Dose Of N-nitrosodiethylamine Research Articles

Mechanisms of hepatoprotective effects of white and black garlic extracts against preneoplastic lesions induced by N-nitrosodiethylamine in rats

Introduction: Garlic has many pharmacological properties such as antibacterial, anti-inflammatory, and antioxidant activities. This study aimed to investigate the possible mechanisms of the hepatoprotective effect of white garlic extract (WGE) and black garlic extract (BGE) against preneoplastic lesions induced by N-nitrosodiethylamine (NDEA) in rats. Methods: Forty-two rats were randomly distributed into six equal groups. Oral administration of WGE and BGE began 3 weeks before injection of NDEA. Group 1 was kept as the negative control, while the other groups were injected by a single intraperitoneal dose of NDEA in the 3rd week, followed by 2 subcutaneous injections/week of CCl4 till six weeks to induce preneoplastic lesions. Group 2 kept positive control and groups 3, 4, 5, and 6 were given WGE and BGE each of them at 250 and 500 mg/kg, respectively, for six weeks from the beginning. Serum liver enzymes, total protein, albumin (Alb), total bilirubin (TBil), and antioxidant enzymes were measured. Malondialdehyde (MDA), glutathione (GSH), tumor biomarkers, along with the content of 8-hydroxy-2-deoxyguanosine (8-OHdG) in liver DNA were estimated and liver histopathology was performed. Results: WGE and BGE significantly decreased the serum liver enzymes, TBil, tumor biomarkers, lipid peroxidation but increased total protein levels. The extracts significantly increased antioxidant enzymes, decreased 8-OHdG content in liver DNA, and alleviated histopathological lesions in the liver. Conclusion: The results affirm the hepatoprotective effect of WGE and BGE against NDEA-induced preneoplastic lesions in rats. This effect may be due to inhibition of lipid peroxidation, reduction of tumor biomarkers, enhancement of antioxidant enzymes, or reduction of 8-OHdG content in liver DNA.

Silibinin Inhibits the Hepatocellular Carcinoma in NDEA-Induced Rodent Carcinogenesis Model: An Evaluation through Biochemical and Bio-Structural Parameters

The present study was aimed to investigate the chemopreventive potential of Silibinin (SIB) against N-nitrosodiethylamine (NDEA)-induced hepatocellular carcinoma (HCC) in wistar rats. Thirty experimental animals were subjected to partial hepatectomy (PH) and after 24 hours of stabilization period a single dose of NDEA (100 mg/kg b.w., i.p) was administered to each animal followed by CCl4 (1 ml/kg b.w., s.c.). The effect of SIB (25 and 50 mg/kg/day, i.p.) on NDEA-induced HCC was determined after 2 weeks of treatment (6th to 8th week after PH, in the promotional stage of tumor development). NDEA treatment to rats resulted in significant decrease in body weight and increase in liver weight along with levels of transaminases, γ-glutamyl transferase, lipoprotein, glycoproteins. Hepatic and serum malondialdehyde content, enzymatic and non-enzymatic antioxidant levels were also altered in HCC bearing animals without treatment. Bio-structural components (such as amide bands of proteins, symmetric phosphate stretching of nucleic acids, methylene chains in membrane lipids, methyl to methylene ratio of carbohydrates and proteins etc.) were marked in NDEA-treated groups of animals by analysing changes in the position and intensities of the peaks in Fourier transform infrared spectroscopy (FTIR). Immunohistochemical staining of Ki67 and histopathological analysis were also carried out in order to support the study. SIB treatment could attenuate NDEA-induced hepatocarcinogenesis by improving the biochemical and bio-structural changes of the hepatic tissue near normal levels in a dose dependent manner. Taken together, this study reveals that SIB may have potential as a multi-functional drug candidate for cancer therapy.

