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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. [ABSTRACT FROM PUBLISHER]

Oltipraz and related dithiolethiones constitute an important class of chemopreventive agents that enhance the expression of carcinogen detoxication and antioxidant genes. Dose-response studies were undertaken to characterize the cancer chemopreventive activities of several dithiolethiones that are at least as active as oltipraz as inducers. Inhibition of formation of pre-neoplastic lesions and formation of DNA adducts in livers of rats exposed to aflatoxin B1 (AFB1) was monitored. In the tumorigenesis experiment, the dithiolethiones were orally gavaged 3 days/week for 3 successive weeks and at four doses ranging from 0.03 to 0.3 mmol/kg body wt. AFB1 was gavaged beginning 1 week after the start of the dithiolethiones and for two successive weeks. The burden of AFB1-induced putative pre-neoplastic lesions (glutathione S-transferase-placental isoform positive foci) was quantified by light microscopy. Reduction in AFB-DNA adduct burden was assessed 24 h following the first dose of AFB1. Both the parent 1,2-dithiole-3-thione (D3T) and its 5-tert-butyl derivative were more potent inhibitors than oltipraz against these endpoints, while two of the seven tested analogs were slightly less inhibitory. D3T, the most potent dithiolethione of this series, was examined by microarray analysis for induction of hepatic genes at an intermediate chemopreventive dose (0.1 mmol/kg). Transcript levels of eight genes, including two known to detoxify aflatoxin, namely, glutathione S-transferase A5 (GSTA5) and AFB1 aldehyde reductase (AFAR) were elevated. Western analysis indicated that induction of hepatic GSTA5 and AFAR were directly related to the dose of D3T. At the highest dose of D3T (0.3 mmol/kg), protein levels of GSTA5 and AFAR were induced by 7- and 27-fold, respectively. While efficacy in humans has yet to be tested, D3T is clearly more potent than oltipraz and serves as a useful molecular probe for determining the key events associated with protection by this class of agents.

Phase II detoxifying enzymes like NAD(P)H (quinone acceptor)oxidoreductase1 (NQO1), glutathione S-transferases (GST), and UDP-glucuronyltransferases (UGT) may play an important role in preventing carcinogen-induced cancers. Inducers of these enzymes have been shown to inhibit carcinogen-induced colon tumors in rat and mouse models. However, it has not been clearly demonstrated that NQO1 contributes to this effect. We examined the effect of NQO1 inducers on colon carcinogenesis using an aberrant crypt foci (ACF) rat model. Sprague-Dawley rats were fed control diet or diet containing 400 ppm dimethyl fumarate or 200 ppm oltipraz for 7 days, and Phase II enzymes in rat colon and liver were measured. Dimethyl fumarate significantly increased NQO1 and GST activities in colon and liver but did not increase UGT activities in these tissues. In contrast, oltipraz significantly increased NQO1 activities in colon and liver and produced a small increase in GST activity in the liver but did not increase GST activity in the colon or UGT activities in the liver or colon. Sprague Dawley rats were fed control diet or diet containing 200 ppm oltipraz and then treated with the carcinogens azoxymethane or methyl nitrosourea. Both carcinogens produced ACF in all of the rat colons, but rats fed oltipraz diet had significantly fewer ACF than those fed control diet. This protective effect was reversed in rats treated with the NQO1 inhibitor, dicoumarol. However, treatment with oltipraz did not alter the distribution of crypt multiplicities in the ACF. These studies demonstrated that induction of NQO1 plays a significant role in inhibiting initiation of carcinogen-induced ACF in Sprague-Dawley rats. This provides the first direct evidence that NQO1 may play a role in preventing colon cancer. The study also found that oltipraz added to the diet of Sprague-Dawley rats selectively increased NQO1 activity in colon mucosa with no increase in GST and UGT activities in these tissues. Thus, this model will be useful for further investigating the role of NQO1 in prevention of colon cancer.

