Dose Of Diethylnitrosamine Research Articles

The modulation of rat liver carcinogenesis by perfluorooctanoic acid, a peroxisome proliferator

Prefluorooctanoic acid (PFOA) is a peroxisome proliferator. The aim of this study was to test for its ability to act as a positive modulator of hepatocarcinogenesis, in the so-called biphasic (initiation by diethylnitrosamine 200 mg/kg ip followed by treatment with the suspected modulators) and triphasic (initiation by the same dose of diethylnitrosamine followed by a selection procedure for 2 weeks consisting of giving 2-acetylaminofluorene and in the middle of this treatment a single dose of CCl 4 followed by treatment with the suspected modulators) protocols of liver carcinogenesis. In both protocols treatment with PFOA increased the incidence of malignant hepatocellular carcinoma (HCC). As compared to phenobarbital, the modulating effect of PFOA is more pronounced in a biphasic than in the triphasic protocol. In parallel with positive modulation of HCC, PFOA also selectively induced the peroxisomal acyl-CoA oxidase activity and, to a lesser extent, catalase activity.

Butylated hydroxytoluene lacks the activity of phenobarbital in enhancing diethylnitrosamine-induced mouse liver carcinogenesis

The effect of the food additive butylated hydroxytoluene (BHT) as an enhancer of liver carcinogenesis in mice was investigated. Liver carcinogenesis was initiated by intraperitoneal injection of diethylnitrosamine (DEN) in male B6C3F 1 mice at 100 or 200 μmol/kg body weight once a week for 10 weeks (total exposure 1000 or 2000 μmol/kg body weight). After an exposure-free recovery interval of 4 weeks, groups of mice were fed either basal diet or diets containing either 5000 ppm BHT or 500 ppm phenobarbital (PB), as a positive control, for 24 weeks. Exposure to the initiating doses of DEN alone induced no liver foci at 10 weeks or at 14 weeks after the recovery period, but at termination at 38 weeks, foci and adenomas were present in a dose-related incidence. In the groups given BHT after DEN/recovery, the incidence and the multiplicity of liver foci and adenomas were not different from those in mice given only DEN/recovery, whereas, in the groups given PB after DEN, liver lesions were increased by 1.7–3.0-fold. In conclusion, BHT had no promoting or syncarcinogenic effect on DEN-induced mouse liver carcinogenesis, whereas under the same conditions, PB acted as an enhancer.

Diethylnitrosamine-induced hepatocarcinogenesis in rats: A theoretical study

Multiple data sets on hepatocarcinogenesis in rats resulting from pulse (single) or continuous exposure to diethylnitrosamine (DEN) are analyzed within the framework of a two-mutation carcinogenesis model in order to identify the underlying biological processes that control the pharmacodynamics of DEN-induced liver cancer. Our findings indicate: (1) Predictions of the two-mutation oncogenic model are consistent with empirical data on DEN-induced hepatocarcinogenesis. (2) The probability of the first genetic alteration (initiation) is linearly dependent on applied dose and decays exponentially following a pulse (single) dose or cessation of exposure. (3) The probability of initiation is proportional to the number of O 4-ethyldeoxythymidine DNA adducts resulting from DEN exposure, indicating that these adducts are the likely promutagenic lesions in DEN-induced hepatocarcinogenesis. (4) The mitotic rates of initiated and transformed cells are nonlinear with dose. (5) The average growth rate of initiated hepatocytes as a function of DEN dose is related to Druckery's slope. (6) The probability of the second genetic event (transformation) is independent of applied dose, suggesting that it is the result of a spontaneous genetic alteration.

