Single Dose Of Dimethylnitrosamine Research Articles

Harman and Norharman Suppressed but NaNO2 Enhanced the Development of Preneoplastic Liver Cell Foci in 2-Amino-3,8-Dimethylimidazo[4,5-f]Quinoxaline (MeIQx)-Treated Rats

The modifying potentials of two heterocyclic amines, harman and norharman, as well as sodium nitrite (NaNO2), on 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced rat liver carcinogenesis were investigated using a medium-term liver bioassay system. Harman (500 ppm in diet), norharman (500 ppm in diet) or NaNO2 (0.1% in drinking water) were given with or without MeIQx (300 ppm in diet) for 6 weeks after initiation with a single dose of dimethylnitrosamine (DEN). The appearance of MeIQx-induced glutathione S-transferase placental form positive foci in the liver was significantly decreased in the harman and norharman cases (p< 0.001), but it was significantly increased by NaNO2 (p< 0.001). These chemicals, however, did not modify DEN-liver carcinogenesis in the absence of MeIQx. Neither harman nor norharman affected mRNA expression of CYP1A1 and 1A2 in the liver, with or without MeIQx administration, whereas NaNO2 significantly enhanced CYP1A1 mRNA levels together with MeIQx.

Expression of type I and type III collagens during the course of dimethylnitrosamine-induced hepatic fibrosis in rats.

We wished to clarify the mechanisms that account for the increase in hepatic collagen accumulation during hepatic fibrosis. The gene expression of type I and type III procollagens and matrix metalloproteinase-1 (MMP-1) was measured by Northern blot analysis; immunolocalization of both types of collagen was estimated by indirect immunohistochemical assay; and the hepatic content of collagen and malondialdehyde (MDA), a product of lipid peroxidation, were assayed in hepatic fibrosis induced in rats with a single dose of dimethylnitrosamine (DMN). During the experimental period, more type I procollagen mRNA was found than type III procollagen mRNA. The immunoreactive intensity of type I collagen was greater in necrotic areas near central veins 3 days after DMN treatment than it was on day 9, whereas the type III collagen immunodeposition for the latter period of the hepatic fibrosis was stronger than it was on day 3. As compared with controls, hepatic collagen content increased significantly after 3 days and continued, increasing gradually, as did type I and III procollagen mRNA levels. On day 14, fibrosis was greatest and both types of procollagen gene expression were at their highest, and type I and III procollagen mRNA levels and hepatic collagen content increased as the dosage of DMN was raised. MMP-1 mRNA levels increased early in hepatic fibrogenesis, and increased on day 14 when DMN dosages were low. Hepatic MDA levels increased rapidly for 3 days after DMN treatment, remaining significantly higher than control values and showing a significant increase even in response to low DMN doses on day 14. Our results suggested that fibrotic liver collagen content may make its first notable increase due in part to the balance between type I collagen and MMP-1 expression rates. Also, lipid peroxidation may be important in the mechanism of hepatofibrogenesis.

Morphological and immunohistochemical characteristics of dimethylnitrosamine-induced malignant mesenchymal renal tumor in F-344 rats

Mesenchymal renal tumors in F-344 newborn rats were induced by a single dose of dimethylnitrosamine. The induced tumors were successfully transplanted into adult rats under the renal capsule. Neither the primary nor the transplanted neoplasms from various generations of grafts changed their morphological features during the tumor passage, having the same cellularity with high mitotic activity and the tendency to invade the host kidney rapidly. On the basis of lectin histochemistry and immunohistology, the tumor proved to be a mesenchymal neoplasm without any obvious capacity of the proliferating cells to differentiate into any well-known organoid element normally found in mature renal parenchyma. However, the proliferating neoplastic cells were found to have a strong vimentin positivity with desmin expression. Ultrastructurally, myofilaments with attachment bodies characteristic of smooth muscle cells were generally present in various amounts in many tumor cells. In addition, on the basis of the physiological data and on kidney/tumor renin activity obtained, it is interesting to note that the tumor-graft-invaded kidneys retained their enzyme activity, despite the obvious loss of renal tissue including glomeruli. However, the immunohistochemical findings with anti-renin antibody have clearly shown that this is not due to a renin-producing tumor but rather to the surviving (probably) non-neoplastic arterioles retaining the capacity to produce renin. Although these arterioles have mostly been found next to necrotic areas, commonly occurring in dimethylnitrosamine-induced transplantable renal tumors, the question of a possible physiological role of renin in tumor necrosis or in angiogenesis has remained open.

