomes were resuspended in 3 weight-vol of 0.1 M potassium phosphate buffer, pH 7.0, as described previously (17). Only the cytosols were prepared from the kidneys, lungs, colon mucosa and mammary glands; the mammary fat pads were pulverized while frozen in liquid nitrogen before homogenization (7). In order to minimize repeated freezing and thawing, the final microsomal suspensions and/or cytosol fractions from each organ were divided into several aliquots and stored at 75°C until assay. Microsomal and cytosolic proteins were determined by the method of Lowry et al. (18).
Enzyme assays
Phase I enzymes.
In all assays, spectral determinations were performed using a Beckman DU-7 spectrophotometer (Palo Alto, CA) or a PerkinElmer 650-10S fluorescence spectrophotometer (Norwalk, CT). Cytochrome P450 and cytochrome b5 concentrations were measured using microsomal proteins adjusted to a concentration of 0.20.5 mg/ml in 0.05 M potassium phosphate, pH 7.5 (19,20). The NADPH-cytochrome c reductase activity of microsomes was determined according to the methods described by Phillips and Langdon (21). 7-Alkoxyresorufin dealkylase activity was used to determine CYP1A1, CYP1A2 and CYP2B1 levels (17). Pentoxyresorufin-O-dealkylase (CYP2B1) activity was assayed by fluorimetric measurement of resorufin formation, as described by Burke and Mayer (22) and Lubet et al. (23). The reaction mixture consisted of 2.0 ml of 50 mM Tris buffer (pH 7.5), 25 mM MgCl2, 10 µM pentoxyresorufin and 125 µM NADPH, and the increase in fluorescence (excitation wavelength = 522 nm, emission wavelength = 586 nm) caused by resorufin was recorded for 45 min. Ethoxyresorufin-O-dealkylation (CYP1A1) and methoxyresorufin-O-dealkylation (CYP1A2) (24) activities were measured in the same way as described for pentoxyresorufin-O-dealkylation, except that the substrate concentration was 1.7 µM for ethoxyresorufin and 5 µM for methoxyresorufin. N-nitrosodimethylamine-N-demethylase (CYP2E1) activity was assayed based on the procedure described previously (17).
Phase II enzymes.
The UDPglucuronosyltransferase (UDPGT) activity of microsomal protein was determined using p-NP as the substrate (25,26) as modified by Sohn et al. (17). The glutathione S-transferase (GST) activity of cytosols prepared from liver, kidney, lung, colon and mammary gland was determined using the method of Habig and Jakoby (27). For the total activity, CDNB was used as substrate; the reaction mixture consisted of 1 mM GSH and 1 mM substrate in 1 ml of 0.1 M potassium phosphate buffer, pH 6.5. The reaction was initiated by addition of 550 µg cytosolic protein and the increase in absorbance at 340 nm caused by the formation of CDNBGSH conjugates was recorded for 4 min. The activity was calculated using an extinction coefficient of 9.6 mM1cm1. For the determination of various isozymes, specific substrates were used (28). The
-class isozyme was monitored using 68 µM 5-androsten-3,7-dione as substrate and the activity was calculated using an extinction coefficient of 16.3 mM1cm1 at 248 nm. The µ-class isozyme was monitored using 1 mM of DCNB as the substrate, and an extinction coefficient of 8.5 mM1cm1 at 345 nm. The
-class isozyme activity was determined using 0.2 mM of ethacrynic acid as the substrate, and a molar extinction coefficient of 5 mM1cm1 at 270 nm.
GSH peroxidase activity was measured by the method of Wendel (29). The selenium-dependent GSH peroxidase was measured using hydrogen peroxide as substrate, and the combined activities of selenium-dependent and -independent GSH peroxidase were monitored using t-butyl hydroperoxide as substrate. NADP produced during the reduction of GSSG was followed by loss in absorbance at 340 nm using an extinction coefficient of 6.2 mM1cm1. The assay mixture consisted of 25 mM potassium phosphate buffer, pH 7.0, 1 mM GSH, 0.25 mM NADPH, 1 U/ml glutathione reductase, cytosolic enzyme and 1.2 mM of substrate, either hydrogen peroxide or t-butyl hydroperoxide.
