Chemoprotection by organosulfur inducers of phase 2 enzymes: dithiolethiones and dithiins.

One of the major mechanisms of protection against carcinogenesis, mutagenesis, and other forms of toxicity mediated by carcinogens is the induction of enzymes involved in their metabolism, particularly phase 2 enzymes such as glutathione S-transferases, UDP-glucuronosyl transferases, and quinone reductases. Animal studies indicate that induction of phase 2 enzymes is a sufficient condition for obtaining chemoprevention and can be achieved by administering any of a diverse array of naturally-occurring and synthetic chemopreventive agents. Alliaceous and cruciferous plants are rich in organosulfur compounds with inducer activity. Indeed, monitoring of enzyme induction has led to the recognition or isolation of novel, potent chemopreventive agents such as 1,2-dithiole-3-thiones, dithiins and the isothiocyanate sulforaphane. For example, oltipraz, a substituted 1,2-dithiole-3-thione originally developed as an antischistosomal agent, possesses chemopreventive activity against different classes of carcinogens targeting multiple organs. Mechanistic studies in rodent models for chemoprevention of aflatoxin B1 (AFB1)-induced hepatocarcinogenesis by oltipraz indicates that increased expression of phase 2 genes is of central importance, although inhibition of phase 1 activation of aflatoxin B1 can also contribute to protection. Exposure of rodents to 1,2-dithiole-3-thiones triggers nuclear accumulation of the transcription factor Nrf2 and its enhanced binding to the Antioxidant Response Element, leading to transcriptional activation of a score of genes involved in carcinogen detoxification and attenuation of oxidative stress. Nrf2-deficient mice fail to induce many of these genes in response to oltipraz and the impact of this genotype on the chemopreventive efficacy of dithiolethiones is currently under investigation. To test the hypothesis that enzyme induction is a useful strategy for chemoprevention in humans, three key elements are necessary: a candidate agent, an at-risk population and modulatable intermediate endpoints. Towards this end, a placebo-controlled, double blind clinical trial of oltipraz was conducted in residents of Qidong, P.R. China who are exposed to dietary aflatoxins and who are at high risk for the development of liver cancer. Oltipraz significantly enhanced excretion of a phase 2 product, aflatoxin-mercapturic acid, a derivative of the aflatoxin-glutathione conjugate, in the urine of study participants administered 125 mg oltipraz by mouth daily. Administration of 500 mg oltipraz once a week led to a significant reduction in the excretion of the primary oxidative metabolite of AFB1, aflatoxin M1, when measured shortly after drug administration. While this study highlighted the general feasibility of inducing phase 2 enzymes in humans, a longer term intervention is addressing whether protective alterations in aflatoxin metabolism can be sustained for extended periods of time in this high-risk population. Food-based approaches to chemoprotection, targeted both to the general population and high-risk individuals, offer many practical advantages compared to the use of pharmaceutical agents. Thus, identification and utilization of naturally-occurring organosulfur chemoprotectors including dithiins should be a high priority.

Enhanced cytotoxicity of mitomycin C in human tumour cells with inducers of DT-diaphorase.

DT-diaphorase is a two-electron reducing enzyme that activates the bioreductive anti-tumour agent, mitomycin C (MMC). Cell lines having elevated levels of DT-diaphorase are generally more sensitive to MMC. We have shown that DT-diaphorase can be induced in human tumour cells by a number of compounds, including 1,2-dithiole-3-thione. In this study, we investigated whether induction of DT-diaphorase could enhance the cytotoxic activity of MMC in six human tumour cell lines representing four tumour types. DT-diaphorase was induced by many dietary inducers, including propyl gallate, dimethyl maleate, dimethyl fumarate and sulforaphane. The cytotoxicity of MMC was significantly increased in four tumour lines with the increase ranging from 1.4- to threefold. In contrast, MMC activity was not increased in SK-MEL-28 human melanoma cells and AGS human gastric cancer cells, cell lines that have high base levels of DT-diaphorase activity. Toxicity to normal human marrow cells was increased by 50% when MMC was combined with 1,2-dithiole-3-thione, but this increase was small in comparison with the threefold increase in cytotoxicity to tumour cells. This study demonstrates that induction of DT-diaphorase can increase the cytotoxic activity of MMC in human tumour cell lines, and suggests that it may be possible to use non-toxic inducers of DT-diaphorase to enhance the efficacy of bioreductive anti-tumour agents.