Investigation into the effect of DHEA on renal carcinogenesis induced in the rat by a single dose of DMN

Because long-term oral administration of the adrenal steroid dehydroepiandrosterone (DHEA) has previously been shown to inhibit the development of spontaneous breast cancer and chemically induced lung, colon, skin, and liver tumors in various mouse and rat strains, the effect of DHEA on the development of rat kidney tumors by a single dose of 30 mg/kg dimethylnitrosamine (DMN) was tested. DHEA was administered in the diet for a 26-week period commencing 2 weeks after DMN treatment. DHEA administration caused a reduction in body weight gain in accordance with its known antiobesity activity. However, it did not exert any inhibitory effect on either renal mesenchymal or cortical epithelial tumor induction by DMN, nor did it alter the average survival time. There was a statistically significant increase in the incidence of renal adenocarcinomas in the DHEA-treated group but not of renal adenomas. The results were discussed in relation to the mesodermal origin of kidney and the potency of single-dose systems of experimental cancer induction.

Glucagon and insulin for the treatment of hepatic failure in dimethylnitrosamine-intoxicated rats.

When rats received dimethylnitrosamine every 24 h until death, plus hormone treatment after the first 24 h, the survival was enhanced between 100 and 140 h compared with the control rats, with attenuated derangements of prothrombin time and serum albumin levels at 120 h. In rats given a single dose of dimethylnitrosamine, hepatic DNA synthesis peaked at 48 h. The synthesis was increased after hormone treatment when started immediately, but not when delayed for 24 h. Hormone treatment for 3 days starting 24 h after a single dose of dimethylnitrosamine produced a rapid normalization of decreased hepatic protein content on day 9, although hepatic DNA content was not affected. These results suggest that this treatment is effective for hepatic failure, and the promotion of restoration of liver function is a contributing factor to its effect, in addition to the stimulation of hepatocyte proliferation.

High frequency, single-dose model of renal adenoma/carcinoma induction using dimethylnitrosamine in Crl:(W)BR rats.

Following discovery that the type of kidney neoplasm induced in protein-deprived Wistar rats by a single dose of dimethylnitrosamine (DMN) was age-dependent, this study aimed to refine the system in order to develop a high frequency model for the induction of cortical epithelial tumors with low mesenchymal tumor incidence. Using outbred female Crl:(W)BR (Charles River Wistar) rats, DMN at a dose of 30 mg/kg was administered at 9-10 weeks of age following a 5-day period of high-carbohydrate/no-protein diet. From a total of 49 rats, 43 survived the early toxicity and 91% of these developed renal tumors. Mesenchymal tumors were present in only 9% of the tumor-bearing animals. In contrast, 70% of the rats developed epithelial tumors of the tubules classifiable on a size and histological basis as adenocarcinomas/carcinomas in response to DMN. A further 21% of rats had smaller proliferative lesions designated as adenomas, making the total cortical epithelial tumor incidence in excess of 90%. The malignant potential of the epithelial tumors was underscored by the presence of metastatic invasion, mainly involving the lungs, in 15% of the tumor-bearing rats. Metastatic behavior correlated with progressive growth of the carcinomas over a period of time exceeding 1.5 years to dimensions usually exceeding 2.9 cm diameter. Of the tumors approaching or exceeding this size, the metastatic rate was almost 50%. Thus, the administration of DMN to 65-70 days old, protein-deprived Wistar rats provides a potent, single dose model for the study of renal epithelial carcinogenesis with insignificant mesenchymal tumor induction and without the continuing toxicity which perturbs regimens based on repeated or continuous exposures.