Immunodetection of CYP2E1 by western blot
Following the method of Laemmli (30), solubilized liver microsomal proteins (1 µg) were separated by gel electrophoresis using a 12% polyacrylamide gel. The proteins were transferred to 0.45-µ nitrocellulose membranes, and immunodetection was performed with the Amersham International kit (RPN 259; Buckinghamshire, UK). The latter utilizes an anti-rat P450 IIE1 (primary) rabbit antibody, an anti-rabbit Ig-biotinylated species-specific (secondary) donkey antibody, and a streptavidinhorseradish peroxidase conjugate which catalyzes the oxidation of luminol in the presence of H2O2 and an enhancer. For detection of the bands, Hyperfilm-ECL high-performance luminescence detection film from Amersham was used with an exposure time of 1 min.
Analysis of selenium in plasma and tissues by atomic absorption spectrometry (AAS)
In order to determine the selenium content of plasma and tissues, 0.5 ml plasma samples or 1.0 ml aliquots of tissue homogenates reserved from the liver, kidney, and mammary gland, were digested, and total selenium contents were determined using AAS as described previously (31,32). Briefly, a 0.51.0 ml sample was mixed with 2 ml concentrated HNO3, digested using a commercial microwave digestion system (MDS-81D; CEM, Matthews, NC), and then diluted to a final volume of 10 ml with distilled water. For AAS, a Varian AA-1475 atomic absorption spectrophotometer (Varian Techtron, Springvale, Australia) equipped with a GTA-95 graphite tube atomizer was used. For each analysis, a 40 µl sample was mixed with 10 µl of reduced-Pd matrix modifier (3.33 g PdCl2/l, 2% hydroxylamine hydrochloride and 2% HCl).
Statistical analysis
All results are presented as means ± SD of four determinations, each derived from a separate animal. Statistical significance between the control group and each of the experimental diet groups was determined using the unpaired two-tailed Student's t-test.
 |
Results
|
---|
In general, the effects of XSC isomers on phase I and phase II enzymes in several organs at the lower dose levels (5 p.p.m. as Se) were either statistically non-significant or followed a trend similar to that of the higher dose levels (15 p.p.m. as Se). Thus, for brevity, only the data obtained with the higher dose levels are described in detail.
Effects of XSC isomers on phase I enzyme activities in the liver
Although the average final body weights of the animals given diets containing o-XSC or m-XSC were lower than those of the control group, the differences were not statistically significant. Among the three isomers, only o-XSC was found to cause an increase in the total cytochrome P450 content in liver microsomes compared with the control group (from 0.41 ± 0.04 to 0.48 ± 0.04 nmol/mg protein, P < 0.05). However, certain parameters of phase I metabolism, such as cytochrome b5 content, NADPH-cytochrome c reductase, CYP1A1 and CYP1A2 activities, were not altered by any of the treatments. On the other hand, CYP2B1 decreased significantly (from 7.3 ± 1.6 to 4.7 ± 1.1 pmol resorufin formed/min/mg protein, P < 0.05) when o-XSC was fed. The decrease appears to be specific to this particular isozyme, and could be specific inhibition of protein synthesis or to the binding of o-XSC or its metabolite to the isozyme. o-XSC was found to be a good inducer of CYP2E1, as evidenced by the significant increase in N-nitrosodimethylamine N-demethylase activity (from 0.29 ± 0.09 to 0.67 ± 0.04 nmol HCHO formed/min/mg protein, P < 0.01). Feeding m-XSC, p-XSC or p-XTC had no significant effect on this P450 isozyme. The increase in N-nitrosodimethylamine N-demethylase activity caused by o-XSC feeding was also significant (P < 0.05) at the lower dose level (5 p.p.m. as Se). An increase in CYP2E1 protein by o-XSC in rat liver was also clearly demonstrated by the western blot immunodetection technique (Figure 2
).

