Nitrofurantoin cytotoxicity. In vitro assessment of risk based on glutathione metabolism.

Nitrofurantoin, a commonly used urinary tract antiseptic, has been associated with idiosyncratic pulmonary and hepatic damage. We have used human lymphocytes in vitro to explore the biochemical basis of susceptibility to nitrofurantoin toxicity. The drug itself did not damage the cells as assessed by trypan blue dye exclusion. In the presence of a mouse hepatic microsomal drug-activating system, however, nitrofurantoin metabolites produced dose dependent toxicity to the lymphocytes. Inhibition of the enzyme epoxide hydrolase did not enhance toxicity; the metabolite thus does not appear to be a furan epoxide. Binding of reactive metabolites to cell macromolecules may lead directly to cell death, or in vivo, by acting as haptens to secondary immunologic responses. The metabolite caused a dose-dependent depletion of lymphocyte glutathione content. Cells from a patient with glutathione synthetase deficiency showed markedly enhanced nitrofurantoin toxicity. The findings suggest that glutathione plays a major role in protecting cells from nitrofurantoin-induced damage, and that studies of lymphocyte toxicity and glutathione metabolism in patients experiencing idiosyncratic reactions to nitrofurantoin may lead to elucidation of the biochemical and genetic basis of drug susceptibility.

Reduction of atherosclerosis by administration of dehydroepiandrosterone. A study in the hypercholesterolemic New Zealand white rabbit with aortic intimal injury.

Dehydroepiandrosterone (DHEA) is an endogenous steroid that blocks carcinogenesis, retards aging, and exerts antiproliferative properties. In vitro, it is a potent inhibitor of glucose-6-phosphate dehydrogenase, the first committed step of the pentose phosphate pathway. In man, serum levels of DHEA and its sulfate peak in early adulthood and drop markedly with age. Epidemiologic evidence indicates that low levels of DHEA or its sulfate conjugate are linked to an increased risk of developing cancer or of death from cardiovascular disease. Like cancer, atherosclerosis is a proliferative process characterized by both initiation and promotion phases. This similarity provided a framework in which to study the antiatherogenic effects of DHEA. Rabbits were randomly assigned to four groups. Two groups of rabbits received aortic endothelial injury by balloon catheter and were fed a 2% cholesterol diet for 12 wk. DHEA, 0.5%, was incorporated into the diet of one group receiving the 2% cholesterol diet and endothelial injury and also into the diet of one of the control groups. Animals were killed after 12 wk and aortas, hearts, and livers were studied. Plasma samples were analyzed for total cholesterol, VLDL, LDL, HDL, triglycerides, DHEA, and DHEA-sulfate levels. The atherogenic insult resulted in severe atherosclerosis in animals not treated with DHEA. In those receiving DHEA there was an almost 50% reduction in plaque size (P = 0.006), inversely related to the serum level of DHEA attained. Fatty infiltration of the heart and liver were also markedly reduced. These beneficial actions were not attributable to differences in body weight gain, food intake, total plasma cholesterol or distribution of cholesterol among the VLDL, LDL, or HDL fractions. The results show that high levels of plasma DHEA inhibit the development of atherosclerosis and they provide an important experimental link to the epidemiologic studies correlating low DHEA-sulfate plasma levels with an enhanced risk of cardiovascular mortality.

High-dose chemotherapy with reinfusion of purged autologous bone marrow following dose-intense induction as initial therapy for metastatic breast cancer.

We assessed the toxicity and efficacy of high-dose chemotherapy consolidation with reinfusion of purged autologous bone marrow in women with metastatic breast cancer responding to a dose-intense outpatient regimen. Thirty women with hormone-unresponsive metastatic breast cancer, previously untreated with adjuvant doxorubicin or with any chemotherapy for metastatic disease, were treated with cyclophosphamide, methotrexate, doxorubicin, fluorouracil, vincristine, and leucovorin for 16 weeks. Twenty-four patients responded to therapy; 8 showed a complete response, and 16 showed a partial response. These patients proceeded to the next phase of the protocol, ie, marrow harvest and treatment with 6000 mg/m2 cyclophosphamide and 800 mg/m2 thiotepa given over 4 days. Harvested marrow was purged with 100 micrograms/mL 4-hydroperoxycyclophosphamide, and all patients engrafted satisfactorily. The predominant side effects were myelosuppressive and gastrointestinal, and there were no deaths from toxic effects. Three of the 16 patients who showed a partial response after the outpatient phase of treatment achieved a complete response after high-dose therapy. The partial response seen in two more patients converted to a complete response at all sites except bone. The median time to disease progression for all patients in this study was 13 months, and the median survival was 22 months. Four of the original 30 patients remained without disease progression a median of 27 months from entry into the study. This study indicates that this dose-intense regimen can be safely administered, even with the use of purged marrow, with an acceptable toxicity profile. This approach results in a high response rate in women with metastatic breast cancer and could form the basis for a regimen to be tested in the high-risk adjuvant setting.