Cytogenetic changes during the early stages of liver carcinogenesis in Chinese hamster: An in vivo-in vitro comparison

Cytogenetic changes were investigated during the early stages of hepatic adenocarcinoma development in Chinese hamsters injected with a single dose of dimethylnitrosamine (DMN). An in vivo-in vitro comparison was made from 7 to 35 weeks after injection. A partial hepatectomy was used to stimulate mitosis for in vivo analysis, and the excised liver, grown to the primary culture stage, was used for chromosome analysis in vitro. Aneuploidy, tetraploidy, and chromosome aberrations increased significantly in the hepatic cells of DMN-treated animals in vivo, with no significant change over the 7- to 35-week period. No differences, however, could be detected between the primary cultures of control and DMN-treated animals because of an inherent tendency for all cultures to develop aneuploid stem lines at an early stage in culture. A preferential involvement of chromosome #6 in the single trisomic state was demonstrated in vitro and to a minor extent in vivo. The relevance of increased aneuploidy in early carcinogenesis and the differences between the in vivo and in vitro results are discussed.

Production of Kidney Tumors in Rats With Low Dose of Dimethylnitrosamine After Partial Hepatectomy

The occurrence of kidney tumors in inbred Wistar rats as a result of dimethylnitrosamine (DMN) administration at different intervals after partial hepatectomy was studied. The schedule for DMN administration was determined on the basis of the levels of liver dimethylnitrosamine demethylase (DMN-d) at different intervals after the operation. DMN-d was 62% of the control values at 24 hours. 72% at 72 hours, and 96% at 92 hours after the operation. At these intervals a single dose of DMN (10 mg/kg body wt) was given to partially hepatectomized animals and to untreated controls. At termination, when the animals were 94 weeks old, no kidney tumors were found in the control animals, whereas 23 of 54 animals (43%) that had been given injections of DMN 24 hours after partial hepatectomy developed kidney tumors. Kidney tumor incidence was 28 or 12%, respectively, when the carcinogen was administered 72 of 92 hours after the operation. The kidney tumor incidence and the activity of liver DMN-d were inversely related.

Induction of Kidney Tumors by a Single Dose of Dimethylnitrosamine: Dose Response and Influence of Diet and Benzo- (a)pyrene Pretreatment

Induction of Kidney Tumors by a Single Dose of Dimethylnitrosamine: Dose Response and Influence of Diet and Benzo- (a)pyrene Pretreatment

Alkylation of DNA and tissue specificity in nitrosamine carcinogenesis.

A peculiarity of nitrosamines is the high degree of cell and organ specificity in inducing tumors. There is substantial evidence that the initiation of the carcinogenesis process by carcinogens of this group is linked to the metabolic competence of the target tissue or cell to convert these carcinogens into mutagenic metabolites and to the binding of those metabolites to cellular DNA. Alkylation occurs in the DNA at the N-1, N-3, and N-7 positions of adenine; the N-3, N-7, and O6 of guanine; the N-3, and O2 of cytosine; and the N-3, O4, and O2 of thymine; and the phosphate groups. The initial proportion of each DNA adduct depends upon the alkylating agent used. The various DNA adducts are lost to a variable extent from DNA in vivo by spontaneous release of bases and/or by specific DNA repair processes. Studies conducted in vitro and vivo indicate that alkylation at the oxygen atoms of DNA bases is more critical than alkylation at other positions in the mutagenesis and carcinogenesis induced by N-nitroso compounds. In particular, tissues in which tumors occur more frequently after a pulse dose of nitrosamine are those in which O6-alkylguanine persists longest in DNA, presumably resulting in an increased probability that a miscoding event (mutation) will take place during DNA synthesis. The more rapid removal of O6-methylguanine from the DNA of liver (as compared with extrahepatic tissues) of rats has been associated with the absence of tumor production in this organ by a single dose of dimethylnitrosamine; however, a significant incidence of liver tumors is observed if the same dose is given 24 hr after partial hepatectomy, and tumors are induced by such a dose of dimethylnitrosamine in the liver of hamsters, which has a low capacity to remove O6-methylguanine from its DNA. These data also indicate that the rate of disappearance of 7-methylguanine from the liver or extrahepatic tissues is independent of the dose of dimethylnitrosamine; whereas O6-methylguanine is lost from DNA more rapidly after a low dose of this nitrosamine. It has been shown that in liver the removal of O6-methylguanine but not other DNA adducts, from DNA can be affected by pretreating the animals with N-nitroso compounds. The modulation of DNA repair processes observed after a single dose and after chronic treatment with nitrosamines is discussed in relation to the tissue-specific carcinogenic effect of this group of carcinogens.

Induction of kidney tumours by a single dose of dimethylnitrosamine: dose response and influence of diet and benzo(a)pyrene pretreatment.