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 2. Western blot analysis of liver microsomal CYP2E1 content of rats fed XSC isomers or p-XTC. The amounts of protein used are 1.25 µg for the isoniazid-induced microsomes (positive control from the commercial kit) and 1 µg for the microsomes obtained from rats fed the control diet or the diets containing XSC isomers (15 p.p.m. as Se) or p-XTC (90 µmol/kg).
|
|
Effects of XSC isomers on UDPGT and GST activities in the liver
All three XSC isomers were found to enhance UDPGT and GST activities to varying degrees. The UDPGT activities in the liver microsomes were significantly increased in rats fed o-, m- and p-XSC (15 p.p.m. as Se) compared with the control (20.7 ± 2.2 nmol p-NP consumed/min/mg protein); the effect being the greatest with o-XSC (82%), followed by m-XSC (46%) and p-XSC (21%). The sulfur analog p-XTC was ineffective, and for o-XSC the activity was increased (P < 0.05) even at 5 p.p.m. Se level. The GST activity of the liver cytosols (nmol CDNBGSH formed/min/mg protein) were significantly increased in the o-XSC (1160 ± 49) and p-XSC (1007 ± 97) groups compared with the control (738 ± 119). For m-XSC, an increase was observed, but it was not statistically significant.
Effects of XSC isomers on the activities of the
, µ and
GST isozymes in the liver and extrahepatic tissues
We used substrate-specific assays to examine the effects of organoselenium compounds in five different organs, namely liver, kidneys, lungs, colon and mammary glands. As shown in Table IA
, the total GST activity, as monitored using CDNB as the substrate, was increased not only in the liver, but also in the extrahepatic tissues by the XSC isomers, although the amount of increase varied. In the kidney and the lung, all three XSC isomers significantly increased the total GST activities, whereas in the colon and the mammary gland, a significant increase was noted only with o- and m-XSC. p-XTC had no effect on these activities in any organs examined. With o-XSC, the total GST activity increased ~3-fold in the kidney cytosol and ~2-fold in the mammary gland, whereas the degree of increase in the colon and the lung was much smaller. The
-class isozyme activity, which was assayed by using 5-androsten-3,17-dione as the substrate, showed a slight but a significant increase in the case of o-XSC in the kidney and the mammary gland, and with p-XSC, in the lung (Table IB
). When the µ-class isozyme was monitored using DCNB as the substrate (Table IC
), the activities in the liver, kidney and mammary gland increased significantly with both dietary o- and m-XSC; with o-XSC, the activity in the kidney cytosol increased ~5-fold even at the lower dose level (data not shown). The analysis of the
-class isozyme using ethacrynic acid as the substrate (Table ID
) indicated that o-XSC is the only compound that increased this enzyme activity in the liver and the kidney, whereas all three isomers appeared to be effective inducers for the colon and the lung enzymes. In the case of the colon, the
-class GST enzyme activity was also increased by p-XTC. This is the only instance in which we noted any significant changes in enzyme levels caused by feeding p-XTC.
View this table:
[in this window]
[in a new window]
|
Table I. Effect of 1 week of feeding XSC isomers on the GST isozyme activities in the cytosols of female CD rat organs determined by using substrate-specific assaysa
|
|
Effects of XSC isomers on the activities of GSH peroxidases in the liver and extrahepatic tissues
We examined the effects of feeding the XSC isomers on the activities of the two GSH peroxidase enzymes by using either hydrogen peroxide (Se-dependent GSH peroxidase only) or t-butyl hydroperoxide (both Se-dependent and -independent) as substrates. When the Se-dependent GSH peroxidase was assayed, no significant differences from controls were observed in any of the organs (liver, kidney, lung and mammary gland) except the colon. For the colon, all three XSC isomers, but not p-XTC, significantly increased the activity by 4566% over the control value (100 ± 14 nmol NADPH oxidized/min/mg protein). However, when the Se-dependent plus Se-independent GSH peroxidases were assayed, in addition to the colon, activities in the mammary gland were also increased by all three isomers. Only o- and m-XSC were effective in increasing the total GSH peroxidase activity in the kidneys (data not shown).
