Induction of phase II enzymes by 3H-1,2-dithiole-3-thione: doseresponse study in rats
Rex Munday1 and
Christine M. Munday
AgResearch, Ruakura Agricultural Research Centre, Private Bag 3123, Hamilton, New Zealand
1 To whom correspondence should be addressed Email: rex.munday{at}agresearch.co.nz
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Abstract
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Derivatives of 3H-1,2-dithiole-3-thione (D3T) are known to protect against a variety of chemical carcinogens. There is evidence that this chemoprotective effect depends, at least in part, on the ability of these compounds to increase tissue activities of phase II detoxification enzymes. In the present study, D3T was dosed to rats at daily doses of between 0.98 and 125 µmol/kg/day for 5 days. The activity of two phase II enzymes, quinone reductase and glutathione S-transferase, were then assayed in the liver, spleen, kidneys, lungs, heart, urinary bladder, forestomach, glandular stomach, duodenum, jejunum, ileum, caecum and colon plus rectum of the animals. D3T was particularly effective in increasing enzyme activities in the stomach and duodenum, with significant effects being recorded at a dose-level of only 0.98 µmol/kg/day. At slightly higher dose-levels, increases were recorded in other segments of the small and large intestine and in the urinary bladder. D3T caused enlargement of the liver, kidneys, stomach and intestinal tract of the animals at the higher dose-levels, but no other toxic effects were recorded. D3T is a very effective inducer of phase II enzymes, showing significant effects at lower dose-levels than any other compound for which doseresponse data are available. The inductive potency of D3T makes it a most promising candidate for use as a chemoprotective agent.
Abbreviations: D3T, 3H-1,2-dithiole-3-thione; GST, glutathione S-transferases; OR, quinone reductase
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Introduction
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The concept of chemoprotectionthe blockade or suppression of carcinogenesiswas first delineated by Wattenberg in 1966 (1). Since then, many substances, of both natural and synthetic origin, have been shown to protect animals against a variety of chemical carcinogens, and the mechanism of this effect has been extensively investigated (24). One important process in chemoprotection involves modulation of the activity of the so-called phase II enzymes, which convert carcinogens to inactive metabolites that are readily excreted from the body, thus preventing their reaction with DNA. The phase II enzymes, which include quinone reductase (QR; DT-diaphorase, NAD[P]H:quinone acceptor oxidoreductase, EC 1.6.99.2), the glutathione S-transferases (GST; EC 2.5.1.18), epoxide hydrolase (EC 3.3.2.3) and UDP-glucuronosyltransferase (EC 2.4.1.17) are highly inducible in both animals and humans (5,6), and a strong inverse relationship exists between tissue levels of these enzymes and susceptibility to chemical carcinogenesis (7,8).
Certain derivatives of 3H-1,2-dithiole-3-thione (Figure 1) have been shown to induce phase II enzymes and to protect against chemical carcinogenesis in animals. The most extensively studied compound of this class is oltipraz (Figure 1, R1 = pyrazinyl, R2 = methyl). The ability of this compound to induce phase II enzymes was first reported by Ansher, Dolan and Bueding (9), and these authors suggested that oltipraz would be an effective chemoprotective agent. The accuracy of this prediction was demonstrated by Wattenberg and Bueding (10), who showed that administration of oltipraz to mice protected against the neoplasia induced by benzo[a]pyrene, diethylnitrosamine and uracil mustard. Later studies have confirmed the chemoprotective action of oltipraz, which is now known to afford protection against a wide range of carcinogens affecting skin, liver, breast, lung, trachea, pancreas, stomach, urinary bladder and colon (11,12). The ability of this compound to induce phase II enzymes has also been confirmed, with increased enzyme activities being recorded in the liver, lungs, kidneys, stomach, small intestine and colon of mice and rats (1317). Oltipraz also increased the excretion of phase II metabolites of aflatoxin in individuals naturally exposed to this carcinogen, indicating that this dithiolethione is also capable of increasing the levels of cancer-protective enzymes in man (18).
Oltipraz is particularly effective in preventing aflatoxin B1-induced hepatocellular carcinoma (12), and was chosen for investigation as a potential chemoprotective agent for humans in a population in rural China with a high intake of aflatoxin and concomitant high risk of liver cancer. Both phases I and II trials have been undertaken, but because of the high cost of oltipraz, and the side-effects of this substance, a large-scale phase III trial was considered impracticable (18). For this reason, other derivatives of 3H-1,2-dithiole-3-thione are now under investigation, in order to identify cheaper chemoprotective agents, with fewer side-effects, for human use (18).