Cell proliferation and tumour promotion by ethinyl estradiol in rat hepatocarcinogenesis

A two-stage model of hepatocarcinogenesis is used to study the effect of exposure time to ethinyl estradiol (EE) on promotion of preneoplastic lesions in rat liver induced by diethylnitrosamine (DEN). Young male and female Sprague-Dawley rats initiated by a single dose of DEN (100 mg/kg) were subjected to different times of EE administration incorporated into the diet at 10 p.p.m. (0.5 mg/kg x day). Animals were killed 1 year after initiation. Whereas macroscopic tumours were rarely seen in animals with short exposure (3 or 4 months) or in only-initiated controls, all the animals under a long period of administration (8 months) showed macroscopic tumours. Morphometric studies on glutathione-S-transferase (GST) positive preneoplastic lesions revealed an increase in the mean size of foci and nodules corresponding to 8 months of treatment, whereas no changes were observed between animals with short exposure and only-initiated controls. No differences were seen in the incidence of these lesions between any of the protocols. In addition to an acute hyperplastic effect on non-initiated liver described earlier, our preliminary results suggest cytotoxicity and an enhancement of the liver cell turnover after several months of continuous EE administration. These results taken together suggest that promotion of hepatocarcinogenesis by EE largely depends on the time of exposure to the compound and that chronic effects on the liver cell turnover may play an important role in its ability to promote hepatocarcinogenesis.

Hepatotoxicity Induced by Diethylnitrosamine Causes no Significant Disturbances of Systemic Glucose Homeostasis in Rats

In rats, a moderately hepatotoxic single dose of diethylnitrosamine (DEN) 100 mg/kg causing depletion of liver glycogen, elevation of aspartate aminotransferase and decreased liver uptake of 3-O-methylglucose, resulted in substantial changes in insulin and glucagon balance. Two days after DEN, insulin binding to liver membranes and insulin removal by the liver were sharply reduced whereas its binding to muscle and adipocyte membranes remained unaltered. Serum insulin (random and after an overnight fast) remained normal. Intravenous (I.V.) insulin (10 U/kg) caused the usual degree of hypoglycemia that, however, lasted longer than in the control animals. Removal of glucagon by liver was also depressed in spite of its normal binding to hepatocytes, and peripheral serum glucagon was increased three-fold. I.V. glucagon (40 micrograms/kg) resulted in a blunted response of plasma glucose. I.V. glucose tolerance test (1 g/kg) remained normal in spite of the insulin increase to a level twice as high as in the controls, and in spite of nonsuppressed glucagon. These changes were still present after 1-3 months, but disappeared by 6 months. The results demonstrate remarkable ability of homeostatic mechanisms to preserve normal plasma glucose and glucose tolerance in spite of dramatic changes in insulin and glucagon.

The effect of choline and methionine deficiencies on the number and volume percentage of altered hepatic foci in the presence or absence of diethylnitrosamine initiation in rat liver

The ability of methyl-deficient, amino-acid-defined diets to produce enzyme-altered foci was quantitatively determined in the livers of rats treated both with and without an initiating dose of diethylnitrosamine (DEN). Male weanling F-344 rats were fed a complete, amino-acid-defined diet for 1 week. They were then injected i.p. with a single dose of DEN (20 mg/kg body weight) and fed the complete diet for an additional week. Forty animals in each dose group were then maintained for 5-38 weeks on the complete diet (diet 1) or one of the three methyl-deficient diets customarily used in this laboratory: diet 2, devoid of methionine and choline; diet 3, devoid of methionine only; and diet 4, devoid of choline only. In diets 2 and 3, methionine was replaced by equimolar amounts of its metabolic precursor, DL-homocystine. Ten animals per group were killed 8, 12, 17, 24 and 41 weeks after DEN initiation. For 2 weeks prior to being killed, each group was maintained on the complete diet to minimize the histological abnormalities due to acute toxicity of the diets. Serial sections of the livers were obtained, stained sequentially for gamma-glutamyltranspeptidase, ATPase and glucose-6-phosphatase, and the quantitation of the focal lesions scored by these markers was carried out by quantitative stereology. The results indicated that, regardless of the enzyme marker(s) examined, there was a general correspondence between the volume and number of altered hepatic foci (AHF) formed and the previously described tumor-promoting activities of each diet. Thus, while all DEN-treated groups contained significant numbers of AHF 24 weeks after initiation, only the diet-2-fed animals displayed such foci at 8 weeks. Similarly, among the uninitiated rats, only those fed diet 2 exhibited the presence of AHF throughout the experimental period. Interestingly, the livers of uninitiated, choline-deficient rats showed a small number of AHF at 24 and 42 weeks; these foci were not observed at all in the corresponding DEN-untreated animals fed diet 3, deficient in methionine only. The results provide evidence that the carcinogenic effects of the methionine- and choline-deficient diet result more from its strongly promoting effect than from any initiating activity by the diet.