Identification of dithiolethiones with better chemopreventive properties than oltipraz.

Oltipraz and related dithiolethiones are an important class of chemopreventive agents. Studies were undertaken to identify cancer chemopreventive dithiolethiones more active than oltipraz. Largely based upon enzyme induction activities in vitro, 17 dithiolethiones, including oltipraz, were analyzed for their ability to induce hepatic phase II enzyme activities in vivo. Of these compounds, 15 produced greater induction of NAD(P)H:quinone reductase and 11 yielded greater induction of glutathione S-transferase than oltipraz. All 17 dithiolethiones were then tested for their ability to inhibit acute hepatotoxicity by aflatoxin B1 (AFB1), which previously has been shown to be an intermediate predictor of chemopreventive activity. Rats were pretreated with dithiolethiones (0.3 mmol/kg body wt, three times a week per os) and challenged with two acutely toxic doses of AFB1 (0.5 mg/kg body wt, once daily for two successive days per os). Inhibition of hepatotoxicity was measured by changes in body weight gain during AFB1 challenge, reduction in levels of hepatic enzymes in serum and diminution of bile duct cell proliferation. Nine dithiolethiones spanning a range of responses in this toxicity screen were further tested for their ability to prevent AFB1-induced tumorigenicity, as assessed by a reduction in hepatic burden of putative preneoplastic foci. Six dithiolethiones were found to be considerably more effective than oltipraz in preventing AFB1-induced tumorigenesis. In general, dithiolethiones that were very effective in inhibition of acute hepatotoxicity were also found to be effective in prevention of hepatic tumorigenesis.

Induction of DT-diaphorase by 1,2-dithiole-3-thiones in human tumour and normal cells and effect on anti-tumour activity of bioreductive agents.

DT-diaphorase is a two-electron-reducing enzyme that is an important activator of bioreductive anti-tumour agents, such as mitomycin C (MMC) and EO9, and is inducible by many compounds, including 1,2-dithiole-3-thiones (D3Ts). We showed previously that D3T selectively increased DT-diaphorase activity in mouse lymphoma cells compared with normal mouse marrow cells, and also increased MMC or EO9 cytotoxic activity in the lymphoma cells with only minor effects in the marrow cells. In this study, we found that D3T significantly increased DT-diaphorase activity in 28 of 38 human tumour cell lines representing ten tissue types with no obvious relationships between the tumour type, or the base level of DT-diaphorase activity, and the ability of D3T to increase the enzyme activity. Induction of DT-diaphorase activity in human tumour cell lines by 12 D3T analogues varied markedly with the D3T structure. D3T also increased DT-diaphorase activity in normal human bone marrow and kidney cells but the increases were small in these cells. In addition, D3T increased the level of enzyme activity in normal human lung cells. Pretreatment of human tumour cells with D3T analogues significantly increased the cytotoxic activity of MMC or EO9 in these cells, and the level of enhancement of anti-tumour activity paralleled the level of DT-diaphorase induction. In contrast, D3T did not effect the toxicity of EO9 in normal kidney cells. These results demonstrate that D3T analogues can increase DT-diaphorase activity in a wide variety of human tumour cells and that this effect can enhance the anti-tumour activity of the bioreductive agents MMC and EO9.

Induction of DT-diaphorase in cancer chemoprevention and chemotherapy.