Unsubstituted 3H-1,2-dithiole-3-thione (D3T; Figure 1, R1 = R2 = H) is known to protect against the formation of pre-neoplastic changes induced in rat liver by aflatoxin (19), to decrease DNA adduct formation and mammary tumorigenesis by 7,12-dimethylbenz[a]anthracene (20) and to decrease the frequency of mutations in the colon of rats treated with 2-amino-1-methyl-6-phenylimidazole[4,5-b]pyridine (21). It is also an inducer of phase II enzymes in rodents (18,2225), and may therefore be an effective chemoprotective agent.
For a chemoprotective agent to be useful to humans, it is essential that it exerts its beneficial effects at practicable dose-levels. At present, little information is available on the relationship between the dose of D3T and degree of enzyme induction, and the data that are available relate only to liver enzymes (18,22). The dose-levels of D3T that are likely to be effective are therefore unknown. Furthermore, since comparatively few tissues have been examined in animals, it is not known if the inductive effect of D3T is tissue-specific. This is an important point, since other phase II enzyme inducers do show such specificity. For example, aliphatic disulphides increase phase II enzyme activity in the stomach and upper gastrointestinal tract of rats at doses much lower than those required for an effect in other tissues (26), while isothiocyanates have by far their greatest inductive effect in the urinary bladder (27).
In order to provide detailed information on the dose-levels of D3T that are required for phase II enzyme induction, and to investigate the possibility of tissue specificity, a doseresponse study with this compound has been conducted in rats, with assay of QR and GST in a wide range of tissues.
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Materials and methods
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Chemicals
D3T was synthesized by the method of Meinetsberger et al. (28), and its purity and identity confirmed by melting point and high-resolution mass spectrometry. It was stored at 20°C before use. Reagents for enzyme assays were from Sigma.
Animals and housing
Eleven- to twelve-week-old female SpragueDawley rats from the Ruakura colony were randomly allocated to treatment groups. The rats were housed in solid-bottomed cages containing bedding of wood shavings, and were allowed free access to food (Laboratory Chow, Sharpes Animal Feeds, Carterton, NZ) and tap water. Room temperature was maintained at 2123°C under a 12-h lightdark cycle. The body weights of the animals were recorded daily.
Dosing and necropsy
Groups of five rats were dosed by oral intubation with D3T, as freshly prepared solutions in soya oil, each day for 5 days. A uniform dose of solution (2 ml/kg body wt) was employed. The D3T concentrations were adjusted such that the rats received daily doses of 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5 or 125 µmol/kg of the test substance. Ten control rats received soya oil alone. On the sixth day, the rats were anaesthetized with halothane and killed by exsanguination, blood being taken into EDTA-containing tubes from the posterior vena cava. After macroscopic examination, the liver was dissected out and weighed, and a portion of this organ, together with the kidneys, spleen, heart, urinary bladder and lungs were placed in plastic vials. The whole of the gastrointestinal tract was dissected out and separated into forestomach, glandular stomach, duodenum, jejunum, ileum, caecum and colon plus rectum. These tissues were cut longitudinally, and the contents were washed out under running water. They were then gently blotted on absorbent paper and, along with the other organs, stored at 80°C before analysis for QR and GST activity.
Enzyme assay
Tissue samples were weighed and then homogenized in ice-cold 0.2% Triton X-100 using a Polytron tissue homogenizer. In most instances, the whole organ was homogenized in this way. The jejunum, however, was too large to be homogenized as such, and after weighing, the frozen tissue was cut into segments, and a sample of these segments was homogenized. The homogenates were centrifuged for 20 s at 12 000 g, and the supernate assayed at 25°C for QR by the 2,6-dichlorophenol indophenol method of Ernster (29) and for GST by the method of Habig et al. (30) with 1-chloro-2,4-dinitrobenzene as substrate. Enzyme activities were calculated as international units (IU) per gram of tissue. Statistical significance was tested by analysis of variance followed by the StudentNewmanKeuls multiple comparisons test using InStat software (GraphPad Software, San Diego, CA).
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Results and discussion
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All rats remained in good health throughout the experimental period. Body weight gains did not differ among the test and control groups (data not shown), and no macroscopic changes were recorded at necropsy. However, significant changes in relative organ weights were recorded in animals receiving D3T, as discussed below.
Tissue activities of QR (Tables I and II) and GST (Tables III and IV) increased with increasing dose-levels of D3T. As recorded with other phase II enzyme inducers (26,27), the activities of QR tended toward a plateau at the higher dose-levels. Such a plateau was not so pronounced in the case of GST, however, suggesting that even higher levels of induction may be expected at doses above 125 µmol/kg/day.