Protective effect of chokeberry on chemical-induced oxidative stress in rat

Male Wistar rats were treated with chokeberry juice per os, 10 mL/kg/day, for 28 days and a single intraperitoneal (i.p.) dose of N-nitrosodiethylamine (NDEA), 150 mg/kg, or carbon tetrachloride (CCl(4)), 2 ml/kg. The level of hepatic microsomal lipid peroxidation, expressed as thiobarbituric acid reactive substances (TBARS), was increased in animals dosed with NDEA and CCl(4). Juice pretreatment resulted in a significant decrease in TBARS by 53% and 92%, respectively. In rats administered juice alone, 50% decrease in TBARS was noted. The activities of all antioxidant enzymes were decreased in the liver of rats administered either toxicant by 29%-52% as compared to controls. Juice pretreatment resulted in an increase in the activity of catalase, glutathione peroxidase and glutathione reductase by 117%, 56% and 44%, respectively, only in rats challenged with NDEA. Although no response of plasma protein carbonyls to both toxicants was observed, the pretreatment with juice caused a 55% decrease of this parameter in CCl(4)-dosed rats. DNA damage in blood leukocytes induced by either toxicant was slightly reduced, by 24%, in the rats pretreated with juice and administered NDEA. The results of the study showed that pretreatment with chokeberry juice confers some protection against chemical-induced oxidative stress.

Tumor Promotion in Liver of Mice with a Conditional Cx26 Knockout

Connexin (Cx) 26 and 32 are the major gap junction proteins in liver. We recently demonstrated that Cx32 is essential for phenobarbital (PB)-mediated tumor promotion in mouse liver. To investigate whether Cx26 plays a similar role, an initiation-promotion experiment was conducted using mice with a liver-specific knockout of Cx26. Control and Cx26-deficient mice were injected a single dose of N-nitrosodiethylamine (DEN, 90 microg/g b.wt.) at 6 weeks of age and groups of mice were subsequently kept on a PB (0.05%) containing or control diet for 35 weeks. At the end of the experiment, the carcinogenic response in the liver was monitored. Mice from PB treatment groups showed strongly increased liver weights compared with mice treated with DEN alone, which was mostly due to a much higher tumor burden. The tumor response in PB-treated mice of both strains was quite similar, but the number of smaller tumors and of enzyme-altered neoplastic lesions was somewhat larger in PB-treated Cx26 knockout (Cx26 KO) compared with wild-type mice, whereas the volume fraction of enzyme-altered lesions was slightly reduced in PB-treated Cx26-deficient mice. There was no significant difference in tumor prevalence between Cx26 KO and wild-type mice. Altogether our present data show that elimination of Cx26 has only minor effects on chemically induced mouse hepatocarcinogenesis, in striking contrast to the effects seen in Cx32 KO mice.

Enhancement of N-Nitrosodiethylamine-lnitiated Hepatocarcinogenesis by Phenytoin in Male F344/NCr Rats at a Dose Causing Maximal Induction of CYP2B

The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of male F344/NCr rats initiated with a single i.p. dose of N-nitrosodiethylamine (75 mg/kg b.w.) was studied. Beginning 2 weeks post-initiation, the rats received control diet or diet containing 500 or 1500 ppm DPH or 500 ppm phenobarbital. At 52 weeks age, the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0%, 17% (1 ± 0), 42% (1.8 ± 0.8), or 67% (2.5 ± 1.9) in rats exposed to N-nitrosodiethylamine alone, or the carcinogen followed by 500 ppm DPH, 1500 ppm DPH, or 500 ppm phenobarbital, respectively. Between 53 and 79 weeks of age, 39% of rats receiving N-nitrosodiethylamine alone developed multiple (1.5 ± 0.8) hepatocellular adenomas. A similar incidence (41%) occurred in the rats administered the carcinogen followed by 500 ppm DPH. The incidence of hepatocellular adenomas (88% and 89%) was significantly greater in rats exposed to N-nitrosodiethylamine followed by 1500 ppm DPH or 500 ppm phenobarbital, respectively. Multiplicities of hepatocellular adenomas were significantly greater than the control value in rats fed 1500 ppm DPH or 500 ppm phenobarbital (5.9 ± 4.8 and 10.1 ± 6.7, respectively), but not in the rats receiving 500 ppm DPH (2.3 ± 1.6). No rats exposed to N-nitrosodiethylamine alone or the carcinogen followed by 500 ppm DPH developed hepatocellular carcinomas, while hepatocellular carcinomas occurred in 29% or 67% of the rats given 1500 ppm DPH or 500 ppm phenobarbital, respectively, following initiation. Increases in hepatic CYP2B-mediated benzyloxyresorufin O-dealkylation activity in rats exposed to 500 and 1500 ppm DPH for 2 or 23 weeks were ∼50% and ∼100%, respectively, of the maximal induction caused by 500 ppm phenobarbital. Thus, in the rat model, DPH enhanced N-nitrosodiethyl-amine-initiated hepatocarcinogenesis when administered at a dose causing maximal CYP2B induction.