Cell proliferation and promotion of rat liver carcinogenesis: different effect of hepatic regeneration and mitogen induced hyperplasia on the development of enzyme-altered foci

A series of experiments was performed to investigate the effect of different types of cell proliferation on the development of enzyme-altered preneoplastic hepatic foci in male Wistar rats. Animals were given a single dose of diethylnitrosamine (100 mg/kg body weight). After a 2-week recovery period liver cell proliferation was repeatedly induced by four or eight necrogenic doses of carbon tetrachloride (compensatory cell proliferation), or by four or eight treatments with three different liver mitogens, namely lead nitrate, ethylene dibromide and nafenopin (direct hyperplasia). The carcinogen altered hepatocytes were monitored as gamma-glutamyltransferase positive or adenosine triphosphatase negative foci. The results indicate that compensatory cell proliferation induced by both four and eight carbon tetrachloride treatments enhanced the growth of diethylnitrosamine-initiated hepatocytes to enzyme-altered foci. On the contrary, repeated waves of cell proliferation induced by liver mitogens did not result in any significant number of enzyme-altered foci.

Transgenic mice containing hepatitis B virus sequences are more susceptible to carcinogen-induced hepatocarcinogenesis

Transgenic mice containing one copy of hepatitis B virus (HBV) genome without the core gene and expressing hepatitis B surface antigen (HBsAg) were crossed with C3H/He mice. The F1 hybrids (approximately 50% HBV positive and approximately 50% HBV negative) were treated with a single dose of diethylnitrosamine (NDEA) or p-dimethylaminoazobenzene (DAB) given at 7 days of age, or were untreated. Mice were kept under observation without further treatments until 30 weeks old and then killed. Stereological analysis of liver nodules and estimations of their size distribution demonstrated a significative enhancing effect of HBV transgene in both NDEA- and DAB-induced hepatocarcinogenesis in male mice. Hepatocellular adenomas and carcinomas were also more frequent in NDEA-treated HBV-positive than HBV-negative male mice. Female mice showed a lower tumorigenic response than males without significant differences between groups of HBV-positive and HBV-negative mice. It is proposed that the presence of the transgene enhanced carcinogen-induced hepatocarcinogenesis.

Lack of hepatocarcinogenic potential of acetaminophen in rats with liver damage associated with a choline-devoid diet

The potential carcinogenic activity of acetaminophen (paracetamol, APAP) was studied in male F344 rats with pre-existing liver damage induced by a choline-devoid (CD) diet. In a short-term experiment, APAP was administered by intragastric intubation as single doses of 0.5-1.5 g/kg body wt after 4 weeks feeding of CD diet had produced fatty livers in rats. Two-thirds partial hepatectomy was performed 4 h subsequent to the initiating treatment step. After a 2 week recovery period, all rats were subjected to the selection procedure of Cayama et al. and killed at week 9 of the experiment. Quantitative analysis of placental form glutathione S-transferase (GST-P)-positive liver lesion development did not reveal any enhancement by APAP, whereas administration of a non-necrogenic dose of diethylnitrosamine (20 mg/kg body wt) in the same protocol demonstrated significant promotion, confirming the utility of the model for detection of weak carcinogenicity of chemicals. In the second long-term experiment, APAP was fed at doses of 0.45 and 0.9% for 25 weeks following 27 weeks administration of CD diet which produced liver cirrhosis in the rats. Despite a slight enhancement of focal liver lesions positive for gamma-glutamyltranspeptidase (GGT), no significant promotion of GST-P-positive altered foci or nodules was observed. In contrast, continuous feeding of CD diet or 0.5% phenobarbital treatment after generation of cirrhosis with CD diet clearly enhanced the induction of both GST-P and GGT-positive liver lesions. Thus, these results indicate that APAP does not possess significant carcinogenic activity in damaged rat liver.