The combination of P4S10 and hexamethyldisiloxane efficiently converts esters, lactones, amides, lactams, and ketones to their corresponding thiono derivatives. In the presence of elemental sulfur, 3-oxoesters are converted to dithiolethiones by this reagent. Yields are comparable to or superior to those obtained with Lawesson's reagent. The method has the advantage that reagent-derived byproducts may be removed by a simple hydrolytic workup or by filtration through silica gel, rather than by chromatography, as required for Lawesson's reagent.

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.

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.

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.

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.

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.

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)

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.

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.

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.

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.

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.

Five human small cell carcinoma of the lung (SCCL) cell lines selected from 25 established cultures were grown as three-dimensional spheroid tumor models in either spinner culture or in static, agar-coated multiwells. Volume doubling times for the cell lines were approximately 4.5 days. Decreases in spheroid volumes after exposure to a variety of chemotherapeutic agents were used as indicators of drug activity. To further quantify cell killing in SCCL spheroids by chemotherapeutic agents 24 hours after exposure to drugs, a technique was employed that measured maximum levels of incorporation of 125IUdR after continuous labeling for 48 hours. The results of the use of this assay report for SCCL spheroid responses to various concentrations of doxorubicin hydrochloride, cytosine arabinoside, mechlorethamine hydrochloride, cisplatin, or etoposide. Some evidence for an intertumor heterogeneous response to chemotherapy is presented for some of the drugs tested. This assay was also used to characterize a potentiated cell kill when etoposide is combined with cisplatin and to identify activity by a new compound, diazoacetylcholine iodide (DACI), which was synthesized as an agent targeted for SCCL cells.

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.

An animal model for the experimental study of small cell carcinoma of the lung (SCCL; human) has been developed. Tumors arising from continuous cell lines of cultured tumor cells as well as transplantable tumors of human SCCL in nude athymic mice have been characterized as to growth rate and morphology. The effect of antilymphocyte and antithymocyte sera and the sex of the host mouse were evaluated.

Studies were undertaken to determine the identity of an azaserine:DNA adduct. The most probable adduct, 7-carboxymethylguanine, was synthesized. DNA isolated from pancreatic acinar cells treated in culture with [14C]azaserine was hydrolyzed under neutral conditions to liberate N-alkylated purines. The neutral hydrolysate was subjected to high-performance liquid chromatography along with the synthetic standard. One of the radioactive peaks from the treated DNA was found to cochromatograph with 7-carboxymethylguanine in three systems: reverse phase; anion exchange; and ion pair reverse phase. These results suggest that azaserine metabolism in acinar cells results in carboxymethylation of DNA, supporting previously proposed models of azaserine degradation.

Chemoprevention by retinoids of the progression of carcinomas induced in rats by azaserine was evaluated. Wistar/Lewis rats were given 15 weekly injections of azaserine, 10 mg/kg, while fed a chow diet; after the completion of carcinogen treatment, they were fed a chow diet supplemented with four different retinoids at the level of 0.5 to 2 mmol/kg diet for 1 year. The incidence of neoplasms was determined by autopsy and histological study. The incidence of pancreatic carcinoma among a male positive control group (azaserine treated, but not retinoid treated) was 42%. The incidence of pancreatic carcinoma among male rats treated with retinoids was: N-2-hydroxyethylretinamide, 6%; N-4-propionyloxyphenylretinamide, 17%; and retinylidene dimedone, 12%. The incidence in rats fed these three retinoids was significantly (p less than 0.05) below the control group incidence. Thus, these three retinoids appeared to be effective in inhibiting the progression of pancreatic carcinomas in the azaserine-induced model. A similar trend was demonstrated in females, but statistical significance was shown only in rats fed N-2-hydroxyethylretinamide. A fourth retinoid, N-4-carboxyphenylretinamide, was more toxic and less effective in chemoprevention. Since retinoids were fed after exposure to carcinogen, the effect was exerted during the postinitiation phase of carcinogenesis. The ratio of invasive pancreatic carcinomas to localized carcinomas (carcinoma in situ) was clearly higher among non-retinoid-treated rats than among those treated with retinoids. This is consistent with retarded progression in the retinoid-treated groups.