DT-diaphorase (EC 1.6.99.2) is a flavoprotein that catalyses two-electron reduction of quinones, quinone imines, and nitrogen oxides. It is a Phase II detoxifying enzyme that can detoxify chemically reactive metabolites, and may be important in an early cellular defense against tumorigenesis. DT-diaphorase is also an activating enzyme for bioreductive antitumor agents like mitomycin C (MMC) and EO9. DT-diaphorase is induced in many tissues by a wide variety of compounds including dithiolethiones and isothiocyanates. Dithiolethiones are chemoprotective agents against a variety of chemical carcinogens in animal models, and the dithiolethione analogue, oltipraz, is currently in Phase I and Phase II clinical chemoprevention trials. Similarly, the isothiocyanate derivative, sulforaphane, blocks the formation of carcinogen-induced mammary tumors in rats. The low toxicity of these inducers of DT-diaphorase makes them suitable for use as chemopreventive agents in high-risk individuals. Cells with elevated DT-diaphorase levels are generally more sensitive to bioreductive antitumor agents. Thus, we suggested that the antitumor efficacy of bioreductive agents can be enhanced by selective induction of DT-diaphorase in tumor cells compared with normal cells. We showed that 1,2-dithiole-3-thione (D3T) can increase the level of DT-diaphorase activity and the cytotoxic activity of bioreductive agents in mouse lymphoma cells without increasing these activities in normal mouse marrow cells. D3T also increased DT-diaphorase activity in 24 of 33 human tumor cell lines representing nine tissue types with no obvious relationships between the tumor type, or the base level of DT-diaphorase activity, and the ability to increase enzyme activity. A series of dithiolethione analogues and dietary components were also shown to be good inducers of DT-diaphorase in human tumor cells. D3T increased DT-diaphorase activity in normal human bone marrow and kidney cells but the increases were small in these cells. Combination treatment with D3T and EO9 increased cell kill in HL-60 human leukemia cells compared with EO9 alone, but had no effect on EO9 toxicity in normal human kidney cells. Similarly, D3T increased tumor cell kill by EO9 in H661 human lung cancer cells and by MMC in T47D human breast cancer cells. Thus, inducers of DT-diaphorase may play an important role in cancer chemoprevention programs and may also be useful in enhancing the antitumor efficacy of bioreductive agents.

Protection against aflatoxin B1-induced hepatic toxicity as short-term screen of cancer chemopreventive dithiolethiones.

Dithiolethiones are an important class of cancer chemopreventive agents. More than 50 new dithiolethione analogs were synthesized for structure-activity studies. Using selected dithiolethiones, studies were designed to measure protection against the hepatotoxicity of aflatoxin B1 (AFB1) and relate it to the protection against carcinogenicity. Young male F344 rats were pretreated with 0.1 or 0.3 mmol dithiolethiones/kg body wt and challenged with toxic doses of AFB1 (50 micrograms/100 g rat/day) on 2 successive days. One day later, the protection from hepatotoxicity was assessed by measuring serum hepatic enzymes, hepatic necrosis, and degree of bile duct cell proliferation. The ability of these dithiolethiones to prevent AFB1-induced tumorigenicity was assessed by quantifying the hepatic burden of putative preneoplastic lesions [placental glutathione S-transferase (GST-P)-positive foci]. Significant correlations (p < 0.01) were observed between these toxicological indices and GST-P focal burden (alanine aminotransferase, r = 0.943; sorbitol dehydrogenase, r = 0.897; histological index, r = 0.893; bile duct cell proliferation, r = 0.933). These results imply that inhibition of hepatotoxicity affords protection against hepatocarcinogenicity. The extent of protection from acute hepatotoxicity offers a simple, short-term biological endpoint to screen dithiolethiones and related compounds for their chemopreventive properties.

Induction of DT-diaphorase by 1,2-dithiole-3-thione and increase of antitumour activity of bioreductive agents.

Bioreductive antitumour agents are an important new class of anticancer drugs that require activation by reduction. The two-electron reducing enzyme, DT-diaphorase, has been shown to be an important activating enzyme for the bioreductive agents, mitomycin C (MMC) and EO9. Incubation of L5178Y murine lymphoma cells in vitro with 1,2-dithiole-3-thione (D3T) increased the level of DT-diaphorase activity in these cells 22-fold. In contrast, D3T had no effect on the DT-diaphorase level in normal mouse bone marrow cells. Combination therapy with D3T and MMC or EO9, produced a 2- or 7-fold enhancement, respectively, of the cytotoxic activity of these antitumour agents in L5178Y cells. By comparison, D3T did not enhance the activity of MMC in marrow cells and produced only a small increase in EO9 cytotoxicity in these cells. The DT-diaphorase inhibitor, dicoumarol, inhibited the effect of D3T on the antitumour activity of the bioreductive agents, supporting the proposal that the enhanced anticancer activity was due to the elevated enzyme level. These findings suggest that D3T, or other inducers of DT-diaphorase, could be used to enhance the antitumour efficacy of bioreductive antitumour agents.