Serum levels of dehydroepiandrosterone and its sulfate and the risk of developing bladder cancer.

Dehydroepiandrosterone and dehydroepiandrosterone sulfate are endogenous steroids largely produced in the adrenal cortex and excreted in the urine. Many studies have demonstrated that administration of dehydroepiandrosterone to animals protects against a variety of chemical carcinogens. Epidemiological studies suggest that the circulating levels of these steroids in humans are related to the risk of developing some cancers and of dying from atherosclerotic cardiovascular disease. We measured serum levels of both of these steroids in 35 individuals who donated serum to a community-based serum bank in 1974 and who subsequently developed bladder cancer and in 69 matched controls from the same cohort of volunteers. Prediagnostic serum levels of dehydroepiandrosterone and dehydroepiandrosterone sulfate were significantly lower among cases compared with controls. The risk of developing bladder cancer increased monotonically with decreasing serum levels of both steroids. The observed associations were not affected by adjustment for smoking or the time interval between serum collection and diagnosis. These results support a role for dehydroepiandrosterone and/or dehydroepiandrosterone sulfate in the prevention of bladder cancer.

Evaluation of dietary dehydroepiandrosterone for chemoprotection against tumorigenesis in premalignant colonic epithelium of male F344 rats.

Epidemiological and experimental studies suggest that dehydroepiandrosterone (DHEA), an adrenal cortical steroid, has chemoprotective properties. Rat colonic epithelium which had been induced to a premalignant state by the colonic carcinogen azoxymethane was used as a model for patients at high risk of colorectal carcinoma, and the efficacy of dietary DHEA for chemoprotection against tumorigenesis was evaluated. Ten-week-old male F344 rats (n = 100) were given 10 weekly s.c. injections of azoxymethane at a dose of 10 mg/kg/week. One day after the final dose of carcinogen, DHEA was added to the diet of 50 rats (0.5% DHEA chow), and the other rats were used as pair-fed controls. DHEA-fed rats lost body weight throughout the 17-week study, in contrast to their pair-fed controls. Serum DHEA in DHEA-fed rats at the end of the study was 6 times that of controls (120 +/- 30 versus 18 +/- 14 pmol/ml), and serum DHEA sulfate was 23 times that of controls (1311 +/- 13 versus 55 +/- 13 pmol/ml). Addition of DHEA to the diet produced no significant chemoprotection in our model. Tumor-related mortality was somewhat increased in DHEA-fed rats (20% versus 6% in week 16 of DHEA feeding, P not significant). The cumulative prevalence of left colonic tumors, identified by weekly colonoscopic examinations, was somewhat lower in DHEA-fed rats than in controls during weeks 10 through 13 (17% versus 33% in week 12, P not significant), but in week 14 the prevalence in DHEA-fed rats became similar to that in controls (39% versus 41%). Growth curves of autochthonous left colonic tumors, as assessed for 8 weeks by computerized image analysis of colonoscopic photographs, were similar for DHEA-fed and control rats. Prevalence, mean frequency, multiplicity, and diameter of colonic tumors at necropsy of colonoscopically negative rats in week 17 were somewhat lower in the DHEA-fed rats (e.g., prevalence of 47% versus 67%), but the differences from controls were not significant. Parameters of colonic epithelial proliferation after tritiated thymidine incorporation in DHEA-fed rats were similar to those in control rats (labeling index of 8.3 +/- 0.7% versus 8.4 +/- 0.6% in week 17), despite higher serum DHEA and DHEA sulfate levels. Our findings indicate that DHEA did not have significant postinduction chemoprotective activity against azoxymethane-induced colonic tumorigenesis in this model utilizing pair-fed controls. Further preclinical studies appear to be needed before dietary DHEA can be recommended for chemoprotection trials in patients with premalignant colorectal epithelium.