A calcitonin (CT)-producing cell line (DMS53) established from human small cell carcinoma of the lung was grown as three-dimensional multicellular spheroids in spinner culture or on agar in multiwells, and as tumors in nude (athymic) mice. CT release into the media was directly proportional to spheroid volume. The response of these cells following exposures to X-irradiation, Adriamycin, or diazoacetylcholine iodide was assessed by monitoring levels of CT released into the media by individual spheroids. Levels of CT in the blood of nude mice bearing DMS53 xenografts were directly proportional to tumor volume and decreased proportionally with tumor response to X-irradiation and cisplatin treatment. These results suggest that the DMS53 spheroid and xenograft models may be useful systems to monitor responses to therapy utilizing CT as an indicator of tumor burden.

Syrian golden hamsters were treated with N delta-(N-methyl-N-nitrosocarbamoyl)-L-ornithine (MNCO), a nitrosourea amino acid, which has induced a high incidence of breast, kidney and skin neoplasms and a low incidence of pancreatic carcinoma in rats. MNCO induced a few breast and skin carcinomas, and a high incidence of foci of atypical acinar cells and of focal ductular abnormalities in the exocrine pancreas. The latter were similar to lesions observed in rats treated with MNCO. MNCO was less toxic and less effective as a carcinogen in hamster than in the rat.

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.

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.

Four synthetic retinoids were evaluated with regard to chemo-prevention of pancreatic carcinomas in carcinogen-treated hamsters. Syrian golden hamsters were given two injections of N-nitrosobis(2-oxopropyl)amine (20 mg/kg) and then were fed retinoid-supplemented diets for 1 year. The incidence of pancreatic carcinomas was lower in six of eight retinoid-fed groups than in the control group, although the differences were not statistically significant. The lowest incidence was observed in groups fed N-(4-pivaloyloxyphenyl)retinamide and N-(2-hydroxypropyl)retinamide. Testicular atrophy with decreased spermatogenesis was noted in males fed N-(2-hydroxypropyl)retinamide, N-(3-hydroxypropyl)retinamide, and N-(2,3-dihydroxypropyl)retinamide.

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.

Isolated rat and hamster acinar cell suspensions possess the ability to carry out the cytochrome P-450-dependent O-deethylation of 7-ethoxycoumarin, 2-,3-, and 4-hydroxylation of biphenyl and 3-hydroxylation of benzo(a)pyrene. Rat and hamster acinar cells isolated from 5,6-benzoflavone-pretreated animals oxidize all three substrates at measurable rates. These rates are considerably lower (16-210-fold in the rat and 290-2670-fold in the hamster) than those in incubations using hepatocytes isolated from 5,6-benzoflavone-pretreated animals. Hydroxylation of biphenyl at the 2-, 3-, and 4-positions proceeds at similar rates in rat acinar cells. The rate of 3-hydroxybiphenyl formation is barely detectable in hamster acinar cells where the rates of 2- and 4-hydroxybiphenyl formation are slower than in the rat. No detectable oxidation products of 7-ethoxycoumarin, biphenyl, or benzo(a)pyrene are found with acinar cells of either species isolated from untreated and phenobarbital-pretreated animals. The O-deethylation of 7-ethoxycoumarin in rat and hamster acinar cells is decreased in the presence of inhibitors of the cytochrome P-450-dependent monooxygenase system, 7,8-benzoflavone being much more effective than metyrapone. The deethylation product of 7-ethoxycoumarin, 7-hydroxycoumarin, is conjugated with sulfate and glucuronic acid moieties at appreciable rates by acinar cells isolated from both rat and hamster. Pretreatment of rats and hamsters with either 5,6-benzoflavone or phenobarbital has little effect on the rates of conjugation in isolated acinar cell preparations.