Formation of immunochemical advanced glycosylation end products precedes and correlates with early manifestations of renal and retinal disease in diabetes.

Elevated levels of advanced glycosylation end products (AGEs) have been found in multiple tissues in association with diabetic vascular complications and during the microalbuminuric phase of diabetic nephropathy. In this study, we have used an AGE-specific enzyme-linked immunosorbent assay (ELISA) to measure skin AGEs to determine whether elevated levels can be detected before the onset of overt microangiopathy. Subjects with type I diabetes (n = 48) were graded for the degree of nephropathy (normal [23], microalbuminuria [12], or macroalbuminuria [12]) and retinopathy (none [13], background [20], or proliferative [15]). Subgroups with a premicroalbuminuric phase of albumin excretion (< or = 28 mg/24 h, n = 27) or with the earliest stages of retinopathy (n = 27) were identified. A significant increase in tissue AGEs was found as urinary albumin increased during the premicroalbuminuric phase of nephropathy even when the data were adjusted for age and duration of diabetes (P = 0.005). Immunoreactive AGEs also increased as normal renal status advanced to microalbuminuria and macroalbuminuria (P = 0.0001 across groups). Significant elevation of AGEs was also found in association with the earliest stages of clinically evident retinopathy (early background versus minimal grades). In addition, higher AGE levels were found in subjects with proliferative retinopathy when compared with those with less severe retinopathy (P < 0.004 across groups). In contrast, no significant differences were found in tissue AGE levels between groups with or without early retinopathy based on pentosidine or fluorescent AGE measurements, although fluorescent AGEs correlated with albumin excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of phase 2 enzyme induction by oltipraz and other dithiolethiones.

4-Methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (oltipraz) and several other dithiolethiones protect against the acute toxicities of many xenobiotics and are effective inhibitors of experimental carcinogenesis. These protective effects are mediated, in part, through elevation of glutathione S-transferase, NAD(P)H: quinone reductase and UDP-glucuronosyltransferase activities in the liver and other target tissues. The induction of these phase 2 enzymes by oltiprax results from enhanced transcription. In the present study, the molecular mechanisms of these inductions were analyzed utilizing a construct containing a 41 bp enhancer element derived from the 5'-upstream region of the mouse liver glutathione S-transferase Ya subunit gene ligated to the 5' end of the isolated promoter region of this gene, and inserted into a plasmid containing a human growth hormone reporter gene. When this construct was transfected into murine Hepa 1c1c7 hepatoma cells, the concentrations of 25 dithiolethiones and related analogs required to double growth hormone production were determined and spanned a range nearly three orders of magnitude. Concentrations of dithiolethiones required to double the specific activity of NAD(P)H: quinone reductase were also determined in Hepa 1c1c7 cells. There was a positive correlation (r = 0.78) between the potencies of the 21 active compounds as inducers of both NAD(P)H: quinone reductase activity and growth hormone production. Moreover, no dithiolethiones were inactive in only one system. It is probable, therefore, that the induction of NAD(P)H: quinone reductase and other phase 2 enzymes by oltipraz and other dithiolethiones is mediated entirely through the 41 bp enhancer element.

Increased collagen-linked pentosidine levels and advanced glycosylation end products in early diabetic nephropathy.

RATIONALE: Advanced glycosylation end products (AGEs) may play an important role in the development of diabetic vascular sequelae. An AGE cross-link, pentosidine, is a sensitive and specific marker for tissue levels of AGEs. OBJECTIVES: To evaluate the role of AGEs in the development of diabetic nephropathy and retinopathy, we studied pentosidine levels and the clinical characteristics of 48 subjects with insulin-dependent diabetes mellitus. Diabetic nephropathy was classified as normal, microalbuminuria, or gross proteinuria, and retinopathy was graded as none, background, or proliferative. NEWLY OBSERVED FINDINGS: Significant elevation of pentosidine (P = 0.025) was found in subjects with microalbuminuria or gross proteinuria (73.03 +/- 9.47 vs 76.46 +/- 6.37 pmol/mg col) when compared with normal (56.96 +/- 3.26 pmol/mg col). Multivariate analysis to correct for age, duration of diabetes, and gender did not modify the results. Elevated pentosidine levels were also found in those with proliferative when compared with those with background retinopathy (75.86 +/- 5.66 vs 60.42 +/- 5.98 pmol/mg col) (P < 0.05). CONCLUSIONS: Microalbuminuria is associated with elevated levels of pentosidine similar to those found in overt diabetic nephropathy suggesting that elevated AGE levels are already present during the earliest detectable phase of diabetic nephropathy.