Epidermal ribosome accumulation during two-stage skin tumorigenesis.

The increase in interfollicular epidermal ribosomes on the backs of mice initiated with 7,12-dimethylbenz(a)-anthracene and promoted with 12-0-tetradecanoyl-phorbol-13-acetate was disproportionate to the increase in epidermal wet weight, protein, and DNA. Whereas ribosome numbers increased five- to sixfold 48 hr after the first, fourth, or eight application of 12-3-tetradecanoyl-phorbol-13-acetate, epidermal tissue increased only two- to threefold at these times. This disproportionate increase was due to the fact that, concurrent with the increased amount of interfollicular epidermal tissue and cells, ribosomes per g epidermis and per mg DNA increased two to three times normal. The tissue concentration and cellular content of ribosomes were also increased in the epidermal component of induced squamous papillomas. The work of others has demonstrated that, during growth of other tissues and organs, ribosome accumulation is proportionate to accumulation of tissue and/or cells. The results of our study indicate that the epidermis may have unique kinetics of ribosome accumulation during induced growth. Furthermore, these findings suggest the interesting possibility that other tumor-prone surface epithelia, such as the linings of the respiratory and gastrointestinal tracts, have similar kinetics of ribosome accumulation during induced growth.

Inhibition of the conversion of 3T3 fibroblast clones to adipocytes by dehydroepiandrosterone and related anticarcinogenic steroids.

Dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one; DHEA) and related steroids have widespread protective effects against spontaneous and chemically induced tumors, suppress weight gain without affecting food intake, and depress lipogenesis. We have observed that DHEA and 16 alpha-bromoepiandrosterone (16 alpha-bromo-3 beta-hydroxy-5 alpha-androstan-17-one) block the conversion to adipocytes of the 3T3-L1 and 3T3-F442A mouse embryo fibroblast clones. The arrest of lipogenic conversion was assessed by measurements of lipid biosynthesis and the specific activity of cytosolic glycerol-3-phosphate dehydrogenase. In the presence of 215 microM DHEA or 30 microM 16 alpha-bromoepiandrosterone, the increase in glycerol-3-phosphate activity was only 50% of that of fully differentiated control cells. The blocking effects were concentration dependent and were observed only if the differentiation stimuli and the blocking steroid were present simultaneously. Concentrations of these steroids that almost completely blocked conversion to adipocytes were not cytotoxic. Although the relation between structure and blocking activity of steroids is complicated by metabolism of DHEA in these cultures, a strong correlation exists between the structural requirements for blocking differentiation and for inhibition of glucose-6-phosphate dehydrogenase. The 3T3-L1 and 3T3-F442A preadipocyte clones are, therefore, appropriate and convenient model systems for the analysis of the mechanism of the anticarcinogenic effects of DHEA and related steroids.

Relationship of prediagnostic serum levels of dehydroepiandrosterone and dehydroepiandrosterone sulfate to the risk of developing premenopausal breast cancer.

Dehydroepiandrosterone and dehydroepiandrosterone sulfate are steroids which may be associated with the development of breast cancer. To examine the association between serum levels of dehydroepiandrosterone and dehydroepiandrosterone sulfate and the risk of developing premenopausal breast cancer, we measured hormone levels in 15 women who donated blood to a community-based serum bank in 1974 and who subsequently developed premenopausal breast cancer and in 29 matched controls from the same group of volunteers. The mean serum level of dehydroepiandrosterone among cases was 10% lower than among controls. The risk of developing breast cancer for women in the highest tertile compared with the lowest tertile of serum dehydroepiandrosterone was 0.40 with a suggestion of a dose-response trend with increasing levels. No consistent association between dehydroepiandrosterone sulfate and the risk of premenopausal breast cancer was evident. In contrast to postmenopausal breast cancer, a protective effect of dehydroepiandrosterone against premenopausal breast cancer is suggested, but because of the small sample size, the results of this study need to be replicated by others.

Relationship of serum levels of dehydroepiandrosterone and dehydroepiandrosterone sulfate to the risk of developing postmenopausal breast cancer.