Immunofluorescent analysis of γ-glutamyl transpeptidase and glutathione-S-transferase-P during the initial phase of experimental hepatocarcinogenesis

We used double-label immunofluorescence to evaluate the expression of glutathione-S-transferase-P (GST-P) and gamma-glutamyl transferase (gamma GT) in individual liver cells in rats placed on a modified Solt-Farber hepatocarcinogenesis protocol. Four days after treatment with diethylnitrosamine (DEN), single GST-P+ cells were found in the centrilobular and midzonal regions. Most of these were gamma GT-, but the percentage of gamma GT+ cells increased with DEN dose to a high of 32%. At later times (7-8 days) doublets and small clusters of GST-P+ cells predominated; all cells in each pair or cluster displayed the same phenotype (GST-P+ or GST-P+/gamma GT+). Following administration of acetylaminofluorene and partial hepatectomy, lesions were larger and a greater percentage were GST-P+/gamma GT+. However, within these lesions individual cells displayed different phenotypes. These results indicate that the expression of GST-P and gamma GT is discordant in individual cells and small clusters soon after treatment, and suggest that different molecular events may be involved in their expression.

Enhancement of murine Hepatocarcinogenesis by all-transretinoic acid and two synthetic retinamides

Although retinoids have significant chemopreventive activity in many in vivo carcinogenesis models, their influence on experimental hepatocarcinogenesis has received only limited study. The present experiment was designed to determine the effects of three retinoids on hepatocarcinogenesis induced in female B6D2F1 mice by diethylnitrosamine (DEN). Beginning 1 week after a single i.p. dose of 0, 50 or 100 mg DEN per kg body wt, groups of 35 mice received dietary supplements of (per kg diet): 0.1 mmol all-trans-retinoic acid (RA), 0.5 or 1.0 mmol all-trans-ethyl retinamide (ER), 0.5 or 1.0 mmol 13-cis-ethyl retinamide (13-cis-ER), or vehicle only. In mice treated with 100 mg DEN/kg, all three retinoids significantly increased the incidence of liver tumors (benign + malignant) from the level seen in dietary controls; significant increases in the incidence of hepatocellular carcinoma were seen in groups fed ER. At the lower DEN dose, all three retinoids significantly increased the incidence of total liver tumors and of hepatocellular carcinomas. In mice not treated with DEN, liver tumors were seen only in mice fed ER or 13-cis-ER at 1.0 mmol/kg diet. These data indicate that RA, ER and 13-cis-ER can promote liver carcinogenesis in mice when administered at doses that inhibit cancer induction in other tissues. Because chronic exposure to many retinoids results in the deposition and accumulation of both parent compound and metabolites in the liver, these results should suggest caution in the design of clinical chemoprevention trials with this class of compounds.

Cell population kinetics and ploidy rate of early focal lesions during hepatocarcinogenesis in the rat.

We have studied the changes in cell population kinetics and DNA-content of cycling parenchymal cells during the very early steps of rat hepatocarcinogenesis in Faber's protocol. Adult rats were initiated by a single dose of diethylnitrosamine (DENA, 200 mg kg-1), followed 2 weeks later by a 2-week diet of 0.03% 2-acetylaminofluorene (2-AAF) as selection phase. In the middle of selection time, a single necrogenic dose of carbon tetrachloride (CCl4, 2 ml kg-1) was administered by gavage. Twenty four hours thereafter, radiolabelled thymidine (3H-TdR, 1.5 microCi g-1) was given by repeated injections during 24 h. An emergence of small, pyroninophilic ('tigroid') foci was observed at the second, fifth and eighth days after the proliferative stimulus. The focal putative precancerous cells presented a significant higher labelling index (L1) than the non-affected parenchymal cells for all exposure times. However, the labelling intensity decreased from the second to the eighth day after CCl4, suggesting a dilution of the radiolabelled DNA by repeated divisions within the foci. The nuclei of the same foci were analysed for DNA-content by feulgen microdensitometry on neighbouring sections. A gradual reduction of nuclear DNA-content was observed in 66% of the foci at the fifth day and in 100% of foci at the eight day, as compared to surrounding tissue and untreated animals, where labelling and DNA-content remain in the same ratio.