Relationship between glycemic control and collagen-linked advanced glycosylation end products in type I diabetes.

OBJECTIVE: To evaluate the relationship between glycemic control over a 3-yr period and tissue levels of advanced glycosylation end products. The development of renal failure, blindness, and generalized vascular occlusion continue to be the most serious ravages of diabetes. Tissue glycosylation and AGEs are felt to play an important role in the development of these sequelae, but no data are available on the relationship between AGEs and long-term glycemic control. RESEARCH DESIGN AND METHODS: We studied 48 subjects with type I diabetes. Glycemic control was determined by mean levels of HbA1, and AGEs were determined on collagenase digests of skin collagen by fluorescence at excitation/emission readings of 335/385 and 370/440 nm. RESULTS: To evaluate the relationship between glycemic control and AGE levels, control was classified as good (< or = 8.5%), fair (> 8.5% but < or = 10%), or poor (> 10%) on the basis of mean HbA1 levels during 1- and 3-yr periods. Analysis of the mean AGE levels for each level of glycemic control over 1-3 yr showed that AGEs differed significantly across categories of glycemic control (P = 0.04 and 0.003), with the lowest AGE levels associated with good and the highest with poor glycemic control. The relationship also was highly significant when adjusted for age, sex, and duration of diabetes, and when examined by Pearson's correlation coefficients (P = 0.02 and 0.008). CONCLUSIONS: Finding a relationship between glycemic control over 1-3 yr and tissue levels of AGEs suggests that AGEs can be modified and possibly reversed by improved glucose levels.

Potent inhibition of aflatoxin-induced hepatic tumorigenesis by the monofunctional enzyme inducer 1,2-dithiole-3-thione.

1,2-Dithiole-3-thiones are five-membered cyclic sulfur-containing compounds with antioxidant, chemotherapeutic, radioprotective and chemoprotective properties. Several substituted 1,2-dithiole-3-thiones are used medicinally and one of these, oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione], has been recently shown to be an inhibitor of aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Structure-activity studies have been undertaken to probe the mechanisms by which dithiolethiones inhibit carcinogenesis. Such studies revealed that unsubstituted 1,2-dithiole-3-thione was more effective than oltipraz at inhibiting aflatoxin-DNA adduct formation in vivo and at inducing electrophile detoxication enzymes in cell culture. In the present studies the effects of dietary administration of 1,2-dithiole-3-thione on the induction of xenobiotic metabolizing enzymes and inhibition of aflatoxin-induced hepatic tumorigenesis were examined. Male F344 rats were fed graded doses of 1,2-dithiole-3-thione (0.001-0.03%) for 4 weeks. During the second and third weeks of 1,2-dithiole-3-thione feeding, rats were dosed by gavage with 250 micrograms of AFB1/kg five times a week. Rats were then restored to control AIN-76A diet 1 week after cessation of AFB1 dosing. At 4 months, focal areas of hepatocellular alteration were identified and quantified by staining sections of liver for gamma-glutamyltranspeptidase (GGT) activity and glutathione S-transferase P (GST-P) expression. Treatment with 1,2-dithiole-3-thione at the lowest dose (0.001%) reduced by greater than 80% the volume of liver occupied by GGT or GST-P foci; higher dietary concentrations provided greater than 98% reductions in the volume per cent of these markers for presumptive preneoplastic lesions. All dietary concentrations of 1,2-dithiole-3-thione resulted in significant elevations in hepatic GST activities. In accord with the protective effects against tumorigenesis, 4- to 6-fold increases in the specific activities of aflatoxin-glutathione conjugation were observed in cytosols prepared from livers of animals fed 1,2-dithiole-3-thione. By contrast, 1,2-dithiole-3-thione did not have any detectable inductive effects on hepatic microsomal cytochrome P450 levels or activities. Dietary administration of 1,2-dithiole-3-thione also elevated activities of GSTs and other phase II enzymes in several extrahepatic organs. This broad pattern of induction of detoxication enzymes by 1,2-dithiole-3-thione supports the potential widespread use of this compound as a protective agent against chemical carcinogenesis and other forms of electrophile toxicity.