Utilizing the technique of alkaline elution analysis, the ability of N-nitrosomethyl(2-oxopropyl)amine (MOP), a potent pancreatic carcinogen, to damage pancreatic DNA in rats and hamsters was examined. Pancreatic DNA isolated from hamsters exposed for 1 h to MOP given i.p. at doses of 7-60 mg/kg showed dose-related DNA damage. A similar dose-response was observed in the pancreas of rats receiving 20-180 mg MOP/kg, suggesting that hamsters were 2-3 times more sensitive than rats. In contrast to the results obtained in vivo, functionally viable acinar cells from both rat and hamster pancreas, when exposed in vitro to levels of MOP comparable to those in vivo (20-180 micrograms/ml), failed to show dose-related DNA damage. Acinar cells from hamsters pretreated with 5,6-benzoflavone, an inducer of cytochrome P-450 activity, showed greatly enhanced drug-metabolizing capability, but again no DNA damage was observed upon exposure to MOP. Minced hamster or rat pancreas also failed to show DNA damage in response to MOP treatment. When hamsters in which hepatic blood supply was interrupted by ligation were given 60 mg/kg MOP i.v. and sacrificed 15 min later, damage to pancreatic and liver DNA was comparable to that observed in ligated controls which had received saline only. Administration of MOP to sham-operated animals led to extensive DNA damage in both pancreas and liver at 15 min. Analysis by h.p.l.c. showed an almost 2-fold increase in the amount of MOP present in the pancreases of the liver-ligated animals as compared to the sham-operated unligated animals. MOP was absent from the liver of the ligated animals. These experiments strongly suggest that DNA damage by MOP to the pancreatic acinar cells and probably to other pancreatic cell types, as well, requires metabolic activation by the liver.

The effect of feeding four synthetic retinoids was evaluated in carcinogen-treated hamsters. Syrian golden hamsters were injected with 20 mg/kg of N-nitrosobis(2-oxopropyl)amine (BOP) and then fed retinoid-supplemented diets for 39 weeks. All retinoid-treated groups grew more slowly than controls, and average survival was shorter in female retinoid-treated groups. A significant incidence of testicular atrophy was noted in male hamsters fed 2-hydroxyethylretinamide or 4-carboxyphenylretinamide. The incidence of pancreatic carcinomas was lower in 12 of 14 retinoid-fed groups than in the corresponding control groups, although the differences approached significance only in groups fed two of the retinoids--male hamsters fed N-4-propionyloxyphenylretinamide and those fed retinylidene dimedone. A low incidence of carcinoma in the control groups limits the conclusions that can be drawn from this study, but it is of note that there was no evidence of promotion.

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.

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.

One hour following intraperitoneal injection of the pancreatic and liver carcinogen azaserine, 10 mg/kg (0.06 mmol/kg), DNA damage is present in both pancreas and liver of Wistar/Lewis rats as determined with alkaline sucrose gradients. Single injections (0.06 mmol/kg) of either of 2 structural analogues of azaserine, 6-diazo-5-oxo-L-norleucine (DON) and ethyl diazoacetate (EDA), do not damage pancreatic or liver DNA. EDA administered at 0.5 mmol/kg damages liver DNA, but not pancreatic DNA. Total radioactivity in pancreas and liver of Wistar rats 1 h after intravenous injection of [14C]azaserine, (10 microCi/kg), is 2.8 and 1.3 times respectively, the level of 14C-activity in pancreas and liver following injection [14C]EDA. Three to four times as much [14C]EDA localizes in the liver of Wistar rats as in the pancreas. Azaserine (0.06 mmol/kg weekly for 6 weeks) induces atypical acinar cell nodules (AACN) in pancreas of Wistar/Lewis rats. DON (0.06 mmol/kg weekly for 6 weeks) induces an elevated incidence, but low number of AACN in pancreas. EDA (0.10 mmol/kg weekly for 6 weeks) does not induce pancreatic AACN.

The carcinogenicity of the nitrosourea amino acid, N delta-(N-methyl-N-nitrosocarbamoyl)-L-ornithine (MNCO), and its acute cytotoxicity were studied in Wistar rats. MNCO treatment induced a high incidence of neoplasms in breast, kidney, and skin and a lower incidence of tumors in the exocrine pancreas and ear duct. The duration of these studies was limited by toxicity and carcinogenicity to six months in a high dose group which received 1 mmole MNCO/kg IP weekly for 6 months and to 1 year in a group which received 0.33 mmole/kg. In the latter group the incidence of neoplasms was breast, 40%; skin, 52%; kidney, 81%; pancreas, 19%; and ear duct, 4%. All pancreases in this group also contained foci of fibrosis and cyst formation, and atypical acinar cell nodules. Acute cytotoxic effects were noted in pancreas, kidney and skin after a single IP injection of 3 mmoles/kg although this dose caused no deaths acutely.