Dehydroepiandrosterone and dehydroepiandrosterone sulfate are endogenous steroids that are produced in the adrenal cortex. A number of studies have suggested that circulating levels of these hormones are related in some way to the risk of developing breast cancer. We measured serum levels of these steroids in 30 postmenopausal women who donated blood in 1974 for a community-based serum bank and who subsequently, at least 9 years later, developed breast cancer and in 59 matched controls from the same group of volunteers. Significantly elevated serum levels of dehydroepiandrosterone were found among cases prior to diagnosis compared to controls; serum levels of dehydroepiandrosterone sulfate were slightly increased among cases. In controls, current cigarette use was associated with increased serum levels of these steroids, and levels of both steroids decreased with age.

Hepsulfam sensitivity in human breast cancer cell lines: the role of glutathione and glutathione S-transferase in resistance.

Hepsulfam (NSC 329680, 1,7-heptanediol disulfamate) is an alkylating agent that showed excellent activity against mouse and human mammary carcinoma in preclinical studies. We therefore studied the cytotoxicity of this drug in six human breast cancer cell lines (AdrRMCF7, WTMCF7, Hs578T, MDA-MB-231, T47D, and MDA-MB-468). Clonogenic assays of these cell lines showed a range of sensitivity with the 90% inhibitory concentration ranging from 3.1 microM hepsulfam (MDA-MB-468) to 32.3 microM hepsulfam (AdrRMCF7) after 24-h exposure to the drug. To evaluate possible mechanisms responsible for this observed variation in sensitivity to hepsulfam, we have studied glutathione S-transferase (GST) activity and glutathione (GSH) in these cell lines. Total cytoplasmic GST activity correlated with sensitivity; the most sensitive cell lines had the lowest GST activity, while the two most resistant cell lines, AdrRMCF7 and Hs578T, had the highest GST levels of the six cell lines. Western blot analysis showed that the only detectable isoenzyme was GST-pi. The amount of GST-pi isoform correlated with hepsulfam sensitivity in the three most resistant cell lines and was undetectable in the three most sensitive cell lines. Cellular concentrations of GSH did not correlate with hepsulfam sensitivity. However, GSH depletion with buthionine sulfoximine increased sensitivity to hepsulfam in a dose-dependent fashion in all six cell lines. Evaluation by mass spectrometry revealed that glutathione can form conjugates with hepsulfam. We conclude that the GST/GSH detoxication system plays a role in the sensitivity of these breast cancer cell lines to hepsulfam.

Modulation of methylnitrosourea-induced breast cancer in Sprague Dawley rats by dehydroepiandrosterone: dose-dependent inhibition, effects of limited exposure, effects on peroxisomal enzymes, and lack of effects on levels of Ha-Ras mutations.

Dehydroepiandrosterone (DHEA), the major steroid precursor of androgens and estrogens produced in peripheral tissues in primates, is an effective chemopreventive agent in the N-methyl-N-nitrosourea (MNU)-induced rat mammary tumor model. Dietary DHEA (5-600 ppm; 600 mg/kg diet) was administered beginning 1 week before MNU and administered continually throughout the duration of the experiment. The highest dose of DHEA (600 ppm) significantly decreased tumor incidence from 95 to 45% and increased tumor latency and decreased tumor multiplicity from 4.1 to 0.5 tumors/rat. Lower doses of DHEA (5, 24, and 120 ppm) were also effective, decreasing tumor multiplicity by 28, 40, and 55%, respectively, increasing tumor latency in a dose-dependent manner but only minimally affecting final tumor incidence. DHEA in the diet caused a dose-dependent increase in serum levels of DHEA. The 120-ppm dietary dose of DHEA resulted in serum levels of DHEA of approximately 42 pmol/ml levels, similar to those seen in young humans. When we examined whole mounts of mammary glands derived from rats exposed to higher levels of DHEA (600 ppm), we observed a striking increase in lobular development. The doses of DHEA used in these studies (< or =600 ppm) had minimal effects on the induction of fatty acid CoA synthetase, a peroxisome-associated enzyme. In contrast, a dose of 2000 ppm substantially increased levels of peroxisome-associated fatty acid CoA synthetase. The varied and striking efficacy of DHEA was achieved in the absence of any significant effect on body weight gain in the treated rats. Furthermore, tumors from rats treated with MNU alone or rats treated with MNU plus DHEA were examined for the presence of mutations in the Ha-Ras oncogene. There was a slight decrease in the percentage of tumors bearing Ha-Ras mutations in tumors derived from MNU-control rats as contrasted with tumors from MNU-DHEA (120 and 600 ppm)-treated rats. Based on the striking chemopreventive efficacy of continual exposure to DHEA, we examined the effects of more limited exposure to DHEA. Rats were treated with DHEA for a period of 7 weeks immediately before and after MNU injection. Rats were then placed on the control diet for the ensuing 15 weeks. Even this limited exposure to DHEA for a period of 7 weeks profoundly decreased final tumor incidence and multiplicity. Additionally, we examined the effects of intermittent dosing with DHEA. Rats were treated alternatively at 3-week intervals either with diet containing DHEA or with control diet. It was found that this intermittent dosing with DHEA also substantially inhibited the formation of mammary tumors.