Seven days on a protein-free diet increases the susceptibility of rats to the action of DMN as a renal carcinogen. The dose response for the induction of kidney tumours by a single dose of dimethylnitrosamine (DMN) in these rats is reported. The first tumour was not found until 28 weeks after the dose. At 100 weeks the incidence ranged from 22.5% at the lowest dose (20 mg/kg) to 97% at the highest dose (60 mg/kg). The incidence in probits at any time between 50 and 100 weeks was linearly related to the log dose. Epithelial and mesenchymal tumours were produced in an approximate ratio of 2:1. The protein-free diet alters the rate of metabolism of DMN in the rat, and increases the alkylation of nucleic acids by this carcinogen in the kidney. Further treatment of the rat with benzo(a)pyrene can reverse, to some extent, the change in metabolism, but does not reverse the change in alkylation. It is shown that the change in kidney-tumour incidence produced by the change in diet, and by the treatment with benzo(a)pyrene, corresponds to the changes these treatments produce in the alkylation of kidney DNA by the carcinogen.

Induction of kidney tumours by a single dose of dimethylnitrosamine: dose response and influence of diet and benzo(a)pyrene pretreatment

Induction of kidney tumours by a single dose of dimethylnitrosamine: dose response and influence of diet and benzo(a)pyrene pretreatment

The effects of a single dose of dimethylnitrosamine in the Chinese hamster and the persistence of DNA alkylation products in selected tissues

The acute toxicity and carcinogenicity of dimethylnitrosamine (DMN) have been examined in the Chinese hamster (Cricetulus griseus). A single dose of 20 mg/kg or less was not acutely lethal but at 30 mg/kg only a few animals survived. Liver tumours were found in all of the animals surviving two doses of 20 mg/kg and so far, 25 months after treatment, in 80% of the animals given a single dose of 20 mg/kg. Altering the diet to sucrose lumps and 20% glucose in the drinking water ad libitum for 3 days prior to DMN treatment had only a slight effect on acute toxicity and carcinogenicity. In biochemical studies, DNA was extracted from several tissues of animals killed 9 h after DMN administration (20 mg/kg). The amounts of normal and methylated purines were determined after mild acid hydrolysis and Sephadex G-10 chromatography. The highest level of methylated bases was found in the DNA of the liver. Measurement of the persistence of methylated guanines showed that liver, kidney, and lung were able to remove the major product 7-methylguanine from DNA while none of these tissues had any capacity to remove the pro-mutagenic lesion O6-methylguanine. These results are discussed in relation to the mechanism of action and tissue specificity of alkylating carcinogens in this and other experimental animal systems.

DNA repair synthesis in primary cultures of kidneys from BALB/c and C3H mice treated with dimethylnitrosamine

A combined 'in vivo--in vitro' autoradiographic method was employed to examine the DNA repair induced in the kidney by a single dose of dimethylnitrosamine (DMNA). Unscheduled DNA synthesis was found to be dose-dependent in primary kidney cultures of DMNA-treated mice, and practically not detectable in controls. Its amount was positively correlated with the different susceptibility of C3H and BALB/c mice to kidney tumor induction by DMNA. This experimental model appears sensitive and able to provide repeatable results. It may be useful to detect the organotropic activity of a carcinogen toward the kidney.

The phosphorylation of rat liver ribosomes following administration of dimethylnitrosamine

Following a single dose of dimethylnitrosamine (DMNA) the phosphorylation of rat liver ribosomes was strongly enhanced. The incorporation of [ 32P]phosphate into the 40 S subunit was stimulated 5- and 9-fold 2 and 5 hr, respectively, after administration of the drug. The phosphorylation of the large ribosomal subunit was not affected. The enhanced phosphorylation of the small subunit was due to a 20-fold stimulated incorporation of phosphate into ribosomal protein S6, the only one affected by DMNA.