Tissue distribution of total selenium derived from XSC isomers
To determine whether there is a correlation between the effects of the XSC isomers on enzyme activities in various organs and the total selenium levels in those organs, we examined the tissue selenium distribution using AAS (Figure 3
). For o-, m- and p-XSC, the levels of selenium present in the organs of the rats that received the compounds for 1 week appeared to be dose dependent (data on lower dose levels are not shown), whereas the selenium levels in the organs of rats that received p-XTC were essentially the same as those of the controls. In the blood plasma of rats fed 15 p.p.m. (as Se) of XSC isomers, the selenium level increased 80100% over the control values (0.37 ± 0.03 µg/ml) (Figure 3A
). In control rats, the basal level of selenium in the liver or kidneys was 1.15 ± 0.31 or 0.94 ± 0.18 µg/g, respectively, whereas it was below the detection limit (<0.15 µg/g) in the mammary gland. In the liver, the selenium content was the highest after p-XSC treatment (Figure 3B
). At 15 p.p.m. dietary Se, the selenium levels in the livers were 4.51 ± 0.36, 3.96 ± 0.95 and 7.27 ± 1.61 µg/g for o-, m- and p-XSC, respectively. The kidney selenium content was highest after o-XSC feeding at both dose levels (Figure 3C
), whereas the selenium level in the mammary gland appeared to be slightly higher after m-XSC feeding than after o- or p-XSC feeding (Figure 3D
).

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 3. Content of total selenium in various organs of rats fed control or diets containing XSC isomers (15 p.p.m. as Se) or p-XTC (90 µmol/kg) for 1 week. (A) Blood plasma; (B) liver; (C) kidney; (D) mammary gland. The data represent means ± SD of four determinations, each derived from a separate animal. N.D., Se level below the detection limit (<0.15 µg/g tissue).
|
|
 |
Discussion
|
---|
The goals of this study were to characterize XSC chemopreventive compounds with respect to their ability to modulate xenobiotic metabolizing enzyme systems in rat liver and other organs, and to determine whether the modulation is related to the tissue selenium contents. The results indicate that, in female CD rats, XSC isomers are capable of modulating phase I and/or phase II enzyme activities, including CYP2B1, CYP2E1, UDPGT, GST and GSH peroxidases, when given in the diet. This suggests that the chemopreventive efficacy of these compounds at the stage of tumor initiation may partly be the result of their effects on these enzyme systems. The fact that the sulfur analog p-XTC did not significantly influence these enzyme systems (with the exception of the
-class GST activity in the colon; Table ID
) again emphasizes the requirement of selenium in the chemopreventive activities of these compounds, as shown previously (2,4,13,15). The results also indicate that o-XSC is a more potent enzyme inducer than m- or p-XSC. However, a comparison of the total selenium content derived from the three XSC isomers showed that the selenium levels in the liver are highest in rats given p-XSC (Figure 3B
). These results indicate that the magnitude of distribution of these compounds and/or their metabolite(s) to organs is not a major factor in determining their effects on the enzyme activities examined in this study. In previous work (32), we observed that there were marked differences in the excretory patterns of the three XSC isomers/metabolites; o-XSC was more readily absorbed from the gastrointestinal tract than m- and p-XSC on oral administration. The more potent effects of o-XSC on enzyme activities noted in the current study could be caused by, in part, its better absorption and perhaps differential metabolism. It is not known at this time whether the modulation of xenobiotic-metabolizing enzymes by XSC isomers is caused by the compounds themselves or by their metabolites.
Of the P450 isozymes examined, CYP2E1, which is involved in the metabolism of colon carcinogen AOM and methylazoxymethanol (13,14), as well as of various N-nitrosamines (33), was increased significantly only by o-XSC, and the increase was dose-dependent. It is noted that, in this respect, o-XSC is similar to BSC, which is a strong inducer of liver CYP2E1 (13). CYP2E1 expression in the mature rat liver is controlled by enzyme stabilization against degradation as well as by mRNA stabilization (33). That o-XSC does in fact increase CYP2E1 protein in rat liver is shown by western blot immunodetection in Figure 2
. We previously demonstrated that feeding of BSC resulted in decreased AOM-induced colon DNA methylation at the O6 and N7 positions of guanine, and postulated that BSC inhibits AOM-induced colon carcinogenesis by inducing liver CYP2E1, thereby accelerating the liver oxidation of the carcinogen and limiting the transport of more proximate carcinogenic forms of AOM to the colon (13). Although the effects of o-XSC on AOM-induced colon DNA methylation and/or carcinogenesis have not yet been evaluated, we would expect to observe inhibition similar to that obtained with BSC.