Plasma dehydroepiandrosterone and dehydroepiandrosterone sulfate in patients undergoing diagnostic coronary angiography.

Serum levels of DHEA sulfate are inversely associated with cardiovascular death in men, and urinary dehydroepiandrosterone (DHEA) levels are inversely associated with clinical manifestations of coronary artery disease. These observations may be related to the antiproliferative effects of DHEA, resulting in inhibition of atherosclerotic intimal hyperplasia. To examine the relation between these steroids and a direct measure of coronary atherosclerosis, plasma DHEA and DHEA sulfate levels were determined in 206 middle-aged patients (103 men, 103 women) undergoing elective coronary angiography. Plasma DHEA sulfate levels were lower in men with at least one stenosis greater than or equal to 50% compared with those without any stenosis greater than or equal to 50% (4.9 +/- 2.7 versus 6.1 +/- 3.5 nmol/ml, p = 0.05). Levels of DHEA sulfate were also inversely related to the number of diseased coronary vessels (r = -0.20, p = 0.05) and a continuous measure of the extent of coronary atherosclerosis (r = -0.25, p = 0.01) in men. The association between DHEA sulfate levels and extent of coronary artery disease was independent of age and other conventional risk factors for coronary disease. In women, there was no association between plasma DHEA or DHEA sulfate levels and coronary disease. These data demonstrate a consistent, independent, inverse, dose-response relation between plasma DHEA sulfate levels and angiographically defined coronary atherosclerosis in men. Plasma DHEA sulfate may be another important and potentially modifiable risk factor for the development and progression of coronary atherosclerosis.

Glutathione synthetase-deficient lymphocytes and acetaminophen toxicity.

Toxic electrophilic metabolites of acetaminophen are detoxified by conjugation with glutathione. Cellular glutathione content of patients with glutathione synthetase deficiency (5-oxoprolinuria) is 10% to 20% of normal. These patients might be at increased risk for acetaminophen toxicity. The hypothesis was tested by challenging lymphocytes from normals and a patient with glutathione synthetase deficiency in vitro with acetaminophen metabolites generated by a mouse hepatic microsomal drug-metabolizing system. For toxicity to be manifested in normal cells, glutathione content had to be depleted to less than 20% of control values at high acetaminophen concentrations (500 and 1,500 micrograms/ml), concentrations similar to blood levels in massive overdose and associated with hepatotoxicity in vivo. The patient's cells had only 14% of normal glutathione content, and exhibited more toxicity at 12.5 micrograms/ml acetaminophen (within the therapeutic range) as normals at maximum concentrations. The in vitro system may be of value in screening drugs potentially hazardous for glutathione synthetase-deficient patients, for exploring the role of glutathione in the detoxification of xenobiotics, and for examining glutathione protective mechanisms in patients with idiosyncratic cytotoxic drug reactions.

The relationship of serum dehydroepiandrosterone and its sulfate to subsequent cancer of the prostate.

Levels of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S) in sera collected and frozen in 1974 were studied among 81 prostate cancer cases diagnosed in the following 12 years and 81 age- and race-matched controls. Although mean levels of DHEA were 11% lower among cases than controls and DHEA-S levels were 12% lower than among controls, no dose-response association was noted for either DHEA or DHEA-S. It seems unlikely that serum levels of DHEA or DHEA-S are important risk factors for prostate cancer.

Serum dehydroepiandrosterone and dehydroepiandrosterone sulfate and the subsequent risk of developing colon cancer.