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Table I. QR activities in the spleen, liver, kidneys, heart, lungs and urinary bladder of rats receiving D3T at various dose-levels
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Table II. QR activities in the glandular stomach, forestomach, duodenum, jejunum, ileum, caecum, colon plus rectum of rats receiving D3T at various dose-levels
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Table III. GST activities in the spleen, liver, kidneys, heart, lungs and urinary bladder of rats receiving D3T at various dose-levels
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Table IV. GST activities in the glandular stomach, forestomach, duodenum, jejunum, ileum, caecum, colon plus rectum of rats receiving D3T at various dose-levels
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At 125 µmol/kg/day, significant increases in QR and/or GST were observed in every one of the 13 rat tissues examined. The greatest effects were seen in the kidneys, urinary bladder, forestomach and intestine. Smaller increases in enzyme activity were seen in the liver, glandular stomach, lung, spleen and heart. Within the intestine, the degree of enzyme induction by D3T tended to decrease from the proximal to the distal portions of the tract, with the duodenum showing the greatest effect with both QR and GST.
With decreasing dose-levels of D3T, the number of tissues showing significant increases in phase II enzyme activity diminished. However, at the lowest level of administration (0.98 µmol/kg/day, equivalent to
0.13 mg of D3T/rat/day) significant effects were still seen with QR and/or GST in the stomach (both glandular stomach and forestomach) and in the duodenum. Significant increases in enzyme activity were recorded in the urinary bladder at 3.91 µmol/kg/day, and in the liver, kidney, lung, jejunum, ileum, caecum and colon plus rectum of the rats at 7.815.6 µmol/kg/day. The heart and spleen were comparatively resistant to the inductive effects of D3T. In most tissues, the dose-levels at which significant effects were recorded were very similar for both QR and GST. In the liver, however, GST activities were significantly increased by D3T at 7.81 µmol/kg/day, while a significant effect on QR was seen only at four times this dose. This does not indicate a true difference in sensitivity, however, but rather a difference in the coefficients of variation of GST and QR activities in the liver.
The observed effects of D3T on hepatic GST activities are consistent with a previous report, in which increased activity was recorded in rats fed D3T at a dietary level of 0.001%, equivalent to
4 µmol/kg/day (22). In the latter study, other tissues were not investigated at this low level, but only at a dietary level providing a dose of
110 µmol/kg/day. The recorded increases in GST activities in the kidney, lungs, forestomach and the small intestine (taken as a whole) were similar to those seen in the present investigation at 125 µmol/kg/day. It is interesting to note that dosing methodology appears to have little effect upon the inductive activity of D3T, whether given by gavage, as in the present investigation, or fed in the diet, as in the study by Kensler et al. (22).
Although little comparative data are available, it would appear that the tissue specificity of D3T is different to that of oltipraz. The latter compound is reported to have much less effect on phase II enzyme activities in the small intestine and urinary bladder than in the liver (16,17). These data suggest that results on one dithiolethione cannot be directly extrapolated to another, and further work on dithiolethione derivatives, assaying a larger range of tissues in a single experiment, is required in order provide more information on the tissue specificity of this class of compound.
No significant effects on blood packed cell volumes were recorded in rats receiving D3T at any dose-level (Table V). This result is in accord with experiments with oltipraz, in which no haematological effects were seen at a dose-level of 130 µmol/kg/day (31). At a dose of 260 µmol/kg/day, however, oltipraz caused a decrease in packed cell volume. Haematological studies on D3T at higher dose-levels would be of interest.
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Table V. Blood packed cell volumes and relative weights of spleen, liver, kidneys, heart, lungs and urinary bladder of rats receiving D3T at various dose-levels
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Oltipraz has consistently been shown to cause liver enlargement in rodents (3234), which is associated with hepatocytic hypertrophy (31). Renal enlargement has also been recorded (31). In the present study, D3T similarly caused enlargement of the kidneys at 125 µmol/kg/day and of the liver at 15.6 µmol/kg/day or above (Table V). The higher dose-levels of D3T also caused significant enlargement of the stomach and gastrointestinal tract (Table VI). Liver enlargement is often associated with enzyme induction (35), but this is unlikely to account for the effects of D3T on the intestine, since other phase II inducers, while having a pronounced effect on intestinal enzyme activity, cause no increase in weight (26,27). Intestinal enlargement is an uncommon toxicological change, and histological examination of the gastrointestinal tract of animals receiving D3T is required in order to cast light on its cause and possible significance with regard to the use of D3T in humans.
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Table VI. Relative weights of glandular stomach, forestomach, duodenum, jejunum, ileum, caecum and colon plus rectum of rats receiving D3T at various dose-levels
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D3T appears to be the most effective inducer of phase II enzymes so far described in vivo, showing significant effects at lower dose-levels than other compounds for which dose-response data are available (26,27). Increased enzyme activities were recorded in the stomach and intestine of rats at very low dose-levels of D3T, suggesting that this compound may be particularly effective against carcinogens targeting these tissues. While further study on the toxicology of D3T is required, the potency of this compound in inducing phase II enzymes makes it a most promising candidate for use in chemoprotection.
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Acknowledgments
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This work was funded by the Waikato Medical Research Foundation.
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Received March 2, 2004;
accepted March 31, 2004.