Chronic dosing of oltipraz in people at increased risk for colorectal cancer.

The dithiolethione oltipraz is being developed as a chemopreventive agent for many malignancies, including colorectal cancer, on the basis of its in vivo protective activity against chemically induced tumors in a variety of animal models. This protection has been associated with an enhanced capacity to detoxify reactive carcinogens and, more recently, with increased DNA repair. In a previous single-dose study, elevated detoxification gene expression was observed in the days after oltipraz dosing. Now, in this clinical study, we evaluated the effects of oltipraz when given over a 3-month period. Fourteen individuals with increased risk for colorectal cancer were randomly assigned to one of two oral doses (125 or 250 mg/m2) of oltipraz twice weekly for 12 weeks. Two of seven subjects at the 250 mg/m2 dosage required dose reductions, owing to significant fatigue. The 125 mg/m2 dose level was well tolerated by all patients. Blood or colon tissue (or both) for evaluation of glutathione, glutathione S-transferase, DT-diaphorase activity, and DT-diaphorase mRNA expression were obtained prior to treatment and at weeks 6, 12, and 16. No significant modulation of phase II detoxification enzymes was seen at either dose studied during this period. Phase II trials evaluating a tolerable regimen of oltipraz (as demonstrated in this study) and other possible mechanisms that may be responsible for the protective activity of oltipraz should be pursued.

Depletion of colonic detoxication enzyme activity in mice with dextran sulphate sodium-induced colitis.

BACKGROUND: The increased risk of colonic malignancies in individuals with ulcerative colitis has prompted a search for early biomarkers of disease progression. AIM: To characterize Phase II detoxication enzyme expression during acute and chronic colitis. The mouse model of dextran sulphate sodium (DSS)-induced colitis represents a relevant system with which to sequentially evaluate the spectrum of biochemical changes associated with colorectal cancer risk. METHODS: Acute and chronic colitis were induced in Swiss Webster mice by administering DSS in the drinking water (5%) for 1-4 cycles. Each cycle consisted of 7 days DSS and 14 days of water. The glutathione S-transferase (GST) activity, gamma-glutamylcysteine synthetase (gamma-GCS) activity and glutathione content of the colonic tissues were determined at various time points throughout the experiment. Alterations in GST isozyme expression were confirmed by Western and Northern blot. RESULTS: GST activity was reduced significantly in the colon by the end of Cycle 1 (84% of control values). Specific activities continued to decrease with subsequent cycles of DSS exposure. By the end of Cycle 4, glutathione levels and gamma-GCS activity had reached 29% and 56% of control, respectively. CONCLUSIONS: These data suggest that detoxication enzyme depletion is associated with both acute and chronic colitis and may be an important event in the progression of ulcerative colitis to colon cancer.

Disruption of c-Fos leads to increased expression of NAD(P)H:quinone oxidoreductase1 and glutathione S-transferase.

Regulation of the basal and induced expression of detoxifying enzymes such as NAD(P)H:quinone oxidoreductasel (NQO1) and glutathione S-transferase (GST) by the antioxidant response element (ARE) is important for cellular protection against oxidative stress. The ARE contains AP1 and AP1-like elements and is known to bind to several leucine zipper proteins including c-Fos. Previous studies (Venugopal, R., and Jaiswal, A.K. (1996) Proc. NatL Acad. Sci. USA 93, 14960-14965) have shown that overexpression of c-Fos in transfected cells leads to repression of ARE-mediated gene expression. In the present report, we used c-Fos-/- mice and investigated the physiological (in vivo) role of c-Fos in repression of the NQO1 and GST genes expression. The analysis of enzyme activity levels showed significant increases in NQO1 and GST activities in several tissues of c-Fos-/- mice, as compared with wild type (c-Fos+/+) mice. The increases in enzyme activities were supported by Wetern analysis of respective proteins. Western analyses showed significant increases in the expression of NQO1 in kidney, liver and skin tissues of c-Fos-/- mice, as compared with wild type (c-Fos+/+) controls. Western analyses also demonstrated an increased expression of the GST Ya gene in kidney and liver tissues of the c-Fos-/-mice. These results confirm a negative (repressive) role for c-Fos in the expression of NQO1, GST Ya, and other detoxifying enzyme genes.

Preclinical and clinical evaluation of broccoli supplements as inducers of glutathione S-transferase activity.