In the case of phase II enzymes, all three XSC isomers were found to significantly increase the activity of liver UDPGT. The increase was greatest with o-XSC, followed by m-XSC and then by p-XSC. The induction of UDPGT by organo-selenium compounds may contribute to increased rates of detoxification of chemical carcinogens, and may explain, in part, the reduction of DMBAmammary DNA binding by o-, m- and p-XSC (4), and the inhibition of NNK-induced lung tumorigenesis in mice by p-XSC (9,10). In fact, the order of potency of the XSC isomers in increasing UDPGT in the liver appears to correlate with their degree of inhibition of DMBA binding to DNA in rat mammary tissues (4).
GST enzymes have the important function of conjugating reactive electrophilic metabolites of carcinogens and other xenobiotics with glutathione (34,35). Reaction of the electrophiles with glutathione reduces the chances that they will react with more critical macromolecules such as DNA. The ability to induce GST is an important attribute of many cancer chemopreventive compounds (36). When specific substrates were used to assay the different isozyme classes, we found that the effects of organoselenium compound feeding varied with the organs. The
-class isozyme is known to be important for the detoxification of aflatoxin B1-8,9-oxide; the µ-class isozyme is involved in the glutathione conjugation of epoxides of DMBA, B[a]P and 1-nitropyrene; and the
-class enzyme is also known to be involved in the conjugation of B[a]Pdiol-epoxide (34,37,38). The significant increase in the activities of the µ-class isozyme in the liver, kidney and especially in the mammary gland (Table IC
) is noteworthy, because the µ-class isozyme or GST subunit 3 is important in the detoxification of DMBA (38). Chae et al. (4) observed a significant reduction of DMBADNA binding in mammary tissues of rats given o- and m-XSC. The increase of the µ-class isozyme by o- and m-XSC might have facilitated the detoxification of DMBA metabolites by GSH conjugation. The tissue-specific pattern of GST activities we observed after administration of organo-selenium compounds is similar to the results of Christensen et al. (39,40), who also noted that dietary inorganic selenium differentially affected various isozymes of GST in different rat tissue.
GSH peroxidase is an important protective antioxidant enzyme that catalyzes the reduction of organic peroxides, such as lipid peroxides, to the corresponding alcohols, and of hydrogen peroxide to water, thus decreasing cellular oxidative stress. Oxidative damage has been implicated in the initiation and post-initiation stages of the carcinogenic process (41,42). The increased formation of 8-oxodeoxyguanosine, usually an indication of the oxidative DNA damage by a tobacco-specific lung carcinogen NNK (42,43) and a mammary carcinogen DMBA (41,44) suggests that increased protection from oxidative damage by induction of GSH peroxidase might provide an important chemopreventive strategy against these carcinogens. In this regard, the significant increase of GSH peroxidase (Se-dependent plus -independent) in the mammary gland by all three XSC isomers observed in this study is noteworthy. Recently, p-XSC was shown to reduce the levels of NNK-induced 8-oxodeoxyguanosine in the lung of A/J mice (43). Reddy et al. (3) previously reported the induction of Se-dependent GSH peroxidase in rat colon by p-XSC; our data corroborate their results and also demonstrate that o- and m-XSC are more effective than the p-isomer. Although the level of GSH peroxidase (at least in rat liver) is regulated, within certain limits, by selenium intake, the precise mechanisms are unknown, but may involve stabilization of the mRNA for the enzyme (39). How regulation occurs in extrahepatic organs has not, to our knowledge, been described in the literature.