Enhancement of N-nitrosodiethylamine-initiated hepatocarcinogenesis by phentoin in male F344/NCr rats at a dose causing maximal induction of CYP2B.

The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of male F344/NCr rats initiated with a single i.p. dose of N-nitrosodiethylamine (75 mg/kg b.w.) was studied. Beginning 2 weeks post-initiation, the rats received control diet or diet containing 500 or 1,500 ppm DPH or 500 ppm phenobarbital. At 52 weeks age, the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0%, 17% (1 +/- 0), 42% (1.8 +/- 0.8), or 67% (2.5 +/- 1.9) in rats exposed to N-nitrosodiethylamine alone, or the carcinogen followed by 500 ppm DPH, 1,500 ppm DPH, or 500 ppm phenobarbital, respectively. Between 53 and 79 weeks of age, 39% of rats receiving N-nitrosodiethylamine alone developed multiple (1.5 +/- 0.8) hepatocellular adenomas. A similar incidence (41%) occurred in the rats administered the carcinogen followed by 500 ppm DPH. The incidence of hepatocellular adenomas (88% and 89%) was significantly greater in rats exposed to N-nitrosodiethylamine followed by 1,500 ppm DPH or 500 ppm phenobarbital, respectively. Multiplicities of hepatocellular adenomas were significantly greater than the control value in rats fed 1,500 ppm DPH or 500 ppm phenobarbital (5.9 +/- 4.8 and 10.1 +/- 6.7, respectively), but not in the rats receiving 500 ppm DPH (2.3 +/- 1.6). No rats exposed to N-nitrosodiethylamine alone or the carcinogen followed by 500 ppm DPH developed hepatocellular carcinomas, while hepatocellular carcinomas occurred in 29% or 67% of the rats given 1,500 ppm DPH or 500 ppm phenobarbital, respectively, following initiation. Increases in hepatic CYP2B-mediated benzyloxyresorufin O-dealkylation activity in rats exposed to 500 and 1,500 ppm DPH for 2 or 23 weeks were approximately 50% and approximately 100%, respectively, of the maximal induction caused by 500 ppm phenobarbital. Thus, in the rat model, DPH enhanced N-nitrosodiethyl-amine-initiated hepatocarcinogenesis when administered at a dose causing maximal CYP2B induction.

Dose-related increases in quantitative values for altered hepatocytic foci and cytochrome P-450 levels in the livers of rats exposed to phenobarbital in a medium-term bioassay.

The dose-response relationship between liver tumor promoting activity and cytochrome P-450 (CYP) induction by phenobarbital sodium (PB) was investigated using the liver medium-term bioassay system of Ito. Two weeks after a single dose of N-nitrosodiethylamine (DEN) (200 mg/kg body weight, i.p.), rats were given PB at dietary levels of 500, 250, 125, 60, 30, 15 and 8 parts per million (ppm) for 6 weeks. All rats were subjected to partial hepatectomy at week 3, and were killed at week 8. Quantitative values for glutathione S-transferase placental form positive hepatocytic (GST-P+) foci were increased in the high dose groups dose-dependently. In contrast, the values in the low dose groups were rather lower than that of the control. CYP2B1, 2C6 and 3A2 were predominantly immunostainable in hepatocytes around the central vein. While Western blotting revealed CYP2B1 and 2C6 proteins to be increased with strict dose-dependence, CYP3A2 was only elevated at high doses. Thus, a good correlation between increase of GST-P+ foci and CYP3A2 induction was observed, as well as with CYP2B1 and 2C6 in high dose groups.