Modifications of Hepatic Microsomal 9-Oxidation ofN-2-Fluorenylacetamide in Response to Gonadectomy and Treatment of Rats with Phenobarbital or Diethylnitrosamine

1. The hepatic microsomal 9-hydroxylation of N-2-fluorenylacetamide (2-FAA) is greater in the presence of male, or absence of female, hormones. Thus, 9-hydroxy-2-FAA was the major microsomal metabolite of male rats, which formed 6-fold greater amounts than did female rats. One week after gonadectomy, the amount of 9-hydroxy-2-FAA formed by male rats was decreased by 61%, whereas that formed by female rats was increased 1.4-fold. 2. Treatment of rats with phenobarbital (PB) increased 2- to 3-fold the capacities of hepatic microsomes of both sexes (normal and gonadectomized) for 9-hydroxylation of 2-FAA. 3. Hepatic microsomes of male rats also had greater capacities to form 9-oxo-2-FAA, the metabolite of 9-hydroxy-2-FAA, and 6-hydroxy-2-FAA, a newly identified microsomal metabolite of 2-FAA. These metabolites were also decreased by orchidectomy and induced by PB. 4. 9-Hydroxy-2-FAA was a poor substrate for hepatic microsomal UDP-glucuronyltransferase, and conjugation was not induced by treatment of rats with PB. This indicated retention of 9-hydroxy-2-FAA in the liver and/or further metabolism (e.g. to 9-oxo-2-FAA). 5. The formation of 9-oxo-2-FAA from 2-FAA or 9-hydroxy-2-FAA was increased (1.5-fold) two weeks after treatment of male rats with a single i.p. dose of diethylnitrosamine (200 mg/kg), an initiator of hepatocarcinogenesis. 6. Based on the data we suggest that 9-oxidized metabolites of 2-FAA, the preferential formation of which coincides with the susceptibility of the rat to hepatocarcinogenesis, are promoters.

ICRC mouse with congenital mega-esophagus as a model to study esophageal tumorigenesis.

ICRC mouse, an inbred strain, developed at the Cancer Research Institute, Bombay, exhibits mega-esophagus with markedly hyperplastic mucosa. Diethylnitrosamine (DEN) when given in drinking water at the dose of 4 mg/kg body weight/day, induced esophageal papillomas consistently in 100% of the animals, in a short period of 12 weeks. Further, tumors were produced, even at a very low cumulative dose of 28 mg/kg body weight. Development of the esophageal papillomas was dose dependent. DEN even induces esophageal tumors transplacentally in the ICRC F1 progeny. Tobacco acts predominantly as a promoter in this system. ICRC mouse thus provides a much needed animal model to study esophageal tumorigenesis, including the two-stage carcinogenesis. An interesting feature of the study is that initiation could be induced by exposure to low doses of DEN in the intra-uterine life. Tumors develop in such F1 animals only if they are fed tobacco, a predominant promoter, post-natally.