Evaluation of promotion of pancreatic carcinogenesis in rats by benzyl acetate.

Benzyl acetate was found to induce liver tumours and gastric squamous neoplasms in mice in a chronic bioassay conducted through the National Toxicology Program. An increased incidence of acinar cell adenomas of the pancreas of F344 rats was noted in the bioassay, but the significance of these lesions was confounded because the benzyl acetate was given by gavage in corn oil. The use of corn oil as a vehicle has been shown to enhance the growth of such lesions in the rat pancreas. The current studies were undertaken to evaluate benzyl acetate alone as an initiator and promoter of carcinogenesis in the pancreas. Alkaline elution analysis of acinar cell DNA showed no evidence of damage 1 hr after administration of benzyl acetate. Significant stimulation of growth of azaserine-induced foci was observed in a 6-month study, and a low but significant incidence of carcinoma in situ was observed in rats fed benzyl acetate in the diet for 2 yr. These experiments suggest that benzyl acetate is a weak promoter of the growth of carcinogen-induced and spontaneous pre-neoplastic foci in the pancreas.

Potentiation of radiation-induced bacterial cell killing by binuclear rhodium(II) complexes and their amines.

Rhodium(II) binuclear complexes were surveyed for potentiation of radiation-induced cell killing of hypoxic and fully oxic Salmonella typhimurium cells. The Rh2 tetracarbonate ion substantially potentiated hypoxic cell radiation sensitivity. Phosphate interfered with this potentiation. In the latter two respects, radiation potentiation by Rh2 tetracarbonate is similar to that found for Rh2 tetracarboxylates. Amines such as ammonia, methylamine, ethylamine, n-propylamine, and n-butylamine were examined with both Rh2 tetracarbonate and tetraacetate complexes. With Rh2 tetraacetate in phosphate-buffered saline, these amines variably increased radiation potentiation to a maximum of nearly that seen by Rh2 tetraacetate alone in the absence of phosphate. With Rh2 tetracarbonate, particular amines were found to either enhance or restrict radiation potentiation. Results as a whole support the hypothesis that a radiolytic Rh species initiated in a one-electron reduction process external to the cell is responsible for the potentiation by Rh2 complexes in bacteria. Phosphate interference of potentiation by Rh2 tetracetate appears to be limited competitively by amines, suggesting that axial associations of phosphate with the Rh2 center may be involved in the inhibition of radiation potentiation. Of interest in this regard is the finding that 5'-adenosinemonophosphate eliminates the potentiation seen with Rh2 tetraacetate.

Inhibition of pancreatic and liver carcinogenesis in rats by retinoid- and selenium-supplemented diets.

Chemoprevention by a synthetic retinoid, selenium, and these agents in combination during the postinitiation stages of carcinogenesis induced in rats by azaserine was evaluated. Male Lewis rats were given three weekly injections of 30 mg/kg azaserine while being fed a purified diet. One week after completion of carcinogen treatment, groups of rats were switched to the purified diet supplemented with either a retinoid, N-(2-hydroxyethyl)retinamide, at a level of 0.5 or 1 mmol/kg diet, or with 5 ppm sodium selenite, or with a combination of retinoid and selenium. One year after the diet change, the incidence of pancreatic and other neoplasms was determined by autopsy and histologic study. The incidence of pancreatic carcinoma (including carcinoma-in-situ, CIS) among nonretinoid-treated controls was 68%. Since the dietary supplements were fed after completion of exposure to the carcinogen, the effects on both pancreatic and liver carcinogenesis were exerted during the postinitiation phase of carcinogenesis. As in previous studies, the retinoid inhibited the progression of pancreatic carcinogenesis in a dose-related fashion. Selenium alone had no effect. However, the combination of retinoid plus selenium was more effective than retinoid alone, although the increase in inhibition was not large. The retinoid was also found to inhibit liver carcinogenesis induced by azaserine. Selenium, either alone or in combination with retinoid, was ineffective. Finally, testicular atrophy, noted as a toxic effect of retinoids in other studies, was not observed in this work.