Chemoprevention by synthetic retinoids of the progression of carcinomas of the pancreas induced in rats by azaserine was evaluated. Lewis rats were given five weekly injections of azaserine, 30 mg/kg, while being fed a chow diet. Two weeks after completion of carcinogen treatment, groups of rats were fed the chow diet supplemented with four different retinoids at the level of 0.5 to 2 mmol/kg of diet for 1 year. The incidence of pancreatic and other neoplasms was determined by autopsy and histological study. The incidence of localized pancreatic carcinoma among male and female non-retinoid-treated controls was 25 and 17%, respectively. No invasive or metastatic carcinomas were found in the control group. The combined incidence of localized and invasive pancreatic carcinomas among male and female rats treated with retinoids was: N-(4-pivaloyloxyphenyl)retinamide, 4 and 0%; N-(2-hydroxypropyl)retinamide, 14 and 6%; N-(3-hydroxypropyl)retinamide, 16 and 4%; and N-(2,3-dihydroxypropyl)retinamide, 12 and 6%. High- and low-dose groups are combined in this summary of data. Thus, there was a trend towards fewer pancreatic carcinomas among all retinoid-treated groups. The reduction in incidence was significant in both male and female rat groups given N-(4-pivaloyloxyphenyl)retinamide and N-(2,3-dihydroxypropyl)retinamide. The principal evidence of retinoid toxicity was growth failure, which was most severe in animals treated with N-(4-pivaloyloxyphenyl)retinamide, and testicular atrophy, which was most severe among male animals treated with N-(3-hydroxypropyl)retinamide. Among the females, groups treated with three of the four retinoids showed a dose-related increase in incidence of hepatocellular carcinomas. Since the retinoids were fed after the completion of exposure to the carcinogen, the effects on both pancreatic and liver carcinogenesis were exerted during the postinitiation phase of carcinogenesis.

The effects of pyridoxal deficiency on the genotoxicity and nodule inducing ability of azaserine in rat pancreas were examined. Azaserine at a dose of 10 mg/kg body weight which causes substantial DNA damage in normal rat pancreas, failed to induce DNA damage detectable by alkaline elution in the pancreas of pyridoxal-deficient rats. Studies of the distribution of [14C]azaserine in rat tissues revealed that uptake of azaserine in pancreas of pyridoxal deficient rats was not significantly different from that of normal rats. The ability of a structurally unrelated amino acid carcinogen N delta-(N-methyl-N- nitrosocarbamoyl )-L-ornithine to damage rat pancreatic DNA was not affected by pyridoxal deficiency. In another study, the pyridoxal antagonist 4'-deoxypyridoxine was administered i.p. to rats prior to and during azaserine treatment. Four months later, quantitative sterological analysis of atypical acinar cell nodules revealed that there was a significant reduction in the number but not size of nodules in the pancreases of 4'-deoxypyridoxine-treated rats. These results confirm the relationship of the induction of DNA damage by azaserine to its ability to induce pancreatic tumors, and support previous studies of azaserine metabolism, strongly suggesting that the in vivo activation of this carcinogen is pyridoxal dependent.

The carcinogenicity of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) for rat pancreas was evaluated. Two-week-old male LEW rats were given a single ip injection of HPOP, 160 mg/kg body weight; the rats were autopsied 4, 6, or 12 months later. Histologic examination showed that the pancreata contained multiple foci of atypical acinar cells and nodules of atypical acinar cells (AACN), acinar cell adenomas, localized carcinomas, and carcinomas. The incidence of carcinomas was 77%. The carcinomas were composed of poorly differentiated acinar cells and ductlike structures. Pancreatic ducts were unaffected. The prominence of AACN, the histologic type of the neoplasms, and the absence of hyperplastic changes in ductal epithelium suggest that the pancreatic carcinomas were derived from acinar cells. The incidence of liver cell carcinomas and pulmonary adenomas was similar to that of localized pancreatic carcinomas. Neoplasms of other organs were less frequent. HPOP has been shown to induce pancreatic carcinomas in hamsters but has not previously been reported to be a pancreatic carcinogen in rats.