Comparison of three rat liver foci bioassays-incidence of preneoplastic foci initiated by diethylnitrosamine

Three rat liver foci bioassays have been compared with respect to their sensitivity by the histochemical demonstration of preneoplastic foci, and by the biochemical determination of alterations in enzyme activities of serum indicating hepatotoxicity. We studied the initiation/promotion schedules according to Oesterle and Deml (A), and according to Pereira (B, Broad Spectrum Protocol), and the initiation/selection protocol according to Tatematsu et al. (C), with diethylnitrosamine (DEN), given as a single initiating dose of 10 and 30 mg/kg body wt respectively. With all schedules Sprague-Dawley rats, either females, 3 weeks old (A), or males, 6 weeks old (B, C) were used. For promotion polychlorinated biphenyls (A) or phenobarbital (B) were administered. Selection was performed with 2-acetylaminofluorene (C). The rats in schemes (B) and (C) underwent partial hepatectomy one day prior to initiation. The number and total area of foci deficient in adenosine-5'-triphosphatase (ATPase) and positive in gamma-glutamyltranspeptidase (GGTase) was evaluated. In the complete schedule with 30 mg of DEN in system (A) foci incidence exceeded that of the other systems by about 7-fold (ATPase) and 2-fold (GGTase) respectively. The lower dose of DEN and all control experiments resulted in a respective lower foci yield. With scheme (C), but not with schemes (A) and (B), e.g. serum fructose-1.6-bisphosphatase and alkaline phosphatase were increased, suggesting liver cell damage. Thus tested with DEN, scheme (A) is most sensitive and causes a low impairment of animals' welfare.

A carcinogenesis- and tumorigenesis-associated rat fetal protein: an immuno-histochemical and immuno-biochemical study utilizing a new monoclonal antibody, MOFP.

An oncofetal protein (OFP), which is a potential marker for carcinogenesis and tumorigenesis, was evaluated with monoclonal antibodies shown to be specific for the antigen. Treatment of partially hepatectomized rats with a single non-necrogenic dose of diethylnitrosamine induced OFP in the liver. Its concentration, as measured by a dual immuno/bioassay, increased steadily over a 5-week period of observation before reaching a constant level. Immunohistochemical localization of OFP in liver sections from rats treated with N-nitroso-N-diethyl-nitrosamine showed that the factor was primarily localized to the cell cytoplasm in cells of most of the altered hepatic foci although some of this shedding antigen was also extracellular. Monoclonal antibody 17-1A specific for 17-1A antigen, an established surface marker for adenocarcinomas of the gastrointestinal tract, showed a similar distribution in liver from the carcinogen-treated rats, but localized to the cell membrane and cytoplasm. Scattered cells surrounding the altered hepatic foci were also positive for both monoclonal antibodies. Immunolocalization studies showed fetal rat liver and hepatoma were positive for OFP but adult normal or regenerating liver was negative. It was not detected in cells which morphologically could be classified as oval cells. As assessed by immuno/bioassay, the OFP released to the peripheral blood (plasma) of hepato-carcinogen-treated rats increased for 3 weeks, before undergoing a transitory decrease. Circulating antibodies specific for the factor were detected in the blood around 3-5 weeks post-treatment. Development of Western blots of the OFP with antiphosphotyrosine IgG indicates that the marker protein contains phosphotyrosine.

Dose-response relationship of diethylnitrosamine-initiated tumors in neonatal balb/c mice: effect of phenobarbital promotion.

The dose-response of diethylnitrosamine (DENA) initiation of hepatocarcinogenesis was determined in infant Balb/c male mice with and without subsequent phenobarbital treatment. Male Balb/c mice received a single intraperitoneal injection of DENA (0, 2.5, 10.0, 25.0 or 50.0 micrograms/gbw) in saline on day 15 of age. Ninety mice were treated at each dose level. At weaning, mice received either deionized drinking water (45 mice per group) or deionized drinking water containing 500 mg/L sodium phenobarbital (PB) (45 mice per group). Mice from each group were sacrificed 12, 24, and 40 weeks post-weaning. Liver and lung tumors were found in DENA-only-treated and DENA + PB-treated mice. In DENA-only-treated mice, the incidence and number of hepatic adenomas were similar (not dose-dependent) at DENA doses of 10, 25, and 50 micrograms/gbw at each of the 3 sampling times. DENA-only-treated mice did display a time-related increase in hepatic adenoma incidence and number at each dose. In PB-treated mice, the hepatic adenoma number was dependent upon the dose of DENA between 2.5 and 50 micrograms/gbw. PB treatment following DENA administration resulted in a decrease in the time required for the detection of hepatic adenomas and increased the number of hepatic adenomas at most sampling times compared to the mice that received DENA only. Hepatocellular carcinomas (HPC) were detected in mice receiving the highest DENA doses (25 and 50 micrograms/gbw). PB treatment increased the number and incidence of HPC and decreased the time of first detection of HPC.(ABSTRACT TRUNCATED AT 250 WORDS)