Effects of administration of N-nitrosodialkylamines and N-nitrodiethylamine on hepatic UDP-glucuronosyltransferase activity in Wistar rats

N-Nitrosodiethylamine (NEN) and N-nitrodiethylamine (NEA) are carcinogens and in vitro activators of hepatic UDP-glucuronosyltransferase (GT) toward 2-aminophenol (AP) and 4-nitrophenol (NP). In this communication, they were intraperitoneally administered to male Wistar rats for 7 days and GT activities were determined towards AP, NP, phenolphthalein (PH) and testosterone (TS). Administration of 30 or 20 mg/kg dose of NEN caused marked decrease of liver and body weights, and did not affect hepatic GT activities. Injection of 10 mg/kg dose of NEN did not diminish liver and body weights, and increased the maximally activated GT activities toward AP and NP. In contrast, 30 mg/kg dose of NEA, did not affect either liver and body weights or GT activities. N-Nitrosodimethylamine (NMN), which is a carcinogen and a weak in vitro AP GT activator, was more toxic than NEN, and 3.6 mg/kg dose of NMN appears to induce GT toward NP and AP. Administration of 46.5 mg/kg N-nitrosodibutylamine (NBN), which is a carcinogen but not a GT activator, did not affect GT activities or liver body weights.

Sequential study on the synergistic effects of 2,2',4,4',5,5'-hexabromobiphenyl and 3,3',4,4',5,5'-hexabromobiphenyl on hepatic tumor promotion.

A sequential study was completed to determine the effect of polybrominated biphenyl (PBB) congeners on the enhancement of gamma-glutamyl transpeptidase (GGT)-positive altered hepatic foci (AHF) and the development of hepatic nodules (HN) and carcinomas. Female Sprague-Dawley rats were given a single dose of N-nitrosodiethylamine (NDEA) 24 h following a 70% partial hepatectomy. Thirty days later, rats were randomly assigned to groups and fed a basal diet or the basal diet containing 10 p.p.m. 2,2',4,4',5,5'-hexabromobiphenyl (245-HBB), 0.1 p.p.m. 3,3',4,4',5,5'-hexabromobiphenyl (345-HBB) or 10 p.p.m. 245-HBB plus 0.1 p.p.m. 345-HBB for 140 days followed by a basal diet for up to another 310 days. Rats from each group were killed 170, 240 or 480 days after partial hepatectomy. Dietary exposure to 245-HBB and 245-HBB plus 345-HBB enhanced the development of AHF and HN whereas 345-HBB alone did not. The combination of 245-HBB and 345-HBB caused a synergistic effect on the development of AHF and HN. The number of hepatocellular carcinomas was low and evenly distributed among the groups of rats fed diets containing PBB.

Short term induction of preneoplastic nodules in the rat liver. I. The role of 2-AAF as selecting agent

Induction of preneoplastic nodules by a single administration of N-nitrosodiethylamine (NDEA) as initiator and selective stimulation of proliferation by 2-acetylaminofluorene ( 2-AAF) in combination with partial hepatectomy was used to study the effect of different doses of NDEA and 2-AAF on number and size of the nodules. There was a positive relationship between the number of preneoplastic nodules and the dose of NDEA. The marked increased capacity to proliferate with increasing amounts of NDEA suggests an acquired genetically fixed phenomenon. Morphologically there was a striking uniformity of the nodules induced by different doses of NDEA during the first 2 weeks of development, and stimulated by different doses of 2-AAF. This suggests that possible subpopulations were not formed at all during the experimental period of 2 weeks. Alternatively, a genetic heterogenicity may be expressed at a later stage or under different conditions. The effect of 2-AAF reached a plateau at 5 mg/kg body weight; higher concentrations did not influence the number and size of the nodules. Although there was a steady increase in size of the nodules during early development, the number of nodules reached a maximum value at 2 days after partial hepatectomy. It is concluded that 2-AAF only permits selective outgrowth of previously initiated cells by its cytotoxic effects on the surrounding normal tissue and that the carcinogenic properties of 2-AAF in this model do not come to expression.