The binding of [14C] azaserine or its metabolites to DNA and protein in the organs of rats and hamsters was determined at various time after treatment with [14C] azaserine. The specific activity of 14C labelling of DNA and protein was determined. Rat liver DNA and protein were most extensively labelled at 90 min post-injection, but by 24 h the specific activity decreased to the levels found in pancreas and kidney. Thymus contained negligible amounts of radioactivity at all time-points. DNA and protein from hamster pancreas contained more label than did DNA and protein from rat pancreas. The results suggest that factors other than DNA binding play a role in determining the species and organ specificity of azaserine.

The enzyme gamma-glutamyl transpeptidase (GGT) is characteristically present at high levels in mammalian cells that are vulnerable in vivo to the selectively toxic and carcinogenic effects of the naturally occurring diazo amino acid L-azaserine. The possible role of GGT as a determinant of cellular sensitivity to azaserine toxicity was investigated. No correlation was found between GGT activity and the abilities of different cell lines or GGT-deficient cell strains of TuWi, a human nephroblastoma-derived line high in GGT, to accumulate azaserine. However, the thiols glutathione and cysteine were found to inhibit the toxicity of azaserine in cultures of TuWi. In addition, maleate lowered both intracellular and extracellular glutathione levels and enhanced sensitivity of TuWi cells to azaserine, while serine-borate, a potent inhibitor of GGT, increased extracellular glutathione levels and inhibited azaserine toxicity. Since extracellular glutathione accumulation, which may reflect the rate of cellular glutathione turnover, is increased in cultures of azaserine-resistant, GGT-deficient strains of TuWi, we propose that GGT enhances cellular sensitivity to azaserine primarily by increasing the rate of glutathione turnover, thus removing the glutathione from detoxification pathways.

The response of LEW and F344 strain rats to the pancreatic carcinogen azaserine was compared using the size and number of azaserine-induced acidophilic acinar cell foci and nodules as parameters in a 4-month experiment. A second experiment compared the effect of corn oil intake by gavage and dietary routes on the growth of azaserine-induced pancreatic lesions in LEW rats. A third experiment tested the activity of benzyl acetate in regard to its ability to induce acinar cell foci or to promote the growth of such foci in azaserine-treated rats. The results showed that equivalent doses of azaserine induce two to seven times more foci in LEW than in F344 rats, and that LEW rats have a higher incidence of "spontaneous" foci than F344 rats. Azaserine-treated LEW rats that were given 5 mL corn oil/kg body weight 5 days per week by gavage developed more acinar cell foci than rats fed a basal diet (chow). Addition of an equivalent amount of corn oil to chow had a similar effect of enhancing the development of foci. Rats of neither strain developed acinar cell foci when benzyl acetate was given by gavage or in the diet nor was there evidence that benzyl acetate has a significant effect on the development of foci in azaserine-treated rats. These studies also demonstrate that the azaserine/rat model of pancreatic carcinogenesis which was developed in LEW rats can be adapted for use with F344 rats.

Carcinomas of the pancreas, stomach, and breast, as well as mesotheliomas and ovarian stromal tumors, were induced in Syrian golden hamsters treated with N delta-(N-methyl-N-nitrosocarbamoyl)-L-ornithine (MNCO), which has previously been shown to cause pancreatic acinar cell carcinomas in rats. The pancreatic carcinomas in hamsters appeared ductlike. The nonneoplastic and preneoplastic lesions induced in the hamster pancreas included cystic ductal complexes, tubular complexes, intraductal hyperplasia and atypical hyperplasia, focal eosinophilic metaplasia, and foci of atypical acinar cells. High doses of 654 mg MNCO/kg body weight were cytotoxic for acinar cells and caused atrophy of the pancreas. Alkaline elution analysis of DNA from acinar cells treated in culture with MNCO showed an increased rate of elution characteristic of single-strand breaks. A group of hamsters treated with a low dose of N-nitrosobis(2-oxopropyl)amine (BOP) developed pancreatic lesions similar to those seen when a subcarcinogenic dose of MNCO was given. The results suggest that MNCO affects both acinar and ductal cells in the hamster and that the response of the hamster pancreas to MNCO and BOP is similar in many respects.