Tumor frequency and characteristics after a single dose of dimethylnitrosamine or diethylnitrosamine in partially hepatectomized rats

Two groups of 75 rats were injected intraperitoneally with one single dose of 9 mg/kg DMNA and 120 mg/kg DENA, respectively, 28 h after partial hepatectomy. Control groups of non-hepatectomized rats received one single injection of DMNA or DENA at doses of 20 mg/kg and 200 mg/kg, respectively, which were found to be equitoxic to the doses administered to partially hepatectomized rats. One control group of partially hepatectomized rats was injected with saline. Tumors developed in the partially hepatectomized rats injected with nitrosamines with the following frequency: for DMNA: 13% in the liver, 13% in kidney, 4% in lung, 4% at the surgical scar, 9% in hematopoietic tissue and 13% in other organs; for DENA: 11% in liver, 27% in the kidney, 2% at the surgical scar, 2% in hematopoietic tissue, and 11% in other organs. The histological characteristics of these tumors are described. Statistical analysis of the results shows that no significant difference exists in the overall tumor incidence induced by DMNA and DENA and that the difference between liver and kidney tumors induced by DENA is significant, whereas the difference between DMNA- and DENA-induced kidney tumors is less significant. When analyzed by t-test, our results show that death from tumors occurred at an earlier time after DENA than after DMNA treatment and that liver tumors appeared at a significantly earlier time after DENA than after DMNA treatment. Tumors appearing in the same experimental group display similar mean induction times.

Protein chain initiation in vitro by liver cell components from DMNA-treated rats

The mechanism of inhibition of protein synthesis in rat liver after dimethyl-nitrosamine (DMNA) administration was studied at the level of peptide-chain initiation by use of initiation-dependent amino acid incorporating systems. Ribosomal monomers, poly(A)-containing RNA from monosemes and polysemes, and crude initiation factors from microsomes were prepared 2 h after a single dose of DMNA (75 mg/kg), and their activities in the production of new protein chains determined under conditions of nearly linear response. Monosomes and crude initiation factors from DMNA-treated rats were at least as active as those from controls. Preparations of poly(A)-containing RNA had a consistently higher template activity when prepared from polysomes instead of monosomes. However, in neither case was there any loss of activity due to the DMNA treatment. The poly(A)-containing RNA was methylated by DMNA to about the same extent as the 18S and 28S rRNA. The methylation was consistently somewhat higher in the RNA preparations from monosomes than in those from polysomes.

The dose response for the induction of kidney tumours in the rat by a single dose of dimethylnitrosamine.

The dose response for the induction of kidney tumours in the rat by a single dose of dimethylnitrosamine

Increased carcinogenic action of dimethylnitrosamine after prior administration of carbon tetrachloride.

Rats were given a single dose of dimethylnitrosamine (DMN, 20 mg/kg body weight) alone or 42 or 60 hours after a non-lethal hepatotoxic dose of carbon tetrachloride (CC1(4)) and killed 12 months later. DMN alone produced no tumours in the kidney and a few in the liver, but when given 42 hours after CC1(4), tumours formed in the kidneys and the number in the liver was increased. When given after 60 hours, the incidence of kidney tumours was less but that of liver tumours was further increased. A larger dose of DMN (40 mg/kg) was tolerated 42 hours after CC1(4) and enhanced the number of kidney and liver tumours, the latter apparently due to an increased proportion of cholangiomata. Numerous small focal proliferations of atypical liver cells and of bile duct epithelium were observed after treatment with DMN. The incidence of these lesions in the different experimental treatments varied in a similar manner to the liver tumours.

Cellular injury and carcinogenesis. The effect of a protein-free high-carbohydrate diet on the metabolism of dimethylnitrosamine in the rat.

1. Rats fed on a protein-free high-carbohydrate diet for 7 days metabolized dimethylnitrosamine at only 55% the rate of rats fed on a commercial diet. 2. Dimethylnitrosamine was metabolized by liver slices from rats fed on the protein-free diet at less than half the rate attained by slices from rats fed on a commercial diet. But kidney slices from these rats metabolized dimethylnitrosamine at the same rate as kidney slices from rats on a commercial diet. 3. Methylation by dimethylnitrosamine (70mg/kg body wt.) of N-7 of guanine of the liver RNA and DNA of rats fed on a protein-free diet was only slightly higher than in rats fed on a normal diet given 27mg/kg body wt. In contrast, the methylation by dimethylnitrosamine of guanine in kidney nucleic acids of these rats was three times that in the rats fed on a normal diet. 4. In rats fed on a protein-free diet the incidence of kidney tumours produced by a single dose of dimethylnitrosamine is increased.