This purpose of this study was to evaluate whether serum dehydroepiandrosterone (DHEA) and its sulfate conjugate, dehydroepiandrosterone sulfate (DHEAS), are associated with the likelihood of developing colon cancer. A nested case-control study was conducted using the serum bank and cancer registry in Washington County, Maryland. From a population of 20,305 county residents who donated blood in 1974, incident cases of colon cancer that occurred from 1975 to 1991 (n = 117) were matched to one cancer-free control by age, race, and sex. Serum specimens that were stored at -70 degrees C since 1974 were assayed for DHEA and DHEAS. Compared with the controls, the mean serum concentrations of cases were 3% lower for DHEA (P = 0.90) and 13% lower for DHEAS (P = 0.60). When DHEA levels were analyzed according to fourths, no noteworthy associations were observed. Compared with the lowest fourth, the highest fourth of serum DHEAS was nonsignificantly associated with a halving in the risk of colon cancer (odds ratio, 0.50; 95% confidence limits, 0.18, 1.37; Ptrend = 0.22), and further analyses showed the potential protective association was confined largely to males (highest-versus-lowest fourth odds ratio, 0.26; 95% confidence limits, 0.06, 1.16; Ptrend = 0.06). This prospective study does not provide strong evidence that circulating DHEA and DHEAS concentrations are associated with the risk of colon cancer. Among men, DHEAS was associated with a decreased risk of colon cancer, but the association was within the bounds of chance. Further studies are needed to either support or refute the potentially promising lead hinted at by the results for DHEAS.

Serum levels of dehydroepiandrosterone and dehydroepiandrosterone sulfate and the risk of developing gastric cancer.

Although the incidence of gastric cancer varies widely between countries it is nonetheless a leading cause of cancer deaths worldwide. Migration studies indicate that dietary choices are an important exogenous factor. The United States has a very low incidence of gastric cancer, suggesting that exogenous etiological agents are at a minimum and providing a favorable setting for detecting important endogenous etiological factors. Dehydroepiandrosterone and dehydroepiandrosterone sulfate are endogenous steroids produced in the adrenal gland. Epidemiological studies show that the risk of developing specific cancers is related to the serum or urinary levels of these steroids. In addition, dehydroepiandrosterone prevents a variety of spontaneous and chemically induced tumors when administered to laboratory animals. To examine the association between circulating levels of dehydroepiandrosterone and dehydroepiandrosterone sulfate and the development of gastric cancer, we measured the serum levels of these steroids in 13 individuals who donated serum to the Washington County Maryland serum bank in 1974 and who subsequently developed gastric cancer and in 52 matched controls. Prediagnostic serum levels of dehydroepiandrosterone were 38% lower in cases as compared to controls (P = 0.09). The risk of developing gastric cancer increased with decreasing levels of both steroids. Adjustment for confounding factors such as smoking or the interval between blood donation and time to diagnosis did not alter the findings. These results suggest that there may be a role for this steroid in the prevention of gastric cancer.

Oltipraz chemoprevention trial in Qidong, People's Republic of China: study design and clinical outcomes.

In 1995, 234 adults from Qidong, Jiangsu Province, People's Republic of China, where hepatocellular carcinoma is the leading cause of cancer deaths and exposure to dietary aflatoxins is widespread, were enrolled and followed in a Phase II chemoprevention trial. The goals of the study were to define a dose and schedule of oltipraz for reducing levels of validated aflatoxin biomarkers and to characterize dose-limiting toxicities. Healthy eligible individuals, including those infected with hepatitis B virus, were randomized to receive either 125 mg of oltipraz daily, 500 mg of oltipraz weekly, or placebo. Blood and urine specimens were collected to monitor toxicities and evaluate biomarkers over the 8-week intervention period and subsequent 8-week follow-up period. Unique trial aspects included a synchronous follow-up schedule, daily observed administration of all medications, timely international data transference, and use of biomarkers as outcomes. One hundred thirty-two participants took their medications without interruptions, approximately 77% contributed all nine urine samples, and 78% contributed all seven blood samples. Fifty-one participants (21.8%) reported clinical adverse events. An extremity syndrome, developing soon after the start of treatment, was the only event that occurred more frequently (P = 0.002) among the active groups (18.4 and 14.1% of the daily 125 and weekly 500 mg arms, respectively) compared with placebo (2.5%). The oltipraz arms did not differ in symptom type or severity, and there were no indications of exacerbated drug intolerance among the few participants infected with hepatitis B virus. The good compliance with an intense follow-up schedule shows that chemoprevention trials with biomarker end points may be conducted in such populations.