Inbred W/LEW rats and noninbred CD-1 mice were compared for responsiveness to induction of pancreatic adenocarcinomas by azaserine. At 1 year following the first of 15 weekly ip injections of 10 mg azaserine/kg body weight, the rats had a pancreatic adenoma incidence of 71% (12/17) and a pancreatic adenocarcinoma incidence of 35% (6/17), with 1 invasive adenocarcinoma. The mice showed none of these advanced lesions, although numerous pancreatic atypical acinar cell nodules (AACN) were present. An examination of the number and size of the AACN showed the rats to hve more and larger AACN thatn did the mice. The concentration of [14C]azaserine and/or its metabolites was greater in rat pancreas than in mouse pancreas. Alkaline sucrose gradients were used to compare azaserine-induced pancrewtic and liver DNA damage in W/LEW and F344 rats, the CD-1 mouse, the Syrian golden hamster, and the strain 13 guinea pig. DNA damage was detected 1 hour following azaserine administration in both pancreata and livers of all animals tested and persisted for at least 1 week in the pancreata of all animals except the CD-1 mouse; however, neither the degree nor persistence of DNA damage accurately reflected the differing responsiveness of these species to induction of pancreatic AACN or neoplasms by azaserine.

Development of a model of carcinoma of the pancreas in rats was approached by attempting to identify chemicals that (a) behave as mutagens and (b) localize in the pancreas following systemic administration; and then to study the effects of long-term administration. Azaserine was selected because it behaves as a direct-acting mutagen in two bacterial test systems and because tissue distribution studies showed concentration especially in kidney and pancreas. Groups of rats have been given i.p. injections once or twice weekly for 6 months, and rats have been autopsied after 6 to 18 months. During the first year pancreases developed (a) nodules of atypical exocrine cells which seem to represent hyperplastic foci and (b) encapsulated adenomas. After 1 year most pancreases from treated rats are diffusely abnormal and contain many hyperplastic nodules and adenomas, while more than one-quarter have had pancreatic adenocarcimona. Metastases have been observed in lymph nodes, liver, and lung. No carcinomas or adenomas have been observed in control rats. No other organ shows as high an incidence of involvement as pancreas, but renal neoplasms were frequent. Studies with another chemical O-(N-methyl-N-nitroso-beta-alanyl)-L-serine, are at an earlier stage. The tissue distribution of radioactivity following injection of a 14C-labeled sample is similar to that of azaserine; however, this compound is not a direct-acting bacterial mutagen. Rats treated for 6 months twice weekly i.p. have a higher incidence of nodules of atypical acinar cells than did controls, although the number of nodules per rat is few. No adenomas or carcinomas have been found during 13 months of the study. We conclude that azaserine is a carcinogen in rats and causes major abnormalities of growth and differentiation of the exocrine pancreas, including adenocarcinoma in some rats. O-(N-Methyl-N-mitroso-beta-alanyl)-L-serine had less effect than azaserine on pancreatic growth and differentiation.

Sixteen continuous tumor-cell cultures have been isolated from 91 tissue specimens from patients with small-cell carcinoma of the lung. Biopsy and autopsy specimens of primary and metastatic tumors have been utilized. The developing cell lines were recognized by proliferation of tumor cells in the culture from one to 14 weeks after explanation and have been maintained for up to four years. Primary lung tumor, bone marrow aspirations, pleural effusions and other metastases have all been productive explant material for the development of cell lines. Their human origin has been demonstrated by chromosome and/or isoenzyme analysis. Dense core vesicles, characteristically found in small-cell tumor cells were observed by electron microscopic examination of cultured cells. Growth rates in vitro have been measured and the in vitro cycle time in tumors of one cell line (DMS 79) has been compared with in vivo cycle time in tumors arising from DMS 79 cells in nude athymic mice.