In summary, we have investigated the effects of dietary o-, m- and p-XSC isomers on the xenobiotic metabolizing enzyme systems in female CD rats. The results indicate that individual XSC isomers are capable, although to varying degree, of modulating specific phase I and/or phase II enzyme activities involved in the activation and/or detoxification of chemical carcinogens, and provide some mechanistic basis for the cancer chemopreventive efficacy of these organoselenium compounds at the stage of tumor initiation.
 |
Acknowledgments
|
---|
The authors appreciate the technical assistance of Mr H.Li and the staff of the Research Animal Facility. This work was supported by grant CA46589 from the National Cancer Institute.
 |
Notes
|
---|
1 To whom correspondence should be addressed 
 |
References
|
---|
-
Reddy,B.S., Wynn,T.T., El-Bayoumy,K., Upadhyaya,P., Fiala,E.S. and Rao,C.V. (1996) Evaluation of organoselenium compounds for potential chemopreventive properties in colon cancer. Anticancer Res., 16, 11231128.[ISI][Medline]
-
Nayini,J.R., Sugie,S., El-Bayoumy,K., Rao,C.V., Rigotty,J., Sohn,O.-S. and Reddy,B.S. (1991) Effect of dietary benzylselenocyanate on azoxymethane-induced colon carcinogenesis in male F344 rats. Nutr. Cancer, 15, 129139.[ISI][Medline]
-
Reddy,B.S., Rivenson,A., Kulkarni,N., Upadhyaya,P. and El-Bayoumy,K. (1992) Chemoprevention of colon carcinogenesis by synthetic organo-selenium compound 1,4-phenylenebis(methylene)selenocyanate. Cancer Res., 52, 56355640.[Abstract]
-
Chae,Y.-H., Upadhyaya,P. and El-Bayoumy,K. (1997) Structureactivity relationships among the ortho-, meta- and para-isomers of phenylenebis-(methylene)selenocyanate (XSC) as inhibitors of 7,12-dimethylbenz-[a]anthraceneDNA binding in mammary glands of female CD rats. Oncol. Rep., 4, 10671071.[ISI]
-
El-Bayoumy,K., Upadhyaya,P., Chae,Y.-H., Sohn,O.S., Rao,C.V., Fiala,E.S. and Reddy,B.S. (1995) Chemoprevention of cancer by organoselenium compounds. J. Cell. Biochem., 22 (suppl.), 92100.
-
Ip,C., El-Bayoumy,K., Upadhyaya,P., Ganther,H., Vadhanavikit,S. and Thompsom,H. (1994) Comparative effect of inorganic and organic selenocyanate derivatives in mammary cancer chemoprevention. Carcinogenesis, 15, 187192.[Abstract]
-
El-Bayoumy,K., Chae,Y.-H., Upadhyaya,P., Meschter,C., Cohen,L.A. and Reddy,B.S. (1992) Inhibition of 7,12-dimethylbenz[a]anthracene-induced tumors and DNA adduct formation in the mammary glands of female SpragueDawley rats by the synthetic organoselenium compound 1,4-phenylenebis(methylene)selenocyanate. Cancer Res., 52, 24022407.[Abstract]
-
El-Bayoumy,K. (1985) Effects of organoselenium compounds on induction of mouse forestomach tumors by benzo[a]pyrene. Cancer Res., 45, 36313635.[Abstract]
-
El-Bayoumy,K., Upadhyaya,P., Desai,D., Amin,S. and Hecht,S.S. (1993) Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone tumorigenicity in mouse lung by the synthetic organoselenium compound 1,4-phenylenebis(methylene)selenocyanate. Carcinogenesis, 14, 11111113.[Abstract]
-
Prokopczyk,B., Amin,S., Desai,D.H., Kurtzke,C., Upadhyaya,P. and El-Bayoumy,K. (1997) Effects of 1,4-phenylenebis(methylene)selenocyanate and selenomethionine on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced tumorigenesis in A/J mouse lung. Carcinogenesis, 18, 18551857.[Abstract]
-