Previous studies suggest that cruciferous vegetables may provide protection against carcinogen exposure by inducing detoxification enzymes. ICR(Ha) mice were gavaged with broccoli tablets (1 g/kg), and colon tissues were collected after treatment. Glutathione S-transferase (GST) activity was assayed and peaked on days 1 and 2 after treatment, respectively (P = 0.03). Elevations in GST activity were attributed to the increased expression of mu and pi. These data supported a clinical assessment of broccoli supplements. Twenty-nine subjects at increased risk for colorectal cancer were randomized to group 1 (no cruciferous vegetables) or group 2 (broccoli supplements, 3 g/day) for 14 days. Blood samples and colon biopsies were obtained pre- and postintervention. No significant difference was observed between the GST activities of the control and broccoli supplementation groups posttreatment. Mean lymphocyte GST activity was 107% of baseline in the broccoli supplementation group (range, 79-158%) and 102% of baseline in the control group (range, 75-158 percent;). Correlation of the GST activities of blood lymphocytes and colon mucosa taken simultaneously suggested that the GST activity of blood lymphocytes may be used as a biomarker of the responsiveness of colon tissue to chemopreventive regimens. Future clinical studies evaluating cruciferous vegetables should consider using concentrated dietary supplements in subjects with a previous history of colorectal cancer.

Modulation of gene expression in subjects at risk for colorectal cancer by the chemopreventive dithiolethione oltipraz.

Prolonged exposure to mutagenic substances is strongly associated with an individual's risk of developing colorectal cancer. Clinical investigation of oltipraz as a chemopreventive agent is supported by its induction of the expression of detoxication enzymes in various tissues, and its protective activity against the formation of chemically induced colorectal tumors in animals. The goals of the present study were: to determine if oltipraz could induce detoxicating gene expression in human tissues; to identify effective non-toxic doses for more extensive clinical testing; and to establish a relationship between effects in the colon mucosa and those in a more readily available tissue, the peripheral mononuclear cell. 24 evaluable patients at high risk for colorectal cancer were treated in a dose-finding study with oltipraz 125, 250, 500, or 1,000 mg/m2 as a single oral dose. Biochemical analysis of sequential blood samples and colon mucosal biopsies revealed increases in glutathione transferase activity at the lower dose levels. These effects were not observed at the higher doses. More pronounced changes were observed in detoxicating enzyme gene expression in both tissues at all doses. Peripheral mononuclear cell and colon mRNA content for gamma-glutamylcysteine synthetase (gamma-GCS) and DT-diaphorase increased after dosing to reach a peak on day 2-4 after treatment, and declined to baseline in the subsequent 7-10 d. The extent of induction of gene expression in colon mucosa reached a peak of 5.75-fold for gamma-GCS, and a peak of 4.14-fold for DT-diaphorase at 250 mg/m2 ; higher doses were not more effective. Levels of gamma-GCS and DT-diaphorase correlated closely (P < or = 0.001) between peripheral mononuclear cells and colon mucosa both at baseline and at peak. These findings demonstrate that the administration of minimally toxic agents at low doses may modulate the expression of detoxicating genes in the tissues of individuals at high risk for cancer. Furthermore, peripheral mononuclear cells may be used as a noninvasive surrogate endpoint biomarker for the transcriptional response of normal colon mucosa to drug administration.

Glutathione S-transferase activity and glutathione S-transferase mu expression in subjects with risk for colorectal cancer.

The glutathione S-transferases (alpha, mu, and pi), a family of Phase II detoxication enzymes, play a critical role in protecting the colon mucosa by catalyzing the conjugation of dietary carcinogens with glutathione. We investigated the efficacy of using the glutathione S-transferase (GST) activity of blood lymphocytes and GST-mu expression as biomarkers of risk for colorectal cancer. GST activity was measured in the blood lymphocytes of control individuals (n = 67) and in the blood lymphocytes (n = 60) and colon tissue (n = 34) of individuals at increased risk for colon cancer. Total GST activity was determined spectrophotometrically with the use of 1-chloro-2,4-dinitrobenzene as a substrate. The ability to express the um subclass of GST was determined with the use of an ELISA. Although interindividual variability in the GST activity of blood lymphocytes was greater than 8-fold (range, 16.7-146.8 nmol/min/mg), the GST activity of blood lymphocytes and colon tissue within an individual was constant over time and was unrelated to sex, age, or race. The GST activity of blood lymphocytes from high-risk individuals was significantly lower than that of blood lymphocytes from control individuals (P < or = 0.004). No association was observed between the frequency of GST-mu phenotype and risk for colorectal cancer. Blood lymphocytes from high-risk individuals unable to express GST-mu had lower levels of GST activity than did those from control subjects with the GST-mu null phenotype; however, this difference was significant in male subjects only (P < or = 0.006). Analysis of paired samples of blood lymphocytes and colon tissue indicated a strong correlation between the GST activity of the two tissue types (Spearman's rank correlation, r = 0.87; P < or = 0.0001). The GST activity of blood lymphocytes may be used to identify high-risk individuals with decreased protection from this Phase II detoxication enzyme who may benefit from clinical trials evaluating GST modulators as chemopreventive agents for colorectal cancer. The GST activity of blood lymphocytes may also be used in colorectal cancer chemoprevention trials to monitor the responsiveness of colon tissue to regimens that modify Phase II detoxication enzymes.