Pancreatic carcinogenesis in azaserine-treated rats: inhibition by a solvent mixture in the diet.

The growth of azaserine-induced foci and nodules in a 4-month experiment and the incidence of carcinomas in a 15-month experiment were greater in LEW/CrlBR inbred rats fed a purified diet (AIN-76A) than in rats fed a natural-ingredient diet (chow). Addition of a mixture of several solvents to either diet reduced the incidence of adenocarcinomas in the pancreas in the long-term study but failed to reduce the number or size of pancreatic atypical acinar cell foci in the experiments of 4 months and 6 months (chow only) duration. Apparently, some component of the solvent mixture inhibits a late stage in the development of pancreatic carcinoma. Glyceryl monooleate and propylene glycol are implicated as the components of the mixture most likely to be responsible for the inhibitory effect, but neither the identity of the critical component nor the mechanism of the inhibition is known. The solvent mixture also contained ethanol and trioctanoin.

In vivo and in vitro genotoxicity of selected compounds toward rodent pancreas.

Using the technique of alkaline elution analysis, the ability of 11 known or suspected pancreatic carcinogens to damage the DNA of pancreatic acinar cells when administered to rats and hamsters was examined. The two species respond differently to several agents. In selected instances, DNA damage was also assessed in cultured pancreatic acinar cells exposed in vitro to the agents. Comparisons of DNA damage produced in vivo with that produced in vitro gave useful information on the role of pancreatic metabolism in activating pancreatic carcinogens. Finally, information germane to the question of the cell of origin for pancreatic cancer was obtained.

gamma-Glutamyltransferase activity in atypical acinar cell nodules of rat pancreas.

The biochemical and histochemical measurement of the enzyme gamma-glutamyltransferase (GGT) was undertaken in normal rat pancreas and in rat pancreas containing azaserine-induced preneoplastic nodules. A steady decrease in pancreatic GGT activity was observed in the normal animals as they aged from 5 to 34 weeks. The azaserine-induced nodules contained a lower average GGT activity than the control pancreas although a 10-fold variation was noted in the GGT activity of individual nodules. A significant increase in concentrations of both reduced glutathione and oxidized glutathione was noted in pancreatic nodules from azaserine-treated rats compared to concentrations found in both control pancreas from untreated rats and internodular pancreas from azaserine-treated rats. A pancreatic acinar cell carcinoma contained low GGT activity--similar to that found in large nodules and about 10% of the level found in control pancreas. Pancreatic GGT levels were higher in 5- and 7-week-old rats fed chow than in rats fed a purified diet, but this effect of chow was not observed at 34 weeks of age. Feeding a purified diet supplemented with a retinoid, N-2-hydroxyethylretinamide (2-HER), for a period of 2 weeks did not influence the GGT activity level in either normal pancreas or in the azaserine-induced nodules. While decreased GGT activity does not serve as a marker for all atypical acinar cell nodules, deficient activity with concomitant increased glutathione levels appears to correlate generally with increased growth potential.

Mutagenicity of L-azaserine for V79 cells in a pancreatic acinar cell-mediated mutagenesis assay.

The mutagenicity of azaserine was determined in a pancreatic acinar cell-mediated mutagenesis assay using V79 cells as the responder cell line. The mutation frequency of V79 cells was increased in direct culture with azaserine as well as in coculture with rat and hamster pancreatic acinar cells. Although slightly higher mutation frequencies were seen with coculture, the mutation frequency induced by azaserine in coculture was not significantly enhanced over that observed in direct culture. Thus, azaserine cannot be used as a positive control to monitor the level of acinar cell metabolism in such cell-mediated mutagenesis assays. Statistical analysis suggested that hamster acinar cell cocultures were more effective at increasing the mutation frequency of azaserine as compared to rat acinar cell cocultures. Hamster acinar cell cocultures, but not rat acinar cell cocultures, increased the mutagenicity of azaserine in a dose-response fashion. These results suggest that azaserine may be a pancreatic carcinogen for the hamster as well as the rat.