Tanaka,T., Makita,H., Kawabata,K., Mori,H. and El-Bayoumy,K. (1997) 1,4-Phenylenebis(methylene)selenocyanate exerts exceptional chemopreventive activity in rat tongue carcinogenesis. Cancer Res., 57, 36443648.[Abstract]
-
Wattenberg,L.W. (1985) Chemoprevention of cancer. Cancer Res., 45, 18.[ISI][Medline]
-
Fiala,E.S., Joseph,C., Sohn,O.S., El-Bayoumy,K. and Reddy,B.S. (1991) Mechanism of benzylselenocyanate inhibition of azoxymethane-induced colon carcinogenesis in F344 rats. Cancer Res., 51, 28262830.[Abstract]
-
Sohn,O.S., Ishizaki,H., Yang,C.S. and Fiala,E.S. (1991) Metabolism of azoxymethane, methylazoxymethanol and N-nitrosodimethylamine by cytochrome P450IIE1. Carcinogenesis, 12, 127131.[Abstract]
-
Fiala,E.S., Sohn,O.S., Li,H., El-Bayoumy,K. and Sodum,R.S. (1997) Inhibition of 2-nitropropane-induced rat liver DNA and RNA damage by benzyl selenocyanate. Carcinogenesis, 18, 18091815.[Abstract]
-
Shimada,T., El-Bayoumy,K., Upadhyaya,P., Sutter,T.R., Guengerich,F.P. and Yamazaki,H. (1997) Inhibition of human cytochrome P450-catalyzed oxidation of xenobiotics and procarcinogens by synthetic organoselenium compounds. Cancer Res., 57, 47574764.[Abstract]
-
Sohn,O.S., Surace,A., Fiala,E.S., Richie,J.P.Jr, Colosimo,S., Zang,E. and Weisburger,J.H. (1994) Effects of green tea and black tea on hepatic xenobiotic metabolizing systems in the male F344 rat. Xenobiotica, 24, 119127.[ISI][Medline]
-
Lowry,O.H., Rosebrough,N.H., Farr,A.L. and Randall,R.J. (1951) Protein measurement with folin-phenol reagent. J. Biol. Chem., 193, 265275.[Free Full Text]
-
Omura,T. and Sato,R. (1964) The carbon monoxide binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem., 239, 23702378.[Free Full Text]
-
Estabrook,R.W. and Werringloer,J. (1978) The measurement of difference spectra: application to the cytochromes of microsomes. Meth. Enzymol., 52, 212220.[Medline]
-
Phillips,A. and Langdon,R. (1962) Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization and kinetic studies. J. Biol. Chem., 237, 26522660.[Free Full Text]
-
Burke,M.D. and Mayer,R.T. (1974) Direct fluorimetric assay of a microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene. Drug Metab. Dispos., 2, 583588.[ISI][Medline]
-
Lubet,R.A., Mayer,R.T., Cameron,J.W., Nims,R.W., Burke,M.D., Wolff,T. and Guengerich,F.P. (1985) Dealkylation of pentoxyresorufin: a rapid sensitive assay for measuring induction of cytochrome(s)-P-450 by phenobarbital and other xenobiotics in the rat. Arch. Biochem. Biophys., 238, 4348.[ISI][Medline]
-
Nerukar,P.V., Park,S.S., Thomas,P.E., Nims,R.W. and Lubet,R.A. (1993) Methoxyresorufin and benzoyloxyresorufin: substrates preferentially metabolized by cytochromes P4501A2 and 2B, respectively, in the rat and mouse. Biochem. Pharmacol., 46, 933943.[ISI][Medline]
-
Isselbacher,K.J., Chrabas,M.F. and Quinn,R.C. (1962) The solubilization and partial purification of a glucuronyl transferase from rabbit liver microsomes. J. Biol. Chem., 237, 30333036.[Free Full Text]
-
Bock,K.W., Burchell,B., Dutton,G.J., Hanninen,O., Mulder,G.J., Owens,I.S., Siest,G. and Tephly,T.R. (1983) UDPglucuronyltransferase activities: guidelines for consistent interim terminology and assay conditions. Biochem. Pharmacol., 32, 953955.[ISI][Medline]
-
Habig,W.H. and Jakoby,W.B. (1981) Assay of differentiation of glutathione S-transferase. Meth. Enzymol., 77, 398405.[Medline]
-
Mannervik,B., Alin,P., Guthenberg,C., Jensson,H., Tahir,M.K., Warholm,M. and Jornvall,H. (1985) Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties. Proc. Natl Acad. Sci. USA, 82, 72027206.[Abstract]