C-H-ras and c-K-ras gene hypomethylation in the livers and hepatomas of rats fed methyl-deficient, amino acid-defined diets.

The extent of methylation of the c-H-ras and c-K-ras oncogenes was compared in neoplastic and preneoplastic livers of rats fed one of several methyl-deficient, amino acid-defined diets for 18 months, with or without a preceding initiating dose of diethylnitrosamine (DEN). The restriction endonucleases MspI, HpaII, HhaI, PaeR71 and XhoI were used for studying the extent and pattern of DNA methylation. The results indicated that both c-H-ras and c-K-ras oncogenes were hypomethylated in all DNA samples derived from both neoplastic and preneoplastic livers of rats fed any of the methyl-deficient diets used, regardless of whether or not the rats had received an initiating dose of DEN. It thus appears that dietary methyl deficiency does indeed lead to hypomethylation of ras genes in the DNAs of the resulting tumors. However, the significance of this hypomethylation in the tumorigenic process is not clearly understood.

The role of N-sulfation in the N-hydroxy-2-acetylaminofluorene-mediated outgrowth of diethylnitrosamine-initiated hepatocytes to gamma-glutamyltranspeptidase-positive foci in male rat liver.

The ability of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to promote the appearance of gamma-glutamyltranspeptidase-positive (GGT+) foci initiated by diethylnitrosamine (DEN) was studied in a slightly modified Solt and Farber protocol. This protocol consisted of the following treatments: initiation with a single dose of DEN followed by selection/promotion with several non-necrogenic doses of N-OH-AAF and partial hepatectomy. Treatment with N-OH-AAF resulted in a 25-fold increase of the liver volume occupied by GGT+ cells as compared to controls. The role of N-sulfation of N-OH-AAF in the GGT+ foci-selecting activity of N-OH-AAF was studied using the sulfotransferase inhibitor pentachlorophenol (PCP). Inhibition of the N-sulfation pathway with PCP during selection with N-OH-AAF resulted in a greater than 80% decrease in the volume occupied by GGT+ cells, without effects on the number of GGT+ foci generated with this protocol. Also, PCP reduced the number of so called oval and bile duct cells generated by the N-OH-AAF/partial hepatectomy treatment. It is concluded that N-sulfation of N-OH-AAF is responsible for the N-OH-AAF-mediated outgrowth of DEN-initiated hepatocytes to preneoplastic GGT+ foci.

Carcinogenicity of diethylnitrosamine in vitamin‐A‐deficient mice

The effect of suboptimal levels of dietary vitamin A on diethylnitrosamine (DEN) carcinogenesis was studied in BALB/c and Swiss mice. Two different dietary regimens were employed to induce vitamin A deficiency and DEN was administered by gavage at 2 dose levels: 0.6 mg/kg as a single dose and 200 mg/kg in 4 divided doses. Shark liver oil (SLO) which was the main source of vitamin A in the standard diet, was deleted in one regimen and reduced to 25% in the other. The mice maintained on the former diet were given a high dose of DEN and those on the latter diet received a low dose. In both strains the deficient mice had a greater tumour incidence than those on standard diet with a marginal reduction in the latent period. At the low level of DEN there was shift in organotrophy, i.e. from liver in controls to lung in the vitamin-A-deficient mice of BALB/c strain. With the higher dose, lung adenomas predominated in deficient as well as control groups in both the strains. Forestomach carcinomas appeared in deficient mice and not in the controls.