Oltipraz chemoprevention trial in Qidong, People's Republic of China: modulation of serum aflatoxin albumin adduct biomarkers.

In 1995, 234 adults from Qidong, People's Republic of China, were enrolled and followed in a Phase IIa 4-methyl-5-(N-2-pyrazinyl)-1,2-dithiole-3-thione (oltipraz) chemoprevention trial. Residents of this area are at high risk for development of hepatocellular carcinoma, in part due to consumption of aflatoxin-contaminated foods. The intervention was a randomized, placebo-controlled, double-blind study. Elements of the study design and clinical outcomes have been recently published (Jacobson et al, Cancer Epidemiol. Biomark. Prev., 6: 257-265, 1997). The primary objective was to conduct a preliminary assessment of the ability of oltipraz to modulate levels of a validated biomarker of aflatoxin exposure and of the risk of hepatocellular carcinoma by determining levels of aflatoxin-albumin adducts in sera. Healthy eligible individuals were randomized into three arms to receive p.o. 125 mg of oltipraz daily, 500 mg of oltipraz weekly, or placebo for 8 weeks. There were no consistent changes in biomarker levels in the placebo arm over the 16-week observation period, nor was any apparent effect observed in the arm receiving 125 mg of oltipraz each day. However, individuals receiving 500 mg of oltipraz once a week for 8 weeks showed a triphasic response to oltipraz. No effect was observed during the 1st month of the intervention, whereas a significant (P = 0.001) diminution in adduct levels was observed during the 2nd month of active intervention and during the lst month of follow-up. A partial rebound in adduct levels toward baseline values was observed during the 2nd month postintervention. Linear regression models up to week 13 confirmed a significant (P = 0.008) weekly decline of biomarker levels in the group receiving 500 mg of oltipraz once a week. However, despite these effects relative to baseline values within the 500-mg weekly arm, there were no statistically significant differences in biomarker trajectories between treatment arms. The genotype for glutathione S-transferase M1, an oltipraz-inducible isoform involved in the detoxification of aflatoxin B1, did not appear to affect either baseline levels or rates of decline in the biomarker. A follow-up Phase IIb trial with a longer intervention period will be necessary to determine the full extent to which aflatoxin biomarker burden can be reduced and whether diminution of biomarkers can be sustained over the long term.

Megestrol acetate-induced differentiation of 3T3-L1 adipocytes in vitro.

We recently observed significant weight gain in patients with advanced breast cancer treated with high-dose megestrol acetate. However, the mechanisms of this effect are completely unknown. As the analysis of the action of steroids in vivo is complex, we chose to examine the effects of megestrol acetate on the differentiation of a preadipocyte clone (L1) of the Swiss 3T3 mouse fibroblast cell line. Addition of insulin, fetal bovine serum, dexamethasone, and methyl-isobutylxanthine to confluent 3T3-L1 cells induced adipocyte differentiation, as verified by morphologic studies and analysis of the activity of the enzyme glycerol-3-phosphate dehydrogenase (G-3-PD), a specific and sensitive indicator of lipocyte function. Substitution of megestrol acetate for dexamethasone also resulted in greatly increased lipocyte differentiation. Tumor necrosis factor (TNF) alpha blocked the adipocyte differentiation induced by the combination of insulin, dexamethasone, and methyl-isobutylxanthine. Addition of megestrol acetate failed to reverse the inhibitory effect of TNF. Thus, megestrol acetate, in vitro, is a potent inducer of lipocyte differentiation. Further studies on the regulation of adipocyte differentiation and function in this model system may be important to clarify megestrol acetate's mechanism of action.

Aspects of in vitro placental perfusion: effects of hyperoxia and phenol red.

The study of a number of parameters of placental function indicated that the perfused human placental lobe maintained its structural and functional integrity when PO2 levels in buffer perfusate were near physiological values, despite low O2 consumption. High O2 content in the perfusate may reduce placental transfer either through a direct vasoconstrictor effect or in combination with the destruction of vascular cyclo-oxygenase, resulting in the reduced synthesis of the vasodilator prostacyclin. A similar mechanism may be involved in the reduction of placental transfer observed in the presence of phenol red. These studies suggest that aspects of in vitro methodologies which may relate to prostaglandin production deserve careful consideration and further study.