-
Wendel,A. (1981) Glutathione peroxidase. Meth. Enzymol., 77, 325333.[Medline]
-
Laemmli,U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227, 680685.[ISI][Medline]
-
Sohn,O.S., Blackwell,L., Mathis,J., Asaad,W.W., Reddy,B.S. and El-Bayoumy,K. (1991) Excretion and tissue distribution of selenium following treatment of male F344 rats with benzylselenocyanate or sodium selenite. Drug Metab. Dispos., 19, 865870.[Abstract]
-
Sohn,O.S., Li,H., Surace,A., El-Bayoumy,K., Upadhyaya,P. and Fiala,E.S. (1995) Contrasting patterns of selenium excretion by female CD rats treated with chemically related chemopreventive organic selenocyanate compounds. Anticancer Res., 15, 18491856.[ISI][Medline]
-
Yang,C.S., Yoo,J.-S.H., Ishizaki,H. and Hong,J. (1990) Cytochrome P450IIE1: roles in nitrosamine metabolism and mechanisms of regulation. Drug Metab. Rev., 22, 147159.[ISI][Medline]
-
Ketterer,B., Meyer,D.J. and Clark,A.G. (1988) Soluble glutathione transferase isozymes. In Sies,H. and Ketterer,B. (eds) Glutathione Conjugation. Mechanism and Biological Significance. Academic Press, London, pp. 73135.
-
Commadeur,J.N.M., Stijntjes,G.J. and Vermeulen,N.P.E. (1995) Enzymes and transport systems involved in the formation and disposition of glutathione S-conjugates. Role in bioactivation and detoxication mechanisms of xenobiotics. Pharmacol. Rev., 4, 271330.
-
McLellan,L.I., Kerr,L.A., Cronshaw,A.D. and Hayes,J.D. (1991) Regulation of mouse glutathione S-transferases by chemoprotectors. Biochem. J., 276, 461469.[ISI][Medline]
-
Zangar,R.C., Springer,D.L., McCrary,J.-A., Novak,R.F., Primiano,T. and Buhler,D.R. (1992) Changes in adult metabolism of aflatoxin B1 in rats neonatally exposed to diethylstilbestrol. Alterations in
-class glutathione S-transferases. Carcinogenesis, 13, 23752379.[Abstract]
-
Elegbede,J.A., Maltzman,T.H., Elson,C.E. and Gould,M.N. (1993) Effects of anticarcinogenic monoterpenes on phase II hepatic metabolizing enzymes. Carcinogenesis, 14, 12211223.[Abstract]
-
Christensen,M.J. and Burgener,K.W. (1992) Dietary selenium stabilizes glutathione peroxidase mRNAs in rat liver. J. Nutr., 122, 16201626.[ISI][Medline]
-
Christensen,M.J., Nelson,B.L. and Wray,C.D. (1994) Regulation of glutathione S-transferase gene expression and activity by dietary selenium Biochem. Biophys. Res. Commun., 202, 271277.[ISI][Medline]
-
Frenkel,K., Wei,L. and Wei,H. (1995) 7,12-Dimethylaminobenz[a]anthracene induces oxidative DNA modification in vivo. Free Rad. Biol. Med., 19, 373380.[ISI][Medline]
-
Chung,F.-L. and Xu,Y. (1992) Increased 8-oxodeoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with the tobaccospecific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Carcinogenesis, 13, 12691272.[Abstract]
-
Rosa,J.G., Prokopczyk,B., Desai,D.H., Amin,S.G. and El-Bayoumy,K. (1998) Elevated 8-hydroxy-2'-deoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and inhibition by dietary 1,4-phenylenebis(methylene)-selenocyanate. Carcinogenesis, 19, 17831788.[Abstract]
-
Chae,Y.-H., Rosa,J., Williams,L.K., Desai,D., Amin,S., Fiala,E. and El-Bayoumy,K. (1998) Induction of oxidative DNA damage by 7,12-dimethylaminobenz[a]anthracene and 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine in rats. Proc. Am. Assoc. Cancer. Res., 39, 490.
Received July 9, 1998;
revised October 23, 1998;
accepted December 1, 1998.